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nasdaq:regn
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Regeneron Pharmaceuticals
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Jan 12th, 2021 12:00AM
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Apr 29th, 2019 12:00AM
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https://www.uspto.gov?id=US10888601-20210112
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Use of a VEGF antagonist to treat angiogenic eye disorders
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The present invention provides methods for treating angiogenic eye disorders by sequentially administering multiple doses of a VEGF antagonist to a patient. The methods of the present invention include the administration of multiple doses of a VEGF antagonist to a patient at a frequency of once every 8 or more weeks. The methods of the present invention are useful for the treatment of angiogenic eye disorders such as age related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, branch retinal vein occlusion, and corneal neovascularization.
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10888601
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1. A method for treating age related macular degeneration in a patient in need thereof, comprising intravitreally administering, to said patient, an effective amount of aflibercept which is 2 mg approximately every 4 weeks for the first 3 months, followed by 2 mg approximately once every 8 weeks or once every 2 months.
2. The method of claim 1, wherein the age-related macular degeneration is neovascular (wet).
3. The method of claim 2 wherein the patient loses less than 15 letters of Best Corrected Visual Acuity (BCVA) score.
4. The method of claim 3 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
5. The method of claim 2 wherein the patient gains at least 15 letters of Best Corrected Visual Acuity (BCVA) score.
6. The method of claim 5 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
7. The method of claim 1, wherein approximately every 4 weeks comprises approximately every 28 days or approximately monthly.
8. The method of claim 7, wherein the age-related macular degeneration is neovascular (wet).
9. The method of claim 8 wherein exclusion criteria for the patient include (1) active intraocular inflammation; or (2) active ocular or periocular infection.
10. A method for treating diabetic macular edema in a patient in need thereof, comprising intravitreally administering, to said patient, an effective amount of aflibercept which is 2 mg approximately every 4 weeks for the first 5 injections followed by 2 mg approximately once every 8 weeks or once every 2 months.
11. The method of claim 10, wherein approximately every 4 weeks comprises approximately every 28 days or approximately monthly.
12. The method of claim 10, further comprising, after 20 weeks, administering, via intravitreal injection, 2 mg of aflibercept once every 4 weeks.
13. The method of claim 10 wherein the patient loses less than 15 letters of Best Corrected Visual Acuity (BCVA) score.
14. The method of claim 13 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
15. The method of claim 10 wherein the patient gains at least 15 letters of Best Corrected Visual Acuity (BCVA) score.
16. The method of claim 15 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
17. The method of claim 10 wherein exclusion criteria for the patient include (1) active intraocular inflammation; or (2) active ocular or periocular infection.
18. A method for treating diabetic retinopathy in a patient in need thereof, comprising intravitreally administering, to said patient, an effective amount of aflibercept which is 2 mg approximately every 4 weeks for the first 5 injections followed by 2 mg approximately once every 8 weeks or 2 months.
19. The method of claim 18, wherein approximately every 4 weeks comprises approximately every 28 days or approximately monthly.
20. The method of claim 19 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
21. The method of claim 18, further comprising, after 20 weeks, administering, via intravitreal injection, 2 mg of aflibercept once every 4 weeks.
22. The method of claim 18 wherein the patient loses less than 15 letters of Best Corrected Visual Acuity (BCVA) score.
23. The method of claim 18 wherein the patient gains at least 15 letters of Best Corrected Visual Acuity (BCVA) score.
24. The method of claim 23 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
25. The method of claim 18 wherein exclusion criteria for the patient include (1) active intraocular inflammation; or (2) active ocular or periocular infection.
26. A method for treating diabetic retinopathy in a patient with diabetic macular edema, who is in need of such treatment, comprising intravitreally administering, to said patient, an effective amount of aflibercept which is 2 mg approximately every 4 weeks for the first 5 injections followed by 2 mg approximately once every 8 weeks or 2 months.
27. The method of claim 26, wherein approximately every 4 weeks comprises approximately every 28 days or approximately monthly.
28. The method of claim 26, further comprising, after 20 weeks, administering, via intravitreal injection, 2 mg of aflibercept once every 4 weeks.
29. The method of claim 26 wherein the patient loses less than 15 letters of Best Corrected Visual Acuity (BCVA) score.
30. The method of claim 29 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
31. The method of claim 26 wherein the patient gains at least 15 letters of Best Corrected Visual Acuity (BCVA) score.
32. The method of claim 31 wherein Best Corrected Visual Acuity (BCVA) is according to Early Treatment Diabetic Retinopathy Study (ETDRS) letter score.
33. The method of claim 26 wherein exclusion criteria for the patient include (1) active intraocular inflammation; or (2) active ocular or periocular infection.
34. A method for treating an angiogenic eye disorder in a patient in need thereof, said method comprising administering to the patient an effective sequential dosing regimen of a single initial dose of a VEGF antagonist, followed by one or more secondary doses of the VEGF antagonist, followed by one or more tertiary doses of the VEGF antagonist;
wherein each secondary dose is administered 4 weeks after the immediately preceding dose; and
wherein each tertiary dose is administered 8 weeks after the immediately preceding dose;
wherein the VEGF antagonist is a receptor-based chimeric molecule comprising
an immunoglobin-like (Ig) domain 2 of a first VEGF receptor which is VEGFR1 and an Ig domain 3 of a second VEGF receptor which is VEGFR2, and a multimerizing component.
35. The method of claim 34 wherein the VEGF antagonist is aflibercept.
36. The method of claim 35 wherein exclusion criteria for the patient include (1) active intraocular inflammation; or (2) active ocular or periocular infection.
37. The method of claim 34, wherein all doses of the VEGF antagonist are administered to the patient by intraocular administration.
38. The method of claim 37, wherein the intraocular administration is intravitreal administration.
39. The method of claim 38, wherein all doses of the VEGF antagonist comprise from about 0.5 mg to about 2 mg of the VEGF antagonist.
40. The method of claim 39, wherein all doses of the VEGF antagonist comprise 0.5 mg of the VEGF antagonist.
41. The method of claim 39, wherein all doses of the VEGF antagonist comprise 2 mg of the VEGF antagonist.
42. The method of claim 34, wherein the angiogenic eye disorder is selected from the group consisting of: age related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, branch retinal vein occlusion, and corneal neovascularization.
43. The method of claim 34 wherein the angiogenic eye disorder is age related macular degeneration.
44. The method of claim 43 wherein all doses of VEGF antagonist comprise 0.5 mg of the VEGF antagonist.
45. The method of claim 43 wherein all doses of VEGF antagonist comprise 2.0 mg of the VEGF antagonist.
46. The method of claim 34 wherein the angiogenic eye disorder is diabetic retinopathy.
47. The method of claim 34, wherein the angiogenic eye disorder is diabetic macular edema.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of Ser. No. 16/159,282 filed Oct. 12, 2018, which is a continuation of Ser. No. 15/471,506 filed Mar. 28, 2017, now U.S. Pat. No. 10,130,691 issued Nov. 20, 2018, which is a continuation of Ser. No. 14/972,560 filed Dec. 17, 2015, now U.S. Pat. No. 9,669,069 issued Jun. 6, 2017, which is a continuation of Ser. No. 13/940,370 filed Jul. 12, 2013, now U.S. Pat. No. 9,254,338 issued Feb. 9, 2016, which is a continuation-in-part of International Patent Application No. PCT/US2012/020855, filed on Jan. 11, 2012, which claims the benefit of U.S. Provisional Application No. 61/432,245, filed on Jan. 13, 2011, 61/434,836, filed on Jan. 21, 2011, and 61/561,957, filed on Nov. 21, 2011, the contents of which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to the field of therapeutic treatments of eye disorders. More specifically, the invention relates to the administration of VEGF antagonists to treat eye disorders caused by or associated with angiogenesis.
BACKGROUND
Several eye disorders are associated with pathological angiogenesis. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from the CNV causes macular edema and collection of fluid beneath the macula resulting in vision loss. Diabetic macular edema (DME) is another eye disorder with an angiogenic component. DME is the most prevalent cause of moderate vision loss in patients with diabetes and is a common complication of diabetic retinopathy, a disease affecting the blood vessels of the retina. Clinically significant DME occurs when fluid leaks into the center of the macula, the light-sensitive part of the retina responsible for sharp, direct vision. Fluid in the macula can cause severe vision loss or blindness. Yet another eye disorder associated with abnormal angiogenesis is central retinal vein occlusion (CRVO). CRVO is caused by obstruction of the central retinal vein that leads to a back-up of blood and fluid in the retina. The retina can also become ischemic, resulting in the growth of new, inappropriate blood vessels that can cause further vision loss and more serious complications. Release of vascular endothelial growth factor (VEGF) contributes to increased vascular permeability in the eye and inappropriate new vessel growth. Thus, inhibiting the angiogenic-promoting properties of VEGF appears to be an effective strategy for treating angiogenic eye disorders.
FDA-approved treatments of angiogenic eye disorders such as AMD and CRVO include the administration of an anti-VEGF antibody called ranibizumab (Lucentis®, Genentech, Inc.) on a monthly basis by intravitreal injection.
Methods for treating eye disorders using VEGF antagonists are mentioned in, e.g., U.S. Pat. Nos. 7,303,746; 7,306,799; 7,300,563; 7,303,748; and US 2007/0190058. Nonetheless, there remains a need in the art for new administration regimens for angiogenic eye disorders, especially those which allow for less frequent dosing while maintaining a high level of efficacy.
BRIEF SUMMARY OF THE INVENTION
The present invention provides methods for treating angiogenic eye disorders. The methods of the invention comprise sequentially administering multiple doses of a VEGF antagonist to a patient over time. In particular, the methods of the invention comprise sequentially administering to the patient a single initial dose of a VEGF antagonist, followed by one or more secondary doses of the VEGF antagonist, followed by one or more tertiary doses of the VEGF antagonists. The present inventors have surprisingly discovered that beneficial therapeutic effects can be achieved in patients suffering from angiogenic eye disorders by administering a VEGF antagonist to a patient at a frequency of once every 8 or more weeks, especially when such doses are preceded by about three doses administered to the patient at a frequency of about 2 to 4 weeks. Thus, according to the methods of the present invention, each secondary dose of VEGF antagonist is administered 2 to 4 weeks after the immediately preceding dose, and each tertiary dose is administered at least 8 weeks after the immediately preceding dose. An example of a dosing regimen of the present invention is shown in FIG. 1. One advantage of such a dosing regimen is that, for most of the course of treatment (i.e., the tertiary doses), it allows for less frequent dosing (e.g., once every 8 weeks) compared to prior administration regimens for angiogenic eye disorders which require monthly administrations throughout the entire course of treatment. (See, e.g., prescribing information for Lucentis® [ranibizumab], Genentech, Inc.).
The methods of the present invention can be used to treat any angiogenic eye disorder, including, e.g., age related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, corneal neovascularization, etc.
The methods of the present invention comprise administering any VEGF antagonist to the patient. In one embodiment, the VEGF antagonist comprises one or more VEGF receptor-based chimeric molecule(s), (also referred to herein as a “VEGF-Trap” or “VEGFT”). An exemplary VEGF antagonist that can be used in the context of the present invention is a multimeric VEGF-binding protein comprising two or more VEGF receptor-based chimeric molecules referred to herein as “VEGFR1R2-FcΔC1 (a)” or “aflibercept.”
Various administration routes are contemplated for use in the methods of the present invention, including, e.g., topical administration or intraocular administration (e.g., intravitreal administration).
Aflibercept (EYLEA™, Regeneron Pharmaceuticals, Inc) was approved by the FDA in November 2011, for the treatment of patients with neovascular (wet) age-related macular degeneration, with a recommended dose of 2 mg administered by intravitreal injection every 4 weeks for the first three months, followed by 2 mg administered by intravitreal injection once every 8 weeks.
Other embodiments of the present invention will become apparent from a review of the ensuing detailed description.
BRIEF DESCRIPTION OF THE FIGURE
FIG. 1 shows an exemplary dosing regimen of the present invention. In this regimen, a single “initial dose” of VEGF antagonist (“VEGFT”) is administered at the beginning of the treatment regimen (i.e. at “week 0”), two “secondary doses” are administered at weeks 4 and 8, respectively, and at least six “tertiary doses” are administered once every 8 weeks thereafter, i.e., at weeks 16, 24, 32, 40, 48, 56, etc.).
DETAILED DESCRIPTION
Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term “about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Dosing Regimens
The present invention provides methods for treating angiogenic eye disorders. The methods of the invention comprise sequentially administering to a patient multiple doses of a VEGF antagonist. As used herein, “sequentially administering” means that each dose of VEGF antagonist is administered to the patient at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of a VEGF antagonist, followed by one or more secondary doses of the VEGF antagonist, followed by one or more tertiary doses of the VEGF antagonist.
The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the VEGF antagonist. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of VEGF antagonist, but will generally differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of VEGF antagonist contained in the initial, secondary and/or tertiary doses will vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
In one exemplary embodiment of the present invention, each secondary dose is administered 2 to 4 (e.g., 2, 2½, 3, 3½, or 4) weeks after the immediately preceding dose, and each tertiary dose is administered at least 8 (e.g., 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of VEGF antagonist which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
In one exemplary embodiment of the present invention, a single initial dose of a VEGF antagonist is administered to a patient on the first day of the treatment regimen (i.e., at week 0), followed by two secondary doses, each administered four weeks after the immediately preceding dose (i.e., at week 4 and at week 8), followed by at least 5 tertiary doses, each administered eight weeks after the immediately preceding dose (i.e., at weeks 16, 24, 32, 40 and 48). The tertiary doses may continue (at intervals of 8 or more weeks) indefinitely during the course of the treatment regimen. This exemplary administration regimen is depicted graphically in FIG. 1.
The methods of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of a VEGF antagonist. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 4 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 8 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. For example, the present invention includes methods which comprise administering to the patient a single initial dose of a VEGF antagonist, followed by one or more secondary doses of the VEGF antagonist, followed by at least 5 tertiary doses of the VEGF antagonist, wherein the first four tertiary doses are administered 8 weeks after the immediately preceding dose, and wherein each subsequent tertiary dose is administered from 8 to 12 (e.g., 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12) weeks after the immediately preceding dose. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
Vegf Antagonists
The methods of the present invention comprise administering to a patient a VEGF antagonist according to specified dosing regimens. As used herein, the expression “VEGF antagonist” means any molecule that blocks, reduces or interferes with the normal biological activity of VEGF.
VEGF antagonists include molecules which interfere with the interaction between VEGF and a natural VEGF receptor, e.g., molecules which bind to VEGF or a VEGF receptor and prevent or otherwise hinder the interaction between VEGF and a VEGF receptor. Specific exemplary VEGF antagonists include anti-VEGF antibodies, anti-VEGF receptor antibodies, and VEGF receptor-based chimeric molecules (also referred to herein as “VEGF-Traps”).
VEGF receptor-based chimeric molecules include chimeric polypeptides which comprise two or more immunoglobulin (Ig)-like domains of a VEGF receptor such as VEGFR1 (also referred to as Flt1) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also contain a multimerizing domain (e.g., an Fc domain which facilitates the multimerization [e.g., dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is a molecule referred to as VEGFR1R2-FcΔC1(a) which is encoded by the nucleic acid sequence of SEQ ID NO:1. VEGFR1R2-FcΔC1(a) comprises three components: (1) a VEGFR1 component comprising amino acids 27 to 129 of SEQ ID NO:2; (2) a VEGFR2 component comprising amino acids 130 to 231 of SEQ ID NO:2; and (3) a multimerization component (“FcΔC1(a)”) comprising amino acids 232 to 457 of SEQ ID NO:2 (the C-terminal amino acid of SEQ ID NO:2 [i.e., K458] may or may not be included in the VEGF antagonist used in the methods of the invention; see e.g., U.S. Pat. No. 7,396,664). Amino acids 1-26 of SEQ ID NO:2 are the signal sequence.
The VEGF antagonist used in the Examples set forth herein below is a dimeric molecule comprising two VEGFR1R2-FcΔC1(a) molecules and is referred to herein as “VEGFT.” Additional VEGF receptor-based chimeric molecules which can be used in the context of the present invention are disclosed in U.S. Pat. Nos. 7,396,664, 7,303,746 and WO 00/75319.
Angiogenic Eye Disorders
The methods of the present invention can be used to treat any angiogenic eye disorder. The expression “angiogenic eye disorder,” as used herein, means any disease of the eye which is caused by or associated with the growth or proliferation of blood vessels or by blood vessel leakage. Non-limiting examples of angiogenic eye disorders that are treatable using the methods of the present invention include age-related macular degeneration (e.g., wet AMD, exudative AMD, etc.), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO; e.g., macular edema following CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV; e.g., myopic CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, and diabetic retinopathies.
Pharmaceutical Formulations
The present invention includes methods in which the VEGF antagonist that is administered to the patient is contained within a pharmaceutical formulation. The pharmaceutical formulation may comprise the VEGF antagonist along with at least one inactive ingredient such as, e.g., a pharmaceutically acceptable carrier. Other agents may be incorporated into the pharmaceutical composition to provide improved transfer, delivery, tolerance, and the like. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the antibody is administered. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa., 1975), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in the context of the methods of the present invention, provided that the VEGF antagonist is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Powell et al. PDA (1998) J Pharm Sci Technol. 52:238-311 and the citations therein for additional information related to excipients and carriers well known to pharmaceutical chemists.
Pharmaceutical formulations useful for administration by injection in the context of the present invention may be prepared by dissolving, suspending or emulsifying a VEGF antagonist in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there may be employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule if desired.
Modes of Administration
The VEGF antagonist (or pharmaceutical formulation comprising the VEGF antagonist) may be administered to the patient by any known delivery system and/or administration method. In certain embodiments, the VEGF antagonist is administered to the patient by ocular, intraocular, intravitreal or subconjunctival injection. In other embodiments, the VEGF antagonist can be administered to the patient by topical administration, e.g., via eye drops or other liquid, gel, ointment or fluid which contains the VEGF antagonist and can be applied directly to the eye. Other possible routes of administration include, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral.
Amount of VEGF Antagonist Administered
Each dose of VEGF antagonist administered to the patient over the course of the treatment regimen may contain the same, or substantially the same, amount of VEGF antagonist. Alternatively, the quantity of VEGF antagonist contained within the individual doses may vary over the course of the treatment regimen. For example, in certain embodiments, a first quantity of VEGF antagonist is administered in the initial dose, a second quantity of VEGF antagonist is administered in the secondary doses, and a third quantity of VEGF antagonist is administered in the tertiary doses. The present invention contemplates dosing schemes in which the quantity of VEGF antagonist contained within the individual doses increases over time (e.g., each subsequent dose contains more VEGF antagonist than the last), decreases over time (e.g., each subsequent dose contains less VEGF antagonist than the last), initially increases then decreases, initially decreases then increases, or remains the same throughout the course of the administration regimen.
The amount of VEGF antagonist administered to the patient in each dose is, in most cases, a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means a dose of VEGF antagonist that results in a detectable improvement in one or more symptoms or indicia of an angiogenic eye disorder, or a dose of VEGF antagonist that inhibits, prevents, lessens, or delays the progression of an angiogenic eye disorder. In the case of an anti-VEGF antibody or a VEGF receptor-based chimeric molecule such as VEGFR1R2-FcΔC1(a), a therapeutically effective amount can be from about 0.05 mg to about 5 mg, e.g., about 0.05 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 1.0 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg, about 1.2 mg, about 1.25 mg, about 1.3 mg, about 1.35 mg, about 1.4 mg, about 1.45 mg, about 1.5 mg, about 1.55 mg, about 1.6 mg, about 1.65 mg, about 1.7 mg, about 1.75 mg, about 1.8 mg, about 1.85 mg, about 1.9 mg, about 2.0 mg, about 2.05 mg, about 2.1 mg, about 2.15 mg, about 2.2 mg, about 2.25 mg, about 2.3 mg, about 2.35 mg, about 2.4 mg, about 2.45 mg, about 2.5 mg, about 2.55 mg, about 2.6 mg, about 2.65 mg, about 2.7 mg, about 2.75 mg, about 2.8 mg, about 2.85 mg, about 2.9 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg of the antibody or receptor-based chimeric molecule.
The Amount of VEGF Antagonist Contained within the Individual Doses May be Expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, the VEGF antagonist may be administered to a patient at a dose of about 0.0001 to about 10 mg/kg of patient body weight.
Treatment Population and Efficacy
The methods of the present invention are useful for treating angiogenic eye disorders in patients that have been diagnosed with or are at risk of being afflicted with an angiogenic eye disorder. Generally, the methods of the present invention demonstrate efficacy within 104 weeks of the initiation of the treatment regimen (with the initial dose administered at “week 0”), e.g., by the end of week 16, by the end of week 24, by the end of week 32, by the end of week 40, by the end of week 48, by the end of week 56, etc. In the context of methods for treating angiogenic eye disorders such as AMD, CRVO, and DME, “efficacy” means that, from the initiation of treatment, the patient exhibits a loss of 15 or fewer letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart. In certain embodiments, “efficacy” means a gain of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) letters on the ETDRS chart from the time of initiation of treatment.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
The exemplary VEGF antagonist used in all Examples set forth below is a dimeric molecule having two functional VEGF binding units. Each functional binding unit is comprised of Ig domain 2 from VEGFR1 fused to Ig domain 3 from VEGFR2, which in turn is fused to the hinge region of a human IgG1 Fc domain (VEGFR1R2-FcΔC1(a); encoded by SEQ ID NO:1). This VEGF antagonist is referred to in the examples below as “VEGFT”. For purposes of the following Examples, “monthly” dosing is equivalent to dosing once every four weeks.
Example 1: Phase I Clinical Trial of Intravitreally Administered VEGF Receptor-Based Chimeric Molecule (VEGFT) in Subjects with Neovascular AMD
In this Phase I study, 21 subjects with neovascular AMD received a single intravitreal (IVT) dose of VEGFT. Five groups of three subjects each received either 0.05, 0.15, 0.5, 2 or 4 mg of VEGFT, and a sixth group of six subjects received 1 mg. No serious adverse events related to the study drug, and no identifiable intraocular inflammation was reported. Preliminary results showed that, following injection of VEGFT, a rapid decrease in foveal thickness and macular volume was observed that was maintained through 6 weeks. At Day 43 across all dose groups, mean excess retinal thickness [excess retinal thickness=(retinal thickness −179μ)] on optical coherence tomography (OCT) was reduced from 119μ to 27μ as assessed by Fast Macular Scan and from 194μ to 60μ as assessed using a single Posterior Pole scan. The mean increase in best corrected visual acuity (BCVA) was 4.75 letters, and BCVA was stable or improved in 95% of subjects. In the 2 highest dose groups (2 and 4 mg), the mean increase in BCVA was 13.5 letters, with 3 of 6 subjects demonstrating improvement of 3 lines.
Example 2: Phase II Clinical Trial of Repeated Doses of Intravitreally Administered VEGF Receptor-Based Chimeric Molecule (VEGFT) in Subjects with Neovascular AMD
This study was a double-masked, randomized study of 3 doses (0.5, 2, and 4 mg) of VEGFT tested at 4-week and/or 12-week dosing intervals. There were 5 treatment arms in this study, as follows: 1) 0.5 mg every 4 weeks, 2) 0.5 mg every 12 weeks, 3) 2 mg every 4 weeks, 4) 2 mg every 12 weeks and 5) 4 mg every 12 weeks. Subjects were dosed at a fixed interval for the first 12 weeks, after which they were evaluated every 4 weeks for 9 months, during which additional doses were administered based on pre-specified criteria. All subjects were then followed for one year after their last dose of VEGFT. Preliminary data from a pre-planned interim analysis indicated that VEGFT met its primary endpoint of a statistically significant reduction in retinal thickness after 12 weeks compared with baseline (all groups combined, decrease of 135μ, p<0.0001). Mean change from baseline in visual acuity, a key secondary endpoint of the study, also demonstrated statistically significant improvement (all groups combined, increase of 5.9 letters, p<0.0001). Moreover, patients in the dose groups that received only a single dose, on average, demonstrated a decrease in excess retinal thickness (p<0.0001) and an increase in visual acuity (p=0.012) at 12 weeks. There were no drug-related serious adverse events, and treatment with the VEGF antagonists was generally well-tolerated. The most common adverse events were those typically associated with intravitreal injections.
Example 3: Phase I Clinical Trial of Systemically Administered VEGF Receptor-Based Chimeric Molecule (VEGFT) in Subjects with Neovascular AMD
This study was a placebo-controlled, sequential-group, dose-escalating safety, tolerability and bioeffect study of VEGFT by IV infusion in subjects with neovascular AMD. Groups of 8 subjects meeting eligibility criteria for subfoveal choroidal neovascularization (CNV) related to AMD were assigned to receive 4 IV injections of VEGFT or placebo at dose levels of 0.3, 1, or 3 mg/kg over an 8-week period.
Most adverse events that were attributed to VEGFT were mild to moderate in severity, but 2 of 5 subjects treated with 3 mg/kg experienced dose-limiting toxicity (DLT) (one with Grade 4 hypertension and one with Grade 2 proteinuria); therefore, all subjects in the 3 mg/kg dose group did not enter the study. The mean percent changes in excess retinal thickness were: −12%, −10%, −66%, and −60% for the placebo, 0.3, 1, and 3 mg/kg dose groups at day 15 (ANOVA p<0.02), and −5.6%, +47.1%, and −63.3% for the placebo, 0.3, and 1 mg/kg dose groups at day 71 (ANOVA p<0.02). There was a numerical improvement in BCVA in the subjects treated with VEGFT. As would be expected in such a small study, the results were not statistically significant.
Example 4: Phase III Clinical Trials of the Efficacy, Safety, and Tolerability of Repeated Doses of Intravitreal VEGFT in Subjects with Neovascular Age-Related Macular Degeneration
A. Objectives, Hypotheses and Endpoints
Two parallel Phase III clinical trials were carried out to investigate the use of VEGFT to treat patients with the neovascular form of age-related macular degeneration (Study 1 and Study 2). The primary objective of these studies was to assess the efficacy of IVT administered VEGFT compared to ranibizumab (Lucentis®, Genentech, Inc.), in a non-inferiority paradigm, in preventing moderate vision loss in subjects with all subtypes of neovascular AMD.
The secondary objectives were (a) to assess the safety and tolerability of repeated IVT administration of VEGFT in subjects with all sub-types of neovascular AMD for periods up to 2 years; and (b) to assess the effect of repeated IVT administration of VEGFT on Vision-Related Quality of Life (QOL) in subjects with all sub-types of neovascular AMD.
The primary hypothesis of these studies was that the proportion of subjects treated with VEGFT with stable or improved BCVA (<15 letters lost) is similar to the proportion treated with ranibizumab who have stable or improved BCVA, thereby demonstrating non-inferiority.
The primary endpoint for these studies was the prevention of vision loss of greater than or equal to 15 letters on the ETDRS chart, compared to baseline, at 52 weeks. Secondary endpoints were as follows: (a) change from baseline to Week 52 in letter score on the ETDRS chart; (b) gain from baseline to Week 52 of 15 letters or more on the ETDRS chart; (c) change from baseline to Week 52 in total NEI VFQ-25 score; and (d) change from baseline to Week 52 in CNV area.
B. Study Design
For each study, subjects were randomly assigned in a 1:1:1:1 ratio to 1 of 4 dosing regimens: (1) 2 mg VEGFT administered every 4 weeks (2Q4); (2) 0.5 mg VEGFT administered every 4 weeks (0.5Q4); (3) 2 mg VEGFT administered every 4 weeks to week 8 and then every 8 weeks (with sham injection at the interim 4-week visits when study drug was not administered (2Q8); and (4) 0.5 mg ranibizumab administered every 4 weeks (RQ4). Subjects assigned to (2Q8) received the 2 mg injection every 4 weeks to week 8 and then a sham injection at interim 4-week visits (when study drug is not to be administered) during the first 52 weeks of the studies. (No sham injection were given at Week 52).
The study duration for each subject was scheduled to be 96 weeks plus the recruitment period. For the first 52 weeks (Year 1), subjects received an IVT or sham injection in the study eye every 4 weeks. (No sham injections were given at Week 52). During the second year of the study, subjects will be evaluated every 4 weeks and will receive IVT injection of study drug at intervals determined by specific dosing criteria, but at least every 12 weeks. (During the second year of the study, sham injections will not be given.) During this period, injections may be given as frequently as every 4 weeks, but no less frequently than every 12 weeks, according to the following criteria: (i) increase in central retinal thickness of ≥100 μm compared to the lowest previous value as measured by optical coherence tomography (OCT); or (ii) a loss from the best previous letter score of at least 5 ETDRS letters in conjunction with recurrent fluid as indicated by OCT; or (iii) new or persistent fluid as indicated by OCT; or (iv) new onset classic neovascularization, or new or persistent leak on fluorescein angiography (FA); or (v) new macular hemorrhage; or (vi) 12 weeks have elapsed since the previous injection. According to the present protocol, subjects must receive an injection at least every 12 weeks.
Subjects were evaluated at 4 weeks intervals for safety and best corrected visual acuity (BCVA) using the 4 meter ETDRS protocol. Quality of Life (QOL) was evaluated using the NEI VFQ-25 questionnaire. OCT and FA examinations were conducted periodically.
Approximately 1200 subjects were enrolled, with a target enrollment of 300 subjects per treatment arm.
To be eligible for this study, subjects were required to have subfoveal choroidal neovascularization (CNV) secondary to AMD. “Subfoveal” CNV was defined as the presence of subfoveal neovascularization, documented by FA, or presence of a lesion that is juxtafoveal in location angiographically but affects the fovea. Subject eligibility was confirmed based on angiographic criteria prior to randomization.
Only one eye was designated as the study eye. For subjects who met eligibility criteria in both eyes, the eye with the worse VA was selected as the study eye. If both eyes had equal VA, the eye with the clearest lens and ocular media and least amount of subfoveal scar or geographic atrophy was selected. If there was no objective basis for selecting the study eye, factors such as ocular dominance, other ocular pathology and subject preference were considered in making the selection.
Inclusion criteria for both studies were as follows: (i) signed Informed consent; (ii) at least 50 years of age; (iii) active primary subfoveal CNV lesions secondary to AMD, including juxtafoveal lesions that affect the fovea as evidenced by FA in the study eye; (iv) CNV at least 50% of total lesion size; (v) early treatment diabetic retinopathy study (ETDRS) best-corrected visual acuity of: 20/40 to 20/320 (letter score of 73 to 25) in the study eye; (vi) willing, committed, and able to return for all clinic visits and complete all study-related procedures; and (vii) able to read, understand and willing to sign the informed consent form (or, if unable to read due to visual impairment, be read to verbatim by the person administering the informed consent or a family member).
Exclusion criteria for both studies were as follows: 1. Any prior ocular (in the study eye) or systemic treatment or surgery for neovascular AMD except dietary supplements or vitamins. 2. Any prior or concomitant therapy with another investigational agent to treat neovascular AMD in the study eye, except dietary supplements or vitamins. 3. Prior treatment with anti-VEGF agents as follows: (a) Prior treatment with anti-VEGF therapy in the study eye was not allowed; (b) Prior treatment with anti-VEGF therapy in the fellow eye with an investigational agent (not FDA approved, e.g. bevacizumab) was allowed up to 3 months prior to first dose in the study, and such treatments were not allowed during the study. Prior treatment with an approved anti-VEGF therapy in the fellow eye was allowed; (c) Prior systemic anti-VEGF therapy, investigational or FDA/Health Canada approved, was only allowed up to 3 months prior to first dose, and was not allowed during the study. 4. Total lesion size >12 disc areas (30.5 mm2, including blood, scars and neovascularization) as assessed by FA in the study eye. 5. Subretinal hemorrhage that is either 50% or more of the total lesion area, or if the blood is under the fovea and is 1 or more disc areas in size in the study eye. (If the blood is under the fovea, then the fovea must be surrounded 270 degrees by visible CNV.) 6. Scar or fibrosis, making up >50% of total lesion in the study eye. 7. Scar, fibrosis, or atrophy involving the center of the fovea. 8. Presence of retinal pigment epithelial tears or rips involving the macula in the study eye. 9. History of any vitreous hemorrhage within 4 weeks prior to Visit 1 in the study eye. 10. Presence of other causes of CNV, including pathologic myopia (spherical equivalent of −8 diopters or more negative, or axial length of 25 mm or more), ocular histoplasmosis syndrome, angioid streaks, choroidal rupture, or multifocal choroiditis in the study eye. 11. History or clinical evidence of diabetic retinopathy, diabetic macular edema or any other vascular disease affecting the retina, other than AMD, in either eye. 12. Prior vitrectomy in the study eye. 13. History of retinal detachment or treatment or surgery for retinal detachment in the study eye. 14. Any history of macular hole of stage 2 and above in the study eye. 15. Any intraocular or periocular surgery within 3 months of Day 1 on the study eye, except lid surgery, which may not have taken place within 1 month of day 1, as long as it was unlikely to interfere with the injection. 16. Prior trabeculectomy or other filtration surgery in the study eye. 17. Uncontrolled glaucoma (defined as intraocular pressure greater than or equal to 25 mm Hg despite treatment with anti-glaucoma medication) in the study eye. 18. Active intraocular inflammation in either eye. 19. Active ocular or periocular infection in either eye. 20. Any ocular or periocular infection within the last 2 weeks prior to Screening in either eye. 21. Any history of uveitis in either eye. 22. Active scleritis or episcleritis in either eye. 23. Presence or history of scleromalacia in either eye. 24. Aphakia or pseudophakia with absence of posterior capsule (unless it occurred as a result of a yttrium aluminum garnet [YAG] posterior capsulotomy) in the study eye. 25. Previous therapeutic radiation in the region of the study eye. 26. History of corneal transplant or corneal dystrophy in the study eye. 27. Significant media opacities, including cataract, in the study eye which might interfere with visual acuity, assessment of safety, or fundus photography. 28. Any concurrent intraocular condition in the study eye (e.g. cataract) that, in the opinion of the investigator, could require either medical or surgical intervention during the 96 week study period. 29. Any concurrent ocular condition in the study eye which, in the opinion of the investigator, could either increase the risk to the subject beyond what is to be expected from standard procedures of intraocular injection, or which otherwise may interfere with the injection procedure or with evaluation of efficacy or safety. 30. History of other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding giving reasonable suspicion of a disease or condition that contraindicates the use of an investigational drug or that might affect interpretation of the results of the study or render the subject at high risk for treatment complications. 31. Participation as a subject in any clinical study within the 12 weeks prior to Day 1. 32. Any systemic or ocular treatment with an investigational agent in the past 3 months prior to Day 1. 33. The use of long acting steroids, either systemically or intraocularly, in the 6 months prior to day 1. 34. Any history of allergy to povidone iodine. 35. Known serious allergy to the fluorescein sodium for injection in angiography. 36. Presence of any contraindications indicated in the FDA Approved label for ranibizumab (Lucentis®). 37. Females who were pregnant, breastfeeding, or of childbearing potential, unwilling to practice adequate contraception throughout the study. Adequate contraceptive measures include oral contraceptives (stable use for 2 or more cycles prior to screening); IUD; Depo-Provera®; Norplant® System implants; bilateral tubal ligation; vasectomy; condom or diaphragm plus either contraceptive sponge, foam or jelly.
Subjects were not allowed to receive any standard or investigational agents for treatment of their AMD in the study eye other than their assigned study treatment with VEGFT or ranibizumab as specified in the protocol until they completed the Completion/Early Termination visit assessments. This includes medications administered locally (e.g., IVT, topical, juxtascleral or periorbital routes), as well as those administered systemically with the intent of treating the study and/or fellow eye.
The study procedures are summarized as follows:
Best Corrected Visual Acuity:
Visual function of the study eye and the fellow eye were assessed using the ETDRS protocol (The Early Treatment Diabetic Retinopathy Study Group) at 4 meters. Visual Acuity examiners were certified to ensure consistent measurement of BCVA. The VA examiners were required to remain masked to treatment assignment.
Optical Coherence Tomography:
Retinal and lesion characteristics were evaluated using OCT on the study eye. At the Screen Visit (Visit 1) images were captured and transmitted for both eyes. All OCT images were captured using the Zeiss Stratus OCT™ with software Version 3 or greater. OCT images were sent to an independent reading center where images were read by masked readers at visits where OCTs were required. All OCTs were electronically archived at the site as part of the source documentation. A subset of OCT images were read. OCT technicians were required to be certified by the reading center to ensure consistency and quality in image acquisition. Adequate efforts were made to ensure that OCT technicians at the site remained masked to treatment assignment.
Fundus Photography and Fluorescein Angiography (FA):
The anatomical state of the retinal vasculature of the study eye was evaluated by funduscopic examination, fundus photography and FA. At the Screen Visit (Visit 1) funduscopic examination, fundus photography and FA were captured and transmitted for both eyes. Fundus and angiographic images were sent to an independent reading center where images were read by masked readers. The reading center confirmed subject eligibility based on angiographic criteria prior to randomization. All FAs and fundus photographs were archived at the site as part of the source documentation. Photographers were required to be certified by the reading center to ensure consistency and quality in image acquisition. Adequate efforts were made to ensure that all photographers at the site remain masked to treatment assignment.
Vision-Related Quality of Life:
Vision-related QOL was assessed using the National Eye Institute 25-Item Visual Function Questionnaire (NEI VFQ-25) in the interviewer-administered format. NEI VFQ-25 was administered by certified personnel at a contracted call center. At the screening visit, the sites assisted the subject and initiated the first call to the call center to collect all of the subject's contact information and to complete the first NEI VFQ-25 on the phone prior to randomization and IVT injection. For all subsequent visits, the call center called the subject on the phone, prior to IVT injection, to complete the questionnaire.
Intraocular Pressure:
Intraocular pressure (10P) of the study eye was measured using applanation tonometry or Tonopen. The same method of 10P measurement was used in each subject throughout the study.
C. Results Summary (52 Week Data)
The primary endpoint (prevention of moderate or severe vision loss as defined above) was met for all three VEGFT groups (2Q4, 0.5Q4 and 2Q8) in this study. The results from both studies are summarized in Table 1.
TABLE 1
VEGFT
Ranibizumab
0.5 mg
VEGFT
VEGFT
0.5 mg monthly
monthly
2 mg monthly
2 mg every 8
(RQ4)
(0.5Q4)
(2Q4)
weeks[a] (2Q8)
Maintenance of vision* (% patients losing <15 letters) at week 52
versus baseline
Study 1
94.4%
95.9%**
95.1%**
95.1%**
Study 2
94.4%
96.3%**
95.6%**
95.6%**
Mean improvement in vision* (letters) at 52 weeks versus
baseline (p-value vs RQ4)***
Study 1
8.1
6.9 (NS)
10.9 (p < 0.01)
7.9 (NS)
Study 2
9.4
9.7 (NS)
7.6 (NS)
8.9 (NS
[a]Following three initial monthly doses
*Visual acuity was measured as the total number of letters read correctly on the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart.
**Statistically non-inferior based on a non-inferiority margin of 10%, using confidence interval approach (95.1% and 95% for Study 1 and Study 2, respectively)
***Test for superiority
NS = non-significant
In Study 1, patients receiving VEGFT 2 mg monthly (2Q4) achieved a statistically significant greater mean improvement in visual acuity at week 52 versus baseline (secondary endpoint), compared to ranibizumab 0.5 mg monthly (RQ4); patients receiving VEGFT 2 mg monthly on average gained 10.9 letters, compared to a mean 8.1 letter gain with ranibizumab 0.5 mg dosed every month (p<0.01). All other dose groups of VEGFT in Study 1 and all dose groups in Study 2 were not statistically different from ranibizumab in this secondary endpoint.
A generally favorable safety profile was observed for both VEGFT and ranibizumab. The incidence of ocular treatment emergent adverse events was balanced across all four treatment groups in both studies, with the most frequent events associated with the injection procedure, the underlying disease, and/or the aging process. The most frequent ocular adverse events were conjunctival hemorrhage, macular degeneration, eye pain, retinal hemorrhage, and vitreous floaters. The most frequent serious non-ocular adverse events were typical of those reported in this elderly population who receive intravitreal treatment for wet AMD; the most frequently reported events were falls, pneumonia, myocardial infarction, atrial fibrillation, breast cancer, and acute coronary syndrome. There were no notable differences among the study arms.
Example 5: Phase II Clinical Trial of VEGFT in Subjects with Diabetic Macular Edema (DME)
In this study, 221 patients with clinically significant DME with central macular involvement were randomized, and 219 patients were treated with balanced distribution over five groups. The control group received macular laser therapy at baseline, and patients were eligible for repeat laser treatments, but no more frequently than at 16 week intervals. The remaining four groups received VEGFT by intravitreal injection as follows: Two groups received 0.5 or 2 mg of VEGFT once every four weeks throughout the 12-month dosing period (0.5Q4 and 2Q4, respectively). Two groups received three initial doses of 2 mg VEGFT once every four weeks (i.e., at baseline, and weeks 4 and 8), followed through week 52 by either once every 8 weeks dosing (2Q8) or as needed dosing with very strict repeat dosing criteria (PRN). Mean gains in visual acuity versus baseline were as shown in Table 2:
TABLE 2
Mean change in
Mean change in
visual acuity
visual acuity
at week 24
at week 52
versus baseline
versus baseline
n
(letters)
(letters)
Laser
44
2.5
−1.3
VEGFT 0.5 mg
44
8.6**
11.0**
monthly (0.5Q4)
VEGFT 2 mg monthly
44
11.4**
13.1**
(2Q4)
VEGFT 2 mg every 8
42
8.5**
9.7**
weeks[a] (2Q8)
VEGFT 2 mg as
45
10.3**
12.0**
needed[a] (PRN)
[a]Following three initial monthly doses
**p < 0.01 versus laser
In this study, the visual acuity gains achieved with VEGFT administration at week 24 were maintained or numerically improved up to completion of the study at week 52 in all VEGFT study groups, including 2 mg dosed every other month
As demonstrated in the foregoing Examples, the administration of VEGFT to patients suffering from angiogenic eye disorders (e.g., AMD and DME) at a frequency of once every 8 weeks, following a single initial dose and two secondary doses administered four weeks apart, resulted in significant prevention of moderate or severe vision loss or improvements in visual acuity.
Example 6: A Randomized, Multicenter, Double-Masked Trial in Treatment Naïve Patients with Macular Edema Secondary to CRVO
In this randomized, double-masked, Phase 3 study, patients received 6 monthly injections of either 2 mg intravitreal VEGFT (114 patients) or sham injections (73 patients). From Week 24 to Week 52, all patients received 2 mg VEGFT as-needed (PRN) according to retreatment criteria. Thus, “sham-treated patients” means patients who received sham injections once every four weeks from Week 0 through Week 20, followed by intravitreal VEGFT as needed from Week 24 through Week 52. “VEGFT-treated patients” means patients who received VEGFT intravitreal injections once every four weeks from Week 0 through Week 20, followed by intravitreal VEGFT as needed from Week 24 through Week 52. The primary endpoint was the proportion of patients who gained ≥15 ETDRS letters from baseline at Week 24. Secondary visual, anatomic, and Quality of Life NEI VFQ-25 outcomes at Weeks 24 and 52 were also evaluated.
At Week 24, 56.1% of VEGFT-treated patients gained ≥15 ETDRS letters from baseline vs 12.3% of sham-treated patients (P<0.0001). Similarly, at Week 52, 55.3% of VEGFT-treated patients gained ≥15 letters vs 30.1% of sham-treated patients (P<0.01). At Week 52, VEGFT-treated patients gained a mean of 16.2 letters vs 3.8 letters for sham-treated patients (P<0.001). Mean number of injections was 2.7 for VEGFT-treated patients vs 3.9 for sham-treated patients. Mean change in central retinal thickness was −413.0 μm for VEGFT-treated patients vs −381.8 μm for sham-treated patients. The proportion of patients with ocular neovascularization at Week 24 were 0% for VEGFT-treated patients and 6.8% for sham-treated patients, respectively; at Week 52 after receiving VEGFT PRN, proportions were 0% and 6.8% for VEGFT-treated and sham-treated. At Week 24, the mean change from baseline in the VFQ-25 total score was 7.2 vs 0.7 for the VEGFT-treated and sham-treated groups; at Week 52, the scores were 7.5 vs 5.1 for the VEGFT-treated and sham-treated groups.
This Example confirms that dosing monthly with 2 mg intravitreal VEGFT injection resulted in a statistically significant improvement in visual acuity at Week 24 that was maintained through Week 52 with PRN dosing compared with sham PRN treatment. VEGFT was generally well tolerated and had a generally favorable safety profile.
Example 7: Dosing Regimens
Specific, non-limiting examples of dosing regimens within the scope of the present invention are as follows:
VEGFT 2 mg (0.05 mL) administered by intravitreal injection once every 4 weeks (monthly).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 12 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 12 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 12 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 16 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 16 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 16 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 20 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 20 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 20 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 24 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 24 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 24 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 28 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks.
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 28 weeks, followed by 2 mg (0.05 mL) via intravitreal injection on a less frequent basis based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 28 weeks, followed by 2 mg (0.05 mL) via intravitreal injection administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
VEGFT 2 mg (0.05 mL) administered by intravitreal injection as a single initial dose, followed by additional doses administered pro re nata (PRN) based on visual and/or anatomical outcomes (as assessed by a physician or other qualified medical professional).
Variations on the above-described dosing regimens would be appreciated by persons of ordinary skill in the art and are also within the scope of the present invention. For example, the amount of VEGFT and/or volume of formulation administered to a patient may be varied based on patient characteristics, severity of disease, and other diagnostic assessments by a physician or other qualified medical professional.
Any of the foregoing administration regimens may be used for the treatment of, e.g., age-related macular degeneration (e.g., wet AMD, exudative AMD, etc.), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO; e.g., macular edema following CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV; e.g., myopic CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, etc.
SEQUENCES
SEQ ID NO: 1 (DNA sequence having 1377
nucleotides):
ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTG
TCTGCTTCTCACAGGATCTAGTTCCGGAAGTGATACCGGTAGACCTTTCG
TAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGG
GAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTT
AAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCT
GGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATA
GGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAA
CTATCTCACACATCGACAAACCAATACAATCATAGATGTGGTTCTGAGTC
CGTCTCATGGAATTGAACTATCTGTTGGAGAAAAGCTTGTCTTAAATTGT
ACAGCAAGAACTGAACTAAATGTGGGGATTGACTTCAACTGGGAATACCC
TTCTTCGAAGCATCAGCATAAGAAACTTGTAAACCGAGACCTAAAAACCC
AGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGT
GTAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTGGGCT
GATGACCAAGAAGAACAGCACATTTGTCAGGGTCCATGAAAAGGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
TGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT
ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG
AGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO: 2 (polypeptide sequence haying 458
amino acids):
MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGR
ELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEI
GLLICEATVNGHLYKTNYLTHRQINTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDG
VTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
SLSLSPGK
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying FIGURES. Such modifications are intended to fall within the scope of the appended claims.
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16397267
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Aug 18th, 2020 12:00AM
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Feb 21st, 2019 12:00AM
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https://www.uspto.gov?id=US10745471-20200818
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Method of treating osteoarthritis with an antibody to NGF
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Methods are disclosed for treating osteoarthritis in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-human NGF antibody, or antigen-binding fragment thereof, wherein at least one symptom associated with osteoarthritis is prevented, ameliorated or improved.
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10745471
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1. An injectable formulation, comprising:
a polysorbate;
a diluent; and
a human antibody or antigen-binding fragment thereof that specifically binds human nerve growth factor (NGF), wherein the antibody comprises a.) a heavy chain CDR1 (HCDR1) domain comprising SEQ ID NO: 86, a heavy chain CDR2 (HCDR2) domain comprising SEQ ID NO: 88, and a heavy chain CDR3 (HCDR3) domain comprising SEQ ID NO: 90; and b.) a light chain CDR1 (LCDR1) domain comprising SEQ ID NO: 94; a light chain CDR2 (LCDR2) domain comprising SEQ ID NO: 96, and a light chain CDR3 (LCDR3) domain comprising SEQ ID NO: 98.
2. The injectable formulation of claim 1, wherein the human antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 100 and 108.
3. The injectable formulation of claim 1, wherein the human antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 92, 102, and 110.
4. The injectable formulation of claim 1, wherein the human antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 100 and 108 and a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 102 and 110.
5. The injectable formulation of claim 1, wherein the human antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR sequence pairs are selected from the group consisting of SEQ ID NO: 108 and 92; SEQ ID NO: 100 and 102; and SEQ ID NO: 108 and 110.
6. The injectable formulation of claim 1, wherein the antibody or antigen binding fragment thereof has a KD of about 5 pM or less, as measured by surface plasmon resonance; and
wherein the antibody or antigen binding fragment thereof binds human NGF with an affinity of about 2-10-fold higher than the antibody or fragment binds rat and mouse NGF.
7. The injectable formulation of claim 6, wherein the antibody or antigen binding fragment thereof has a KD of less than 0.5 pM.
8. An injectable formulation comprising:
a human NGF inhibitor, wherein the NGF inhibitor is a human antibody or antigen-binding fragment of an antibody that specifically binds human nerve growth factor (NGF), a second therapeutic agent which is a human interleukin-1 (IL-1) inhibitor; and
a pharmaceutically acceptable carrier,
wherein the antibody comprises a.) a heavy chain CDR1 (HCDR1) domain comprising SEQ ID NO: 86, a heavy chain CDR2 (HCDR2) domain comprising SEQ ID NO: 88, and a heavy chain CDR3 (HCDR3) domain comprising SEQ ID NO: 90; and b.) a light chain CDR1 (LCDR1) domain comprising SEQ ID NO: 94; a light chain CDR2 (LCDR2) domain comprising SEQ ID NO: 96, and a light chain CDR3 (LCDR3) domain comprising SEQ ID NO: 98.
9. The injectable formulation of claim 8, wherein the human antibody or antigen-binding fragment thereof, specifically binds human nerve growth factor (NGF) with KD of 5 pM or less as measured by surface plasmon resonance; and
wherein the antibody or fragment thereof binds human NGF with an affinity of about 2-10-fold higher than the antibody or fragment binds rat and mouse NGF.
10. The injectable formulation of claim 9, wherein the antibody or antigen binding fragment thereof has a KD of less than 0.5 pM.
11. The injectable formulation of claim 8, further comprising:
a second therapeutic agent.
12. The injectable formulation of claim 11, wherein the second therapeutic agent is selected from the group consisting of an IL-1 and an IL-6 antagonist.
13. A method of treating pain associated with an NGF-related condition or disease wherein said pain is inhibited, ameliorated, or reduced by inhibition of NGF, comprising:
injecting a subject suffering from pain with an injectable formulation, comprising:
a polysorbate;
a diluent; and
0.01 to 20 mg/kg body weight of the subject of a human antibody or antigen-binding fragment thereof which specifically binds human nerve growth factor (NGF), wherein the antibody or antigen-binding fragment thereof comprises a.) a heavy chain CDR1 (HCDR1) domain comprising SEQ ID NO: 86, a heavy chain CDR2 (HCDR2) domain comprising SEQ ID NO: 88, and a heavy chain CDR3 (HCDR3) domain comprising SEQ ID NO: 90; and b.) a light chain CDR1 (LCDR1) domain comprising SEQ ID NO: 94; a light chain CDR2 (LCDR2) domain comprising SEQ ID NO: 96, and a light chain CDR3 (LCDR3) domain comprising SEQ ID NO: 98.
14. The method of claim 13, wherein the pain associated with the NGF-related condition or disease is inhibited without significant impairment of motor coordination, and wherein human antibody or antigen-binding fragment thereof is in an amount of 0.02 to about 7 mg/kg body weight of the subject.
15. The method of claim 13, wherein the NGF-related condition or disease is selected from the group consisting of inflammatory pain, post-operative incision pain, neuropathic pain, fracture pain, gout joint pain, post-herpetic neuralgia, pain resulting from burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, and pains associated with sickle cell crises.
16. The method of claim 15, wherein the NGF-related condition is post-herpetic neuralgia.
17. The method of claim 13, further comprising administering a second therapeutic agent.
18. The method of claim 17, wherein the second therapeutic agent is an IL-1 or an IL-6 antagonist.
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18
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FIELD OF THE INVENTION
The present invention is related to therapeutic methods for treating osteoarthritis in a human patient in need thereof, with an antibody or antigen-binding fragment of an antibody specific for human nerve growth factor (NGF) and pharmaceutical compositions containing the antibody or antibody fragment.
STATEMENT OF RELATED ART
While numerous analgesic medications are currently available, the adequate relief of pain remains an unmet medical need for many acute and chronic pain states. The limitations of currently available analgesic therapies include adverse central nervous system (CNS) effects, nausea and vomiting, gastrointestinal (GI) bleeding and ulceration, idiosyncratic cardiovascular events attributed to drugs that suppress cyclooxygenase-2, renal toxicity, abuse potential and others that span the spectrum of drug toxicity.
Osteoarthritis is a progressive, chronic disease in which pain is often a key limiting factor and for which acceptable long-term therapy does not yet exist. Current long-term therapies such as non-steroidal anti-inflammatory drugs (NSAIDs) and celecoxib can be problematic due to specific side effects and potential health risks such as GI bleeding and increased risk of cardiovascular events. In addition, these medications must be taken daily to maintain their analgesic effects. As the prevalence of OA in patients aged older than 65 years is 60% in men and 70% in women and continually rising, the search for additional treatment options with fewer associated side-effects is ongoing.
Neurotrophins are a family of peptide growth factors that play a role in the development, differentiation, survival and death of neuronal and non-neuronal cells. The first neurotrophin to be identified was nerve growth factor (NGF), and its role in the development and survival of both peripheral and central neurons during the developing nervous system has been well characterized. In the adult, NGF is a pain mediator that sensitizes neurons and is not required as a survival factor.
NGF activity is mediated through two different membrane-bound receptors, the TrkA receptor and the p75 common neurotrophin receptor. The NGF/TrkA system appears to play a major role in the control of inflammation and pain, since it is upstream of several relevant molecular pathways. Mast cells, for example, are capable of producing NGF, but are also induced by NGF to release inflammatory mediators. Nerve growth factor expression is known to be upregulated in injured and inflamed tissues in conditions such as cystitis, prostatitis, and chronic headache.
Selective antagonism of NGF by a fully-human high-affinity monoclonal antibody (mAb) has the potential to be effective without the adverse side effects of traditional analgesic drugs, since it works through a different physiological mechanism of action. Human genetic studies that show that people suffering from a loss of deep pain perception have mutations in TrkA (HSAN IV) or NGF (HSAN V). In addition, NGF is known to be elevated in the synovial fluid of patients suffering from rheumatoid arthritis and other types of arthritis.
Anti-NGF antibodies are described in, for example, EP1575517; WO 01/78698, WO 02/096458, WO 2004/032870; U.S. Pat. Nos. 7,601,818; 7,449,616; 7,655,232; US patent application publications 2009/0155274; 2009/0208490; 2008/033157; 2008/0107658; 2005/0074821; 2004/0237124, and 2004/0219144.
BRIEF SUMMARY OF THE INVENTION
In a first aspect, the invention features methods for preventing, inhibiting, ameliorating and/or treating at least one of the symptoms associated with osteoarthritis in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a fully human anti-NGF antibody, or antigen-binding fragment thereof, wherein at least one of the symptoms of osteoarthritis is prevented, inhibited, ameliorated or improved. In specific embodiments, the antibody or antigen-binding fragment of an antibody to be used in the method of the invention is a fully human antibody comprising heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 4, 20, 24, 28, 44 and 48. In more specific embodiments, the HCVR is selected from the group of SEQ ID NO: 20, 24 and 48. In one specific embodiment, the HCVR is SEQ ID NO:24. In specific embodiments, the antibody or antigen-binding fragment thereof to be used in the present invention is a fully human antibody comprising light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 12, 22, 26, 36, 46 and 50. In more specific embodiments, the LCVR is selected from the group of SEQ ID NO: 22, 26 and 50. In one specific embodiment, the LCVR is SEQ ID NO:26.
In specific embodiments, the antibody or fragment thereof comprises a HCVR and LCVR (HCVR/LCVR) sequence pair selected from the group consisting of SEQ ID NO: 4/12, 20/22, 24/26, 28/36, 44/46 and 48/50. In more specific embodiments, the HCVR/LCVR sequence pair is selected from the group consisting of SEQ ID NO: 20/22, 24/26 and 48/50. In one specific embodiment, the HCVR/LCVR sequence pair is SEQ ID NO:24/26.
In a second aspect, the invention features a method of treating, inhibiting, ameliorating, or reducing the occurrence of osteoarthritis in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-human NGF antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises heavy and light chain complementary determining regions (HCDR and LCDR) from HCVR/LCVR sequence pairs selected from the group consisting of SEQ ID NO: 4/12, 20/22, 24/26, 28/36, 44/46 and 48/50. In more specific embodiments, the antibody or antibody fragment comprise CDRs from HCVR/LCVR sequence pairs selected from the group consisting of SEQ ID NO: 20/22, 24/26 and 48/50. In one specific embodiment, the CDRs are from the sequence pair of SEQ ID NO: 24/26. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18 (SEQ ID NO:53) wherein X1 is Ala or Ser, X2 is Thr or Lys, X3 is Glu or lie, X4 is Phe or Gly, X5 is Val or Gly, X6 is Val or Trp, X7 is Val or Phe, X8 is Thr or Gly, X9 is Asn or Lys, X10 is Phe or Leu, X11 is Asp or Phe, X12 is Asn or Ser, X13 is Ser or absent, X14 is Tyr or absent, X15 is Gly or absent, X16 is Met or absent, X17 is Asp or absent, and X18 is Val or absent; and the LCDR3 comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO:56) wherein X1 is Gln, X2 is Gln, X3 is Tyr, X1 is Asn, X5 is Arg or Asn, X6 is Tyr or Trp, X7 is Pro, X8 is Tyr or Trp, and X9 is Thr.
In another embodiment, the antibody or antigen binding fragment thereof further comprises a HCDR1 sequence comprising the formula X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:51), wherein X1 is Gly, X2 is Phe, X3 is Thr or Asn, X4 is Phe or Leu, X5 is Thr or Asp, X6 is Asp or Glu, X7 is Tyr or Leu, and X8 is Ser or Ala; a HCDR2 sequence comprising the formula X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:52), wherein X1 is lie or Phe, X2 is Asp or Ser, X3 is Pro or Trp, X4 is Glu or Asn, X5 is Asp or Ser, X6 is Gly, X7 is Thr or Glu, X8 is Thr or Ile; a LCDR1 sequence comprising the formula X1-X2-X3-X4-X5-X6 (SEQ ID NO:54) wherein X1 is Gln, X2 is Ala or Ser, X3 is Val or lie, X4 is Arg or Thr, X5 is Asn or Tyr, and X6 is Asp or Asn; and a LCDR2 sequence comprising the formula X1-X2-X3 (SEQ ID NO:55) wherein X1 is Gly or Ala, X2 is Ala, and X3 is Ser or Phe.
In a third aspect, the invention features a method of treating, inhibiting or ameliorating osteoarthritis in a subject in need thereof, or at least one symptom associated with osteoarthritis, comprising administering to the subject a therapeutically effective amount an antibody or antigen-binding fragment thereof comprising a HCDR3 selected from the group consisting of SEQ ID NO: 10 and 34, and a LCDR3 selected from the group consisting of SEQ ID NO: 18 and 42. In a more specific embodiment, the HCDR3/LCDR3 are selected from the sequence pair groups consisting of SEQ ID NO: 10/18 and 34/42.
In a further embodiment, the antibody or fragment thereof comprises heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) selected from the group consisting of SEQ ID NO: 6, 8, 10, 30, 32, 34; and 14, 16, 18, 38, 40, 42, respectively. In one embodiment, the antibody or fragment thereof comprises CDR sequences SEQ ID NO: 6, 8, 10, 14, 16 and 18.
In various embodiments of a method of the invention, administration of the antibody or antigen-binding fragment of an antibody is by, for example, subcutaneous or intravenous administration, or administration locally at the site of disease. In one embodiment, the
In a fourth aspect, the invention features a method of treating, inhibiting, ameliorating, or reducing the occurrence of osteoarthritis in a subject in need thereof, or at least one symptom associated with osteoarthritis, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof in combination with a second therapeutic agent. Examples of a second therapeutic agent having applications in the method of the present invention include, but are not limited to, a second NGF antibody, a non-steroidal anti-inflammatory drug (NSAID), an oral or injectable glucocorticoid, an opioid, tramadol, an alpha-2-delta ligand and hyaluronic acid.
In one embodiment, an antibody or antigen-binding fragment thereof having applications in a method of the present invention is administered as an initial dose of at least approximately about 0.1 mg to about 800 mg. In certain embodiments, an antibody or antigen-binding fragment thereof having applications in a method of the present invention is administered as an initial dose of at least approximately about 5 to about 100 mg. In other embodiments, an antibody or antigen-binding fragment thereof having applications in a method of the present invention is administered as an initial dose of at least approximately about 10 to about 50 mg. In specific embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that is approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least one day; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
A particular example of an antibody or antigen-binding fragment thereof having applications in a method of the present invention is mAb1 (HCVR/LCVR SEQ ID NO:24/26).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the clinical study flowchart.
DETAILED DESCRIPTION
Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be defined in and limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
Anti-NGF Antibodies and Antigen-Binding Fragments Thereof
The method of the invention relates to the use of an anti-NGF antibody or antibody fragment that specifically binds NGF. The term “human nerve growth factor” or “NGF”, as used herein, refers to human NGF having the nucleic acid sequence shown in SEQ ID NO:1 and the amino acid sequence of SEQ ID NO:2, or a biologically active fragment thereof.
The term “specifically binds,” means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a equilibrium dissociation constant of about 1×10−6 M or smaller. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
An “antibody” is an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (CH) comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). 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 HCVR and LCVR 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. In certain embodiments of the invention, the FRs of the anti-NGF antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
The fully-human anti-NGF antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
The present invention also includes anti-NGF antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-NGF antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 20 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
The term “antigen-binding fragment” of an antibody (or “antibody-binding 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., NGF). An antibody fragment may include, for example, a Fab fragment, a F(ab′)2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR.
In certain embodiments, an antibody or antibody fragment of the invention may be conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. A therapeutic moiety that is a cytotoxin includes any agent that is detrimental to cells.
In certain embodiments, the antibody or antibody fragment for use in the method of the invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide. An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation, such as an H95R modification (by IMGT exon numbering; H435R by EU numbering), which reduces or abolishes Protein A binding. The second CH3 may further comprise an Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.
Therapeutic Administration and Formulations
The invention provides methods of using therapeutic compositions comprising anti-NGF antibodies or antigen-binding fragments thereof. The therapeutic compositions of the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations are available to the skilled artisan such as those that can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
The dose may vary depending upon the age and the weight of a subject to be administered, target disease, conditions, route of administration, and the like. When the antibody of the present invention is used for treating various conditions and diseases associated with NGF, including inflammatory pain, neuropathic and/or nociceptive pain, hepatocellular carcinoma, breast cancer, liver cirrhosis, and the like, in an adult patient, it is advantageous to administer the antibody of the present invention either intravenously or subcutaneously, normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.
Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533). In certain situations, the pharmaceutical composition can be delivered in a controlled release system, for example, with the use of a pump or polymeric materials. In another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.
The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, local injection, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousands Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.
Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules, pre-filled syringes or auto-injectors), suppositories, etc. The amount of the aforesaid antibody contained is generally about 0.1 to 800 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 1 to 250 mg or about 10 to 100 mg for the other dosage forms.
By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
In specific embodiments of the therapeutic methods of the invention, a subject suffering from osteoarthritis may be treated with a combination of an antibody or antibody fragment of the invention and optionally with at least a second therapeutic agent. Examples of a second therapeutic agent having applications in a method of the present invention include, but are not limited to, a non-steroidal anti-inflammatory drug (NSAID), an oral or injectable glucocorticoid, an opioid, tramadol, an alpha-2-delta ligand or hyaluronic acid.
EXAMPLES
The following examples are put forth so as to further provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be understood. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. The statistical analyses were conducted according to mixed Factorial ANOVA with Bondferroni post hoc or Tukey HSD post hoc tests.
Example 1. Study of Anti-NGF Antibody in Patients with Osteoarthritis of the Knee
A double-blind study in which patients with osteoarthritis (OA) of the knee are randomized to 1 of 4 treatment arms (3 active and 1 placebo) was conducted. Randomization was stratified by Baseline walking knee pain score (>7 and ≤7). Each patient received a dose of a fully human anti-NGF mAb (mAb1) or placebo at baseline (Day 1) and at week 8 (Day 57) for a total of 2 doses.
The doses evaluated were 0.03, 0.1, or 0.3 mg/kg administered intravenously (IV). Approximately 53 patients were enrolled in each treatment arm.
Patients of the target population were asked to discontinue their current pain medications prior to the baseline visit and for the duration of the study (end of week 24 [Day 169]). Rescue medication (acetaminophen) was allowed during this time (a maximum of 4 g per day, but not for more than 4 consecutive days). Low-dose aspirin (up to 325 mg/day) was also allowed. The duration of the washout period prior to baseline (Day 1) was determined by the half-life of the medication (approximately 5 half-lives).
Patients received study drug on Day 1 (baseline) and at week 8 (Day 57). Patients were followed for 16 weeks after the second infusion, until the end of week 24 (Day 169), for a total study duration of 24 weeks for each patient.
Safety and tolerability of mAb1 was assessed by evaluating the incidence of treatment-emergent adverse events (TEAEs) from Day 1 to the end of week 24 (Day 169) or study withdrawal, by patient medical history, physical examination, monitoring of vital signs and ECGs, clinical laboratory testing, and neurological assessments of sensory (tactile, pain, and vibration) and motor (muscle strength, and reflex) function.
The effect of mAb1 on walking knee pain was assessed using the numerical rating scale (NRS). Patients were asked to report the average intensity of their walking knee pain daily for the duration of the 24-week study. Changes in OA status were assessed using the WOMAC (pain, stiffness and function subscales). The patient's assessment of overall treatment effect was assessed by the Patient Global Impression of Change (PGIC). The patient's assessment of quality of life (QOL) was assessed using the SF-12 Scale.
Serum samples were collected for PK analysis, anti-mAb1 antibody evaluation, and exploratory proteomic and gene expression (RNA) analysis.
Patients completed the study when they received 2 doses of mAb1 or placebo and completed all scheduled safety and efficacy assessments to week 24 (Day 169).
Target Population.
Eligible patients for this study were men and women between 40 and 75 years of age, with a diagnosis of OA of the knee and who have experienced moderate to severe knee pain for an average period of ≥3 months.
Inclusion Criteria.
A patient met the following criteria, to be eligible for inclusion in the study: (1) Men and women≥40 and ≤75 years of age; (2) Diagnosis of OA of the knee according to American College of Rheumatology (ACR) criteria, and experiencing moderate to severe pain in the index knee for at least 3 months prior to the screening visit; (3) Kellgren-Lawrence grade 2-3 radiographic severity of the index knee at or within 6 months prior to Screening; (4) No new chronic medications introduced within the past 30 days. This criterion does not apply to the use of acetaminophen as rescue medication; (5) Walking knee pain levels at Screening and Baseline≥4 on the NRS; (6) Willingness to discontinue currently used pain medications (for 5 half-lives) prior to the baseline visit and throughout the study; (7) Body weight<110 kg; (8) Willing, and able to return for all clinic visits and complete all study-related procedures; (9) Able to read and understand and willing to sign the informed consent form; (10) Able to read, understand, and complete study-related questionnaires.
Exclusion Criteria.
A patient who met any of the following criteria was excluded from the study: (1) Significant concomitant illness including, but not limited to, cardiac, renal, neurological, endocrinological, metabolic or lymphatic disease that would adversely affect the patient's participation in the study; (2) Patients with joint replacement in the affected knee; (3) Patients with peripheral neuropathy due to any reason; (4) Known Human Immunodeficiency Virus (HIV) antibody, Hepatitis B surface antigen (HBsAg), and/or Hepatitis C antibody (HCV) at the screening visit by history or testing; (5) Known sensitivity to doxycycline or mAb therapeutics; (6) Other medical or psychiatric conditions that could, in the opinion of the Investigator or Sponsor, compromise protocol participation; (7) Participation in any clinical research study evaluating another investigational drug or therapy within 3 weeks or at least 5 half-lives, whichever was longer, of the investigational drug, prior to the screening visit; (8) Previous exposure to an anti-NGF antibody; (9) Women who are pregnant or nursing; (10) Sexually active men or women of childbearing potential who were unwilling to practice adequate contraception during the study (adequate contraceptive measures included stable use of oral contraceptives or other prescription pharmaceutical contraceptives for 2 or more cycles prior to screening; intrauterine device [IUD]; bilateral tubal ligation; vasectomy; condom or diaphragm plus either contraceptive sponge, foam or jelly); (11) Women of childbearing potential who had either a positive serum pregnancy test result at screening or a positive urine pregnancy test result at baseline. (Women had to be amenorrheic for at least 12 months in order to be considered post-menopausal); (12) Current or prior substance abuse, alcohol abuse, or abuse of prescription pain medication.
Investigational Treatment.
Sterile mAb1 Drug Product 20 mg/ml was provided in an aqueous buffered vehicle, pH 5.0, containing 10 mM acetate, 20% (w/v) sucrose and 1% (w/v) PEG 3500. Drug was supplied in a 5 ml glass vial.
Reference Treatment.
Placebo was supplied in matched vials containing the same volume of aqueous buffered vehicle (pH 5.0), but with no active protein.
Dose Administration and Schedule.
Study drug (mA1 or placebo) was administered on baseline (Day 1) and at week 8 (Day 57). Prior to IV administration, the pharmacist or designee withdrew the required amount of study drug (depending on the patient's dose and weight) from a single-use vial and injected it into an infusion bag of normal saline for infusion. Calculations to determine the volume to be withdrawn were provided in the Site Study Manual.
Method of Treatment Assignment.
Randomization was in a 1:1:1:1 ratio between the 4 treatment arms. On Day 1, patients were randomized to receive either mAb1 at a dose of 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, or placebo (in a 1:1:1:1 ratio) according to a pre-determined central randomization scheme. Randomization was stratified by baseline walking knee pain scores (>7 and ≤7).
Data Collection.
Study assessments and procedures are shown in the Study Flowchart (FIG. 1). For early termination patients, all week 24 (End-of-study) assessments were performed when the patient returned to the clinic for the final visit. All visits after Day 1 were scheduled within a ±2-day window. X-Ray of knee affected with OA (semi-flexed) was taken only if existing film was not available within 6 months of screening date. At baseline (Day 1) and on Day 57, vital signs were measured immediately prior to dosing, at 15-minute intervals during the infusion, at the end of the infusion, and 1, 2, and 4 hours after the completion of the infusion. Average walking knee pain was assessed at all clinic visits using the NRS. In-between visits, patients were asked to report the average intensity of their walking knee pain DAILY via the IVRS. On Day 1 and week 8 (Day 57), samples were collected prior to the start of the infusion, immediately post-infusion, and at 1, 2, and 4 hours post-infusion.
Visit Descriptions.
Screening/Day −14 to −3: Informed consent was obtained before performing or initiating any study-related procedures. The following information was collected: Inclusion/exclusion criteria; Demographics; Medical history and concurrent illnesses including any pre-dose symptoms or ongoing AEs; Concomitant medications; The following procedures and assessments were conducted: X-Ray of knee affected with OA (semi-flexed) which was taken only if an existing film was not available within 6 months prior to screening; Physical examination; Vital signs, height and weight; ECG; Serum pregnancy test for women of childbearing potential; Hematology; Serum Chemistry; Urinalysis. After screening was completed and a patient was deemed eligible to participate, a discussion was held with the patient to discuss the need to stop their current analgesic medications for a specified number of days prior to the baseline visit. The duration of this washout period was based upon the half-life of the medication(s). In addition, patients were told that they must remain off their medications for the duration of the study.
Treatment Period.
Baseline/Day 1: At this visit subjects were randomized to a study treatment and received either study drug or placebo. The following information was collected prior to the administration of study drug: Concomitant medications; Presence of any AEs; The following procedures and assessments were conducted prior to the administration of study drug: Vital signs (measured immediately prior to dosing, at 15-minute intervals during the infusion, at the end of the infusion, and 1, 2, and 4 hours post infusion); Urine pregnancy test for women of childbearing potential; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; QOL Questionnaire; Blood sample collection for PK analysis (samples were collected prior to infusion, immediately post-infusion, and at 1, 2 and 4 hours post infusion); Blood sample for anti-mAb1 antibody assessment; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Instruction in use of IVRS.
Week 1/Day 8 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; Blood sample collection for PK analysis; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Review of compliance with IVRS.
Week 2/Day 15 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were completed: Vital signs; Neurological Evaluation; Walking knee pain; WOMAC; Blood sample collection for PK analysis; Review of compliance with IVRS.
Week 4/Day 29 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Review of compliance with IVRS.
Week 8/Day 57 (±2 Days).
Patients received the second dose of study drug on Day 57. Prior to receiving study drug, the following information was collected: Concomitant medications; Presence of any AEs; The following procedures and assessments were also conducted prior to the administration of study drug: Physical examination; Vital signs and weight (vital signs measured immediately prior to dosing, at 15-minute intervals during the infusion, at the end of the infusion, and 1, 2, and 4 hours post infusion); ECG; Urine pregnancy test for women of childbearing potential; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis (samples were collected prior to infusion, immediately post-infusion, and at 1, 2 and 4 hours post infusion); Blood sample for mAb1 antibody assessment; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Review of compliance with IVRS.
Week 10/Day 71 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Neurological evaluation; Walking knee pain; WOMAC; Blood sample collection for PK analysis; Review of compliance with IVRS.
Week 12/Day 85 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Hematology; Serum Chemistry; Urinalysis; Neurological valuation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis; Blood sample for mAb1 antibody assessment; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Review of compliance with IVRS. Week 16/Day 113 (±2 days). The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis; Review of compliance with IVRS.
Week 20/Day 141 (±2 Days).
The following information was collected: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Vital signs; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis; Review of compliance with IVRS.
End of Study Assessments (Week 24/Day 169) (±2 Days).
The following information was collected during the end-of-study visit: Concomitant medications; Presence of any AEs. The following procedures and assessments were conducted: Physical examination; Vital signs and weight; ECG; Urine pregnancy test for women of childbearing potential; Hematology; Serum Chemistry; Urinalysis; Neurological evaluation; Walking knee pain; WOMAC; Patient Global Impression of Change; QOL Questionnaire; Blood sample collection for PK analysis; Blood sample for anti-mAb1 antibody assessment; Blood sample for exploratory proteomic and gene expression (RNA) analysis; Review of compliance with IVRS.
Walking Knee Pain.
The key efficacy endpoint in this study was the mean change from baseline in walking knee pain using the NRS at each study visit until Week 24. The baseline value was defined as the NRS value from Visit 2 and the weekly NRS value up to Week 24 (End of Study [EOS]) were defined as the average of daily assessments measured during the week. Patients reviewed the intensity of their knee pain with the appropriate study site personnel during their scheduled clinic visits and this information was recorded in the eCRF. Patients were also asked to record the average intensity of their walking knee pain daily, using the IVRS system, during their participation in the study. Daily assessment and recording of walking knee pain was performed at the same time each day when possible. The Numerical Rating Scale (NRS) instructed the patient to rate their pain on a 0-10 pain rating scale, 0 means no pain and 10 means the worst possible pain. The middle of the scale (around 5) was considered to be moderate pain. A value of 2 or 3 was considered to be mild pain, but a value of 7 or higher was considered to be severe pain.
Western Ontario and McMaster Osteoarthritis Index (WOMAC).
The WOMAC Index was used to assess patients with OA of the hip or knee using 24 parameters in three areas: pain (5 items), stiffness (2 items), and function (17 items). It can be used to monitor the course of a disease or to determine effectiveness of medications. Patients completed the WOMAC (pain, stiffness and function subscales) at baseline (Day 1), week 1 (Day 8), week 2 (Day 15), week 4 (Day 29), week 8 (Day 57), week 10 (Day 71), week 12 (Day 85), week 16 (Day 113), week 20 (Day 141), and at the end-of-study (week 24 [Day 169]). Patients were asked to score each of the 24 parameters using the scale shown in Table 1. The patient was asked to rate each statement on a Likert item, ranging from 0 (none) to 10 (extreme). Pain dimension subscale was calculated as the average score of Q1 to Q5 (Thumboo et al (2001), Osteoarthritis Cartilage, July; 9(5):440-6.). Stiffness dimension subscale was calculated as the average score of Q6 and Q7. Function dimension subscale was calculated as the average score of Q8 to Q24. Standardized total scale will be calculated as the average score from all 24 questions. Change from baseline in the above subscales and the standardized total scale to each measurement visit was analyzed.
TABLE 1
Response
Points
none
0
slight
1
moderate
2
severe
3
extreme
4
WOMAC parameters: Pain: 1. Walking on a flat surface; 2. Stair climbing; 3. Nocturnal (at night, lying in bed); 4. Rest (sitting or lying down); 5. Weight bearing (standing upright). Stiffness: 6. Morning stiffness; 7. Stiffness occurring later in the day. Function: 8. Difficulty descending stairs; 9. Difficulty ascending stairs; 10. Rising from sitting; 11. Standing; 12. Bending to floor (to pick something up); 13. Walking on a flat surface; 14. Getting in or out of car; 15. Going shopping; 16. Putting on socks; 17. Rising from bed; 18. Taking off socks; 19. Lying in bed; 20. Getting in and out of the bathtub; 21. Difficulty sitting (for a period of time); 22. Getting on or off toilet; 23. Heavy domestic duties; 24. Light domestic duties.
Patient Global Impression of Change (PGIC).
The PGIC is a patient-rated assessment of response to treatment on a 7-point Likert scale and was completed at week 1 (Day 8), week 4 (Day 29), week 8 (Day 57), week 12 (Day 85), week 16 (Day 113), week 20 (Day 141), and at the end-of-study (week 24 [Day 169]). The recall period for this scale was 1 week. The patient responded to the question “Compared to a week ago, how would you rate your overall status?” by selecting an option from 1. Very Much Improved; 2. Much Improved; 3. Minimally Improved; 4. No Change; 5. Minimally Worse; 6. Much Worse; 7. Very Much Worse.
Quality of Life Questionnaire.
The SF-12 is a patient-rated, 12-question assessment of QOL. It is a validated, shorter version of the commonly used SF-36. Both scales assess important QOL domains relevant to patients suffering from a wide range of medical conditions. The SF-12 was completed at week 1 (Day 8), week 4 (Day 29), week 8 (Day 57), week 12 (Day 85), week 16 (Day 113), week 20 (Day 141), and at the end-of-study (week 24 [Day 169]). QOL: A. In general, would you say your health is: Excellent (1), Very Good (2), Good (3), Fair (4), Poor (5). B. Does your health now limit you in these activities? If so, how much? C. Moderate Activities, such as moving a table, pushing a vacuum cleaner, bowling, or playing golf: Yes, Limited A Lot (1), Yes, Limited A Little (2), No, Not Limited At All (3). 3. Climbing several flights of stairs: Yes, Limited A Lot (1), Yes, Limited A Little (2), No, Not Limited At All (3). D. During the past 4 weeks have you had any of the following problems with your work or other regular activities as a result of your physical health? 4. Accomplished less than you would like: Yes (1), No (2). 5. Were limited in the kind of work or other activities: Yes (1), No (2). E. During the past 4 weeks, were you limited in the kind of work you do or other regular activities as a result of any emotional problems (such as feeling depressed or anxious)? 6. Accomplished less than you would like: Yes (1), No (2). 7. Didn't do work or other activities as carefully as usual: Yes (1), No (2). 8. During the past 4 weeks, how much did pain interfere with your normal work (including both work outside the home and housework)? Not At All (1). A Little Bit (2), Moderately (3), Quite A Bit (4), Extremely (5). F. The next three questions are about how you feel and how things have been during the past 4 weeks. For each question, please give the one answer that comes closest to the way you have been feeling. How much of the time during the past 4 weeks—9. Have you felt calm and peaceful? All of the Time (1), Most of the Time (2), A Good Bit of the Time (3), Some of the Time (4), A Little of the Time (5), None of the Time (6). 10. Did you have a lot of energy? All of the Time (1), Most of the Time (2), A Good Bit of the Time (3), Some of the Time (4), A Little of the Time (5), None of the Time (6). 11. Have you felt downhearted and blue? All of the Time (1), Most of the Time (2), A Good Bit of the Time (3), Some of the Time (4), A Little of the Time (5), None of the Time (6). 12. During the 4 weeks, how much of the time has your physical health or emotional problems interfered with your social activities (like visiting with friends, relatives, etc.)? All of the Time (1), Most of the Time (2), A Good Bit of the Time (3), Some of the Time (4), A Little of the Time (5), None of the Time (6). This questionnaire yields an 8-scale profile of functional health and well-being scores as well as psychometrically based physical and mental health summary measures which are physical component summary (PCS) and mental component summary (MCS), respectively. Change from baseline in the standardized summary scores (MCS and PCS) to each measurement visit was analyzed.
Neurological Evaluation.
1. Evaluation of Sensory function: A neurological evaluation of sensory function assesses tactile sense (light touch), pain sensation (pin prick or other) and vibration sense (tuning fork). 2. Evaluation of Motor function: A neurological evaluation of motor function assesses muscle strength (movement of upper and lower limbs against resistance) and reflexes (upper and lower limbs e.g., tricep and patellar tendons). If changes in sensation or motor function were observed or elicited during the study, they were monitored closely by the Investigator. If these changes became persistent, evolved or became severe in intensity, the Investigator referred the patient to a neurologist for a more comprehensive diagnostic evaluation. 3. Persistence of symptoms: For the purpose of this protocol, “persistence” of sensory or motor symptoms was defined as “lasting for a period of 2 weeks and with no improvement in severity.” Persistence of symptoms for 2 weeks or longer triggered an examination of the patient and a referral for neurological consultation, if deemed appropriate. In addition, the Investigator referred any patient at any time for a neurologic consultation if felt to be clinically indicated. 4. Evolution of symptoms: Evolution of symptoms in any timeframe triggered a neurological examination. For example, if a sensory change of “numbness” or “pins and needles” evolved into more dysesthestic or allodynic sensations such as “burning” or “painful”, it did not matter when it occurred during the course of the study or how long it took for the change to occur. Any patient who experienced such a change was referred for a thorough neurological assessment whenever a change like this was reported. In addition, as noted above, the Investigator referred any patient for a neurologic consultation at any time, if it was felt to be clinically indicated. Clinical neurological assessments of sensory and motor function were conducted at baseline (Day 1), and at week 1 (Day 8), week 2 (Day 15), week 4 (Day 29), week 8 (Day 57), week 10 (Day 71), week 12 (Day 85), week 16 (Day 113), week 20 (Day 141), and at the end-of-study (week 24 [Day 169]).
Pharmacokinetic and Antibody Sample Collection.
Drug Concentration Measurements and Samples. Serum samples for PK measurements were collected at every study visit beginning at baseline (Day 1), and at week 1 (Day 8), week 2 (Day 15), week 4 (Day 29), week 8 (Day 57), week 10 (Day 71), week 12 (Day 85), week 16 (Day 113), week 20 (Day 141), and at the end of study visit (week 24/Day 169). On study treatment days (Day 1 and week 8 [Day 57]), samples were collected prior to the start of the infusion, immediately post infusion, and at 1, 2, and 4 hours post-infusion.
Antibody Measurements and Samples.
Serum samples were collected for analysis of antibodies to mAb1 prior to dosing at baseline (Day 1), after administration of the second dose (week 8 [Day 57]), at week 12 (Day 85), and at the end of study (week 24 [Day 169]).
Use and Storage of Exploratory Serum and RNA Samples.
Exploratory samples were collected to study NGF, mAb1, pain, OA and inflammation. Ribonucleic acid samples were collected for exploratory microarray expression profiling. All samples were coded to maintain patient confidentiality. Remaining RNA samples after profiling were stored for future analyses. Serum samples were stored and may be used for future proteomics analyses.
Analysis of Efficacy Data
Key Efficacy Endpoint: Walking Knee Pain
Mean weekly change in NRS of walking knee pain from baseline was analyzed using a mixed-effect model repeated measure (MMRM) approach. The MMRM analyses was implemented via PROC MIXED in SAS by fitting changes from baseline at all post randomization visits in the treatment period up to Week 24.
The statistical inference on the primary efficacy variable, mean change from baseline to Week 24 in pain intensity was derived from this model using an appropriate contrast.
The model included factors (fixed effects) for treatment, baseline-NRS stratum (>7 and ≤7), visit, treatment-by-visit interaction, and baseline value as a covariate. The factor visit with nominal visits has 24 levels (e.g., Week 1 to Week 24).
An unstructured correlation matrix was used to model the within-patient errors. Parameters were estimated using restricted maximum likelihood method with the Newton-Raphson algorithm. Denominator degrees of freedom were estimated using Satterthwaite's approximation. Least squares means (LS-means) estimates at each week by treatment group are provided, as well as the differences of these estimates versus placebo, with their corresponding standard errors and associated 95% confidence intervals. Student t-tests were used to determine the statistical significance of the comparison of each mAb1 dose versus placebo. In addition, data and change from baseline were summarized by treatment group using descriptive statistics (mean, median, standard deviation, minimum and maximum) by visits based on Observed Cases (OC). Graphical presentations will be used to illustrate trends over time.
If the algorithm does not converge or any other computational issue occurs, the mean weekly change in NRS of walking knee pain from baseline was analyzed using an Analysis of Covariance (ANCOVA) approach. The ANCOVA analyses was implemented via PROC Mixed in SAS by fitting changes from baseline at all post randomization visits in the treatment period up to Week 24. In the event that the mixed model assumptions did hold, rank-based ANCOVA was performed. In the event that the ANCOVA assumptions did not hold, rank-based ANCOVA was performed.
The mean weekly NRS was calculated as the average of the reported daily NRS within the week (prorated mean). If the mean weekly change in NRS of walking knee pain from baseline for a specific week was missing, the MMRM handled missing data by incorporating all available data at any weekly time points for each patient into the analysis and utilizing all existing correlations between the weekly time points. For the ANCOVA approach, the last existing value prior to this week was used (Last Observation Carried Forward [LOCF] procedure).
Proportions of patients with 30% or more reduction (30% responder rate) and 50% responder rate from baseline at each week were summarized and plotted by the treatment group. Fisher's exact test was applied to compare each treatment group with placebo group.
Other Efficacy Endpoints
WOMAC Index
Change from baseline in 3 subscales (pain, stiffness and function) and the standardized total scale to each measurement visit was analyzed similarly as for the key efficacy variable.
In the MMRM or ANCOVA model, the factor visit with nominal visits has 9 levels (e.g., Week 1, Week 2, Week 4, Week 8, Week 10, Week 12, Week 16, Week 20 and Week 24).
Dimension scores were computed if at least 50% of items were available within the corresponding dimension. LOCF procedure was used for the missing data imputation for ANCOVA approach.
PGIC
PGIC at each measurement visit, as a multinomial repeated measure with 7 categories, was analyzed as for the key efficacy variable. The model excluded the baseline and the factor visit with nominal visits had 7 levels (e.g., Week 1, Week 4, Week 8, Week 12, Week 16, Week 20 and Week 24). Due to the nature of non-normality, Minimum Variance Quadratic Unbiased Estimation (MIVQUE) method was specified in the SAS Proc Mixed to estimate the covariance parameters.
If the algorithm did not converge or any other computational issue occurred, an analysis of variance (ANOVA) model was applied. The ANOVA analyses were implemented via PROC Mixed in SAS at all post randomization visits in the treatment period up to Week 24. In the event that the ANOVA assumptions did not hold, rank-based ANOVA was performed.
LOCF procedure was used for the missing data imputation for the ANOVA approach.
Quality of Life Questionnaire (SF-12)
Change from baseline in the standardized summary scores (MCS and PCS) to each measurement visit were analyzed similarly as for the key efficacy variable.
In MMRM or ANCOVA model, the factor visit with nominal visits had 6 levels (e.g., Week 4, Week 8, Week 12, Week 16, Week 20 and Week 24).
Total scores were computed if at least 50% of items were available. The missing items were imputed by the mean of available items. Dimension scores were computed if at least 50% of items were available within the corresponding dimension.
LOCF procedure was used for the missing data imputation in the ANCOVA approach.
Results
Key Efficacy Endpoint: Walking Knee Pain Assessed Using the Numerical Rating Scale (NRS)
The effect of mAb1 on walking knee pain was assessed using the NRS, as described above.
The results of this landmark analysis, which are summarized in Table 2, indicate that mAb1 provided clinically relevant pain relief for Walking Knee Pain compared to placebo at both the Week 8 and Week 16 evaluations. The effect at Week 24 (16 weeks after the second dose administration) was diminished compared to the earlier timepoints. At Week 8, the 0.1 and 0.3 mg/kg doses were statistically significantly different from placebo at the 5% significance level in the change from baseline. At Week 16, the two lower doses were statistically different from placebo (0.03 and 0.1 mg/kg). None of the doses evaluated were statistically different from placebo at the Week 24 evaluation. As this exploratory timepoint was 16 weeks after the final dose administration, this loss of effect was consistent with the plasma elimination half-life of the drug
TABLE 2
NRS of Walking Knee Pain from Baseline to Week 8, 16 and
24 ---Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Baseline
N
55
53
53
54
Mean (SD)
6.4
(1.69)
6.6
(1.65)
6.5
(1.53)
6.6
(1.47)
Median
6.0
7.0
7.0
7.0
Min:Max
4:10
4:10
4:10
4:9
Week 8
Original NRS
N
53
50
51
50
Mean (SD)
4.3
(2.23)
3.9
(2.34)
3.2
(2.22)
3.1
(2.32)
Median
4.3
3.8
3.7
2.4
Min:Max
0:8
0:9
0:8
0:9
Change from baseline
N
53
50
51
50
Mean (SD)
−2.1
(2.08)
−2.8
(2.29)
−3.3
(2.61)
−3.6
(2.48)
Median
−2.3
−2.6
−3.0
−3.9
Min:Max
−6:2
−9:2
−9:1
−8:2
Difference vs. placebo [1]
LS Means (SE)
−0.7
(0.43)
−1.2
(0.42)
−1.3
(0.43)
95% CI
−1.5:0.1
−2.0:−0.4
−2.1:−0.4
P-value
0.0981
0.0053
0.0035
Week 16
Original NRS
N
45
48
45
42
Mean (SD)
3.8
(2.34)
3.2
(2.08)
3.1
(2.38)
3.2
(2.69)
Median
4.1
3.2
3.0
2.9
Min:Max
0:10
0:7
0:8
0:9
Change from baseline
N
45
48
45
42
Mean (SD)
−2.5
(2.15)
−3.4
(2.24)
−3.4
(2.58)
−3.3
(2.55)
Median
−2.3
−3.1
−3.6
−3.4
Min:Max
−7:2
−8:2
−8:2
−8:2
Difference vs. placebo [1]
LS Means (SE)
−1.1
(0.46)
−1.0
(0.46)
−0.9
(0.47)
95% CI
−2.0:−0.1
−1.9:−0.1
−1.8:0.0
P-value
0.0229
0.0267
0.0631
Week 24
Original NRS
N
33
39
39
35
Mean (SD)
3.7
(2.44)
4.1
(2.42)
3.2
(2.04)
3.7
(2.70)
Median
4.0
4.0
3.0
4.0
Min:Max
0:8
0:9
0:8
0:8
Change from baseline
N
33
39
39
35
Mean (SD)
−2.4
(2.24)
−2.5
(2.23)
−3.3
(2.09)
−2.8
(2.76)
Median
−2.7
−2.2
−3.0
−3.0
Min:Max
−8:2
−7:1
−8:1
−8:3
Difference vs. placebo [1]
LS Means (SE)
−0.1
(0.49)
−0.8
(0.49)
−0.5
(0.50)
95% CI
−1.1:0.8
−1.8:0.1
−1.5:0.4
P-value
0.7736
0.0894
0.2804
Model Effects
P-Value
Treatment
0.0337
Baseline
0.0015
Time
<0.0001
Time-by-T-reatment
<0.0001
Baseline NRS Stratum
0.0120
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
Other Efficacy Endpoints
WOMAC Pain Subscale and Function Subscale
The WOMAC Index was used to assess patients with OA of the hip or knee using 24 parameters in three areas: pain (5 items), stiffness (2 items), and function (17 items).
The results of these analyses are summarized in Table 3 (Pain Subscale) and Table 4 (Function Subscale).
As shown in Table 3, the baseline mean WOMAC Pain Subscale scores ranged from 5.7 to 6.4 with the mean score in the patient group given mAb1 at 0.03 mg/kg being the smallest. Treatment effect in terms of the LS mean difference vs. placebo in the group given 0.03 mg/kg of mAb1 was the smallest. For the groups given 0.1 mg/kg and 0.3 mg/kg of mAb1, the LS mean differences vs. placebo were similar and ranged from −0.7 to −1.4. The p-values indicate that the results were statistically significant at Week 8 and Week 16, but not at Week 24.
TABLE 3
WOMAC Pain Subscale from Baseline to Week 8, 16 and
24--- Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Baseline
N
55
52
53
54
Mean (SD)
5.9
(1.79)
5.7
(1.77)
6.1
(1.75)
6.4
(1.97)
Median
6.2
5.5
6.2
6.8
Min:Max
1:9
2:10
2:9
3:10
Week 8
Original WOMAC pain subscale
N
51
50
50
46
Mean (SD)
4.0
(1.90)
3.1
(2.05)
2.7
(2.10)
2.6
(2.33)
Median
4.0
2.8
2.3
2.1
Min:Max
0:8
0:9
0:7
0:7
Change from baseline
N
51
49
50
46
Mean (SD)
−1.9
(1.74)
−2.6
(2.01)
−3.4
(2.54)
−3.5
(2.42)
Median
−1.4
−2.6
−3.1
−3.3
Min:Max
−7:2
−7:3
−8:1
−9:3
Difference vs. placebo [1]
LS Means (SE)
−0.9
(0.39)
−1.4
(0.39)
−1.3
(0.39)
95% CI
−1.7:−0.1
−2.2:−0.7
−2.1:−0.5
P-value
0.0228
0.0003
0.0010
Week 16
Original WOMAC pain subscale
N
44
47
44
41
Mean (SD)
3.5
(2.31)
2.9
(2.15)
2.6
(2.15)
2.8
(2.38)
Median
3.6
2.4
2.3
2.4
Min:Max
0:10
0:10
0:8
0:8
Change from baseline
N
44
47
44
41
Mean (SD)
−2.4
(2.18)
−2.7
(1.89)
−3.4
(2.53)
−3.2
(2.24)
Median
−1.9
−2.4
−3.4
−3.4
Min:Max
−8:1
−7:1
−8:2
−9:2
Difference vs. placebo [1]
LS Means (SE)
−0.6
(0.42)
−1.1
(0.42)
−0.8
(0.42)
95% CI
−1.4:0.2
−1.9:−0.3
−1.7:−0.0
P-value
0.1486
0.0090
0.0488
Week 24
Original WOMAC pain subscale
N
38
46
42
37
Mean (SD)
3.4
(2.15)
3.6
(2.30)
3.1
(2.31)
3.1
(2.47)
Median
3.4
3.3
2.7
2.4
Min:Max
0:8
0:8
0:9
0:8
Change from baseline
N
38
46
42
37
Mean (SD)
−2.4
(2.19)
−2.0
(2.15)
−2.9
(2.46)
−2.8
(2.26)
Median
−2.0
−1.8
−2.5
−3.0
Min:Max
−8:1
−7:2
−8:3
−9:2
Difference vs. placebo [1]
LS Means (SE)
−0.1
(0.45)
−0.7
(0.46)
−0.7
(0.47)
95% CI
−1.0:0.8
−1.6:0.2
−1.6:0.3
P-value
0.8648
0.1513
0.1601
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
As shown in Table 4, the baseline mean WOMAC Function Subscale scores were similar and ranged from 5.9 to 6.2. Treatment effect in terms of the LS mean differences vs. placebo for mAb1 0.03 mg/kg group was the smallest. For the two groups of patients given mAb1 at 0.1 mg/kg and 0.3 mg/kg, the treatment effects were similar and ranged from −0.6 to −1.6. The p-values were statistically significant for the week 8 duration and the week 16 duration, respectively, but not for the week 24 duration. For the group of patients given mAb1 at 0.03 mg/kg, the p-value was statistically significant for the week 8 duration and had a marginal value for the week 16 duration (p=0.0693), but was not significant for the week 24 duration.
TABLE 4
WOMAC Function Subscale from Baseline to Week 8, 16 and
24 --- Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Baseline
N
55
52
53
54
Mean (SD)
5.9
(1.75)
5.9
(1.83)
6.2
(1.67)
6.2
(2.07)
Median
6.2
5.9
6.2
6.6
Min:Max
2:9
2:10
3:9
1:10
Week 8
Original WOMAC function subscale
N
51
50
50
46
Mean (SD)
4.1
(2.08)
3.1
(2.09)
2.8
(2.14)
2.6
(2.44)
Median
4.5
2.6
2.4
1.5
Min:Max
0:8
0:10
0:7
0:8
Change from baseline
N
51
49
50
46
Mean (SD)
−1.8
(1.95)
−2.8
(2.07)
−3.4
(2.32)
−3.4
(2.57)
Median
−1.4
−3.1
−3.2
−3.5
Min:Max
−7:2
−7:2
−9:1
−9:5
Difference vs. placebo [1]
LS Means (SE)
−1.2
(0.41)
−1.6
(0.40)
−1.4
(0.40)
95% CI
−2.0:−0.4
−2.4:−0.8
−2.2:−0.6
P-value
0.0037
0.0001
0.0005
Week 16
Original WOMAC function
subscale
N
44
47
44
41
Mean (SD)
3.6
(2.26)
3.0
(2.21)
2.7
(2.26)
2.7
(2.43)
Median
3.2
2.9
2.5
1.5
Min:Max
0:9
0:9
0:8
0:8
Change from baseline
N
44
47
44
41
Mean (SD)
−2.3
(2.30)
−2.9
(1.78)
−3.4
(2.28)
−3.1
(2.18)
Median
−1.5
−2.9
−3.5
−3.3
Min:Max
−8:1
−7:1
−9:1
−9:4
Difference vs. placebo [1]
LS Means (SE)
−0.8
(0.41)
−1.1
(0.41)
−0.9
(0.42)
95% CI
−1.6:0.1
−1.9:−0.3
−1.8:−0.1
P-value
0.0693
0.0071
0.0245
Week 24
Original WOMAC function
subscale
N
38
46
42
37
Mean (SD)
3.4
(2.15)
3.6
(2.35)
3.2
(2.33)
3.0
(2.43)
Median
3.4
3.3
2.7
2.7
Min:Max
0:8
0:8
0:9
0:7
Change from baseline
N
38
46
42
37
Mean (SD)
−2.4
(2.29)
−2.3
(2.05)
−2.9
(2.30)
−2.6
(2.40)
Median
−2.0
−2.3
−2.6
−3.0
Min:Max
−8:1
−7:3
−9:2
−9:4
Difference vs. placebo [1]
LS Means (SE)
−0.3
(0.45)
−0.7
(0.45)
−0.6
(0.46)
95% CI
−1.2:0.6
−1.5:0.2
−1.5:0.3
P-value
0.5214
0.1499
0.1748
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
PGIC
The results of the patient-rated assessment of response to treatment (PGIC) are shown in Table 5.
TABLE 5
Patients Global Impression of Change (PGIC) at Week 8, 16
and 24--- Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Week 8
PGIC
N
51
50
50
46
Mean (SD)
3.1
(1.21)
2.3
(1.00)
2.1
(1.10)
2.1
(0.98)
Median
3.0
2.0
2.0
2.0
Min:Max
1:6
1:4
1:5
1:4
Difference vs. placebo [1]
LS Means (SE)
−0.8
(0.21)
−1.0
(0.21)
−0.9
(0.21)
95% CI
−1.2:−0.4
−1.4:−0.6
−1.4:−0.5
P-value
0.0002
<0.0001
<0.0001
Week 16
PGIC
N
44
47
43
41
Mean (SD)
2.8
(1.32)
2.2
(0.95)
2.5
(1.26)
2.4
(1.16)
Median
3.0
2.0
2.0
2.0
Min:Max
1:7
1:5
1:6
1:5
Difference vs. placebo [1]
LS Means (SE)
−0.7
(0.24)
−0.4
(0.24)
−0.5
(0.25)
95% CI
−1.1:−0.2
−0.9:0.1
−1.0:−0.1
P-value
0.0056
0.1168
0.0297
Week 24
PGIC
N
38
46
42
37
Mean (SD)
2.7
(0.94)
2.8
(1.41)
2.7
(1.40)
2.5
(1.07)
Median
3.0
3.0
2.0
2.0
Min:Max
1:5
1:6
1:6
1:6
Difference vs. placebo [1]
LS Means (SE)
−0.1
(0.26)
−0.1
(0.27)
−0.3
(0.28)
95% CI
−0.6:0.5
−0.6:0.5
−0.8:0.2
P-value
0.8274
0.8091
0.2710
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
For all 3 mAb1 treatment groups at week 8, the LS means difference vs. placebo ranged from −1.0 to −0.8 and were statistically significant at the 5% level. Results of the patient groups given mAb1 at 0.03 mg/kg and 0.3 mg/kg as compared with placebo were statistically significant at week 16. None of the three mAb1 groups was significantly different from placebo at week 24.
Quality of Life Questionnaire (SF-12)
The results of the analyses from the Quality of Life questionnaire are shown in Table 6 (Physical Component Score) and Table 7 (Mental Component Score).
TABLE 6
SF-12 Physical Component Score (PCS) from Baseline to Week 8,
16 and 24 --- Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Baseline
N
54
52
53
53
Mean (SD)
34.0
(8.06)
32.0
(8.97)
32.3
(9.65)
33.6
(8.82)
Median
31.9
32.6
30.7
33.2
Min:Max
19:53
14:57
9:55
16:53
Week 8
Original PCS
N
51
50
49
46
Mean (SD)
37.4
(8.98)
40.5
(9.24)
41.5
(8.46)
42.9
(8.43)
Median
35.6
41.1
42.6
43.4
Min:Max
16:58
22:60
22:56
22:56
Change from baseline
N
50
49
49
45
Mean (SD)
2.9
(6.64)
8.3
(8.18)
9.0
(9.42)
8.4
(9.29)
Median
2.1
6.6
9.3
7.3
Min:Max
−10:19
−7:23
−9:40
−12:36
Difference vs. placebo [1]
LS Means (SE)
4.4
(1.47)
5.1
(1.46)
5.6
(1.49)
95% CI
1.5:7.3
2.2:8.0
2.6:8.5
P-value
0.0034
0.0006
0.0002
Week 16
Original PCS
N
44
46
43
41
Mean (SD)
40.3
(8.82)
41.7
(9.72)
41.0
(9.31)
43.2
(9.57)
Median
39.5
40.3
40.5
42.2
Min:Max
24:59
22:62
21:63
26:61
Change from baseline
N
43
46
43
40
Mean (SD)
6.1
(8.43)
9.7
(8.78)
8.4
(10.57)
9.2
(10.74)
Median
5.7
8.3
8.1
8.0
Min:Max
−11:23
−6:30
−8:42
−8:38
Difference vs. placebo [1]
LS Means (SE)
3.0
(1.72)
1.9
(1.73)
3.6
(1.76)
95% CI
−0.4:6.4
−1.5:5.3
0.1:7.1
P-value
0.0854
0.2647
0.0415
Week 24
Original PCS
N
38
46
42
37
Mean (SD)
40.3
(9.89)
39.2
(10.39)
40.2
(9.72)
38.9
(9.45)
Median
40.7
39.8
41.5
38.2
Min:Max
23:58
18:61
16:61
24:62
Change from baseline
N
37
46
42
36
Mean (SD)
6.6
(9.02)
7.3
(10.09)
7.2
(9.54)
4.8
(10.52)
Median
5.9
6.5
4.7
3.6
Min:Max
−11:25
−10:36
−11:35
−17:43
Difference vs. placebo [1]
LS Means (SE)
1.2
(1.86)
1.1
(1.89)
−0.6
(1.95)
95% CI
−2.5:4.8
−2.6:4.9
−4.4:3.3
P-value
0.5340
0.5444
0.7759
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
The baseline means SF-12 PCS were similar among the four groups. The LS mean vs. placebo at week 4 on PCS for the patient groups given mAb1 at 0.03 mg/kg, 0.1 mg/kg, and 0.3 mg/kg were 4.4, 5.1, and 5.6, respectively, with p-values of 0.0034, 0.0006, and 0.0002, respectively. The p-value was significant for the group given mAb1 at 0.3 mg/kg at week 16, but not for the other two groups. All p-values were non-significant at week 24.
TABLE 7
SF-12 Mental Component Score (MCS) from Baseline to Week 8,
16 and 24 --- Observed Data Using MMRM (Full Analysis Set)
mAb1
mAb1
mAb1
Placebo
(0.03 mg/kg)
(0.1 mg/kg)
(0.3 mg/kg)
Week
(N = 55)
(N = 53)
(N = 53)
(N = 54)
Baseline
N
54
52
53
53
Mean (SD)
51.7
(11.89)
51.8
(11.73)
51.4
(10.96)
51.3
(11.63)
Median
51.8
54.7
53.2
51.6
Min:Max
17:71
27:69
25:68
24:71
Week 8
Original MCS
N
51
50
49
46
Mean (SD)
54.2
(9.43)
54.2
(9.01)
54.0
(8.55)
53.7
(10.18)
Median
56.6
55.4
56.5
55.1
Min:Max
30:69
33:70
31:67
25:69
Change from baseline
N
50
49
49
45
Mean (SD)
2.7
(9.70)
1.9
(9.49)
3.2
(9.06)
1.8
(9.61)
Median
0.9
0.9
1.6
1.1
Min:Max
−19:33
−19:21
−11:37
−20:25
Difference vs. placebo [1]
LS Means (SE)
−0.3
(1.50)
0.3
(1.49)
−0.7
(1.53)
95% CI
−3.3:2.6
−2.7:3.2
−3.7:2.3
P-value
0.8169
0.8557
0.6597
Week 16
Original MCS
N
44
46
43
41
Mean (SD)
55.4
(8.16)
52.5
(9.79)
53.0
(9.17)
52.2
(10.00)
Median
57.4
54.7
54.2
53.7
Min:Max
34:67
25:68
22:67
27:71
Change from baseline
N
43
46
43
40
Mean (SD)
3.5
(11.28)
−0.4
(10.26)
2.5
(7.66)
0.2
(8.91)
Median
1.3
−0.2
1.4
−0.2
Min:Max
−19:33
−38:28
−12:16
−13:17
Difference vs. placebo [1]
LS Means (SE)
−3.1
(1.57)
−1.8
(1.58)
−2.9
(1.61)
95% CI
−6.2:−0.0
−4.9:1.3
−6.1:0.3
P-value
0.0488
0.2624
0.0749
Week 24
Original MCS
N
38
46
42
37
Mean (SD)
53.5
(10.09)
50.9
(9.97)
54.9
(9.60)
54.8
(9.07)
Median
55.1
53.6
55.3
57.5
Min:Max
16:68
21:70
35:72
33:70
Change from baseline
N
37
46
42
36
Mean (SD)
1.4
(9.43)
−1.4
(10.35)
4.5
(8.04)
2.7
(10.69)
Median
0.0
−3.1
3.8
0.7
Min:Max
−16:31
−27:23
−16:25
−14:26
Difference vs. placebo [1]
LS Means (SE)
−2.9
(1.66)
1.8
(1.68)
1.1
(1.74)
95% CI
−6.2:0.3
−1.5:5.1
−2.3:4.5
P-value
0.0775
0.2912
0.5316
Note:
SD = standard deviation, CI = confidence interval, SE = standard error.
[1] The difference between each mAb1 treatment group and placebo in term of change from baseline.
The baseline mean SF-12 Mental Component Score (MCS) was similar among the four groups. The p-values indicate non-significant results at all time points for all groups except for the group given mAb1 at 0.3 mg/kg at week 16 (p=0.0415).
SUMMARY
The results of the key efficacy analysis showed that the 2 higher mAb1 doses (0.1 mg/kg and 0.3 mg/kg) consistently demonstrated significant treatment effects as compared with placebo up to week 16 on most efficacy endpoints.
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16282120
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Jul 6th, 2021 12:00AM
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Aug 18th, 2020 12:00AM
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https://www.uspto.gov?id=US11053280-20210706
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Anti-VEGF protein compositions and methods for producing the same
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The present disclosure pertains to compositions comprising aflibercept and methods for producing such compositions in chemically defined media and using chromatography to reduce amounts of certain aflibercept variants.
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11053280
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1. A method of producing aflibercept, comprising:
(a) producing a clarified harvest of cells cultured in a chemically defined medium (CDM);
(b) binding aflibercept from said clarified harvest to a Protein A resin, wherein said aflibercept includes variants that have at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine and a combination thereof;
(c) eluting said aflibercept of step (b) forming an affinity eluate, wherein said eluate has a first color;
(d) subjecting said eluate comprising aflibercept to an anion exchange chromatography (AEX) column; and
(e) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color, and wherein said first color of said affinity eluate is a more intense yellow brown color than said second color of said flowthrough fraction when said affinity eluate and flowthrough fraction protein concentrations are normalized.
2. The method of claim 1, wherein said cells are selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.
3. The method of claim 1, wherein said oxidized amino acid residue is histidine.
4. The method of claim 1, wherein said oxidized amino acid residue is tryptophan.
5. A method of producing aflibercept from a clarified harvest of a cell cultured in a chemically defined medium (CDM), comprising:
(a) binding aflibercept from said clarified harvest to a Protein A resin;
(b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate comprises acidic species of aflibercept;
(c) subjecting said eluted aflibercept to an anion exchange (AEX) chromatography column; and
(d) collecting one or more flowthrough fractions, and wherein the percent of acidic species of aflibercept in said affinity eluate of step (b) is greater than the percent of acidic species of aflibercept in said one or more flowthrough fractions of (d) when the concentrations of protein in said affinity eluate and flowthrough fractions are normalized to 10.0 g/L, and
wherein said acidic species of aflibercept correspond to peaks that elute earlier than a main peak in a cation exchange chromatography (CEX) chromatogram of aflibercept, and wherein the chromatogram is generated using a first mobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 and a second mobile phase of 40 mM sodium phosphate, 100 mM sodium chloride, pH 9.0 (mobile phase B), and wherein the chromatogram is generated using detection at 280 nm.
6. The method of claim 5, wherein said acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine and a combination thereof.
7. The method of claim 5, further comprising after binding aflibercept from said clarified harvest, subjecting aflibercept to one or more further chromatographic steps selected from the group consisting of: cation exchange chromatography (CEX), hydrophobic interactive chromatography, size exclusion chromatography and a combination thereof.
8. The method of claim 5, wherein said cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.
9. The method of claim 6, wherein said cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.
10. The method of claim 6, wherein said acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue of tryptophan.
11. The method of claim 6, wherein said acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue of histidine.
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11
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/065,012, filed on Aug. 13, 2020, the content of which is incorporated herein by reference in its entirety. This application also claims priority to and the benefit of Provisional Patent Application No. 62/944,635, filed on Dec. 6, 2019.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 18, 2021, is named 070816-02200 SL.txt and is 148,897 bytes in size.
FIELD
The present invention generally pertains to anti-VEGF compositions and methods for producing the same.
BACKGROUND
Protein-based biopharmaceutical compositions have emerged as important products for research, the treatment of ophthalmological diseases, cancer, autoimmune disease, and infection, as well as other diseases and disorders. Biopharmaceuticals represent one of the fastest growing product segments of the pharmaceutical industry.
A class of cell-derived dimeric mitogens with selectivity for vascular endothelial cells has been identified and designated vascular endothelial cell growth factor (VEGF).
Persistent angiogenesis may cause or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy and neovascular glaucoma. An inhibitor of VEGF activity would be useful as a treatment for such diseases and other VEGF-induced pathological angiogenesis and vascular permeability conditions, such as tumor vascularization. The angiopoietins and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be largely specific for vascular endothelial cells.
Several eye disorders are associated with pathological angiogenesis. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from the CNV causes macular edema and collection of fluid beneath the macula resulting in vision loss. Diabetic macular edema (DME) is another eye disorder with an angiogenic component. DME is the most prevalent cause of moderate vision loss in patients with diabetes and is a common complication of diabetic retinopathy, a disease affecting the blood vessels of the retina. Clinically significant DME occurs when fluid leaks into the center of the macula, the light-sensitive part of the retina responsible for sharp, direct vision. Fluid in the macula can cause severe vision loss or blindness.
Various VEGF inhibitors, such as the VEGF trap EYLEA® (aflibercept), have been approved to treat such eye disorders.
SUMMARY
The present invention relates to anti-VEGF proteins including the VEGF trap protein aflibercept, which is a fusion protein. The instant invention also pertains to a new anti-VEGF protein, the aflibercept MiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise noted). Disclosed herein are methods of making these anti-VEGF proteins, including production modalities that provide efficient and effective means to produce the proteins of interest. In one aspect, the instant invention is directed towards the use of chemically defined media (CDM) to produce anti-VEGF proteins. In a particular aspect, the CDMs of interest are those that, when used, produce a protein sample wherein the sample has a yellow-brown color and may comprise oxidized species. Still further in the present application, protein variants of aflibercept and VEGF MiniTrap are disclosed together with attendant production methods.
Production of Aflibercept
The present disclosure describes the production of aflibercept using a cell culture medium. In one embodiment, the cell culture medium is a chemically defined medium (“CDM”). CDM is often used because it is a protein-free, chemically-defined formula using no animal-derived components and there is certainty as to the composition of the medium. In another embodiment, the cell culture medium is a soy hydrolysate medium.
In one embodiment, a method of producing a recombinant protein comprises: (a) providing a host cell genetically engineered to express a recombinant protein of interest; (b) culturing the host cell in a CDM under suitable conditions in which the cell expresses the recombinant protein of interest; and (c) harvesting a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is selected from the group consisting of aflibercept and recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), an aflibercept scFv and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.
In one aspect of the present embodiment, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells and BHK.
Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium, Ham's Nutrient Mixture, Excell medium, and IS CHO-CD medium. Other CDMs known to those skilled in the art are also contemplated to be within the scope of the present invention. In a particular aspect, a suitable CDM is CDM1B (Regeneron) or Excell Advanced Medium (SAFC).
In one embodiment, a clarified harvest sample from a CDM culture comprising aflibercept is subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, Protein A. In a further aspect, the eluate of the affinity procedure exhibits a certain color, for example, the eluate can exhibit a yellow-brown color. As described in more detail infra, color can be assessed using (i) the European Color Standard “BY” in which a qualitative visual inspection is made or (ii) a colorimetric assay, CIE L*, a*, b* (or CIELAB), which is more quantitative than the BY system. However, in either case, color assessment between multiple samples should be normalized against protein concentration in order to assure a meaningful assessment. For example, referring to Example 9 below, the Protein A eluate has a “b*” value of around 2.52 which corresponds to a BY value of approximately BY5 (when measured at a concentration of 5 g/L protein in the protein A eluate). If the color of the Protein A eluate is to be compared to another sample, then the comparison should be made against the same protein concentration. The b* value in the CIELAB color space is used to expresses coloration of the samples and covers blue (−) to yellow (+). A higher b* value of a sample compared to another indicates a more intense yellow-brown coloration in the sample compared to the other.
In one embodiment, aflibercept is produced from a host cell genetically engineered to express aflibercept using CDM. In one aspect, other species or variants of aflibercept are also produced. These variants include aflibercept isoforms that comprise one or more oxidized amino acid residues collectively referred to as oxo-variants. A clarified harvest sample produced using CDM comprising aflibercept as well as its oxo-variants can be subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, a Protein A column. When a sample extracted from an affinity eluate, which may or may not manifest a yellow-brown color, is analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more oxidized variants of aflibercept may be detected. Certain amino acid residues of a modified aflibercept are shown to be oxidized including, but not limited to, histidine and/or tryptophan residues. In one aspect, the variants can include oxidation of one or more methionine residues as well as other residues, see infra.
In another aspect, the variants can include oxidation of one or more tryptophan residues to form N-formylkynurenine. In a further aspect, the variants can include oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In a particular aspect, the protein variants can include oxidation of one or more tryptophan residues to form di-hydroxyl tryptophan. In a particular aspect, the protein variants can include oxidation of one or more tryptophan residues to form tri-hydroxyl tryptophan.
In another aspect, the variants can include one or more modifications selected from the group consisting of: deamidation of, for example, one or more asparagines; one or more aspartic acids converted to iso-aspartate and/or Asn; oxidation of one or more methionines; oxidation of one or more tryptophans to N-formylkynurenine; oxidation of one or more tryptophans to mono-hydroxyl tryptophan; oxidation of one or more tryptophans to di-hydroxyl tryptophan; oxidation of one or more tryptophans to tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation of one or more arginines; removal of C-terminal glycine; and presence of one or more non-glycosylated glycosites.
In another embodiment, the invention is directed to methods for producing aflibercept. In one aspect, a clarified harvest sample comprising aflibercept and its variants are subjected to a capture step such as Protein A affinity chromatography. Subsequent to the affinity step, an affinity eluate can be subjected to ion exchange chromatography. The ion exchange chromatography can be either cation or anion exchange chromatography. Also contemplated to be within the scope of the present embodiment is mixed-mode or multimodal chromatography as well as other chromatographic procedures which are discussed further below. In a particular aspect, the ion exchange chromatography is anion exchange chromatography (AEX). Suitable conditions for employing AEX include, but are not limited to, Tris hydrochloride at a pH of about 8.3 to about 8.6. Following equilibration using, for example, Tris hydrochloride at a pH of about 8.3 to about 8.6, the AEX column is loaded with sample. Following the loading of the column, the column can be washed one or multiple times using, for example, the equilibrating buffer. In a particular aspect, the conditions used can facilitate the differential chromatographic behavior of aflibercept and its oxidized variants such that a fraction comprising aflibercept absent significant amounts of oxo-variants can be collected in a flowthrough fraction while a significant portion of oxo-variants are retained on the solid-phase of the AEX column and can be obtained upon stripping the column—see Example 2 below, FIG. 11. Referring to FIG. 11 and Example 2, changes in oxo-variants can be observed between the different production steps. For example, this change can be illustrated by data in the “Tryptophan Oxidation Level (%)” section, specifically, the “W138(+16)” column. There it can be observed that the oxo-variants (specifically, oxo-tryptophan) went from about 0.131% in a load sample to about 0.070% in a flowthrough sample following AEX chromatography (AEX separation 2), indicating that there was a reduction in oxo-variants of aflibercept using AEX.
Use of ion exchange can be used to mitigate or minimize color. In one aspect of the present embodiment, a clarified harvest sample is subjected to capture chromatography, for example, using Protein A affinity chromatography. The affinity column is eluted and has a first color with a particular BY and/or b* value assigned thereto. This Protein A eluate is then subjected to ion exchange chromatography such as anion exchange chromatography (AEX). The ion exchange column is washed and the flowthrough is collected and has a second color having a particular BY and/or b* value assigned thereto. In a particular aspect, the color value (either “BY” or “b*”) of the first color differs from the second color. In a further aspect, the first color of the Protein A eluate has a more yellow-brown color as compared to the second color of the AEX flowthrough as reflected by the respective BY and/or b* value. Typically, there is a reduction in yellow-brown color of the second color following AEX when compared to the first color of the Protein A eluate. For example, the use of anion exchange reduced the yellow-brown color observed in a Protein A eluate sample from a b* value of about 3.06 (first color) to about 0.96 (second color) following AEX—see Example 2, Table 2-3 below.
In one aspect of the embodiment, the pH of both the equilibration and wash buffers for the AEX column can be from about 8.30 to about 8.60. In another aspect, the conductivity of both the equilibration and wash buffers for the AEX column can be from about 1.50 to about 3.00 mS/cm.
In one aspect of the embodiment, the equilibration and wash buffers can be about 50 mM Tris hydrochloride. In one aspect, the strip buffer comprises 2 M sodium chloride or 1 N sodium hydroxide or both (see Table 2-2).
The present embodiment can include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography (HIC), affinity chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and/or ultra/diafiltration as well as other well-known chromatographic steps.
In one embodiment, the anti-VEGF protein is glycosylated at one or more asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6 G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation. In one aspect, the anti-VEGF protein can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
In one aspect, glycosylation profile of a composition of an anti-VEGF protein is as follows: about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (see Example 6). In one aspect, the anti-VEGF protein has Man5 glycosylation at about 32.4% of asparagine 123 residues and/or about 27.1% of asparagine 196 residues.
In one embodiment, the process can further comprise formulating a drug substance using a pharmaceutically acceptable excipient. In one aspect of the embodiment, the pharmaceutically acceptable excipient can be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative. Other well-known excipients to the skilled artisan are within the purview of this embodiment.
In one aspect of the embodiment, the formulation can be suitable for administration to a human subject. In particular, administration can be affected by intravitreal injection. In one aspect, the formulation can have about 40 to about 200 mg/mL of the protein of interest.
The formulation can be used as a method of treating or preventing angiogenic eye disorders which can include: age-related macular degeneration (e.g., wet or dry), macular edema, macular edema following retinal vein occlusion, retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathies (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in a subject that does not suffer from DME).
Production of VEGF MiniTrap
The present disclosure describes the production of a modified version of aflibercept wherein the Fc portion is removed or absent and is referred to as aflibercept MiniTrap or VEGF MiniTrap. This MiniTrap can be produced in cell culture medium including a chemically defined medium (CDM) or soy hydrolysate medium.
In one embodiment, the MiniTrap is produced using CDM. In one aspect of MiniTrap production, full length aflibercept is produced using a suitable host and under suitable conditions and is further processed whereby the Fc portion is enzymatically removed thus yielding MiniTrap. Alternatively, a gene encoding MiniTrap (e.g., a nucleotide sequence encoding aflibercept absent its Fc portion) can be produced under suitable conditions using a suitable host cell.
In one embodiment, a method for manufacturing MiniTrap includes producing a full-length aflibercept fusion protein followed by cleavage of the Fc region. In one aspect, the method involves producing a recombinant protein, namely a full-length aflibercept fusion protein (see, U.S. Pat. No. 7,279,159, the entire teaching of which is incorporated herein by reference), comprising: (a) providing a host cell genetically engineered to express full length aflibercept; (b) culturing the host cell in CDM under suitable conditions in which the cell expresses the full length aflibercept; (c) harvesting a preparation of the full length aflibercept produced by the cell; and (d) subjecting the full length aflibercept to enzymatic cleavage specific for removing the Fc portion of the fusion protein. In another aspect, a nucleotide sequence encoding aflibercept minus its Fc portion is expressed from a suitable host cell under suitable conditions well known to those skilled in the art (see U.S. Pat. No. 7,279,159).
In one aspect of the present embodiment, the aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells and BHK.
Suitable CDMs include Dulbecco's Modified Eagle's (DME) medium, Ham's Nutrient Mixture, EX-CELL medium (SAFC), and IS CHO-CD medium (Irvine). Other CDMs known to those skilled in the art are also contemplated to be within the scope of the present invention. In a particular aspect, a suitable CDM is CDM1B (Regeneron) or Excell medium (SAFC).
In one aspect, during the production of MiniTrap, a sample comprising a protein of interest (i.e., aflibercept fusion protein and/or MiniTrap) along with its variants (including oxo-variants) can exhibit certain color properties—a yellow-brown color. For example, an eluate sample from an affinity chromatography step can exhibit a certain yellow-brown color measured using the BY and/or b* system (see Examples 2 and 9 below). Exemplary sources for a “sample” may include an affinity chromatography, such as Protein A, eluate; the sample may be obtained from a flowthrough fraction of ion exchange chromatography procedure; it may also be obtained from the strip of an ion exchange column—there are other sources during a production process well known to those skilled in the art from which a sample may be analyzed. As mentioned above and described further below, color can be assessed using (i) the European Color Standard “BY” in which a qualitative visual inspection is made or (ii) a colorimetric assay, CIELAB, which is more quantitative than the BY system. However, in either case, color assessment between multiple samples should be normalized, for example, using protein concentration, in order to assure a meaningful assessment between samples.
In one aspect of the present embodiment, a full-length aflibercept fusion protein can be subjected to enzymatic processing (“cleavage activity”) in order to generate a VEGF MiniTrap, for example, using proteolytic digestion employing a protease or enzymatically active variant thereof. In one aspect of this embodiment, the protease can be an immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS). In another aspect, the protease can be thrombin trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain, pronase, or protease from Aspergillus saitoi. In one aspect, the protease can be a cysteine protease. In a particular aspect of the embodiment, the protease can be IdeS. In another aspect, the protease can be a variant of IdeS. Non-limiting examples of variants of IdeS are described infra and include a polypeptide having an amino acid sequence as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the protease can be immobilized on agarose or another suitable matrix.
In one aspect, a protein of interest (together with its variants) is produced using CDM. In a particular aspect, the protein of interest includes aflibercept or MiniTrap. The variants comprise one or more oxidized amino acid residues, collectively oxo-variants. Examples of oxidized residues include, but are not limited to, one or more histidine and/or tryptophan residues. Other oxidized residues have also been detected using LC-MS and are described below, such as oxidized methionine. Subsequent chromatography such as AEX can be used to isolate these oxo-variants from the protein of interest in a given sample and are described herein.
In one aspect, the variants can include oxidation of one or more tryptophan residues to form N-formylkynurenines. In a further aspect, the variants can include oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In a particular aspect, the protein variants can include oxidation of one or more tryptophan residues to form di-hydroxyl tryptophan. In a particular aspect, the protein variants can include oxidation of one or more tryptophan residues to form tri-hydroxyl tryptophan.
In another aspect, the oxo-variants can include one or more modifications selected from the group consisting of: deamidation of one or more asparagine residues; one or more aspartic acids converted to iso-aspartate and/or asparagine; oxidation of one or more methionine residues; oxidation of one or more tryptophan residues to form N-formylkynurenine; oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan; oxidation of one or more tryptophan residues to form di-hydroxyl tryptophan; oxidation of one or more tryptophan residues to form tri-hydroxyl tryptophan; Arg 3-deoxyglucosonation of one or more arginine residues; removal of C-terminal glycine; and presence of one or more non-glycosylated glycosites.
In one embodiment, the method of manufacturing a MiniTrap protein comprises (a) capturing a full-length aflibercept fusion protein on a first chromatographic platform and (b) cleaving the aflibercept thereby forming a MiniTrap protein, i.e., aflibercept absent its Fc domain. In one aspect, the first chromatographic support comprises an affinity chromatography media, an ion-exchange chromatography media, or a hydrophobic interaction chromatography media. In a particular aspect, the first chromatographic platform comprises an affinity chromatography platform such as a Protein A. In a further aspect, the protein of capture step (a) is eluted from the first chromatography platform prior to cleavage step (b). In a still further aspect, a second capture step follows cleavage step (b). In a particular aspect, this second capture step can be facilitated by affinity chromatography such as Protein A affinity chromatography. The flowthrough of this second capture step (comprising MiniTrap) has a first color, for example, a yellow-brown color and measured having a particular BY and/or b* value—see, e.g., Example 9 below. Additionally, LC-MS analysis of this second capture flowthrough may demonstrate the presence of oxo-variants wherein one or more residues of MiniTrap are oxidized (see Example 9 below).
In a further aspect, the second capture flowthrough can be subjected to ion exchange chromatography such as AEX. This AEX column can be washed using a suitable buffer and an AEX flowthrough fraction can be collected comprising essentially MiniTrap. This AEX flowthrough fraction can have a second color that is of a yellow-brown coloration having a particular BY and/or b* value. In a further aspect, the first color (flowthrough from second capture step) and second color (flowthrough of the ion exchange procedure) have different colors as measured either by the BY and/or b* system. In one aspect, the second color demonstrates a diminished yellow-brown color when compared to the first color using either a BY and/or b* value following AEX.
In another embodiment, the cleavage activity of step (b) can be performed using a chromatographic column wherein the cleavage activity, for example, an enzyme activity, is affixed or immobilized to a column matrix. The column used in step (b) can comprise one or more of the proteases already alluded to and more fully described below.
In one embodiment, the ion-exchange chromatography procedure can comprise an anion-exchange (AEX) chromatography media. In another aspect, the ion-exchange chromatography media comprises a cation exchange (CEX) chromatography media. Suitable conditions for employing AEX include, but are not limited to, Tris hydrochloride at a pH of about 8.3 to about 8.6. Following equilibration using, for example, Tris hydrochloride at a pH of about 8.3 to about 8.6, the AEX column is loaded with sample. Following the loading of the column, the column can be washed one or multiple times using, for example, the equilibrating buffer. In a particular aspect, the conditions used can facilitate the differential chromatographic behavior of MiniTrap and its oxo-variants using AEX such that the MiniTrap is substantially in the flowthrough fraction while the oxo-variants are substantially retained on the AEX column and can be collected by stripping the column (see Example 9 below).
In one example, samples from different stages of production were analyzed for color and presence of oxo-variants. Referring to Example 9, the affinity flowthrough pool (flowthrough from a second Protein A affinity step) had a first b* value of about 1.58 (see Table 9-3). This second affinity flowthrough was subjected to AEX. The AEX flowthrough had a second b* value of about 0.50, indicating a significant reduction in yellow-brown color following the use of AEX. Stripping of the AEX column yielded a strip sample and a third b* value of about 6.10 was observed, indicating that this strip sample had a more yellow-brown color when compared to either the load or flowthrough.
Referring again to Example 9, oxo-variant analysis was also performed. Samples analyzed were the affinity flowthrough pool (second Protein A affinity eluate), AEX flowthrough, and AEX strip. Referring to Table 9-5 and Table 9-6, changes in oxo-variants can be observed between the different production steps. For example, this change can be illustrated by data in the “Tryptophan Oxidation Level (%)” section, specifically, the “W58(+16)” column. There it can be observed that the oxo-variants (specifically, oxo-tryptophan) went from about 0.055% in a load sample to about 0.038% in a flowthrough sample following AEX chromatography, indicating that there was a reduction in oxo-variants following AEX. The AEX strip was analyzed and the percent oxo-tryptophan species was found to be about 0.089%. When this strip value was compared to the load (as well as the flowthrough), it appeared that a significant portion of this oxo-variant was retained on the AEX column.
The present embodiment can include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and/or ultra/diafiltration.
One embodiment of the present invention is directed to a method for regenerating a chromatography column comprising a resin. In one aspect of the embodiment, the resin has an immobilized hydrolyzing agent. In yet another aspect of the embodiment, the resin comprises an immobilized protease enzyme. In still another aspect of the embodiment, the resin is a FabRICATOR® resin or a mutant of the resin. In one aspect of the embodiment, the method of regenerating a column comprising a resin improves reaction efficiency of the resin.
In one aspect of the embodiment, a method of regenerating a column comprising a resin includes incubating the column resin with acetic acid. In one aspect, the concentration of acetic acid used is from about 0.1 M to about 2 M. In one aspect, the concentration of acetic acid is about 0.5 M. In one aspect, the resin is incubated for at least about 10 minutes. In another aspect, the resin is incubated for at least about 30 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 50 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 100 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 200 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 300 minutes.
Optionally, the column resin is further incubated with guanidine hydrochloride (Gu-HCl). In one aspect, Gu-HCl absent acetic acid is used to regenerate the column resin. The concentration of Gu-HCl employed is from about 1 N to about 10 N. In another aspect, the concentration of Gu-HCl is about 6 N. In a further aspect, the column resin can be incubated for at least about 10 minutes with the regenerative agents (acetic acid, Gu-HCl). In yet another aspect, the resin is incubated for at least about 30 minutes. In still another aspect, the resin is incubated for at least about 50 minutes. In yet another aspect, said resin is incubated for at least about 100 minutes.
In one embodiment, the column comprising a resin is stored in ethanol. In one aspect, the column is stored in ethanol, wherein the ethanol percentage is from about 5% v/v to about 20% v/v. In a particular aspect, the column is stored using 20% v/v ethanol.
In one embodiment, the process can further comprise formulating the VEGF MiniTrap using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient can be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative. Other well-known excipients to the skilled artisan are within the purview of this embodiment.
The formulation of the present invention is suitable for administration to a human subject. In one aspect of the present embodiment, administration can be effected by intravitreal injection. In one aspect, the formulation can have about 40 to about 200 mg/mL of the protein of interest. In a particular aspect, the protein of interest is either aflibercept or aflibercept MiniTrap.
The formulation can be used in a method of treating or preventing angiogenic eye disorders which can include: age-related macular degeneration (e.g., wet or dry), macular edema, macular edema following retinal vein occlusion, retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathies (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in a subject that does not suffer from DME).
Variants of IdeS
The present disclosure describes the use of IdeS (FabRICATOR) (SEQ ID NO.: 1) or other polypeptides which are IdeS variants (SEQ ID NO.: 2 to 16) to produce a VEGF MiniTrap. IdeS (SEQ ID NO.: 1) includes asparagine residues at position 87, 130, 182 and/or 274 (shown as “N*” bolded and italicized in SEQ ID NO.: 1 below). The asparagine at these positions may be mutated to an amino acid other than asparagine to form IdeS variants (and the mutated amino acid(s) are shown as italicized and underscored amino acid(s)):
SEQ ID NO.: 1
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQL
DSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVK
EGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGK
ALGLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKY
FVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 2
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYF
VGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 3
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYF
VGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 4
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQL
DSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYF
VGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 5
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQL
DSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVK
EGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGK
ALGLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYF
VGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 6
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLDS
KLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 7
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 8
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 9
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 10
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 11
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQL
DSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKE
GSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKA
LGLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFV
GVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 12
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLDS
KLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEGS
KDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALG
LSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYFVG
VNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 13
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLDS
KLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFVG
VNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 14
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFVG
VNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 15
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATA
GNMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLD
SKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEG
SKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKAL
GLSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFVG
VNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
SEQ ID NO.: 16
MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTS
VWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF GKDDLLCGAATAG
NMLHWWFDQNKDQIKRYLEEHPEKQKINF GEQMFDVKEAIDTKNHQLDS
KLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI GYRLSLTNHGPTPVKEGS
KDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALG
LSHTYANVRINHVINLWGADFDS GNLKAIYVTDSDSNASIGMKKYFVGV
NSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN
In one embodiment, the polypeptide has an isolated amino acid sequence comprising at least 70% sequence identity over a full length of an isolated amino acid sequence as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the isolated amino acid sequence has at least about 80% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has at least about 90% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has about 100% sequence identity over a full length of the isolated amino acid sequence. In one aspect, the polypeptide can be capable of cleaving a target protein into fragments. In a particular aspect, the target protein is an IgG. In another aspect, the target protein is a fusion protein. In yet another aspect, the fragments can comprise a Fab fragment and/or an Fc fragment.
The present disclosure also includes an isolated nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising at least 70% sequence identity over a full length of the isolated amino acid sequence as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the isolated amino acid sequence has at least about 80% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has at least about 90% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has about 100% sequence identity over a full length of the isolated amino acid sequence. In one aspect, the polypeptide can be capable of cleaving a target protein into fragments. In a particular aspect, the target protein is an IgG. In another particular aspect, the target protein is a fusion protein. In yet another particular aspect, the fragments can comprise a Fab fragment and/or an Fc fragment.
The present disclosure also includes a vector which comprises a nucleic acid encoding a polypeptide having an isolated amino acid sequence comprising at least 70% sequence identity over a full length of the isolated amino acid sequence as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector can be a plasmid. In one aspect, the isolated amino acid sequence has at least about 80% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has at least about 90% sequence identity over a full length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has about 100% sequence identity over a full length of the isolated amino acid sequence. In one aspect, the polypeptide can be capable of cleaving a target protein into fragments. In a particular aspect, the target protein is an IgG. In another aspect, the target protein is a fusion protein. In yet another aspect, the fragments can comprise a Fab fragment and/or an Fc fragment.
In one embodiment, the isolated amino acid can comprise a parental amino acid sequence defined by SEQ ID NO.: 1 with an asparagine residue at position 87, 130, 182 and/or 274 mutated to an amino acid other than asparagine. In one aspect, the mutation can confer an increased chemical stability at alkaline pH-values compared to the parental amino acid sequence. In another aspect, the mutation can confer an increase in chemical stability by 50% at alkaline pH-values compared to the parental amino acid sequence. In one aspect, the amino acid can be selected from aspartic acid, leucine, and arginine. In a particular aspect, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another aspect, the asparagine residue at position 130 is mutated to an arginine residue. In yet another aspect, the asparagine residue at position 182 is mutated to a leucine residue. In yet another aspect, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another aspect, the asparagine residues at positions 87 and 130 are mutated. In yet another aspect, the asparagine residues at positions 87 and 182 are mutated. In yet another aspect, the asparagine residues at positions 87 and 274 are mutated. In yet another aspect, the asparagine residues at positions 130 and 182 are mutated. In yet another aspect, the asparagine residues at positions 130 and 274 are mutated. In yet another aspect, the asparagine residues at positions 182 and 274 are mutated. In yet another aspect, the asparagine residues at positions 87, 130 and 182 are mutated. In yet another aspect, the asparagine residues at positions 87, 182 and 274 are mutated. In yet another aspect, the asparagine residues at positions 130, 182 and 274 are mutated. In yet another aspect, the asparagine residues at positions 87, 130, 182 and 274 are mutated.
In a related embodiment, the disclosure includes an isolated nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising a parental amino acid sequence defined by SEQ ID NO.: 1 with asparagine residues at positions 87, 130, 182 and/or 274 mutated to an amino acid other than asparagine—see above. The mutation can confer an increased chemical stability at alkaline pH-values compared to the parental amino acid sequence.
In a further related embodiment, the disclosure includes a vector, which comprises a nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising a parental amino acid sequence defined by SEQ ID NO.: 1 with an asparagine residue at position 87, 130, 182 and/or 274 mutated to an amino acid other than asparagine—see above. The mutation can confer an increased chemical stability at alkaline pH-values compared to the parental amino acid sequence. In one aspect, the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector can be a plasmid.
Affinity-Based Production
The present disclosure also provides methods for reducing host cell proteins as well as other undesirable proteins and nucleic acids during production of an anti-VEGF protein using affinity chromatography.
In one embodiment, a method of producing a recombinant protein comprises: (a) providing a host cell genetically engineered to express a recombinant protein of interest; (b) culturing the host cell under suitable conditions in which the cell expresses the recombinant protein of interest; and (c) harvesting a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (an example of which is disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF proteins.
In one aspect of the present embodiment, the recombinant protein of interest is expressed in a suitable host cell. Non-limiting examples of suitable host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells and BHK.
In one aspect of the present embodiment, the recombinant protein of interest is cultured in a CDM. A suitable CDM includes Dulbecco's Modified Eagle's (DME) medium, Ham's Nutrient Mixture, Excell medium, IS CHO-CD medium, and CDM1B. Other CDMs known to those skilled in the art are also contemplated to be within the scope of the present invention.
The production preparation can comprise at least one contaminant including one or more host cell proteins in addition to the recombinant protein of interest. The at least one contaminant can be derived from cell-substrate, cell culture or a downstream process.
In one embodiment, the invention is directed to methods for producing an anti-VEGF protein from a biological sample using affinity chromatography. In a particular aspect, methods disclosed herein can be used to separate, at least in part, the anti-VEGF protein from one or more host cell proteins and nucleic acids (e.g., DNA) formed during the culture production process of an anti-VEGF protein.
In one aspect, the method can comprise subjecting a biological sample comprising the anti-VEGF protein along with accompanying contaminants to an affinity chromatography under suitable conditions. In a particular aspect, the affinity chromatography can comprise material capable of selectively or specifically binding to the anti-VEGF protein (“capture”). Non-limiting examples of such chromatographic material include: Protein A, Protein G, chromatographic material comprising, for example, protein capable of binding to the anti-VEGF protein, and chromatographic material comprising an Fc binding protein. In a specific aspect, the protein capable of binding to or interacting with the anti-VEGF protein can be an antibody, fusion protein or a fragment thereof. Non-limiting examples of such material capable of selectively or specifically binding to the anti-VEGF protein are described in Example 7.
In one aspect of the present embodiment, the method can comprise subjecting a biological sample comprising an anti-VEGF protein and one or more host cell proteins/contaminants to affinity chromatography under suitable conditions, wherein the affinity chromatography stationary phase comprises a protein capable of selectively or specifically binding to the anti-VEGF protein. In a particular aspect, the protein can be an antibody, a fusion protein, a scFv or an antibody fragment. In a specific aspect, the protein can be VEGF165, VEGF121, or VEGF forms from other species, such as rabbit. For example, as exemplified in Table 7-1 and Table 7-10, using VEGF165 as the protein capable of selectively or specifically binding to or interacting with the anti-VEGF protein led to a successful production of MT5 (an anti-VEGF protein), aflibercept and an anti-VEGF scFv fragment. In another specific aspect, the protein can be one or more of the proteins having an amino acid sequence as shown in SEQ ID NO.: 73-80. Table 7-1 also discloses successful production of MT5 using the proteins having amino acid sequences as shown in SEQ ID NO.: 73-80 as the protein capable of selectively or specifically binding to the anti-VEGF protein (MT5).
In one aspect of the present embodiment, the method can comprise subjecting a biological sample comprising the anti-VEGF protein and one or more host cell proteins/contaminants to affinity chromatography under suitable conditions, wherein the affinity chromatography stationary phase comprises a protein capable of selectively or specifically binding to or interacting with the anti-VEGF protein, wherein the anti-VEGF protein can be selected from aflibercept, VEGF MiniTrap, or an anti-VEGF antibody. In a particular aspect, the VEGF MiniTrap can be obtained from VEGF receptor components; further, it can be formed by recombinant expression of the VEGF MiniTrap in a host cell. Performing the method can reduce the amount of the one or more host cell proteins in the sample. For example, FIG. 35A and FIG. 35B show a significant reduction in total host cell proteins in the sample comprising MT5 (an anti-VEGF protein) on using five different affinity chromatography columns comprising (i) VEGF165 (SEQ ID NO.: 72); (ii) mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (iii) mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain); (iv) mAb3 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light chain) and (v) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) as different proteins capable of selectively or specifically binding to MT5. As seen in FIG. 35A and FIG. 35B, the eluates from each of the affinity-based production processes reduced the host cell proteins from above 7000 ppm to about 25 ppm and to about 55 ppm, respectively.
Suitable conditions for employing affinity chromatography can include, but are not limited to, equilibration of an affinity chromatography column using an equilibration buffer. Following equilibration using, for example, Tris hydrochloride at a pH of about 8.3 to about 8.6, the affinity chromatography column is loaded with a biological sample. Following loading of the column, the column can be washed one or multiple times using, for example, the equilibrating buffer such as Dulbecco's Phosphate-Buffered Saline (DPBS). Other washes including washes employing different buffers can be used before eluting the column. Column elution can be affected by the buffer type and pH and conductivity. Other elution conditions well known to those skilled in the art can be applied. Following elution using one or more types of elution buffers, for example, glycine at a pH of about 2.0 to about 3.0, the eluted fractions can be neutralized with the addition of a neutralizing buffer, for example, 1 M Tris at pH 7.5.
In one aspect of the embodiment, the pH of both the wash and equilibration buffer can be from about 7.0 to about 8.6. In one aspect of the embodiment, the wash buffer can be DPBS. In one aspect, the elution buffer can comprise 100 mM glycine buffer with pH of about 2.5. In another aspect, the elution buffer can be a buffer with a pH of about 2.0 to about 3.0. In one aspect, the neutralizing buffer can comprise 1 M tris with pH of about 7.5.
In one aspect of the present embodiment, the method can further comprise washing the column with a wash buffer. In one aspect of the present embodiment, the method can further comprise eluting the column with an elution buffer to obtain elution fractions. In a particular aspect, the amount of host cell proteins in the eluted fractions is significantly reduced as compared to the amount of host cell proteins in the biological sample, for example, by about 70%, about 80%, 90%, about 95%, about 98%, or about 99%.
The present embodiment can include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography, affinity-based chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and/or ultra/diafiltration.
In one aspect, the glycosylation profile of a composition of an anti-VEGF protein is as follows: about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.
In one aspect of this embodiment, the anti-VEGF protein has Man5 glycosylation at about 32.4% of asparagine 123 residues and/or about 27.1% of asparagine 196 residues. In a specific embodiment, the anti-VEGF protein can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
In one embodiment, the method can further comprise formulating a drug substance using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient can be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative. Other well-known excipients to the skilled artisan are within the purview of this embodiment.
In one aspect of the embodiment, the formulation can be suitable for administration to a human subject. In one aspect of the present embodiment, administration can be effected by intravitreal injection. In one aspect, the formulation can have about 40 to about 200 mg/mL of the protein of interest. In a particular aspect, the protein of interest can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
The formulation can be used in a method of treating or preventing angiogenic eye disorders which can include: age-related macular degeneration (e.g., wet or dry), macular edema, macular edema following retinal vein occlusion, retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreal neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathies (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in a subject that does not suffer from DME).
Synthesis of Oxo-Species
One embodiment of the present invention is directed to one or more methods for oxidized protein species using light. In one aspect of the present embodiment, the protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is aflibercept. In another aspect, the anti-VEGF protein is a VEGF MiniTrap including recombinant VEGF MiniTrap. In yet another aspect of the present embodiment, the anti-VEGF protein is a single-chain variable fragment (scFv).
In one aspect of the present embodiment, a sample comprises a protein of interest, for example, aflibercept fusion protein with minimal or no oxo-variants. The sample is photo-stressed to synthesize oxidized species of aflibercept. In a particular aspect, the sample is photo-stressed by using cool-white light. In another particular aspect, the sample is photo-stressed by using ultraviolet light.
In a specific aspect of the embodiment, a sample comprising aflibercept or another anti-VEGF protein is exposed to cool-white light for about 30 hours to about 300 hours resulting in about 1.5 to about 50-fold increase in modified oligopeptide. These peptides are enzymatically digested and analyzed comprising one or more from the group consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28), RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine that is oxidized to 2-oxo-histidine, wherein C* is a cysteine that is carboxymethylated, wherein M* is an oxidized methionine, wherein W* is an oxidized tryptophan, wherein Y* is an oxidized tyrosine, and wherein F* is an oxidized phenylalanine. The digestion can be performed by proteases alluded to before, for example, trypsin. The oligopeptides can be analyzed using mass spectrometry.
In a specific aspect of the embodiment, a sample comprising aflibercept or other anti-VEGF protein is exposed to ultraviolet light for about 4 hours to about 40 hours resulting in about 1.5 to about 25-fold increase in modified oligopeptide products (obtained on performing digestion) wherein the sample comprises one or more modified oligopeptides selected from the group consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28), RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine that is oxidized to 2-oxo-histidine, wherein C* is a cysteine that is carboxymethylated, wherein M* is an oxidized methionine, wherein W* is an oxidized tryptophan, wherein Y* is an oxidized tyrosine, and wherein F* is an oxidized phenylalanine. The digestion can be performed by proteases alluded to before, for example, trypsin. The oligopeptides can be analyzed using mass spectrometry.
Methods to Minimize Yellow-Brown Color
The present disclosure provides methods for reducing yellow-brown coloration during production of aflibercept, MiniTrap or the like produced in a CDM.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, and then harvesting a preparation comprising the recombinant protein of interest. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159, which is incorporated herein by reference in its entirety), a scFv and other anti-VEGF proteins. In one aspect, the method can produce a preparation of the recombinant protein of interest, wherein the color of the preparation is characterized using the European BY method or the CIELAB method (b*). Additionally, the presence of oxo-variants can be analyzed using, for example, LC-MS.
In one aspect of the present embodiment, mitigation conditions include increasing or decreasing cumulative concentrations of one or more media components, for example, amino acids, metals or antioxidants, including, salts and precursors, corresponding to a reduction in color and protein variants of aflibercept and VEGF MiniTrap. Non-limiting examples of amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In a particular aspect, lowering of cysteine concentration can be effective in reducing the yellow-brown color of a preparation. Cysteine concentration can also affect oxo-variants.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept, and harvesting a preparation of the protein of interest produced by the cell, wherein the suitable conditions are obtained, in part, by lowering the cumulative concentration of cysteine in the CDM to less than or equal to about 10 mM. Examples of suitable media include, but are not limited to, CDM1B, Excell or the like. As used herein, the term “cumulative amount” refers to the total amount of a particular component added to a bioreactor over the course of the cell culture to form the CDM, including amounts added at the beginning of the culture (CDM at day 0) and subsequently added amounts of the component. Amounts of a component added to a seed-train culture or inoculum prior to the bioreactor production (i.e., prior to the CDM at day 0) are also included when calculating the cumulative amount of the component. A cumulative amount is unaffected by the loss of a component over time during the culture (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may nonetheless have different absolute levels, for example, if the component is added to the two cultures at different times (e.g., if in one culture all of the component is added at the outset, and in another culture the component is added over time). A cumulative amount is also unaffected by in situ synthesis of a component over time during the culture (for example, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a given component may nonetheless have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by way of a bioconversion process. A cumulative amount may be expressed in units such as, for example, grams or moles of the component.
As used herein, the term “cumulative concentration” refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the beginning of the production batch, including the contribution to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the beginning of the production batch, and one gram of component X is added at days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters). If, on day 4, an additional one liter of liquid not containing component X were added to the bioreactor, the cumulative concentration would remain 2 g/L. If, on day 5, some quantity of liquid were lost from the bioreactor (for example, through evaporation), the cumulative concentration would remain 2 g/L. A cumulative concentration may be expressed in units such as, for example, grams per liter or moles per liter.
In an aspect of this embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, harvesting a preparation of the protein produced by the cell, wherein the suitable conditions are obtained by lowering the ratio of cumulative cysteine concentration from about 1:10 to 1:29 to a cumulative total amino acid concentration from about 1:50 to about 1:30.
In one embodiment, the method comprises (i) culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept, and (ii) harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions are obtained by lowering the cumulative concentration of iron in the CDM to less than about 55.0 μM. In an aspect of this embodiment, the preparation obtained by this method shows lesser yellow-brown color than the preparation obtained by a method wherein the cumulative concentration of iron in the CDM is more than about 55.0 μM.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept. The method further comprises harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions are obtained by lowering the cumulative concentration of copper in the CDM to less than or equal to about 0.8 μM. In an aspect of this embodiment, the preparation obtained by this method shows lesser yellow-brown color than the preparation obtained by a method wherein the cumulative concentration of copper in the CDM is more than about 0.8 μM.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept, and harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions are obtained by lowering the cumulative concentration of nickel in the CDM to less than or equal to about 0.40 μM. In an aspect of this embodiment, the preparation obtained by this method shows lesser yellow-brown color than the preparation obtained by a method wherein the cumulative concentration of nickel in the CDM is more than about 0.40 μM.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept. The method further comprises harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions are obtained by lowering the cumulative concentration of zinc in the CDM to less than or equal to about 56 μM. In an aspect of this embodiment, the preparation obtained by this method shows lesser yellow-brown color than the preparation obtained by a method wherein the cumulative concentration of zinc in the CDM is more than about 56 μM.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept. The method further comprises harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions are obtained by presence of antioxidants in the CDM in a cumulative concentration of about 0.001 mM to about 10 mM for a single antioxidant and no more than about 30 mM cumulative concentration if multiple antioxidants are added in said CDM. In an aspect of this embodiment, the preparation obtained by this method shows lesser yellow-brown color than the preparation obtained by a method wherein antioxidants are present in the CDM in a cumulative concentration of less than about 0.01 mM or above about 100 mM. Non-limiting examples of the antioxidant can be taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, Vitamin C, Vitamin E, chelating agents, catalase, S-carboxymethyl-L-cysteine, and combinations thereof. Non-limiting examples of chelating agents include aurintricarboxylic acid (ATA), deferoxamine (DFO), EDTA and citrate.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept. The method further comprises harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions include a CDM with a: cumulative concentration of iron in said CDM that is less than about 55 μM, cumulative concentration of copper in said CDM that is less than or equal to about 0.8 μM, cumulative concentration of nickel in said CDM that is less than or equal to about 0.40 μM, cumulative concentration of zinc in said CDM that is less than or equal to about 56 μM, cumulative concentration of cysteine in said CDM that is less than 10 mM; and/or an antioxidant in said CDM in a concentration of about 0.001 mM to about 10 mM for a single antioxidant, and no more than about 30 mM cumulative concentration if multiple antioxidants are added in said CDM.
In one aspect of the present embodiment, the preparation obtained from using suitable conditions results in a reduction in protein variants of aflibercept and VEGF MiniTrap to a desired amount of protein variants of aflibercept and VEGF MiniTrap (referred to as a “target value” of protein variants of aflibercept and VEGF MiniTrap). In a further aspect of this embodiment, the preparation obtained from using suitable conditions results in a reduction in color of the preparations to a desired b* value or BY value (referred to as a “target b* value” “target BY value” respectively) when the preparation of protein, including variants of aflibercept and VEGF MiniTrap are normalized to a concentration of 5 g/L or 10 g/L. In a further aspect of the present embodiment, the target b* value (or target BY value) and/or target value of variants can be obtained in a preparation where the titer increases or does not significantly decrease.
These and other aspects of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 depicts a VEGF MiniTrap generated using an exemplary embodiment, including VEGFR1 (SEQ ID NO.: 34), VEGFR2 (SEQ ID NO.: 36), Hinge domain fragment (SEQ ID NO.: 60) and the cleaved off Fc fragment from aflibercept (SEQ ID NO.: 113).
FIG. 2 depicts a proposed mechanism for histidine oxidation to 2-oxo-histidine (14 Da).
FIG. 3 depicts a proposed mechanism for histidine oxidation to 2-oxo-histidine (16 Da).
FIG. 4 depicts a proposed mechanism for oxidation of tryptophan to N-formylkynurenine and kynurenine.
FIG. 5 depicts an exemplary embodiment for production of aflibercept.
FIG. 6 depicts an exemplary embodiment for production of VEGF MiniTrap.
FIG. 7 depicts an exemplary embodiment for production of aflibercept.
FIG. 8 depicts an exemplary embodiment for production of VEGF MiniTrap.
FIG. 9 depicts a chart of calculated BY standards versus b* value calculated according to an exemplary embodiment.
FIG. 10 depicts results of an experiment performed to evaluate the percentage of 2-oxo-histidines and tryptophan oxidation (where underscoring represents oxidation of the residue) in oligopeptides from protease-digested AEX load and flowthrough, including fragments of reduced and alkylated aflibercept (SEQ ID NO.: 55), including SEQ ID NOS 114-115, 21, 115, 28, 28, 20, 18, 17, 116-117, and 19, respectively, in order of appearance.
FIG. 11 depicts the relative abundance of the peptides identified from the peptide mapping analysis performed using oligopeptides from protease-digested AEX load and flowthrough (where underscoring represents oxidation of the residue in the peptide sequence), including fragments of aflibercept (SEQ ID NO.: 55), including SEQ ID NOS 22, 18, 21, 19-20, 118-119, and 28-29, respectively, in order of appearance.
FIG. 12A depicts a full-view of the chromatogram chart of absorbance versus time (minutes) for MT4 and MT1 at 350 nm.
FIG. 12B depicts an expanded-view of the chromatogram chart of absorbance versus time (16-30 minutes) for MT4 and MT1 at 350 nm, including SEQ ID NOS 21, 28, and 28, respectively, in order of appearance.
FIG. 12C depicts an expanded-view of the chromatogram chart of absorbance versus time (30-75 minutes) for MT4 and MT1 at 350 nm, including SEQ ID NOS 17, 20, 18, and 19, respectively, in order of appearance.
FIG. 13 depicts results of an experiment performed to evaluate the percentage of 2-oxo-histidines (and tryptophan dioxidation) in oligopeptides from protease digested MT1 which has been processed by AEX chromatography and oligopeptides from protease digested MT1 which has been stripped from AEX chromatography, including SEQ ID NOS 21, 28, 17, 20, and 18-19, respectively, in order of appearance.
FIG. 14 depicts results of an experiment performed to compare the acidic species present in different production lots of MT1 and the acidic acid fractions obtained on performing a strong cation exchange (CEX) chromatography, including SEQ ID NOS 21, 28, 28, 17, 20, and 18-19, respectively, in order of appearance.
FIG. 15 depicts an exemplary method for the enrichment of the acidic species and other variants present in cell culture harvest samples using strong cation exchange chromatography.
FIG. 16 depicts the fractions from performing strong cation exchange chromatography according to an exemplary embodiment.
FIG. 17 depicts strong cation exchange chromatograms performed according to an exemplary embodiment for the MT1 production (prior to any production procedure, ≤BY3) subjected to CEX and for enriched variants of desialylated MiniTrap (dsMT1) using a dual salt-pH gradient.
FIG. 18A depicts a 3D chromatogram for unfractionated parental control carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18B depicts a 3D chromatogram for MT1, fraction 1 representing some of the tailing feature for the experiment carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18C depicts a 3D chromatogram for MT1, fraction 2 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18D depicts a 3D chromatogram for MT1, fraction 3 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18E depicts a 3D chromatogram for MT1, fraction 4 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18F depicts a 3D chromatogram for MT1, fraction 5 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 18G depicts a 3D chromatogram for MT1, fraction 6 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 1811 depicts a 3D chromatogram for MT1, fraction 7 feature carried out by strong cation exchange chromatography according to an exemplary embodiment.
FIG. 19 depicts imaged capillary isoelectric focusing (icIEF) electropherograms performed according to an exemplary embodiment for the MT1 production.
FIG. 20 depicts results of a study correlating the exposure of MT1 cool white light or UVA light with the appearance of oxidized amino acid residues, including SEQ ID NOS 114, 114-115, 21, 115, 28, 28, 28, 17, 83, 20, 18, 29, 29, 19, and 22, respectively, in order of appearance.
FIG. 21 depicts the 3D SEC-PDA (size exclusion chromatography coupled to photodiode array detection) chromatograms on CWL-stressed MT1 with absorbance at ˜350 nm (see, e.g., circle highlighting ˜350 nm) according to an exemplary embodiment where A shows the chromatogram at T=0, B shows the chromatogram at 0.5×ICH, C shows the chromatogram at 2.0×ICH, and D shows images of MT1 in vials (normalized to 80 mg/mL) stressed by CWL for different time intervals.
FIG. 22 depicts the 3D SEC-PDA chromatograms on UVA-stressed MT1 with absorbance at ˜350 nm (see, e.g., circle highlighting ˜350 nm) according to an exemplary embodiment where A shows the chromatogram at T=0, B shows the chromatogram at 0.5×ICH, C shows the chromatogram at 2.0×ICH, and D shows images of MT1 in vials (normalized to 80 mg/mL) stressed by UVA for different time intervals.
FIG. 23 A depicts A320/280 absorbance ratios quantitated from SEC-PDA chromatograms for the samples stressed using CWL (top panel) and B depicts a chart of A320/280 absorbance ratios for size variants in the samples stressed using CWL (bottom panel), wherein the samples are stressed according to an exemplary embodiment.
FIG. 24 A depicts A320/280 absorbance ratios quantitated from SEC-PDA chromatograms for the samples stressed using UVA (top panel) and B depicts a chart of A320/280 absorbance ratios for size variants in the samples stressed using UVA (bottom panel), wherein the samples are stressed according to an exemplary embodiment.
FIG. 25 A depicts a scaled estimate of the effect that incubation of various components with aflibercept have on the generation of color (CIE L*, a*, b* predicted b value); and B depicts actual against predicted b value plot.
FIG. 26 depicts the effect of CDMs comprising low cysteine and low metals on the titer of aflibercept (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E).
FIG. 27 is a chart showing prediction profile of the color of the harvest (seen as Day 13 b* values) on increasing/decreasing concentrations of metals and cysteine according to an exemplary embodiment.
FIG. 28 (A-B) depicts the effect of incubation of various components with aflibercept in spent CDM on the generation of color (CIE L*, a*, b* predicted b value) (A); and by a plot of scaled predicted impacts on b value (B).
FIG. 28C depicts the scaled estimated effects of incubation of various components with aflibercept in CDM on the generation of color (CIE L*, a*, b* predicted b value) in a shake flask culture.
FIG. 28D depicts the effect of incubation of hypotaurine and deferoxamine mesylate salt (DFO) with aflibercept in spent CDM on the generation of color (CIE L*, a*, b* predicted “b” value).
FIG. 28E depicts the effect of incubation of various components individually with aflibercept from shake flask culture on the generation of color (CIE L*, a*, b* predicted “b” value).
FIG. 29 is a chart showing the effect of addition of uridine, manganese, galactose and dexamethasone in CDMs on the titer of the aflibercept produced.
FIG. 30 is a chart showing the effect of addition of uridine, manganese, galactose and dexamethasone in CDMs on the viability of cells expressing aflibercept, wherein the aflibercept is produced.
FIG. 31 is a chart showing the effect of addition of uridine, manganese, galactose and dexamethasone in CDMs on the viable cell count of cells expressing aflibercept, wherein the aflibercept is produced.
FIG. 32 is a chart showing a standard curve of absorbance versus host cell protein concentrations (ng/mL) prepared using standard host cell protein solutions from Cygnus 3G (F550).
FIG. 33 is an image of SDS-PAGE analysis performed using non-reducing SDS-PAGE sample buffer.
FIG. 34 is an image of SDS-PAGE analysis performed using reducing SDS-PAGE sample buffer.
FIG. 35A is a chart of total host cell protein detected in loading solution, eluted fractions from affinity chromatography columns 1-3 comprising VEGF165, mAb1 and mAb2, respectively.
FIG. 35B is a chart of total host cell proteins detected in loading solution, eluted fractions from affinity chromatography columns 1, 2, 4 and 5 comprising VEGF165, mAb1, mAb3 and mAb4, respectively.
FIG. 36 depicts the SEC profiles of VEGF MiniTrap A before and B after performing affinity chromatography production.
FIG. 37 depicts a cartoon representation of the kinetic study of VEGF MiniTrap to VEGF165, wherein the VEGF MiniTrap constructs studied were from before and after performing affinity chromatography production according to some exemplary embodiments.
FIG. 38 depicts SPR sensorgrams from the kinetic study of VEGF MiniTrap to VEGF165, wherein the VEGF MiniTrap constructs studied were from before and after performing affinity chromatography production according to some exemplary embodiments.
FIG. 39 is a chart of total host cell protein detected in loading solution, eluted fractions from affinity chromatography columns used repeatedly for columns comprising VEGF165, mAb1 and mAb2.
FIG. 40 depicts the structure of VEGF MiniTrap MT1 (SEQ ID NO.: 46) according to an exemplary embodiment.
FIG. 41 depicts the structure of VEGF MiniTrap MT6 (SEQ ID NO.: 51) according to an exemplary embodiment.
FIG. 42 depicts Total Ion Chromatograms (TIC) of relative absorbance versus time (minutes) for native SEC-MS analysis of MT1, MT5 and MT6 and a zoomed view of the low molecular weight region from the TICs.
FIG. 43 depicts deconvoluted mass spectra of the main peak for MT1 and MT5 to confirm the MiniTrap dimer identity with elucidation for some PTMs, with the N-terminal amino acids indicated (SEQ ID NO.: 120).
FIG. 44 depicts a deconvoluted mass spectrum of the main peak for MT6 to confirm the single chain MiniTrap identity with elucidation for some PTMs.
FIG. 45A depicts a chart of relative absorbance versus time (minutes) for low molecular weight impurities in MT1.
FIG. 45B depicts mass spectra for the low molecular weight impurities in MT1.
FIG. 46 depicts relative absorbance versus time (minutes) for MT1 which shows absence of the FabRICATOR enzyme which was used to cleave aflibercept into MT1.
FIG. 47 depicts relative absorbance versus time (minutes) for low molecular weight impurities in MT5.
FIG. 48 depicts relative absorbance versus time (minutes) for low molecular weight impurities in MT6.
FIG. 49A depicts a chart of absorbance versus time (minutes) obtained on performing HILIC-UV/MS for VEGF MiniTrap MT6, wherein the chart shows the elution of main peak at 21 minutes and O-glycans at around 21.5 minutes.
FIG. 49B depicts a mass spectrum obtained on performing HILIC-UV/MS for VEGF MiniTrap MT6 showing the main peak at 47985.8 Da.
FIG. 49C depicts a mass spectra of O-glycans of VEGF MiniTrap MT6 obtained on performing HILIC-UV/MS.
FIG. 50 is an image of VEGF MiniTrap dimer wherein the disulfide bridge in the hinge region (SEQ ID NO.: 83, 123, 83, and 123) of the VEGF MiniTrap can be parallel or crossed.
FIG. 51 depicts relative abundance of distribution of glycans observed at Asn36 among MT1, MT5 and MT6. Figure discloses SEQ ID NO.: 121.
FIG. 52 depicts relative abundance of distribution of glycans observed at Asn68 among MT1, MT5 and MT6. Figure discloses SEQ ID NOS 101 and 30, respectively, in order of appearance.
FIG. 53 depicts relative abundance of distribution of glycans observed at Asn123 among MT1, MT5 and MT6. Figure discloses SEQ ID NO.: 82.
FIG. 54 depicts relative abundance of distribution of glycans observed at Asn196 among MT1, MT5 and MT6. Figure discloses SEQ ID NOS 103 and 122, respectively, in order of appearance.
FIG. 55 depicts the released N-linked glycan analysis by hydrophilic interaction chromatography (HILIC) coupled to fluorescence detection and mass spectrometry analysis (full scale and stacked).
FIG. 56 depicts HILIC-FLR chromatograms for MT1, MT5 and MT6.
FIG. 57 depicts the released N-linked glycan analysis by HILIC coupled to fluorescence detection and mass spectrometry analysis (full scale, stacked and normalized).
FIG. 58A is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
FIG. 58B is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
FIG. 58C is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6.
FIG. 59 depicts an exemplary production procedure for manufacturing MiniTrap according to an exemplary embodiment.
DETAILED DESCRIPTION
Angiogenesis, the growth of new blood vessels from preexisting vasculature, is a highly orchestrated process that is critical for proper embryonic and postnatal vascular development. Abnormal or pathological angiogenesis is a hallmark of cancer and several retinal diseases where the upregulation of proangiogenic factors, such as vascular endothelial growth factor (VEGF) leads to increases in endothelial proliferation, changes in vasculature morphology, and increased vascular permeability. Elevated levels of VEGF have been found in the vitreous fluid and retinal vasculature of patients with various ocular diseases. Blocking VEGF activity has also become the therapy of choice for treating DME, wet AMD, CNV, retinal vein occlusions, and other ocular diseases where abnormal angiogenesis is the underlying etiology.
As used herein, aflibercept is one such anti-VEGF protein comprising an all-human amino acid sequence comprising the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2 expressed as an inline fusion with a (Fc) of human IgG1. Aflibercept binds all forms of VEGF-A (VEGF) but in addition binds P1GF and VEGF-B. Several other homodimeric VEGF MiniTraps have been generated as enzymatically cleaved products from aflibercept or recombinantly expressed directly from host cell lines. One such example of a VEGF MiniTrap is shown in FIG. 1. In this figure, a terminal lysine is depicted (K); some culture processes remove this terminal lysine while others do not. FIG. 1 illustrates a process whereby the terminal lysine remains. In general, aflibercept encompasses both the presence and absence of the terminal lysine.
As demonstrated herein, the present invention, in part, discloses the production of anti-VEGF proteins (Example 1) using a CDM. Analysis of solutions comprising aflibercept produced using certain CDMs demonstrated a certain color property, such as an intense yellow-brown color. The intensity of the solution's color depended upon the CDM used. Not all CDMs examined produced a sample with a distinct yellow-brown color after the solutions were normalized to a concentration of 5 g/L.
A color, such as yellow-brown, in an injectable therapeutic drug solution can be an undesirable feature. It may be an important parameter employed for determining if a drug product satisfies a predetermined level of purity and quality for a particular therapeutic. A color such as yellow-brown observed along the manufacturing route of a biologic can be caused by chemical modifications of that biologic, degradation products of formulation excipients, or degradation products formed through the reaction of the biologic and formulation excipients. However, such information can be valuable for understanding the cause of the color change. It can also assist in designing short-term as well as long-term storage conditions to prevent modifications facilitating such a color change.
The inventors observed that use of AEX during the production of an anti-VEGF protein solution minimized yellow-brown coloration. Additionally, the inventors discovered that the yellow-brown coloration can be decreased by modifying the cell culture used to produce a recombinant protein, such as aflibercept or a modified aflibercept like MiniTrap.
The present invention encompasses anti-VEGF proteins and their production using CDM. Additionally, the present invention is based on the identification and optimization of upstream and downstream process technologies for protein production.
As demonstrated herein, some of the Examples set forth below describe the production of anti-VEGF proteins (Example 1), production of oxidized species of anti-VEGF proteins (Example 4), methods to reduce oxidized species of anti-VEGF proteins by optimizing culture medium (Example 5) and by optimizing production methods (Example 2).
A number of recent patent applications and granted patents purport to describe various aflibercept species and methods of producing the same, but none describe or suggest the anti-VEGF compositions and methods for producing the same described herein. See, e.g., U.S. application Ser. No. 16/566,847 to Coherus Biosciences Inc., U.S. Pat. No. 10,646,546 to Sam Chun Dang Pharm. Co., Ltd., U.S. Pat. No. 10,576,128 to Formycon AG, International Application No. PCT/US2020/015659 to Amgen Inc., and U.S. Pat. Nos. 8,956,830; 9,217,168; 9,487,810; 9,663,810; 9,926,583; and U.S. Pat. No. 10,144,944 to Momenta Pharmaceuticals, Inc.
I. Explanation of Selected Terms
Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein known to the skilled artisan can be used in the practice of particular embodiments described herein. All publications mentioned are hereby incorporated by reference in their entirety.
The term “a” should be understood to mean “at least one” and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art and where ranges are provided, endpoints are included.
As used herein, the term “angiogenic eye disorder” means any disease of the eye, which is caused by or associated with the growth or proliferation of blood vessels or by blood vessel leakage.
As used herein, the term “chemically defined medium” or “chemically defined media” (both abbreviated “CDM”) refers to a synthetic growth medium in which the identity and concentration of all the ingredients are defined. Chemically defined media do not contain bacterial, yeast, animal, or plant extracts, animal serum, or plasma, although individual plant or animal-derived components (e.g., proteins, polypeptides, etc.) may be added. Chemically defined media may contain inorganic salts such as phosphates, sulfates, and the like needed to support growth. The carbon source is defined, and is usually a sugar such as glucose, lactose, galactose, and the like, or other compounds such as glycerol, lactate, acetate, and the like. While certain chemically defined culture media also use phosphate salts as a buffer, other buffers may be employed such as sodium bicarbonate, HEPES, citrate, triethanolamine, and the like. Examples of commercially available chemically defined media include, but are not limited to, various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various EX-CELLs mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD mediums (FUJIFILM Irvine Scientific), combinations thereof, and the like. Methods of preparing chemically defined culture media are known in the art, for example, in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. Patent Application Publication Nos. 2008/0009040 and 2007/0212770, the entire teachings of which are herein incorporated by reference.
As used herein, the term “cumulative amount” refers to the total amount of a particular component added to a bioreactor over the course of the cell culture to form the CDM, including amounts added at the beginning of the culture (CDM at day 0) and subsequently added amounts of the component. Amounts of a component added to a seed-train culture or inoculum prior to the bioreactor production (i.e., prior to the CDM at day 0) are also included when calculating the cumulative amount of the component. A cumulative amount is unaffected by the loss of a component over time during the culture (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may nonetheless have different absolute levels, for example, if the component is added to the two cultures at different times (e.g., if in one culture all of the component is added at the outset, and in another culture the component is added over time). A cumulative amount is also unaffected by in situ synthesis of a component over time during the culture (for example, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a given component may nonetheless have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by way of a bioconversion process. A cumulative amount may be expressed in units such as, for example, grams or moles of the component.
As used herein, the term “cumulative concentration” refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the beginning of the production batch, including the contribution to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the beginning of the production batch, and one gram of component X is added at days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters). If, on day 4, an additional one liter of liquid not containing component X were added to the bioreactor, the cumulative concentration would remain 2 g/L. If, on day 5, some quantity of liquid were lost from the bioreactor (for example, through evaporation), the cumulative concentration would remain 2 g/L. A cumulative concentration may be expressed in units such as, for example, grams per liter or moles per liter.
As used herein, the term “formulation” refers to a protein of interest that is formulated together with one or more pharmaceutically acceptable vehicles. In one aspect, the protein of interest is aflibercept and/or MiniTrap. In some exemplary embodiments, the amount of protein of interest in the formulation can range from about 0.01 mg/mL to about 600 mg/mL. In some specific embodiments, the amount of the protein of interest in the formulation can be about 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about 170 mg/mL, about 180 mg/mL, about 190 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, about 300 mg/mL, about 325 mg/mL, about 350 mg/mL, about 375 mg/mL, about 400 mg/mL, about 425 mg/mL, about 450 mg/mL, about 475 mg/mL, about 500 mg/mL, about 525 mg/mL, about 550 mg/mL, about 575 mg/mL, or about 600 mg/mL. In some exemplary embodiments, pH of the composition can be greater than about 5.0. In one exemplary embodiment, the pH can be greater than about 5.0, greater than about 5.5, greater than about 6.0, greater than about 6.5, greater than about 7.0, greater than about 7.5, greater than about 8.0, or greater than about 8.5.
As used herein, the term “database” refers to a bioinformatics tool, which provides for the possibility of searching the uninterpreted MS-MS spectra against all possible sequences in the database(s). Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com//proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem (www.thegpm.org/TANDEM/), Protein Prospector (www.prospector.ucsf.edu/prospector/mshome.htm), Byonic (www.proteinmetrics.com/products/byonic) or Sequest (fields.scripps.edu/sequest).
As used herein, the term “ultrafiltration” or “UF” can include a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semi-permeable membrane. Ultrafiltration is described in detail in: LEOS J. ZEMAN & ANDREW L. ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS (1996), the entire teaching of which is herein incorporated. Filters with a pore size of smaller than 0.1 μm can be used for ultrafiltration. By employing filters having such small pore size, the volume of the sample can be reduced through permeation of the sample buffer through the filter while proteins are retained behind the filter.
As used herein, “diafiltration” or “DF” can include a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular-weight material, and/or to cause the rapid change of ionic and/or pH environments. Microsolutes are removed most efficiently by adding solvent to a solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes microspecies from the solution at a constant volume. In certain exemplary embodiments of the present invention, a diafiltration step can be employed to exchange various buffers used in connection with the instant invention, for example, prior to chromatography or other production steps, as well as to remove impurities from the protein preparation. As used herein, the term “downstream process technology” refers to one or more techniques used after the upstream process technologies to produce a protein. Downstream process technology includes, for example, production of a protein product, using, for example, affinity chromatography, including Protein A affinity chromatography as well as affinity chromatography that uses a solid phase having a well-defined molecule like VEGF that can interact with its cognate like a VEGF receptor (VEGFR), ion exchange chromatography, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, or displacement chromatography.
The phrase “recombinant host cell” (or simply “host cell”) includes a cell into which a recombinant expression vector coding for a protein of interest has been introduced. It should be understood that such a term is intended to refer not only to a particular subject cell but to a 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. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In one aspect, eukaryotic cells include protist, fungal, plant and animal cells. In a further aspect, host cells include eukaryotic cells such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. In a particular aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other depositories as well as commercial vendors. Cells that can be used in the processes of the invention include, but not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36: 10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H/IOTI/2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntac cells, SIRC cells, CII cells, and Jensen cells, or derivatives thereof) or any other cell type known to one skilled in the art.
As used herein, the term “host cell proteins” (HCP) includes protein derived from a host cell and can be unrelated to the desired protein of interest. Host cell proteins can be a process-related impurity which can be derived from the manufacturing process and can include three major categories: cell substrate-derived, cell culture-derived and downstream derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from a host organism and nucleic acid (host cell genomic, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ion), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.
In some exemplary embodiments, the host cell protein can have a pI in the range of about 4.5 to about 9.0. In an exemplary embodiment, the p1 can be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
As used herein, the term “hydrolyzing agent” refers to any one or combination of a large number of different agents that can perform digestion of a protein. Non-limiting examples of hydrolyzing agents that can carry out enzymatic digestion include protease from Aspergillus saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N(Asp-N), endoproteinase Arg-C(Arg-C), endoproteinase Glu-C(Glu-C) or outer membrane protein T (OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), thermolysin, papain, pronase, V8 protease or biologically active fragments or homologs thereof or combinations thereof. Non-limiting examples of hydrolyzing agents that can carry out non-enzymatic digestion include the use of high temperature, microwave, ultrasound, high pressure, infrared, solvents (non-limiting examples are ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzymes, and on-chip immobilized enzymes. For a recent review discussing the available techniques for protein digestion, see Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried M. A. Niessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013), the entire teachings of which are herein incorporated). One or a combination of hydrolyzing agents can cleave peptide bonds in a protein or polypeptide, in a sequence-specific manner, generating a predictable collection of shorter peptides. The ratio of hydrolyzing agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of the protein. When the enzyme to substrate ratio is unsuitably high, the correspondingly high digestion rate will not allow sufficient time for the peptides to be analyzed by mass spectrometer, and sequence coverage will be compromised. On the other hand, a low E/S ratio would need long digestion and thus long data acquisition time. The enzyme to substrate ratio can range from about 1:0.5 to about 1:200. As used herein, the term “digestion” refers to hydrolysis of one or more peptide bonds of a protein. There are several approaches to carrying out digestion of a protein in a biological sample using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion. One of the widely accepted methods for digestion of proteins in a sample involves the use of proteases. Many proteases are available and each of them have their own characteristics in terms of specificity, efficiency, and optimum digestion conditions. Proteases refer to both endopeptidases and exopeptidases, as classified based on the ability of the protease to cleave at non-terminal or terminal amino acids within a peptide. Alternatively, proteases also refer to the six distinct classes—aspartic, glutamic, and metalloproteases, cysteine, serine, and threonine proteases, as classified based on the mechanism of catalysis. The terms “protease” and “peptidase” are used interchangeably to refer to enzymes which hydrolyze peptide bonds.
The term “in association with” indicates that components, an anti-VEGF composition of the present invention, along with another agent such as anti-ANG2, can be formulated into a single composition for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component). Components administered in association with each another can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time. Separate components administered in association with each other may also be administered essentially simultaneously (e.g., at precisely the same time or separated by a non-clinically significant time period) during the same administration session. Moreover, the separate components administered in association with each other may be administered to a subject by the same or by a different route, for example, a composition of aflibercept along with another agent such as anti-ANG2, wherein the composition of aflibercept comprises about 15% or less of its variants.
As used herein, the term “liquid chromatography” refers to a process in which a biological/chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, mixed-mode chromatography or hydrophobic chromatography.
As used herein, “affinity chromatography” can include separations including any method by which two substances are separated based upon their affinity to a chromatographic material. It can comprise subjecting the substances to a column comprising a suitable affinity chromatographic media. Non-limiting examples of such chromatographic media include, but are not limited to, Protein A resin, Protein G resin, affinity supports comprising an antigen against which a binding molecule (e.g., antibody) was produced, protein capable of binding to a protein of interest and affinity supports comprising an Fc binding protein. In one aspect, an affinity column can be equilibrated with a suitable buffer prior to sample loading. An example of a suitable buffer can be a Tris/NaCl buffer, pH around 7.0 to 8.0. A skilled artisan can develop a suitable buffer without undue burden. Following this equilibration, a sample can be loaded onto the column. Following the loading of the column, the column can be washed one or multiple times using, for example, the equilibrating buffer. Other washes, including washes employing different buffers, can be used before eluting the column. The affinity column can then be eluted using an appropriate elution buffer. An example of a suitable elution buffer can be an acetic acid/NaCl buffer, pH around 2.0 to 3.5. Again, the skilled artisan can develop an appropriate elution buffer without undue burden. The eluate can be monitored using techniques well known to those skilled in the art, including UV. For example, the absorbance at 280 nm can be employed, especially if the sample of interest comprises aromatic rings (e.g., proteins having aromatic amino acids like tryptophan).
As used herein, “ion exchange chromatography” can refer to separations including any method by which two substances are separated based on differences in their respective ionic charges, either on the molecule of interest and/or chromatographic material as a whole or locally on specific regions of the molecule of interest and/or chromatographic material, and thus can employ either cationic exchange material or anionic exchange material. Ion exchange chromatography separates molecules based on differences between the local charges of the molecules of interest and the local charges of the chromatographic material. A packed ion-exchange chromatography column or an ion-exchange membrane device can be operated in a bind-elute mode, a flowthrough mode, or a hybrid mode. After washing the column or the membrane device with an equilibration buffer or another buffer, product recovery can be achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute can be another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). Anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography. Non-limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange medias or support can include DE23™, DE32™ DE52™, CM-23™, CM-32™, and CM-52™ are available from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-based and -locross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM- and S-SEPHAROSE® and SEPHAROSE® Fast Flow, and Capto™ S are all available from GE Healthcare. Further, both DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL™ DEAE-6505 or M and TOYOPEARL™ CM-650S or M are available from Toso Haas Co., Philadelphia, Pa., or Nuvia S and UNOSphere™ S from BioRad, Hercules, Calif., Eshmuno® S from EMD Millipore, Mass.
As used herein, the term “hydrophobic interaction chromatography resin” can include a solid phase, which can be covalently modified with phenyl, octyl, butyl or the like. It can use the properties of hydrophobicity to separate molecules from one another. In this type of chromatography, hydrophobic groups such as, phenyl, octyl, hexyl or butyl can form the stationary phase of a column. Molecules such as proteins, peptides and the like pass through a HIC (hydrophobic interactive chromatography) column that possess one or more hydrophobic regions on their surface or have hydrophobic pockets and are able to interact with hydrophobic groups comprising a HIC's stationary phase. Examples of HIC resins or support include Phenyl sepharose FF, Capto Phenyl (GE Healthcare, Uppsala, Sweden), Phenyl 650-M (Tosoh Bioscience, Tokyo, Japan) and Sartobind Phenyl (Sartorius corporation, New York, USA).
As used herein, the term “Mixed Mode Chromatography” or “multimodal chromatography” (both “MMC”) includes a chromatographic method in which solutes interact with a stationary phase through more than one interaction mode or mechanism. MMC can be used as an alternative or complementary tool to traditional reversed-phased (RP), ion exchange (IEX) and normal phase chromatography (NP). Unlike RP, NP and IEX chromatography, in which hydrophobic interaction, hydrophilic interaction and ionic interaction respectively are the dominant interaction modes, mixed-mode chromatography can employ a combination of two or more of these interaction modes. Mixed mode chromatography media can provide unique selectivity that cannot be reproduced by single mode chromatography. Mixed mode chromatography can also provide potential cost savings, longer column lifetimes and operation flexibility compared to affinity-based methods. In some exemplary embodiments, mixed mode chromatography media can be comprised of mixed mode ligands coupled to an organic or inorganic support, sometimes denoted a base matrix, directly or via a spacer. The support may be in the form of particles, such as essentially spherical particles, a monolith, filter, membrane, surface, capillaries, etc. In some exemplary embodiments, the support can be prepared from a native polymer such as cross-linked carbohydrate material, such as agarose, agPV, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc. To obtain high adsorption capacities, the support can be porous and ligands are then coupled to the external surfaces as well as to the pore surfaces. Such native polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teachings of which are herein incorporated). Alternatively, the support can be prepared from a synthetic polymer such as cross-linked synthetic polymers, for example, styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides and the like. Such synthetic polymers can be produced according to standard methods, for example, “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teachings of which are herein incorporated). Porous native or synthetic polymer supports are also available from commercial sources, such as such as GE Healthcare, Uppsala, Sweden.
As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector into which a polypeptide or peptide may be characterized. A mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application. In some exemplary embodiments, the mass spectrometer can be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes a technique where structural information on sample molecules is obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules be transformed into a gas phase and ionized so that fragments are formed in a predictable and controllable fashion after the first mass selection step. Multistage MS/MS, or MSn, can be performed by first selecting and isolating a precursor ion (MS2), fragmenting it, isolating a primary fragment ion (MS3), fragmenting it, isolating a secondary fragment (MS4), and so on, as long as one can obtain meaningful information, or the fragment ion signal is detectable. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzers to combine for a certain application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two major categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m/z separation and data acquisition. In tandem-in-time, mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m/z separated in the same physical device. The peptides identified by the mass spectrometer can be used as surrogate representatives of the intact protein and their post translational modifications. They can be used for protein characterization by correlating experimental and theoretical MS/MS data, the latter generated from possible peptides in a protein sequence database. The characterization includes, but is not limited, to sequencing amino acids of the protein fragments, determining protein sequencing, determining protein de novo sequencing, locating post-translational modifications, or identifying post translational modifications, or comparability analysis, or combinations thereof.
As used herein, “Mini-Trap” or “MiniTrap” or “MiniTrap binding molecule” is capable of binding to a VEGF molecule. Such MiniTraps can include (i) chimeric polypeptides as well as (ii) multimeric (e.g., dimeric) molecules comprising two or more polypeptides which are bound non-covalently, for example, by one or more disulfide bridges. MiniTraps can be produced through chemical modification, enzymatic activity, or recombinantly manufactured.
As used herein, “VEGF MiniTrap” or “VEGF MiniTrap binding molecule” can be a molecule or complex of molecules that binds to VEGF and has one or more sets of VEGF receptor Ig-like domains (or variants thereof) (e.g., VEGFR1 Ig domain 2 and/or VEGFR2 Ig domain 3 and/or 4) and a modified or absent multimerizing component (MC), for example, wherein the MC is a modified immunoglobulin Fc. The modification may be the result of proteolytic digestion of a VEGF trap (e.g., aflibercept or conbercept) or direct expression of the resulting polypeptide chains with the shortened MC sequence. (See the molecular structure depicted in FIG. 1.) FIG. 1 is a depiction of a VEGF MiniTrap molecule, which is the product of proteolysis of aflibercept with Streptococcus pyogenes IdeS. The homodimeric molecule is depicted having an Ig hinge domain fragment connected by two parallel disulfide bonds. The VEGFR1 domain, the VEGFR2 domain and the hinge domain fragment (MC) is indicated. The point in aflibercept where IdeS cleavage occurs is indicated with a “//”. The cleaved off Fc fragment from aflibercept is also indicated. A single such chimeric polypeptide, which is not dimerized, may also be a VEGF MiniTrap if it has VEGF binding activity. The term “VEGF MiniTrap” includes a single polypeptide comprising a first set of one or more VEGF receptor Ig domains (or variants thereof), lacking an MC, but fused with a linker (e.g., a peptide linker) to one or more further sets of one or more VEGF receptor Ig domains (or variants thereof). The VEGF binding domains in a VEGF MiniTrap of the present invention may be identical or different from another (see WO2005/00895, the entire teachings of which are herein incorporated).
For example, in an embodiment of the invention, the unmodified immunoglobulin Fc domain comprises the amino acid sequence or amino acids 1-226 thereof:
DKTHTCPX1CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKX2TPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 33;
wherein X1 is L or P and X2 is A or T)
Inhibition of VEGF includes, for example, antagonism of VEGF binding to VEGF receptor, for example, by competition with VEGF receptor for VEGF (e.g., VEGF110, VEGF121 and/or VEGF165) binding. Such inhibition may result in inhibition of VEGF-mediated activation of VEGFR, for example, inhibition of luciferase expression in a cell line (e.g., HEK293) expressing chimeric VEGF Receptor (e.g., a homodimer thereof) having VEGFR extracellular domains fused to IL18Ra and/or IL18Rβ intracellular domains on the cell surface and also having an NFkB-luciferase-IRES-eGFP reporter gene, for example, the cell line HEK293/D9/Flt-IL18Rα/Flt-IL18Rβ as set forth herein.
The VEGF receptor Ig domain components of the VEGF MiniTraps of the present invention can include:
(i) one or more of the immunoglobulin-like (Ig) domain 2 of VEGFR1 (Flt1) (R1D2),
(ii) one or more of the Ig domain 3 of VEGFR2 (Flk1 or KDR) (Flk1D3) (R2D3),
(iii) one or more of the Ig domain 4 of VEGFR2 (Flk1 or KDR) (Flk1D4) (R2D4) and/or
(iv) one or more of the Ig domain 3 of VEGFR3 (Flt4) (F1t1D3 or R3D3).
Immunoglobulin-like domains of VEGF receptors may be referred to herein as VEGFR “Ig” domains. VEGFR Ig domains which are referenced herein, for example, R1D2 (which may be referred to herein as VEGFR1(d2)), R2D3 (which may be referred to herein as VEGFR2(d3)), R2D4 (which may be referred to herein as VEGFR2(d4)) and R3D3 (which may be referred to herein as VEGFR3(d3)) are intended to encompass not only the complete wild-type Ig domain, but also variants thereof which substantially retain the functional characteristics of the wild-type domain, for example, retain the ability to form a functioning VEGF binding domain when incorporated into a VEGF MiniTrap. It will be readily apparent to one of skill in the art that numerous variants of the above Ig domains, which will retain substantially the same functional characteristics as the wild-type domain, can be obtained.
The present invention provides a VEGF MiniTrap polypeptide comprising the following domain structure:
((R1D2)-(R2D3))a-linker-((R1D2)-(R2D3))b;
((R1D2)-(R2D3)-(R2D4))c-linker-((R1D2)-(R2D3)-(R2D4))d;
((R1D2)-(R2D3))e-(MC)g;
((R1D2)-(R2D3)-(R2D4))f-(MC)g;
wherein,
R1D2 is the VEGF receptor 1 (VEGFR1) Ig domain 2 (D2);
R2D3 is the VEGFR2 Ig domain 3;
R2D4 is the VEGFR2 Ig domain 4;
MC is a multimerizing component (e.g., an IgG hinge domain or fragment thereof, for example from IgG1);
linker is a peptide comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, for example, (GGGS)g (SEQ ID NO.: 104);
and,
Independently,
a=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
b=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
c=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
d=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
e=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
f=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and
g=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
In an embodiment of the invention, R1D2 comprises the amino acid sequence:
(SEQ ID NO.: 34)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP
DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII
D.
In one aspect, the R1D2 lacks the N-terminal SDT.
In an embodiment of the invention, R1D2 comprises the amino acid sequence:
(SEQ ID NO.: 35)
PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRI
IWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT.
In an embodiment of the invention, R2D3 comprises the amino acid sequence:
(SEQ ID NO.: 36)
VVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNR
DLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH
EK.
In an embodiment of the invention, R2D4 comprises the amino acid sequence:
(SEQ ID NO.: 37)
PFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIK
AGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG.
In an embodiment of the invention, R2D4 comprises the amino acid sequence:
(SEQ ID NO.: 38)
FVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKA
GHVLTIMEVSERDTGNYTVILTNPIKSEKQSHVVSLVVYVP.
In an embodiment of the invention, a multimerizing component (MC) for use in a VEGF MiniTrap is a peptide, for example, a modified Fc immunoglobulin (e.g., from an IgG1) which is capable of binding to another multimerizing component. In one aspect, an MC is a modified Fc immunoglobulin that includes the immunoglobulin hinge region. For example, in an embodiment of the invention, an MC is a peptide comprising one or more (e.g., 1, 2, 3, 4, 5 or 6) cysteines that are able to form one or more cysteine bridges with cysteines in another MC, for example, DKTHTCPPC (SEQ ID NO.: 39), DKTHTCPPCPPC (SEQ ID NO.: 40), DKTHTCPPCPPCPPC (SEQ ID NO.: 41), DKTHTC(PPC)h, wherein h is 1, 2, 3, 4, or 5 (SEQ ID NO.: 105), DKTHTCPPCPAPELLG (SEQ ID NO.: 60), DKTHTCPLCPAPELLG (SEQ ID NO.: 43), DKTHTC (SEQ ID NO.: 44) or DKTHTCPLCPAP (SEQ ID NO.: 45).
The present invention also provides a VEGF MiniTrap polypeptide comprising the following domain structure:
(R1D2)a-(R2D3)b-(MC)c; or (i)
(R1D2)a-(R2D3)b-(R2D4)c-(MC)d; (ii)
which may be homodimerized with a second of said polypeptides, for example, by binding between the MCs of each polypeptide, wherein
(i) said R1D2 domains coordinate;
(ii) said R2D3 domains coordinate; and/or
(iii) said R2D4 domains coordinate, to form a dimeric VEGF binding domain.
In an embodiment of the invention, the VEGF MiniTrap polypeptide comprises the amino acid sequence:
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN36ITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISN68ATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLN123CTARTELNVGIDFNWEYPSSKHQ
HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK
N196STFVRVHEKDKTHTCPPCPAPELLG
(SEQ ID NO.: 46; MC underscored);
GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGK
RIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVV
LSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLK
TQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHENLS
VAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAG
HVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPGDKTHTCP
LCPAPELLG (SEQ ID NO.: 47, MC underscored);
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN36ITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISN68RTYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLN123CTARTELNVGIDFNWEYPSSKHQ
HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK
N196STFVRVHEKDKTHTCPPC
(SEQ ID NO.: 48; MC underscored);
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN36ITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISN68ATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGEILSVGEKLVLN123CTARTELNVGIDFNWEYPSSKHQ
HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK
N196STFVRVHEKDKTHTCPPCPPC
SEQ ID NO.: 49; MC underscored);
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPN36ITVTLKKFPLDTL
IPDGKRIIWDSRKGFIISN68ATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLN123CTARTELBVGIDFNWEYPSSKHQ
HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK
N196STFVRVHEKDKTHTCPPCPPCPPC
(SEQ ID NO.: 50; MC underscored);
or
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP
DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII
DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNR
DLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE
KDKTHTC-(PPC)x
(MC underscored; wherein x is 1, 2, 3, 4 or 5) (SEQ ID NO.: 106). As discussed, such polypeptides may be multimerized (e.g., dimerized (e.g., homodimerized)) wherein binding between the polypeptides is mediated via the multimerizing components.
In an embodiment of the invention, the VEGFR1 Ig-like domain 2 of the monomeric VEGF MiniTraps of the present invention have N-linked glycosylation at N36 and/or N68; and/or an intrachain disulfide bridge between C30 and C79; and/or, the VEGFR2 Ig-like domain 3 of the monomeric VEGF MiniTraps of the present invention, have N-linked glycosylation at N123 and/or N196; and/or an intrachain disulfide bridge between C124 and C185.
In an embodiment of the invention, the VEGF MiniTrap comprises the structure:
(R1D2)1-(R2D3)1-(G4S)3-(R1D2)1-(R2D3)1 (“(G4S)3” disclosed as SEQ ID NO.: 107);
(R1D2)1-(R2D3)1-(G4S)6-(R1D2)1-(R2D3)1 (“(G4S)6” disclosed as SEQ ID NO.: 108);
(R1D2)1-(R2D3)1-(G4S)9-(R1D2)1-(R2D3)1 (“(G4S)9” disclosed as SEQ ID NO.: 109); or
(R1D2)1-(R2D3)1-(G4S)12-(R1D2)1-(R2D3)1 (“(G4S)12” disclosed as SEQ ID NO.: 110). G45 is -Gly-Gly-Gly-Gly-Ser (SEQ ID NO.: 111)
In an embodiment of the invention, the VEGF MiniTrap comprises the amino acid sequence:
(i)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGR
PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNA
TYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTE
LNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAAS
SGLMTKKNSTFVRVHEK (SEQ ID NO.: 51; linker underscored);
(iii)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTE
GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNG
HLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKH
QHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH
EK (SEQ ID NO.: 52; linker underscored);
(iv)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
GGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPD
GKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIEL
SVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTID
GVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 53;
linker underscored)
(v)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
GGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP
NITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHR
QTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLK
TQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.:
54; linker underscored);
or
(vi)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKEPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDENWEYPSSKHQHKKLVNRDLKTQSGSEMKKELSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEK-(GGGGS)x-
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKG
FIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNC
TARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLY
TCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 112)
(wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15). As discussed herein, these polypeptides may comprise a secondary structure wherein like VEGFR Ig domains associate to form an intra-chain VEGF binding domain (e.g., FIG. 2). In an embodiment of the invention, two or more of such polypeptides multimerize (e.g., dimerize (e.g., homodimerize)) wherein the VEGFR Ig domains of each chain associate with like Ig domains of another chain to form an inter-chain VEGF binding domain.
In a certain embodiment of the invention, a VEGF MiniTrap of the present invention lacks any significant modification of the amino acid residues of a VEGF MiniTrap polypeptide (e.g., directed chemical modification such as PEGylation or iodoacetamidation, for example at the N- and/or C-terminus).
In an embodiment of the invention, the polypeptide comprises a secondary structure wherein like VEGFR Ig domains in a single chimeric polypeptide (e.g., (R1D2)a-(R2D3)b-linker-(R1D2)c-(R2D3)d; or (R1D2)a-(R2D3)b-(R2D4)c-linker-(R1D2)d-(R2D3)e-(R2D4)f) or in separate chimeric polypeptides (e.g., homodimers) coordinate to form a VEGF binding domain. For example, wherein
(i) said R1D2 domains coordinate;
(ii) said R2D3 domains coordinate; and/or
(iii) said R2D4 domains coordinate,
to form a VEGF binding domain. FIG. 2 is a description of a single chain VEGF MiniTrap depicting such domain coordination. The VEGFR1, VEGFR2 and linker domains are indicated. The linker shown is (G4S)6 (SEQ ID NO.: 108). The present invention includes single chain VEGF MiniTraps with a (G4S)3 (SEQ ID NO.: 107); (G4S)9 (SEQ ID NO.: 109) or (G4S)12 (SEQ ID NO.: 110) linker.
In addition, the present invention also provides a complex comprising a VEGF MiniTrap as discussed herein complexed with a VEGF polypeptide or a fragment thereof or fusion thereof. In an embodiment of the invention, the VEGF (e.g., VEGF165) is homodimerized and/or the VEGF MiniTrap is homodimerized in a 2:2 complex (2 VEGFs:2 MiniTraps) and/or VEGF MiniTrap is homodimerized in a 1:1 complex. Complexes can include homodimerized VEGF molecules bound to homodimerized VEGF MiniTrap polypeptides. In an embodiment of the invention, the complex is in vitro (e.g., immobilized to a solid substrate) or is in the body of a subject. The present invention also includes a composition of complexes of a VEGF dimer (e.g., VEGF165) complexed with a VEGF MiniTrap.
As used herein, the term “protein” or “protein of interest” can include any amino acid polymer having covalently linked amide bonds. Examples of proteins of interest include, but are not limited to, aflibercept and MiniTrap. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides.” “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. “Synthetic peptide or polypeptide” refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art. A protein may comprise one or multiple polypeptides to form a single functioning biomolecule. In another exemplary aspect, a protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest can include any of bio-therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In a particular aspect, the protein of interest is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap). Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation” (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are herein incorporated). In some exemplary embodiments, proteins comprise modifications, adducts, and other covalently linked moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels and other dyes, and the like. Proteins can be classified on the basis of compositions and solubility and can thus include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.
In some exemplary embodiments, the protein of interest can be a recombinant protein, an antibody, a bispecific antibody, a multispecific antibody, antibody fragment, monoclonal antibody, fusion protein, scFv and combinations thereof.
As used herein, the term “recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein can be a fusion protein. In a particular aspect, the recombinant protein is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap). In certain exemplary embodiments, the recombinant protein can be an antibody, for example, a chimeric, humanized, or fully human antibody. In certain exemplary embodiments, the recombinant protein can be an antibody of an isotype selected from group consisting of: IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments the antibody molecule is a full-length antibody (e.g., an IgG1) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).
The term “antibody,” as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), 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, and FR4. In different embodiments of the invention, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multi specific antibodies formed from antibody fragments. Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some exemplary embodiments, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some exemplary embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multi-molecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.
The term “bispecific antibody” includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains with each heavy chain specifically binding a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes. BsAbs can be divided into two major classes, those bearing an Fc region (IgG-like) and those lacking an Fc region, the latter normally being smaller than the IgG and IgG-like bispecific molecules comprising an Fc. The IgG-like bsAbs can have different formats such as, but not limited to, triomab, knobs into holes IgG (kih IgG), crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgG-scFv), or κλ-bodies. The non-IgG-like different formats include tandem scFvs, diabody format, single-chain diabody, tandem diabodies (TandAbs), Dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Müller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachings of which are herein incorporated). The methods of producing bsAbs are not limited to quadroma technology based on the somatic fusion of two different hybridoma cell lines, chemical conjugation, which involves chemical cross-linkers, and genetic approaches utilizing recombinant DNA technology. Examples of bsAbs include those disclosed in the following patent applications, which are hereby incorporated by reference: U.S. Ser. No. 12/823,838, filed Jun. 25, 2010; U.S. Ser. No. 13/488,628, filed Jun. 5, 2012; U.S. Ser. No. 14/031,075, filed Sep. 19, 2013; U.S. Ser. No. 14/808,171, filed Jul. 24, 2015; U.S. Ser. No. 15/713,574, filed Sep. 22, 2017; U.S. Ser. No. 15/713,569, field Sep. 22, 2017; U.S. Ser. No. 15/386,453, filed Dec. 21, 2016; U.S. Ser. No. 15/386,443, filed Dec. 21, 2016; U.S. Ser. No. 15/22,343 filed Jul. 29, 2016; and U.S. Ser. No. 15/814,095, filed Nov. 15, 2017. Low levels of homodimer impurities can be present at several steps during the manufacturing of bispecific antibodies. The detection of such homodimer impurities can be challenging when performed using intact mass analysis due to low abundances of the homodimer impurities and the co-elution of these impurities with main species when carried out using a regular liquid chromatographic method.
As used herein “multispecific antibody” refers to an antibody with binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e., bispecific antibodies, bsAbs), antibodies with additional specificities such as trispecific antibody and KIH Trispecific can also be addressed by the system and method disclosed herein.
The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
In some exemplary embodiments, the protein of interest can have a pI in the range of about 4.5 to about 9.0. In one exemplary specific embodiment, the pI can be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some exemplary embodiments, the types of protein of interest in the compositions can be more than one.
In some exemplary embodiments, the protein of interest can be produced from mammalian cells. The mammalian cells can be of human origin or non-human origin can include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells), established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK-cells, PK-15 cells or derivatives thereof), fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H/IOTI/2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK′ (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2/0, NS0, NS1 cells or derivatives thereof).
As used herein, the term “protein alkylating agent” refers to an agent used for alkylating certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.
As used herein, “protein denaturing” can refer to a process in which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation can be carried out using a protein denaturing agent. Non-limiting examples of a protein denaturing agent include heat, high or low pH, reducing agents like DTT (see below) or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturing agents. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples for chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, and salts thereof.
As used herein, the term “protein reducing agent” refers to the agent used for reduction of disulfide bridges in a protein. Non-limiting examples of the protein reducing agents used to reduce the protein are dithiothreitol (DTT), B-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof.
As used herein, the term “variant” of a polypeptide (e.g., of a VEGFR Ig domain) refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced or native amino acid sequence of a protein of interest. A sequence comparison can be performed by, for example, a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment). Variants of a polypeptide (e.g., of a VEGFR Ig domain) may also refer to a polypeptide comprising a referenced amino acid sequence except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.; the entire teachings of which are herein incorporated.
Some variants can be covalent modifications that polypeptides undergo, either during (co-translational modification) or after (post-translational modification “PTM”) their ribosomal synthesis. PTMs are generally introduced by specific enzymes or enzyme pathways. Many occur at the site of a specific characteristic protein sequence (e.g., signature sequence) within the protein backbone. Several hundred PTMs have been recorded and these modifications invariably influence some aspect of a protein's structure or function (Walsh, G. “Proteins” (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853, the entire teachings of which are herein incorporated). In certain exemplary embodiments, a protein composition can comprise more than one type of protein variant of a protein of interest.
Protein variants in the case of aflibercept (and proteins sharing structural characteristics of aflibercept, for example, one or more heavy or light chain regions of aflibercept) can comprise, but are not limited to, oxidation variants which can result from oxidation of one or more amino acid residues occurring at, for example, histidine, cysteine, methionine, tryptophan, phenylalanine and/or tyrosine residues; deamidation variants which can result from deamidation at asparagine residues and/or deoxyglucosonation at arginine residues.
With respect to aflibercept (and proteins sharing structural characteristics of aflibercept, for example, one or more heavy or light chain regions of aflibercept) oxidation variants can comprise oxidation of histidine residue at His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of tryptophan residues at Trp58 and/or Trp138 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of tyrosine residues at Tyr64 (or equivalent positions on proteins sharing certain structural characteristics of aflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); and/or oxidation of methionine residues at Met10, Met20, Met163 and/or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept).
With respect to aflibercept (and proteins sharing structural characteristics of aflibercept, for example, one or more heavy or light chain regions of aflibercept) deamidation variants can comprise deamidation of asparagine residue at Asn84 and/or Asn99 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept).
With respect to aflibercept (and proteins sharing structural characteristics of aflibercept for example, one or more heavy or light chain regions of aflibercept) deoxyglucosonation variant can comprise 3-deoxyglucosonation of arginine residue at Arg5 (or equivalent residue position on proteins sharing certain structural characteristics of aflibercept).
Protein variants can include both acidic species and basic species. Acidic species are typically the variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while basic species are the variants that elute later than the main peak from CEX or earlier than the main peak from AEX.
As used herein, the terms “acidic species,” “AS,” “acidic region,” and “AR,” refer to the variants of a protein which are characterized by an overall acidic charge. For example, in recombinant protein preparations such acidic species can be detected by various methods, such as ion exchange, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing). Acidic species of an antibody may include variants, structure variants, and/or fragmentation variants. Exemplary variants can include, but are not limited to, deamidation variants, afucosylation variants, oxidation variants, methylglyoxal (MGO) variants, glycation variants, and citric acid variants. Exemplary structure variants include, but are not limited to, glycosylation variants and acetonation variants. Exemplary fragmentation variants include any modified protein species from the target molecule due to dissociation of peptide chain, enzymatic and/or chemical modifications, including, but not limited to, Fc and Fab fragments, fragments missing a Fab, fragments missing a heavy chain variable domain, C-terminal truncation variants, variants with excision of N-terminal Asp in the light chain, and variants having N-terminal truncation of the light chain. Other acidic species variants include variants comprising unpaired disulfides, host cell proteins, and host nucleic acids, chromatographic materials, and media components. Commonly, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (See FIGS. 16 and 17).
In certain embodiments, a protein composition can comprise more than one type of acidic species variant. For example, but not by way of limitation, the total acidic species can be categorized based on chromatographic retention time of the peaks appearing. Another example in which the total acidic species can be categorized can be based on the type of variant—variants, structure variants, or fragmentation variant.
The term “acidic species” or “AS” does not refer to process-related impurities. The term “process-related impurity,” as used herein, refers to impurities that are present in a composition comprising a protein, but are not derived from the protein itself. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatographic materials, and media components.
In one exemplary embodiment, the amount of acidic species in the anti-VEGF composition compared to the protein of interest can be at most about 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. Examples of anti-VEGF compositions are discussed in Section III below. In one aspect, the anti-VEGF composition can comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.
Among the chemical degradation pathways responsible for acidic or basic species, the two most commonly observed covalent modifications occurring in proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan, and tyrosine are some of the amino acids that are most susceptible to oxidation: Met and Cys because of their sulfur atoms and His, Trp, and Tyr because of their aromatic rings.
As used herein, the terms “oxidative species,” “OS,” or “oxidation variant” refer to the variants of a protein formed by oxidation. Such oxidative species can also be detected by various methods, such as ion exchange, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing). Oxidation variants can result from oxidation occurring at histidine, cysteine, methionine, tryptophan, phenylalanine and/or tyrosine residues. With respect, in particular, to aflibercept (and proteins sharing structural characteristics of aflibercept e.g., one or more heavy or light chain regions of aflibercept), oxidation variants can comprise oxidation of histidine residue at His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of tryptophan residues at Trp58 and/or Trp138 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of tyrosine residues at Tyr64 (or equivalent positions on proteins sharing certain structural characteristics of aflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); and/or oxidation of methionine residues at Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept).
In one exemplary embodiment, the amount of oxidative species in the anti-VEGF composition compared to the protein of interest can be at most about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. Examples of anti-VEGF compositions are discussed in Section III below. In one aspect, the anti-VEGF composition can comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept or MiniTrap.
Cysteine residues may undergo spontaneous oxidation to form either intra- or intermolecular disulfide bonds or monomolecular byproducts such as sulfenic acid.
Histidine residues are also highly sensitive to oxidation through reaction with their imidazole rings, which can subsequently generate additional hydroxyl species (Li, S, C Schoneich, and RT. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500, the entire teaching of which is herein incorporated). Proposed mechanisms for histidine oxidation are highlighted in FIG. 2 and FIG. 3. Detailed mechanistic studies are available in Anal. Chem. 2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. 21 (2000) 1093-1097, the entire teaching of which is herein incorporated.
Oxidation of methionine can lead to formation of methionine sulfoxide (Li, S, C Schoneich, and RT. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500). The various possible oxidation mechanisms of the methionine residues have been discussed in the literature (Brot, N., Weissbach, H. 1982. The biochemistry of methionine sulfoxide residues in proteins. Trends Biochem. Sci. 7: 137-139, the entire teaching of which is herein incorporated).
Oxidation of tryptophan can give a complex mixture of products. The primary products can be N-formylkynurenine and kynurenine along with mono-oxidation, di-oxidation and/or tri-oxidation products (FIG. 4). Peptides bearing oxidized Trp modifications generally exhibit mass increases of 4, 16, 32 and 48 Da, corresponding to the formation of kynurenine (KYN), hydroxytryptophan (Wox1), and N-formylkynurenine/dihydroxytryptophan (NFK/Wox2, referred to also as “doubly oxidized Trp”), trihydroxytryptophan (Wox3, referred to also as “triply oxidized Trp”), and their combinations, such as hydroxykynurenine (KYNox1, +20 Da). Oxidation to hydroxytryptophan (Wox1) has been discussed in the literature (Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification? J. Am. Soc. Mass Spectrom. 2010 Jul.; 21(7): 1114-1117, the entire teaching of which is herein incorporated). Tryptophan oxidation, but not methionine and histidine oxidation, has been found to produce a color change in protein products (Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores. dx.doi.org/10.1021/ac404218t Anal. Chem. 2014, 86, 6850-6857). Similar to tryptophan, oxidation of tyrosine primarily yields 3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li, S, C Schoneich, and RT. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500).
As used herein, the terms “basic species,” “basic region,” and “BR,” refer to the variants of a protein, for example, an antibody or antigen-binding portion thereof, which are characterized by an overall basic charge, relative to the primary charge variant species present within the protein. For example, in recombinant protein preparations, such basic species can be detected by various methods, such as ion exchange, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing). Exemplary variants can include, but are not limited to, lysine variants, isomerization of aspartic acid, succinimide formation at asparagine, methionine oxidation, amidation, incomplete disulfide bond formation, mutation from serine to arginine, aglycosylation, fragmentation and aggregation. Commonly, basic species elute later than the main peak during CEX or earlier than the main peak during AEX analysis. (Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs. 2012 Sep. 1; 4(5): 578-585. doi: 10.4161/mabs.21328, the entire teaching of which is herein incorporated.)
In certain embodiments, a protein composition can comprise more than one type of basic species variant. For example, but not by way of limitation, the total basic species can be divided based on chromatographic retention time of the peaks appearing. Another example in which the total basic species can be divided can be based on the type of variant—variants, structure variants, or fragmentation variant.
As discussed for acidic species, the term “basic species” does not include process-related impurities and the basic species may be the result of product preparation (referred to herein as “preparation-derived basic species”), or the result of storage (referred to herein as “storage-derived basic species”).
In one exemplary embodiment, the amount of basic species in the anti-VEGF composition compared to the protein of interest can be at most about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. Examples of anti-VEGF compositions are discussed in Section III below. In one aspect, the anti-VEGF composition can comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.
As used herein, “sample matrix” or “biological sample” can be obtained from any step of the bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in the downstream processing, drug substance (DS), or a drug product (DP) comprising the final formulated product. In some other specific exemplary embodiments, the biological sample can be selected from any step of the downstream process of clarification, chromatographic production, viral inactivation, or filtration. In some specific exemplary embodiments, the drug product can be selected from manufactured drug product in the clinic, shipping, storage, or handling.
As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human in need of prevention and/or treatment of a cancer or an angiogenic eye disorder. The subject may have cancer or angiogenic eye disorder or be predisposed to developing cancer or angiogenic eye disorder.
In terms of protein formulation, the term “stable,” as used herein refers to the protein of interest within the formulation being able to retain an acceptable degree of chemical structure or biological function after storage under exemplary conditions defined herein. A formulation may be stable even though the protein of interest contained therein does not maintain 100% of its chemical structure or biological function after storage for a defined amount of time. Under certain circumstances, maintenance of about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of a protein's structure or function after storage for a defined amount of time may be regarded as “stable.”
The term “treat” or “treatment” refers to a therapeutic measure that reverses, stabilizes or eliminates an undesired disease or disorder (e.g., an angiogenic eye disorder or cancer), for example, by causing the regression, stabilization or elimination of one or more symptoms or indicia of such disease or disorder by any clinically measurable degree, for example, with regard to an angiogenic eye disorder, by causing a reduction in or maintenance of diabetic retinopathy severity score (DRSS), by improving or maintaining vision (e.g., in best corrected visual acuity, for example, as measured by an increase in ETDRS letters), increasing or maintaining visual field and/or reducing or maintaining central retinal thickness and, with respect to cancer, stopping or reversing the growth, survival and/or metastasis of cancer cells in the subject. Typically, the therapeutic measure is administration of one or more doses of a therapeutically effective amount of VEGF MiniTrap to the subject with the disease or disorder.
As used herein, the term “upstream process technology,” in the context of protein preparation, refers to activities involving the production and collection of proteins from cells during or following the cell culture of a protein of interest. As used herein, the term “cell culture” refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as the methods and techniques for optimizing the production and collection of the protein of interest. For example, once an expression vector has been incorporated into an appropriate host cell, the host cell can be maintained under conditions suitable for expression of the relevant nucleotide coding sequences, and the collection and production of the desired recombinant protein.
When using the cell culture techniques of the instant invention, a protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In embodiments where the protein of interest is produced intracellularly, particulate debris—either host cells or lysed cells (e.g., resulting from homogenization) can be removed by a variety of means, including, but not limited to, centrifugation or ultrafiltration. Where the protein of interest is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, using an Amicon™ or Millipore Pellicon™ ultrafiltration unit. In one aspect, the protein of interest may be harvested by centrifugation followed by depth filtration and then affinity capture chromatography.
As used herein, a “VEGF antagonist” is any protein or peptide that binds to or interacts with VEGF. Typically, this binding to or interacting with inhibits the binding of VEGF to its receptors (VEGFR1 and VEGFR2), and/or inhibits the biological signaling and activity of VEGF. VEGF antagonists include molecules which interfere with the interaction between VEGF and a natural VEGF receptor, for example, molecules which bind to VEGF or a VEGF receptor and prevent or otherwise hinder the interaction between VEGF and a VEGF receptor. Specific exemplary VEGF antagonists include anti-VEGF antibodies (e.g., ranibizumab [LUCENTIS®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies and the like), and VEGF receptor-based chimeric molecules or VEGF-inhibiting fusion proteins (also referred to herein as “VEGF-Traps” or “VEGF MiniTraps”), such as aflibercept, ziv-aflibercept and a protein having an amino acid having SEQ ID NO.: 60. Other examples of VEGF-Traps are ALT-L9, M710, FYB203 and CHS-2020. Additional examples of VEGF-Traps can be found in U.S. Pat. Nos. 7,070,959; 7,306,799; 7,374,757; 7,374,758; 7,531,173; 7,608,261; 5,952,199; 6,100,071; 6,383,486; 6,897,294 & 7,771,721, which are specifically incorporated herein by reference in their entirety.
VEGF receptor-based chimeric molecules include chimeric polypeptides which comprise two or more immunoglobulin (Ig)-like domains of a VEGF receptor such as VEGFR1 (also referred to as Flt1) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also comprise a multimerizing domain (e.g., an Fc domain which facilitates the multimerization [e.g., dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is a molecule referred to as VEGFR1R2-FcΔC1(a) (also known as aflibercept; marketed under the product name EYLEA®). In certain exemplary embodiments, aflibercept comprises the amino acid sequence set forth as
(SEQ ID NO.: 55)
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKEPLDTL
IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT
NTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDENWEYPSSKHQH
KKLVNRDLKTQSGSEMKKELSTLTIDGVTRSDQGLYTCAASSGLMTKKN
STFVRVHEKDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFMWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
As used herein, “viral filtration” can include filtration using suitable filters including, but not limited to, Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation. It will be apparent to one of ordinary skill in the art to select a suitable filter to obtain desired filtration performance.
II. Color Determination
As used herein, color observed during the production of a recombinant protein, specifically, an anti-VEGF protein, can be measured by various methods. Non-limiting examples include using the iodine color number, hazen color number, gardner color number, lovibond color number, Saybolt color number, Mineral oil color number, European pharmacopoeia color number, US pharmacopoeia color number, CIE L*, a*, b* (or CIELAB), Klett color number, Hess-Ives color number, the yellowness index, ADMI color number, and ASBC and EBC brewery color number. Details on such scales can be found in Application Report No. 3.9 e by Lange, the entire teaching of which is herein incorporated.
Visual color matching on the basis of the European Pharmacopoeia (Ph Eur) (European Color Standards, see European Pharmacopoeia. Chapter 2.2.2. Degree of coloration of liquids. 8th ed. EP, the entire teaching of which is herein incorporated) can include preparing a color reference solution as described in Ph. Eur. (EP 2.2.2. Degree of Coloration of Liquids 2)—three parent solutions for red (cobaltous (II) chloride), yellow (ferrous (III) chloride) and blue colors (cuprous (II) sulphate) and 1% hydrochloric acid, five color reference solutions for yellow (Y), greenish-yellow (GY), brownish-yellow (BY), brown (B) and red (R) hues are prepared. With these five reference solutions in turn, a total of thirty-seven color reference solutions are prepared (Y1-Y7, GY1-GY7, BY1-BY7, B1-B9 and R1-R7). Each reference solution is clearly defined in the CIE-Lab color space, for example, by lightness, hue and chroma. Of the seven yellow-brown standards (BY standards), BY1 is the darkest standard and BY7 is the least dark. Matching a given sample to that of a BY color standard is typically done under diffused daylight. The compositions of European yellow-brown color standards are described in Table 1, below.
TABLE 1
Composition of European Brown-Yellow Standards
Volumes in mL
Reference
Standard
Hydrochloric acid
Solution
Solution BY
(10 g/1 HCl)
BY1
100.0
0.0
BY2
75.0
25.0
BY3
50.0
50.0
BY4
25.0
75.0
BY5
12.5
87.5
BY6
5.0
95.0
BY7
2.5
97.5
Brownish-Yellow Standard Solution (BY): 10.8 g/L FeCl3•6H2O, 6.0 g/L CoCl2•6H2O and 2.5 g/L CuSO4•5H2O
The test for color of liquids is carried out by comparing a test solution with a standard color solution. The composition of the standard color solution is selected depending on the hue and intensity of the color of the test solution. Typically, comparison is carried out in flat-bottomed tubes of colorless, transparent, neutral glass that are matched as closely as possible in internal diameter and in all other respects (e.g., tubes of about 12, 15, 16 or 25 mm diameter). For example, a comparison can be between 2 or 10 mL of the test solution and standard color solution. The depth of liquids, for example, can be about 15, 25, 40 or 50 mm. The color assigned to the test solution should not be more intense than that of the standard color. Color comparisons are typically carried out in diffused light (e.g., daylight) against a white background. Colors can be compared down the vertical axis or horizontal axis of the tubes.
In contrast to the EP color measurement, the USP Monograph 1061 Color-Instrumental Measurement references the use of CIE L*, a*, b* (or CIELAB) color measurement to quantify colors precisely and objectively. A total of twenty color reference solutions (identified sequentially by the letters A to T) are defined in U.S. Pharmacopoeia. The color of the measured sample is automatically correlated to the color reference solutions. This means that the color reference solution that is closest to the sample (i.e., the reference solution with the smallest color difference ΔE* to the color of the sample) is displayed. The ΔL*, Δa* and Δb* values give the quantitative differences between the L*, a* and b* values of the sample and those of the displayed USP solutions. In the CIE L*a*b* coordinate system, L* represents the degree of lightness of a color on a scale of 0-100, with 0 being the darkest and 100 the lightest, a* represents the redness or greenness of a color (positive values of a* represent red, whereas negative values of a* represent green), and b* represents the yellowness or blueness of a sample, with positive values of b* representing yellow and negative values of b* representing blue. Color difference from a standard, or from an initial sample in an evaluation, can be represented by a change in the individual color components ΔL*, Δa*, and Δb*. The composite change, or difference in color, can be calculated as a simple Euclidian distance in space using the formula: dE*=√{square root over ((ΔL*)2+(Δa*)2+(Δb*)2)}. CIE L*, a*, b* color coordinates can be generated, for example, using the Hunter Labs UltrascanPro (Hunter Associates Laboratory, Reston, Va.) or on the BYK Gardner LCS IV (BYK-Gardner, Columbia, Md.). For the Hunter Labs UltraScan Pro, the Didymium Filter Test can be executed for wavelength calibration. The instrument can be standardized in TTRAN with the 0.780-inch port insert and DIW before use; thus, establishing the top (L=100) and bottom (L=0) of the photometric scale using a light trap and black card. See Pack et al., Modernization of Physical Appearance and Solution Color Tests Using Quantitative Tristimulus Colorimetry: Advantages, Harmonization, and Validation Strategies, J. Pharmaceutical Sci. 104: 3299-3313 (2015), the entire teaching of which is herein incorporated. The color of the BY standards can also be expressed under the CIE L*, a*, b* color space (“CIELAB” or “CIELab” color space). See Table 2.
TABLE 2
Characterization of European Brown-Yellow Color Standards
in the CIE L*, a*, b* Color Space
Std.
L*{circumflex over ( )}
a*{circumflex over ( )}
b*{circumflex over ( )}
L*~
a*~
b*~
BY1
93.95
−2.76
28.55
92.84
−3.16
31.15
BY2
94.76
−2.96
22.69
94.25
−3.77
26.28
BY3
96.47
−2.84
16.41
95.92
−3.44
18.52
BY4
97.17
−1.94
9.07
97.67
−2.63
10.70
BY5
98.91
−1.19
4.73
98.75
−1.61
5.77
BY6
99.47
−0.59
2.09
99.47
−0.71
2.38
BY7
99.37
−0.31
1.13
99.71
−0.37
1.17
{circumflex over ( )} Reported by Pack et al.
~Measured experimentally herein-the L* and b* values, for each BY color standard
To enable a high throughput screening for the color assay, the spectrophometric assay method (CIELAB) is a more suitable and quantitative measure than BY color standards. The surrogate assay was further optimized as described in the Example section.
For any of the samples evaluated for color, the protein concentration of the test samples must be standardized for protein concentration in the samples, for example, 5 g/L, 10 g/L and the like for comparison.
III. Anti-VEGF Compositions
There are at least five members of the VEGF family of proteins that regulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (P1GF). Anti-VEGF compositions can comprise a VEGF antagonist, which specifically interacts with one or more members of the VEGF family of proteins and inhibits one or more of its biological activities, for example, its mitogenic, angiogenic and/or vascular permeability activity.
In one embodiment, a method of producing an anti-VEGF protein comprises: (a) providing a host cell genetically engineered to express the anti-VEGF protein; (b) culturing the host cell in a CDM under conditions suitable in which the cell expresses the anti-VEGF protein; and (c) harvesting a preparation of the anti-VEGF protein produced by the cell. In one aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), a scFv and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.
The inventors discovered that manufacturing anti-VEGF proteins (e.g., aflibercept) in certain CDMs produced a biological sample exhibiting a distinctive color. The distinct color properties were observed in different manufacturing steps and even in the final formulation comprising the anti-VEGF protein. As observed in Example 9, for the production of VEGF MiniTrap, culturing cells in a CDM produced anti-VEGF proteins (e.g., aflibercept) with an intense yellow-brown color. The affinity capture step following harvesting also produced an eluate exhibiting a certain color—a yellow-brown color. Further production steps using AEX also exhibited a yellow-brown color, however with reduced intensity.
As described in more detail below, color may be assessed using (i) the European Color Standard BY in which a qualitative visual inspection is made or (ii) a colorimetric assay, CIELAB, which is more quantitative than the BY system. However, in either case, color assessment between multiple samples was normalized against protein concentration in order to assure a meaningful assessment/comparison. For example, referring to Example 9, in particular Table 9-2, the Protein A eluate has a b* value of around 2.52 which corresponds to approximately a BY value of BY5 (when measured at a concentration of 5 g/L protein in the Protein A eluate). If the color of the Protein A eluate is to be compared to another sample, then the comparison should be made using the same protein concentration. Thus, comparing the Protein A eluate to the AEX pool which has a b* value of around 0.74 (when measured at a concentration of 5 g/L protein in the protein A eluate), the method of production shows a substantial reduction in the yellow-brown color of the sample from the Protein A eluate to the AEX pool following AEX chromatography.
Compositions of the present invention can be characterized by a yellow-brown color as discussed herein, for example, no darker/more intense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGF protein or about 10 g/L of the anti-VEGF protein and wherein the composition is obtained as a sample from a clarified harvest or a Protein A eluate of the clarified harvest.
In one embodiment, the compositions of the invention produced using CDM produces a biological sample having a distinct yellow-brown color, wherein the sample may be characterized by a recognized standard color characterization:
(i) no more yellow-brown than European Color Standard BY2;
(ii) no more yellow-brown than European Color Standard BY3;
(iii) no more yellow-brown than European Color Standard BY4;
(iv) no more yellow-brown than European Color Standard BY5;
(v) between European Color Standard BY2 and BY3;
(vi) between European Color Standard BY3 and BY4;
(vii) between European Color Standard BY4 and BY5, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein and wherein the composition is obtained as a sample from a Protein A eluate of a clarified harvest.
In another embodiment, the compositions of the invention produced using a CDM produces a biological sample having a distinct yellow-brown color, wherein the composition is characterized by a recognized standard color characterization in the CIELAB scale:
(i) no more yellow-brown than a b* value of about 22-23;
(ii) no more yellow-brown than a b* value of about 16-17;
(iii) no more yellow-brown than a b* value of 9-10;
(iv) no more yellow-brown than a b* value of 4-5;
(v) no more yellow-brown than a b* value of 2-3;
(vi) between b* value of 17-23;
(vii) between b* value of 10-17;
(viii) between b* value of 5-10;
(ix) between b* value of 3-5; or
(x) between b* value of 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein and wherein the composition is obtained as a sample from a Protein A eluate of a clarified harvest.
In one embodiment, the compositions of the invention produced using CDM can comprise other species or variants of the anti-VEGF protein. These variants include anti-VEGF protein isoforms that comprise one or more oxidized amino acid residues collectively referred to as oxo-variants. The enzymatic digestion of such compositions comprising the anti-VEGF protein and its oxo-variants can comprise one or more of:
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.004-0.013% 2-oxo-histidines,
QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about 0.006-0.028% 2-oxo-histidines,
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.049-0.085% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.057-0.092% 2-oxo-histidines,
TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.008-0.022% 2-oxo-histidines, and/or
IIWDSR (SEQ ID NO.: 56) which comprises about 0.185-0.298% dioxidized tryptophan; or
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008% 2-oxo-histidines,
QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about 0.02% 2-oxo-histidines,
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.06% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07% 2-oxo-histidines, TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01% 2-oxo-histidines, and/or
IIWDSR (SEQ ID NO.: 56) which comprises about 0.23% di-oxo-tryptophans, wherein H* is a histidine that may be oxidized to 2-oxo-histidine and wherein C* is a cysteine which may be carboxymethylated. In a particular embodiment, the anti-VEGF protein is aflibercept. In another embodiment, the anti-VEGF protein is a VEGF MiniTrap.
In one exemplary embodiment of the invention, the compositions of the invention can comprise an anti-VEGF protein, wherein no more than about 1%, no more than about 0.1%, or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of histidine residues of the anti-VEGF protein are 2-oxo-histidine. In such compositions, there can be a heterogeneous population of the anti-VEGF protein variants each having a varying amount of 2-oxo-histidine residues and un-oxidized histidine residues. Thus, the percentage of 2-oxo-histidine anti-VEGF protein in a composition refers to the site-specific 2-oxo-histidines among the anti-VEGF molecules divided by total site-specific histidines in the molecules of the anti-VEGF protein (oxidized plus un-oxidized) times 100. One method to quantitate the level of 2-oxo-histidines in a composition is to digest the polypeptide with a protease (e.g., Lys-C and/or trypsin) and analyze the quantity of 2-oxo-histidines in the resulting peptides by, for example, mass spectrometry (MS).
Before digestion of the anti-VEGF protein, cysteine sulfhydryl groups are blocked by reaction with iodoacetamide (IAM) resulting in a residue represented by the following chemical structure:
Such modification protects free thiols from reforming disulfide bridges and prevents disulfide bond scrambling. The present invention includes compositions (e.g., aqueous compositions) comprising anti-VEGF protein and its variants which, when modified with IAM and digested with protease (e.g., Lys-C and trypsin) and analyzed by mass spectrometry comprise the following peptides:
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.004-0.013% 2-oxo-histidines,
QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about 0.006-0.028% 2-oxo-histidines,
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.049-0.085% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.057-0.092% 2-oxo-histidines,
TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.008-0.022% 2-oxo-histidines, and/or
IIWDSR (SEQ ID NO.: 56) which comprises about 0.185-0.298% dioxidized tryptophan; or
EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18) which comprises about 0.008% 2-oxo-histidines,
QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) which comprises about 0.02% 2-oxo-histidines,
TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20) which comprises about 0.06% 2-oxo-histidines,
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17) which comprises about 0.07% 2-oxo-histidines,
TNYLTH*R (SEQ ID NO.: 21) which comprises about 0.01% 2-oxo-histidines, and/or
IIWDSR (SEQ ID NO.: 56) which comprises about 0.23% di-oxo-tryptophans,
wherein H* is 2-oxo-histidine and wherein C* is carboxymethylated cysteine. In one embodiment of the invention, the peptides are deglycosylated with PNGase F.
The present invention includes compositions comprising anti-VEGF protein, wherein about 0.1%-10% of all histidines of the anti-VEGF protein are modified to 2-oxo-histidine. Further, the color of the composition is no darker/more intense than, for example, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6, or, alternatively, having a b* value, as characterized using CIE L*, a*, b*, of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein. The composition is obtained either as a sample from a clarified harvest or a Protein A eluate of the clarified harvest. Such compositions can be obtained from the clarified harvest when the harvest material is subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, a Protein A affinity column. When an affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more variants may be detected.
The present invention includes compositions comprising anti-VEGF protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to kynurenine. Further, the color of the composition is no darker/more intense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value, as characterized by CIE L*, a*, b*, of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGF protein or about 10 g/L of the anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or a Protein A eluate of the clarified harvest. Such compositions can be obtained from the clarified harvest when subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A affinity column. When an affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
The present invention includes compositions comprising anti-VEGF protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to mono-hydroxyl tryptophan. Further, the color of the composition is no darker/more intense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGF protein or about 10 g/L of the anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or a Protein A eluate of the clarified harvest. Such compositions can be obtained from the clarified harvest when subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A affinity column. When a sample extracted from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
The present invention includes compositions comprising anti-VEGF protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to di-hydroxyl tryptophan. Further, the color is no darker/more intense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized using CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGF protein or about 10 g/L of the anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or a Protein A eluate of the clarified harvest. Such compositions can be obtained from the clarified harvest made using CDM comprising the anti-VEGF protein as well as its oxo-variants subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A affinity column. When a sample extracted from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
The present invention includes compositions comprising anti-VEGF protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to tri-hydroxyl tryptophan. Further, the color of the composition is no darker/more intense than the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L of the anti-VEGF protein or about 10 g/L of the anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or a Protein A eluate of the clarified harvest. Such compositions can be obtained using capture chromatography. The capture step is an affinity chromatography procedure using, for example, a Protein A affinity column. When a sample extracted from the affinity is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
In one embodiment, the compositions of the invention can comprise an anti-VEGF protein, wherein the anti-VEGF protein can comprise modifications of one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to iso-aspartate and/or asparagine; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; the C-terminal glycine is not present; and/or there are one or more non-glycosylated glycosites.
Such compositions can be obtained from a clarified harvest made using CDM comprising the anti-VEGF protein as well as its variants subjected to, for example, a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A column. When a sample extracted from the affinity step is analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
In one exemplary embodiment, the compositions of the invention can comprise an anti-VEGF protein sharing structural characteristics of aflibercept which can be oxidized at one or more of the following: His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); Trp58 and/or Trp138 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); Tyr64 (or equivalent positions on proteins sharing certain structural characteristics of aflibercept); Phe44 and/or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); and/or Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept). Such compositions can be obtained from a clarified harvest made using CDM comprising aflibercept as well as its oxo-variants subjected to a capture chromatography procedure. The capture step can be an affinity chromatography procedure using, for example, a Protein A column. When a sample extracted from the affinity step is analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
In one embodiment, the compositions of the invention can comprise a VEGF MiniTrap having the amino acid sequence of SEQ ID NO.: 46, which can be oxidized at His86, His110, His145, His209, His95, His19 and/or His203; Trp58 and/or Trp138; Tyr64; Phe44 and/or Phe166; and/or Met10, Met 20, Met163 and/or Met192. Such compositions can be obtained from the clarified harvest made using CDM comprising the VEGF MiniTrap as well as its oxo-variants subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A column—when analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.
In some exemplary embodiments, compositions of the present invention can comprise an anti-VEGF protein and its variants (including oxo-variants), wherein the amount of the protein variants in the composition can be at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. Such compositions can be obtained from the clarified harvest made using CDM comprising the anti-VEGF protein as well as its variants subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a Protein A column—when analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected. In one aspect, the color of such a composition is no darker/more intense than, for example, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein.
In other exemplary embodiments, compositions of the present invention can comprise an anti-VEGF protein and its variants, wherein the amount of the protein variants in the composition can be about 0% to about 20%, for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5% and ranges within one or more of the preceding. Such compositions can be obtained performing capture chromatography on a harvest sample. The capture step is an affinity chromatography procedure using, for example, a Protein A column. When a sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected. In one aspect, the color of such a composition is no darker/more intense than, for example, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein.
In one embodiment, compositions of the present invention can comprise an anti-VEGF protein including its acidic species, wherein the amount of the acidic species in the composition can be about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. As discussed supra, such acidic species can be detected by various methods such as ion exchange, for example, WCX (WCX-10 HPLC, a weak cation exchange chromatography), or IEF (isoelectric focusing). Commonly, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see FIG. 16 and FIG. 17). Compositions comprising acidic species can be obtained from biological material such as harvest or affinity produced material using ion exchange chromatography.
In one aspect, the color of such a composition is no darker/more intense than, for example, the European Brown-Yellow Color Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value characterized by CIE L*, a*, b* of about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition comprises about 5 g/L or about 10 g/L. As an example, referring to FIG. 16 and FIG. 17, fractions F1 and F2 represent acidic fractions which comprise the majority of the acidic species. Peaks 1 and 2 of MT1 in FIG. 17 comprise the acidic species and fractions F1 and F2 comprise the majority of the acidic fractions. The fractions comprising such acidic species (F1 and F2) also showed a yellow-brown color compared to other fractions (FIG. 18B and FIG. 18C).
In another embodiment, compositions of the instant invention comprise an anti-VEGF protein including its acidic species, wherein the amount of acidic species in the composition can be about 0% to about 20%, for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5% and ranges within one or more of the preceding. As discussed above, such acidic species can be detected by various methods, such as ion exchange, for example, WCX (WCX-10 HPLC, a weak cation exchange chromatography), or IEF (isoelectric focusing). Typically, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (See FIG. 16 and FIG. 17).
Using a cation exchange column, all peaks eluting prior to the main peak of interest were summed as the acidic region, and all peaks eluting after the protein of interest were summed as the basic region. In exemplary embodiments, the acidic species can be eluted as two or more acidic regions and can be numbered AR1, AR2, AR3 and so on based on a certain retention time of the peaks and on the ion exchange column used.
In one embodiment, compositions can comprise an anti-VEGF protein including acidic species, wherein AR1 is 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein including its acidic species, wherein AR1 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the preceding. As discussed above, such acidic regions can be detected by various methods, such as ion exchange, for example, WCX (WCX-10 HPLC, a weak cation exchange chromatography), or IEF (isoelectric focusing). Commonly, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (See FIG. 16 and FIG. 17).
In another embodiment, compositions can comprise an anti-VEGF protein including acidic species, wherein AR2 is 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein including acidic species, wherein AR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the preceding.
In one embodiment, compositions can comprise an anti-VEGF protein including basic species, wherein the amount of the basic species in the composition can be at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein and its basic species, wherein the amount of the basic species in the composition compared to the anti-VEGF protein can be 0% to about 20%, e.g., about 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5% and ranges within one or more of the preceding.
The basic species can be eluted as two or more basic regions and can be numbered BR1, BR2, BR3 and so on based on a certain retention time of the peaks and ion exchange used.
In one embodiment, compositions can comprise an anti-VEGF protein including its basic species, wherein BR1 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein and its basic species, wherein BR1 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the preceding.
In another embodiment, the composition can comprise an anti-VEGF protein and its basic species, wherein BR2 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein and its basic species of the anti-VEGF protein, wherein BR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the preceding.
In another embodiment, the composition can comprise an anti-VEGF protein and its basic species, wherein BR3 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the preceding. In one aspect, compositions can comprise an anti-VEGF protein and its basic species of the anti-VEGF protein, wherein BR3 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the preceding.
Photo-Induced Oxidation of Aflibercept
In addition to discovering the different color characteristics or variants of the anti-VEGF protein compositions produced using CDM, the inventors also discovered that such compositions can be artificially produced in the laboratory by exposure to light.
Modified, including oxidized, variants of an anti-VEGF composition can be produced by exposing an anti-VEGF protein to cool-white light or ultraviolet light. In one aspect, the anti-VEGF composition can comprise about 1.5 to about 50-fold increase in one or more modified oligopeptides, compared to the sample, wherein the oligopeptides are selected from the group consisting of:
DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIIHMTEGR (SEQ ID NO.: 64), SDTGRPFVEMYSEIPEIIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR (SEQ ID NO.: 28), RIIW*DSR (SEQ ID NO.: 115), IIW*DSRK (SEQ ID NO.: 114), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), wherein H* is a histidine is oxidized to 2-oxo-histidine, wherein C* is a cysteine is carboxymethylated, wherein M* is a oxidized methionine, wherein W* is a oxidized tryptophan, wherein Y* is a oxidized tyrosine, and wherein F* is a oxidized phenylalanine. In a further aspect, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing an anti-VEGF composition to cool-white light for a period of time, for example, about 30 hours. In another aspect, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing a sample to cool-white light for about 75 hours. In yet another aspect, the anti-VEGF composition can comprise about 1.5 to about 20-fold increase in one or more oligopeptides by exposing the sample to cool-white light for about 100 hours. In yet another aspect, the anti-VEGF composition can comprise about 1.5 to about 20-fold increase in one or more oligopeptides by exposing the sample to cool-white light for about 150 hours. In still another aspect, the anti-VEGF composition can comprise about 1.5 to about 50-fold increase in one or more oligopeptides by exposing the sample to cool-white light for about 300 hours—see Example 4 below.
The anti-VEGF composition can comprise about 1.5 to about 3-fold increase in one or more oligopeptides, as described above, by exposing a sample of an anti-VEGF composition to ultraviolet light for about 4 hours. In another aspect, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more oligopeptides by exposing the sample to ultraviolet light for about 10 hours. In yet another aspect, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more oligopeptides by exposing the sample to ultraviolet light for about 16 hours. In yet another aspect, the anti-VEGF composition can comprise about 1.5 to about 25-fold increase in one or more oligopeptides by exposing the sample to ultraviolet light for about 20 hours. In yet another aspect, the anti-VEGF composition can comprise about 1.5 to about 25-fold increase in one or more oligopeptides by exposing the sample matrix to ultraviolet light for about 40 hours. See Example 4.
Glycodiversity—Anti-VEGF Protein Produced Using CDM
The compositions of this invention comprise an anti-VEGF protein, wherein the anti-VEGF protein produced in CDM has a variety of glycodiversity. The different glycosylation profiles of the anti-VEGF protein are within the scope of this invention.
In some exemplary embodiments of the invention, the composition can comprise an anti-VEGF protein glycosylated at one or more asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4 A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation. In one aspect, the protein of interest can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
In one embodiment, the composition can have a glycosylation profile as follows: about 40% to about 50% total fucosylated glycans, about 30% to about 50% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (Example 6).
In one embodiment, the composition can comprise an anti-VEGF protein, wherein the protein of interest has Man5 glycosylation at about 32.4% of asparagine 123 residues and/or about 27.1% of asparagine 196 residues. In one aspect, the protein of interest can be aflibercept, anti-VEGF antibody or VEGF MiniTrap.
In another embodiment, the composition can have about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50% total fucosylated glycans.
In yet another embodiment, the composition can have about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50% total sialylated glycans.
In one embodiment, the composition can have about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% mannose-5.
In another embodiment, the composition can have about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, or about 79% total galactosylated glycans.
In one embodiment, the anti-VEGF protein can have a decreased level of fucosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGF protein can have a decreased level of fucosylated glycans by ranges within one or more of the preceding values, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the level of fucosylated glycans in an anti-VEGF protein produced using a soy hydrolysate.
In one embodiment, the anti-VEGF protein can have a decreased level of sialylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGF protein can have a decreased level of sialylated glycans by ranges within one or more of the preceding values, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the level of sialylated glycans in an anti-VEGF protein produced using a soy hydrolysate.
In another embodiment, the anti-VEGF protein can have a decreased level of galactosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGF protein can have a decreased level of galactosylated glycans by ranges within one or more of the preceding values, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the level of galactosylated glycans in an anti-VEGF protein produced using a soy hydrolysate.
In one embodiment, the anti-VEGF protein can have an increased level of mannosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. The anti-VEGF protein can have an increased level of mannosylated glycans by ranges within one or more of the preceding values, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the level of mannosylated glycans in an anti-VEGF protein produced using a soy hydrolysate.
The compositions described in this section can be produced by several upstream and downstream parameters as described below in sections IV and V, respectively.
IV. Preparation of Compositions Using Upstream Process Technologies
For biologics, the implementation of a robust and flexible upstream process is desirable. An efficient upstream process can lead to desirable production and scale-up of a protein of interest. The inventors discovered that the compositions of the invention comprising an anti-VEGF protein can be produced by modulating conditions during upstream protein production, such as changes in media components of a CDM. Each step in an upstream process may affect quality, purity and quantity of the manufactured protein.
The present disclosure provides evidence for the existence of certain variants of aflibercept and/or MiniTrap produced using CDM. These variants include isoforms that comprise one or more oxidized amino acid residues. Examples of oxidized residues include, but are not limited to, one or more histidine, tryptophan, methionine, phenylalanine or tyrosine residues. The compositions produced by using the modified CDM can produce a preparation of anti-VEGF protein with a desired target value of protein variants of aflibercept and/or MiniTrap. As alluded to above, there can also be a yellow-brownish color associated with fractions produced using a CDM. (As mentioned above, not all CDMs tested by the inventors manifested a distinct discoloration.)
This invention includes culturing a host cell in a modified CDM under suitable conditions in which the cell expresses a recombinant protein of interest followed by harvesting a preparation of the recombinant protein of interest produced by the cell. Such a modified CDM can be used to produce the compositions as described above in Section III.
In one embodiment, the method comprises culturing a host cell in a CDM under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept. The method further comprises harvesting a preparation of the recombinant protein of interest produced by the cell, wherein the suitable conditions include a CDM with a: cumulative concentration of iron in said CDM that is less than about 55 μM, cumulative concentration of copper in said CDM that is less than or equal to about 0.8 μM, cumulative concentration of nickel in said CDM that is less than or equal to about 0.40 μM, cumulative concentration of zinc in said CDM that is less than or equal to about 56 μM, cumulative concentration of cysteine in said CDM that is less than about 10 mM; and/or an antioxidant in said CDM in a concentration of about 0.001 mM to about 10 mM for a single antioxidant and no more than about 30 mM cumulative concentration if multiple antioxidants are added in said CDM.
In one aspect of the present embodiment, the preparation obtained from using suitable conditions results in a reduction in protein variants of aflibercept and VEGF MiniTrap to a desired amount of protein variants of aflibercept and VEGF MiniTrap (referred to as a “target value” of protein variants of aflibercept and VEGF MiniTrap). In a further aspect of this embodiment, the preparation obtained from using suitable conditions results in a reduction in color of the preparations to a desired BY value (referred to as a “target BY value”) when the preparation of protein, including variants of aflibercept and VEGF MiniTrap, are normalized to a concentration of 5 g/L, 10 g/L or even higher.
In a further aspect of the present embodiment, the target BY value and/or target value of variants can be obtained in a preparation where the titer increases or does not significantly decrease (see Example 5).
In some embodiments, the compositions produced by using the modified CDM can produce a preparation of anti-VEGF protein with a desired target BY value, wherein the color of the preparation is characterized as follows:
(i) no more yellow-brown than European Color Standard BY2;
(ii) no more yellow-brown than European Color Standard BY3;
(iii) no more yellow-brown than European Color Standard BY4;
(iv) no more yellow-brown than European Color Standard BY5;
(v) between European Color Standard BY2 and BY3;
(vi) between European Color Standard BY3 and BY4;
(vii) between European Color Standard BY4 and BY5, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein and wherein a sample of the composition can be obtained as a sample from a Protein A eluate of a clarified harvest. As seen in Example 9, Table 9-3 below, the Protein A eluate comprising 5 g/L aflibercept exhibited a yellow-brown color measured as having a b* value of 1.77. Such a sample when produced downstream following AEX had a b* value of 0.50 demonstrating the utility of AEX to lower the yellow-brown coloration of a sample (Table 9-3).
The compositions produced by using the modified CDM can produce a preparation of anti-VEGF protein, wherein the color of the preparation is characterized by a recognized standard color characterization in the CIELAB scale:
(i) no more yellow-brown than a b* value of about 22-23;
(ii) no more yellow-brown than a b* value of about 16-17;
(iii) no more yellow-brown than a b* value of 9-10;
(iv) no more yellow-brown than a b* value of 4-5;
(v) no more yellow-brown than a b* value of 2-3;
(vi) between b* value of 17-23;
(vii) between b* value of 10-17;
(viii) between b* value of 5-10;
(ix) between b* value of 3-5; or
(x) between b* value of 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein and wherein the composition is obtained as a sample from a Protein A eluate of a clarified harvest. See Example 9, Table 9-3.
For components added to the cell culture to form the modified CDM, the term “cumulative amount” refers to the total amount of a particular component added to a bioreactor over the course of the cell culture to form the CDM, including amounts added at the beginning of the culture (CDM at day 0) and subsequently added amounts of the component. Amounts of a component added to a seed-train culture or inoculum prior to the bioreactor production (i.e., prior to the CDM at day 0) are also included when calculating the cumulative amount of the component. A cumulative amount is unaffected by the loss of a component over time during the culture (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may nonetheless have different absolute levels, for example, if the component is added to the two cultures at different times (e.g., if in one culture all of the component is added at the outset, and in another culture the component is added over time). A cumulative amount is also unaffected by in situ synthesis of a component over time during the culture (for example, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a given component may nonetheless have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by way of a bioconversion process. A cumulative amount may be expressed in units such as, for example, grams or moles of the component. The term “cumulative concentration” refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the beginning of the production batch, including the contribution to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the beginning of the production batch, and one gram of component X is added at days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L (i.e., 4 grams divided by 2 liters). If, on day 4, an additional one liter of liquid not containing component X were added to the bioreactor, the cumulative concentration would remain 2 g/L. If, on day 5, some quantity of liquid were lost from the bioreactor (for example, through evaporation), the cumulative concentration would remain 2 g/L. A cumulative concentration may be expressed in units such as, for example, grams per liter or moles per liter.
A. Amino Acids:
In some embodiments, a modified CDM can be obtained by decreasing or increasing cumulative concentrations of amino acids in a CDM. Non-limiting examples of such amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (or salts thereof). The increase or decrease in the cumulative amount of these amino acids in the modified CDM can be of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to the starting CDM, and ranges within one or more of the preceding. Alternatively, the increase or decrease in the cumulative amount of the one or more amino acids in the modified CDM can be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to the unmodified CDM, and ranges within one or more of the preceding (see FIGS. 25-27 and Example 5).
In some embodiments, the modified CDM can be obtained by decreasing the cumulative concentration of cysteine in a CDM. The decrease in the amount of the cysteine in the CDM to form the modified CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, and ranges within one or more of the preceding. Alternatively, the decrease in the cumulative amount of the cysteine in the modified CDM can be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to the CDM, and ranges within one or more of the preceding. In one aspect, the amount of cumulative cysteine in modified CDM is less than about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM or about 10 mM (see FIGS. 25-27 and Example 5).
In some embodiments, the modified CDM can be obtained by replacing at least a certain percentage of cumulative cysteine in a CDM with cystine. The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, and ranges within one or more of the preceding. Alternatively, the replacement can be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to the unmodified CDM, and ranges within one or more of the preceding (see FIGS. 25-27 and Example 5).
In some embodiments, the modified CDM can be obtained by replacing at least a certain percentage of cumulative cysteine in a CDM with cysteine sulfate. The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, and ranges within one or more of the preceding. Alternatively, the replacement can be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to the unmodified CDM, and ranges within one or more of the preceding.
B. Metals:
In some embodiments, the modified CDM can be obtained by decreasing or increasing cumulative concentration of metals in a CDM. Non-limiting examples of metals include iron, copper, manganese, molybdenum, zinc, nickel, calcium, potassium and sodium. The increase or decrease in the amount of the one or more metals in the modified CDM can be of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the unmodified CDM, and ranges within one or more of the preceding. Alternatively, the increase or decrease in the cumulative amount of the one or more metals in the modified CDM can be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to the unmodified CDM, and ranges within one or more of the preceding (see FIGS. 25-27 and Example 5).
C. Antioxidants:
In some embodiments, the modified CDM comprises one or more antioxidants. Non-limiting examples of antioxidants can include taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, Vitamin C, Vitamin E and combinations thereof (see FIG. 28A-E and Example 5).
In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM of taurine, i.e., about 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM of hypotaurine, i.e., about 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM of glycine, i.e., about 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 0.01 μM to about 5 μM of thioctic acid, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM, about 1 μM to about 5 μM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 0.01 M to about 5 mM of glutathione, i.e., about 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 0.01 μM to about 5 μM of hydrocortisone, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM, about 1 μM to about 5 μM, and ranges within one or more of the preceding.
In some embodiments, the modified CDM comprises about 1 μM to about 50 μM of vitamin C, i.e., about 1 μM to about 5 μM, about 5 μM to about 20 μM, about 10 μM to about 30 μM, about 5 μM to about 30 μM, about 20 μM to about 50 μM, about 25 μM to about 50 μM, and ranges within one or more of the preceding.
D. Changes to the Media to Modulate Glycosylation:
This disclosure also includes methods of modulating glycosylation of an anti-VEGF protein by varying cumulative concentrations of certain components in a CDM. Based on the cumulative amounts of components added to the CDM, the total % fucosylation, total % galactosylation, total % sialylation and mannose-5 can be varied.
In exemplary embodiments, the method of modulating glycosylation of an anti-VEGF protein can comprise supplementing the CDM with uridine. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (See Example 6 below).
In some embodiments, the method of modulating glycosylation of an anti-VEGF protein can comprise supplementing a CDM with manganese. In one aspect, the CDM is devoid of manganese before supplementation. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (See Example 6 below).
In some embodiments, the method of modulating glycosylation of an anti-VEGF protein can comprise supplementing a CDM with galactose. In one aspect, the CDM is devoid of galactose before supplementation. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (See Example 6 below).
In some embodiments, the method of modulating glycosylation of an anti-VEGF protein can comprise supplementing a CDM with dexamethasone. In one aspect, the CDM is devoid of dexamethasone before supplementation. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (See Example 6 below).
In some embodiments, the method of modulating glycosylation of an anti-VEGF protein can comprise supplementing a CDM with one or more of uridine, manganese, galactose and dexamethasone. In one aspect, the CDM is devoid of one or more of uridine, manganese, galactose and dexamethasone before supplementation. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. (See Example 6 below).
V. Preparation of Compositions Using Downstream Process Technologies
The compositions comprising an anti-VEGF protein of the invention can be produced by modulating conditions during downstream protein production. The inventors discovered that optimizing the downstream procedures can lead to minimization of certain variants of the anti-VEGF protein as well as discoloration. Optimization of the downstream process may produce a composition with reduced oxo-variants as well as optimized color characteristics.
The downstream process technologies may be used alone or in combination with the upstream process technologies described in Section IV, supra.
A. Anion-Exchange Chromatography:
In some embodiments, a composition of the invention can involve a process comprising: expressing an anti-VEGF protein in a host cell in a CDM, wherein the anti-VEGF protein is secreted from the host cell into the medium and a clarified harvest is obtained. The harvest is subjected to the following steps: (a) loading a biological sample obtained from the harvest onto an anion-exchange chromatography (AEX) column; (b) washing the AEX column with a suitable wash buffer, (c) collecting the flowthrough fraction(s), optionally, (d) washing the column with a suitable strip buffer and (e) collecting stripped fractions.
The flowthrough fractions can comprise oxo-variants of the anti-VEGF protein which are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the anti-VEGF protein sample when compared to the oxo-variants in the stripped fraction of the anion-exchange chromatography column. For example, referring to Table 9-5 and Table 9-6, the flowthrough fractions comprise oxidized variants of anti-VEGF protein where several histidine and tryptophan residues are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and ranges within one or more of the preceding) oxidized when compared against the oxidized variants in the stripped fractions.
The pH of both the equilibration and wash buffers for the AEX column can be from about 8.20 to about 8.60. In another aspect, the conductivity of both the equilibration and wash buffers for the AEX column can be from about 1.50 to about 3.0 mS/cm. In one aspect, the equilibration and wash buffers can be about 50 mM Tris hydrochloride. In one aspect, the strip buffer comprises 2 M sodium chloride or 1 N sodium hydroxide or both (see Table 2-2). Example 2 further illustrates optimizing the concentration and conductivity of the equilibration and wash buffers.
Protein variants can include modifications of one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to iso-aspartate and/or Asn; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; the C-terminal glycine is not present; and/or there are one or more non-glycosylated glycosites.
The protein of interest can be aflibercept, anti-VEGF antibody or a VEGF MiniTrap. The protein variants can be formed by one or more of (i) oxidation of histidines from the histidine residues selected from His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); (ii) oxidation of tryptophan residues selected from tryptophan residues at Trp58 and/or Trp138 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); (iii) oxidation tyrosine residue at Tyr64 (or equivalent positions on proteins sharing certain structural characteristics of aflibercept); (iv) oxidation of phenylalanine residues selected from Phe44 and/or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); and/or (v) oxidation of methionine residues selected from Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept).
The flowthrough fractions can comprise one or more of the following:
(a) a percentage of histidine residues which have been oxidized to 2-oxo-histidine wherein their color characterization is as follows:
(i) no more yellow-brown than European Color Standard BY2;
(ii) no more yellow-brown than European Color Standard BY3;
(iii) no more yellow-brown than European Color Standard BY4;
(iv) no more yellow-brown than European Color Standard BY5;
(v) between European Color Standard BY2 and BY3;
(vi) between European Color Standard BY3 and BY4;
(vii) between European Color Standard BY4 and BY5, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein, and wherein the composition is obtained as a sample from the flowthrough fractions.
(b) a percentage of histidine residues which have been oxidized to 2-oxo-histidine. Further, their color is characterized by having a yellow-brown color which approximates that of BY2, BY3, BY4, BY5, BY6, BY7; or is no darker/more intense than BY2, no darker than BY3, no darker than BY4, no darker than BY5, no darker than BY6, no darker than BY7; or is between that of BY2 and BY3, between that of BY2 and BY4, between that of BY3 and BY4 or between that of BY3 and BY5.
(c) a percentage of histidine residues which have been oxidized to 2-oxo-histidine wherein their color is characterized by a color in the CIE L*, a*, b* color space as follows:
(i) no more yellow-brown than a b* value of about 22-23;
(ii) no more yellow-brown than a b* value of about 16-17;
(iii) no more yellow-brown than a b* value of 9-10;
(iv) no more yellow-brown than a b* value of 4-5;
(v) no more yellow-brown than a b* value of 2-3;
(vi) between b* value of 17-23;
(vii) between b* value of 10-17;
(viii) between b* value of 5-10;
(ix) between b* value of 3-5; or
(x) between b* value of 1-3, wherein the composition comprises about 5 g/L or about 10 g/L of the anti-VEGF protein and wherein the composition is obtained as a sample from the flowthrough fractions.
(d) no more than about 1%, no more than about 0.1%, or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of histidine residues in the composition are oxidized to 2-oxo-histidine. The percentage calculation is described in Section II.
B. Affinity Chromatography:
In some embodiments, compositions of the invention can be produced using a process comprising: expressing an anti-VEGF protein in a host cell wherein anti-VEGF protein is secreted from the host cell into the medium and a clarified harvest is obtained. The harvest is subjected to the following steps, comprising (a) loading a biological sample obtained from the clarified harvest onto an affinity chromatography column, wherein the affinity chromatography comprises a protein capable of selectively or specifically binding to the anti-VEGF protein; (b) washing the affinity chromatography column with a suitable elution buffer, and (c) collecting the eluted fraction(s). For example, as exemplified in Table 7-1 and Table 7-7 through 7-10, using VEGF165 as the protein capable of selectively or specifically binding to the anti-VEGF protein and collecting the eluted fractions as per the method above led to a successful production of MT5 (an anti-VEGF protein), aflibercept and an anti-VEGF scFv fragment. Table 7-1 also discloses successful production of MT5 using (i) mAb 1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (ii) mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain); (iii) mAb3 (a mouse anti-VEGF-R1 mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light chain) and (iv) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) as different proteins capable of selectively or specifically binding to MT5.
With respect to step (a) above, the biological sample to be loaded onto the affinity column can come from a sample in which the clarified harvest can be subjected to chromatography prior to affinity including, but not limited to, ion exchange chromatography (either anion or cation). Other chromatographic procedures well known to the skilled artisan can also be employed prior to use of the affinity step. The important point is that a biological sample comprising an anti-VEGF protein can be subjected to affinity chromatography.
In some embodiments, compositions of the invention can be produced using a process comprising: expressing a VEGF MiniTrap protein in a host cell wherein the VEGF MiniTrap is secreted from the host cell into the medium and wherein the medium can be further processed forming a clarified harvest. This harvest can be further processed by known chromatographic procedures yielding a biological sample comprising a VEGF MiniTrap. This biological sample can be further processed by employing the following steps, comprising (a) loading the biological sample onto an affinity chromatography column, wherein the affinity chromatography comprises a protein capable of selectively or specifically binding to or interacting with the VEGF MiniTrap protein; (b) washing the affinity chromatography column with a suitable elution buffer and (c) collecting the eluted fraction(s). Referring again to Table 7-1, disclosed in this Table is a successful production of MT5 (VEGF MiniTrap) using (i) VEGF165; (ii) mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); (iii) mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain); (iv) mAb3 (a mouse anti-VEGF-R1 mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light chain) and (v) mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) as different proteins capable of selectively or specifically binding to of interacting with MT5.
In one embodiment, affinity chromatography can also be used to isolate other MiniTrap proteins. Following cleavage of an aflibercept, a sample comprising the cleaved aflibercept can be subjected to affinity chromatography using a binder specific for the cleaved aflibercept. In one aspect, the binder can be an antibody or portion thereof.
Cleaving of the aflibercept can be facilitated using proteolytic digestion of aflibercept with, for example, IdeS protease (FabRICATOR) or a variant thereof to generate the VEGF MiniTrap. Cleaving of the aflibercept with IdeS protease or a variant thereof can produce a mixture of products including a Fc fragment and the VEGF MiniTrap. The VEGF MiniTrap can be further processed by using one or more of the production strategies described herein.
In some exemplary embodiments, a protein capable of selectively or specifically binding (“binder”) to or interacting with an anti-VEGF protein, such as aflibercept or MiniTrap, can originate from a human or a mouse.
The affinity production process can further comprise equilibrating an affinity column using an equilibration buffer before loading the biological sample. Exemplary equilibration buffers can be 20 mM sodium phosphate, pH 6-8 (esp. 7.2), 10 mM sodium phosphate, 500 mM NaCl, pH 6-8 (esp. 7.2), 50 mM Tris pH 7-8, DPBS pH 7.4.
The biological sample can be loaded using a suitable buffer, such as, DPBS.
This affinity production process can further comprise washing an affinity column with one or more wash buffers. The column can be washed one or multiple times. Further, the washes can also be collected as wash fractions. The pH of the wash buffer can be from about 7.0 to about 8.60. In one aspect, the wash buffer can be DPBS. In another aspect, the wash buffer can be 20 mM sodium phosphate, pH 6-8 (esp. 7.2), 10 mM sodium phosphate, 500 mM NaCl, pH 6-8 (esp. 7.2), 50 mM Tris pH 7-8, or DPBS pH 7.4.
This affinity process can further comprise washing an affinity column with one or more suitable elution buffers and collecting the eluted fractions. The column can be washed one or multiple times. Non-limiting examples of such a suitable elution buffer includes: ammonium acetate (pH of about 2.0 to about 3.0), acetic acid (pH of about 2.0 to about 3.2), glycine-HCl (pH of about 2.0 to about 3.0), sodium citrate (pH of about 2.0 to about 3.0), citric acid (pH of about 2.0 to about 3.0), potassium isothiocyanate (pH of about 2.0 to about 3.0), or combinations thereof.
In some aspects, the eluted fractions can be neutralized using a neutralizing buffer. An example of such a neutralizing buffer is Tris to Tris-HCl (pH of about 7.0 to about 9.0).
C. IdeS Mutants:
The IdeS protease used for the cleavage of an Fc fusion protein such as aflibercept will rapidly lose enzymatic activity under basic pH conditions, which can limit its use during the manufacture of VEGF MiniTrap. Thus, variants have been developed to be more stable at basic pH, for example, in the presence of a strong base such as NaOH. Such basic conditions can be 0.05 N NaOH for 1 hr or 0.1 N NaOH for 0.5 hr.
In some embodiments, an IdeS mutant can have an amino acid sequence comprising at least about 70% sequence identity over its full length to the amino acid sequences set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its full length to the amino acid sequences mentioned directly above.
In some embodiments, an IdeS mutant can have an isolated nucleic acid molecule encoding a polypeptide with an amino acid sequence comprising at least 70% sequence identity over its full length to the amino acid sequences as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16. In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its full length to the amino acid sequences mentioned directly above.
In some embodiments, the polypeptide can have an amino acid sequence comprising at least 70% sequence identity over its full length to the amino acid sequences as set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16 and can be expressed by a host cell with a suitable vector comprising nucleic acid coding for the identified peptides. In one aspect, the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector can be a plasmid. In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its full length to the amino acid sequences mentioned directly above. In some aspects, an isolated nucleic acid molecule can be used to encode the polypeptide.
In some embodiments, an IdeS mutant can have an amino acid sequence comprising a parental amino acid sequence defined by SEQ ID NO.: 1 (IdeS) with an asparagine residue at position 87, 130, 182 and/or 274 mutated to an amino acid other than asparagine. In one aspect, the mutation can confer an increased chemical stability at alkaline pH-values compared to the parental amino acid sequence. In another aspect, the mutation can confer an increase in chemical stability by 50% at alkaline pH-values compared to the parental amino acid sequence. In one aspect, the amino acid can be selected from aspartic acid, leucine, and arginine. In a particular aspect, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another particular aspect, the asparagine residue at position 130 is mutated to an arginine residue. In yet another particular aspect, the asparagine residue at position 182 is mutated to a leucine residue. In yet another particular aspect, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another particular aspect, the asparagine residues at position 87 and 130 are mutated. In yet another particular aspect, the asparagine residues at position 87 and 182 are mutated. In yet another particular aspect, the asparagine residues at position 87 and 274 are mutated. In yet another particular aspect, the asparagine residues at position 130 and 182 are mutated. In yet another particular aspect, the asparagine residues at position 130 and 274 are mutated. In yet another particular aspect, the asparagine residues at position 182 and 274 are mutated. In yet another particular aspect, the asparagine residues at position 87, 130 and 182 are mutated. In yet another particular aspect, the asparagine residues at position 87, 182 and 274 are mutated. In yet another particular aspect, the asparagine residues at position 130, 182 and 274 are mutated. In yet another particular aspect, the asparagine residues at position 87, 130, 182 and 274 are mutated. In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its full length to the amino acid sequences described above. In some aspects, an isolated nucleic acid molecule can be used to encode the polypeptide.
Those of ordinary skill in the art familiar with standard molecular biology techniques can without undue burden prepare and use IdeS mutants of the present invention. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, supra, which is incorporated herein by reference for any purpose. Enzymatic reactions and production techniques can be performed according to manufacturer's specification or as described herein.
VI. Protein Production Generally
A variety of different production techniques, including, but not limited to, affinity, ion exchange, mixed mode, size exclusion, and hydrophobic interaction chromatography, singularly or in combination, are envisaged to be within the scope of the present invention. These chromatographic steps separate mixtures of proteins of a biological sample on the basis of their charge, degree of hydrophobicity, or size, or a combination thereof, depending on the particular form of separation. Several different chromatography resins are available for each of the techniques alluded to supra, allowing accurate tailoring of the production scheme to a particular protein involved. Each separation method results in the protein traversing at different rates through a column to achieve a physical separation that increases as they pass further through the column or adhere selectively to a separation medium. The proteins are then either (i) differentially eluted using an appropriate elution buffer and/or (ii) collected from flowthrough fractions obtained from the column used, optionally, from washing the column with an appropriate equilibration buffer. In some cases, the protein of interest is separated from impurities (HCPs, protein variants, etc.) when the impurities preferentially adhere to the column and the protein of interest less so, i.e., the protein of interest does not adsorb to the solid phase of a particular column and thus flows through the column. In some cases, the impurities are separated from the protein of interest when they fail to adsorb to the column and thus flow through the column.
The production process may begin at the separation step after the recombinant protein has been produced using upstream production methods described above and/or by alternative production methods conventional in the art. Once a clarified solution or mixture comprising the protein of interest, for example, a fusion protein, has been obtained, separation of the protein of interest from process-related impurities (such as the other proteins produced by the cell (like HCPs), as well as product-related substances, such acidic or basic variants) is performed. A combination of one or more different production techniques, including affinity, ion exchange (e.g., CEX, AEX), mixed-mode (MM), and/or hydrophobic interaction chromatography can be employed. Such production steps separate mixtures of components within a biological sample on the basis of their, for example, charge, degree of hydrophobicity, and/or apparent size. Numerous chromatography resins are commercially available for each of the chromatography techniques mentioned herein, allowing accurate tailoring of the production scheme to a particular protein involved. Each of the separation methods allow proteins to either traverse at different rates through a column achieving a physical separation that increases as they pass further through the column or to adsorb selectively to a separation resin (or medium). The proteins can then be differentially collected. In some cases, the protein of interest is separated from components of a biological sample when other components specifically adsorb to a column's resin while the protein of interest does not.
A. Primary Recovery and Virus Inactivation
In certain embodiments, the initial steps of the production methods disclosed herein involve the clarification and primary recovery of a protein of interest from a biological sample. The primary recovery will include one or more centrifugation steps to separate the protein of interest from a host cell and attendant cellular debris. Centrifugation of the sample can be performed at, for example, but not by way of limitation, 7,000×g to approximately 12,750×g. In the context of large-scale production, such centrifugation can occur on-line with a flow rate set to achieve, for example, a turbidity level of 150 NTU in the resulting supernatant. Such supernatant can then be collected for further processing or in-line filtered through one or more depth filters for further clarification of the sample.
In certain embodiments, the primary recovery may include the use of one or more depth filtration steps to clarify the sample and, thereby, aid in processing the protein of interest. In other embodiments, the primary recovery may include the use of one or more depth filtration steps post centrifugation. Non-limiting examples of depth filters that can be used in the context of the instant invention include the Millistak+X0HC, F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), 3M™ model 30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2 μm filter such as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer or Millipore's Express SHR or SHC filter cartridges typically follows the depth filters. Other filters well known to the skilled artisan can also be used.
In certain embodiments, the primary recovery process can also be a point to reduce or inactivate viruses that can be present in a biological sample. Any one or more of a variety of methods of viral reduction/inactivation can be used during the primary recovery phase of production including heat inactivation (pasteurization), pH inactivation, buffer/detergent treatment, UV and γ-ray irradiation and the addition of certain chemical inactivating agents such as β-propiolactone or, for example, copper phenanthroline as described in U.S. Pat. No. 4,534,972, the entire teaching of which is incorporated herein by reference. In certain exemplary embodiments of the present invention, the sample is exposed to detergent viral inactivation during the primary recovery phase. In other embodiments, the sample may be exposed to low pH inactivation during the primary recovery phase.
In those embodiments where viral reduction/inactivation is employed, a biological sample can be adjusted, as needed, for further production steps. For example, following low pH viral inactivation, the pH of the sample is typically adjusted to a more neutral pH, for example, from about 4.5 to about 8.5, prior to continuing the production process. Additionally, the mixture may be diluted with water for injection (WFI) to obtain a desired conductivity.
B. Affinity Chromatography
In certain exemplary embodiments, it may be advantageous to subject a biological sample to affinity chromatography for production of a protein of interest. The chromatographic material is capable of selectively or specifically binding to or interacting with the protein of interest. Non-limiting examples of such chromatographic material include: Protein A and Protein G. Also included is chromatographic material comprising, for example, a protein or portion thereof capable of binding to or interacting with the protein of interest. In one aspect, the protein of interest is an anti-VEGF protein such as aflibercept, MiniTrap or a protein related thereto.
Affinity chromatography can involve subjecting a biological sample to a column comprising a suitable Protein A resin. When used herein, the term “Protein A” encompasses Protein. A recovered from a native source thereof, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to hind proteins which have a CH2/CH3 region. In certain aspects, Protein A resin is useful for affinity-based production and isolation of a variety of antibody isotypes by interacting specifically with the Fc portion of a molecule should it possess that region.
There are several commercial sources for Protein A resin. One suitable resin is MabSelect™ from GE Healthcare. Suitable resins include, but are not limited to, Mab Select SuRe™, Mab Select SuRe LX, Mab Select, Mab Select SuRe pcc, Mab Select Xtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD Millipore, MapCapture from Life Technologies. A non-limiting example of a suitable column packed with MabSelect™ is an about 1.0 cm diameter×about 21.6 cm long column (17 mL bed volume). A suitable column may comprise a resin such as MabSelect™ SuRe or an analogous resin. Protein A can also be purchased commercially from Repligen, Pharmacia and Fermatech.
An affinity column can be equilibrated with a suitable buffer prior to sample loading. Following loading of the column, the column can be washed one or multiple times using a suitable wash buffer. The column can then be eluted using an appropriate elution buffer, for example, glycine-HCl, acetic acid, or citric acid. The eluate can be monitored using techniques well known to those skilled in the art such as a UV detector. The eluted fractions of interest can be collected and then prepared for further processing.
In one aspect, the eluate may be subjected to viral inactivation, for example, either by detergent or low pH. A suitable detergent concentration or pH (and time) can be selected to obtain a desired viral inactivation result. After viral inactivation, the eluate is usually pH and/or conductivity adjusted for subsequent production steps.
The eluate may be subjected to filtration through a depth filter to remove turbidity and/or various impurities from the protein of interest prior to additional chromatographic polishing steps. Examples of suitable depth filters include, but are not limited to, Millistak+ XOHC, FOHC, DOHC, AIHC, XOSP, and BIHC Pod filters (EMD Millipore), or Zeta Plus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The Emphaze AEX Hybrid Purifier multi-mechanism filter may also be used to clarify the eluate. The eluate pool may need to be adjusted to a particular pH and conductivity in order to obtain desired impurity removal and product recovery from the depth filtration step.
C. Anion Exchange Chromatography
In certain embodiments, a protein of interest is produced by subjecting a biological sample to at least one anion exchange separation step. In one scenario, the anion exchange step can occur following an affinity chromatography procedure (e.g., Protein A affinity). In other scenarios, the anion exchange step can occur before the affinity chromatography step. In yet other protocols, anion exchange can occur both before and after an affinity chromatography step. In one aspect, the protein of interest is either aflibercept or MiniTrap.
The use of an anionic exchange material versus a cationic exchange material is based, in part, on the local charges of the protein of interest. Anion exchange chromatography can be used in combination with other chromatographic procedures such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography as well as other modes of chromatography known to the skilled artisan.
In performing a separation, the initial protein composition (biological sample) can be placed in contact with an anion exchange material by using any of a variety of techniques, for example, using a batch production technique or a chromatographic technique.
In the context of batch production, anion exchange material is prepared in, or equilibrated to, a desired starting buffer. Upon preparation, a slurry of the anion exchange material is obtained. The biological sample is contacted with the slurry to allow for protein adsorption to the anion exchange material. A solution comprising acidic species that do not bind to the AEX material is separated from the slurry by allowing the slurry to settle and removing the supernatant. The slurry can be subjected to one or more washing steps and/or elution steps.
In the context of chromatographic separation, a chromatographic column is used to house chromatographic support material (resin or solid phase). A sample comprising a protein of interest is loaded onto a particular chromatographic column. The column can then be subjected to one or more wash steps using a suitable wash buffer. Components of a sample that have not adsorbed onto the resin will likely flow through the column. Components that have adsorbed to the resin can be differentially eluted using an appropriate elution buffer.
A wash step is typically performed in AEX chromatography using conditions similar to the load conditions or alternatively by decreasing the pH and/or increasing the ionic strength/conductivity of the wash in a step wise or linear gradient manner. In one aspect, the aqueous salt solution used in both the loading and wash buffer has a pH that is at or near the isoelectric point (p1) of the protein of interest. Typically, the pH is about 0 to 2 units higher or lower than the p1 of the protein of interest, however it may be in the range of 0 to 0.5 units higher or lower. It may also be at the p1 of the protein of interest.
The anionic agent may be selected from the group consisting of acetate, chloride, formate and combinations thereof. The cationic agent may be selected from the group consisting of Tris, arginine, sodium and combinations thereof. In a particular example, the buffer solution is a Tris/formate buffer. The buffer may be selected from the group consisting of pyridine, piperazine, L-histidine, Bis-Tris, Bis-Tris propane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol), piperazine, 1,3-diaminopropane and piperidine.
A packed anion-exchange chromatography column, anion-exchange membrane device, anion-exchange monolithic device, or depth filter media can be operated either in bind-elute mode, flowthrough mode, or a hybrid mode wherein proteins exhibit binding to the chromatographic material and yet can be washed from such material using a buffer that is the same or substantially similar to the loading buffer.
In the bind-elute mode, a column or membrane device is first conditioned with a buffer with appropriate ionic strength and pH under conditions where certain proteins will adsorb to the resin-based matrix. For example, during the feed load, a protein of interest can be adsorbed to the resin due to electrostatic attraction. After washing the column or the membrane device with the equilibration buffer or another buffer with a different pH and/or conductivity, the product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).
In the flowthrough mode, a column or membrane device is operated at a selected pH and conductivity such that the protein of interest does not bind to the resin or the membrane while the acidic species will either be retained on the column or will have a distinct elution profile as compared to the protein of interest. In the context of this strategy, acidic species will interact with or bind to the chromatographic material under suitable conditions while the protein of interest and certain aggregates and/or fragments of the protein of interest will flow through the column.
Non-limiting examples of anionic exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Additional non-limiting examples include: Poros 50PI and Poros 50HQ, which are a rigid polymeric bead with a backbone consisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impres and Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymeric base bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymeric resin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is a salt-tolerant chromatographic membrane with a primary amine ligand; Sartobind Q nano, which is a strong anion exchange chromatographic membrane; CUNO BioCap, which is a zeta-plus depth filter media constructed from inorganic filter aids, refined cellulose, and an ion exchange resin; and XOHC, which is a depth-filter media constructed from inorganic filter aid, cellulose, and mixed cellulose esters.
In certain embodiments, the protein load of a sample may be adjusted to a total protein load to the column of between about 50 g/L and about 500 g/L, or between about 75 g/L and about 350 g/L, or between about 200 g/L and about 300 g/L. In other embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded to the column of about 0.5 g/L and about 50 g/L, between about 1 g/L and about 20 g/L, or between about 3 g/L and about 10 g/L. In yet other embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material to the column of about 37 g/L.
Additives such as polyethylene glycol (PEG), detergents, amino acids, sugars, chaotropic agents can be added to enhance the performance of the separation to achieve better separation, recovery and/or product quality.
In certain embodiments, including those relating to aflibercept and/or VEGF MiniTrap, the methods of the instant invention can be used to selectively remove, significantly reduce, or essentially remove at least 10% of protein variants, thereby producing protein compositions that have reduced protein variants.
The protein variants can include modifications of one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to aspartate-glycine and/or Asn-Gly; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; the C-terminal glycine is not present; and/or there are one or more non-glycosylated glycosites. The use of AEX was also observed to reduce oxidized and acidic species of anti-VEGF variants in said affinity eluate. Compared to the affinity eluate, following use of AEX, the flowthrough fraction may show a reduction of at least about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% in oxidized and/or acidic species of anti-VEGF variants.
Protein variants of aflibercept and/or VEGF MiniTrap can include one or more of (i) oxidated histidines from the histidine residues selected from His86, His110, His145, His209, His95, His19 and/or His203; (ii) oxidated tryptophan residues selected from tryptophan residues at Trp58 and/or Trp138; (iii) oxidated tyrosine residue at Tyr64; (iv) oxidated phenylalanine residues selected from Phe44 and/or Phe166; and/or (v) oxidated methionine residues selected from Met10, Met 20, Met163 and/or Met192.
D. Cation Exchange Chromatography
The compositions of the present invention can be produced by subjecting a biological sample comprising a protein of interest to at least one cation exchange (CEX) step. In certain exemplary embodiments, the CEX step will be in addition to an AEX step and occur either before or after the AEX step. In one aspect, the protein of interest is either aflibercept, MiniTrap or a molecule related thereto.
The use of a cationic exchange material versus an anionic exchange material, such as those anionic exchange materials discussed supra, is based, in part, on the local charges of the protein of interest in a given solution and the separation conditions desired. It is within the scope of this invention to employ a cationic exchange step prior to the use of an anionic exchange step, or an anionic exchange step prior to the use of a cationic exchange step. Furthermore, it is within the scope of this invention to employ only a cationic exchange step in combination with other chromatography procedures.
In performing cation exchange, a sample comprising a protein of interest can be contacted with a cation exchange material by using any of a variety of techniques, for example, using a batch production technique or a chromatographic technique, as described above for AEX.
An aqueous salt solution may be used as both a loading and wash buffer having a pH that is lower than the isoelectric point (pI) of the protein of interest. In one aspect, the pH is about 0 to 5 units lower than the pI of the protein. In another aspect, it is in the range of 1 to 2 units lower than the pI of the protein. In yet another aspect, it is in the range of 1 to 1.5 units lower than the pI of the protein.
In certain embodiments, the concentration of the anionic agent in aqueous salt solution is increased or decreased to achieve a pH of between about 3.5 and about 10.5, or between about 4 and about 10, or between about 4.5 and about 9.5, or between about 5 and about 9, or between about 5.5 and about 8.5, or between about 6 and about 8, or between about 6.5 and about 7.5. In one aspect, the concentration of anionic agent is increased or decreased in the aqueous salt solution in order to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for use in the CEX methods include, but are not limited to, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.
In certain embodiments, the conductivity and pH of the aqueous salt solution is adjusted by increasing or decreasing the concentration of a cationic agent. In one aspect, the cationic agent is maintained at a concentration ranging from about 20 mM to about 500 mM, about 50 mM to about 350 mM, about 100 mM to about 300 mM, or about 100 mM to about 200 mM. Non-limiting examples of the cationic agent can be selected from the group consisting of sodium, Tris, triethylamine, ammonium, arginine, and combinations thereof.
A packed cation-exchange chromatography column or a cation-exchange membrane device can be operated either in bind-elute mode, flowthrough mode, or a hybrid mode wherein the product exhibits binding to or interacting with a chromatographic material yet can be washed from such material using a buffer that is the same or substantially similar to the loading buffer (details of these modes are outlined above).
Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationic materials include, but are not limited to: Capto SP ImpRes, which is a high flow agarose bead; CM Hyper D grade F, which is a ceramic bead coated and permeated with a functionalized hydrogel, 250-400 ionic groups μeq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether base matrix with 50-100 μeq/mL ionic capacity; Nuvia C Prime, which is a hydrophobic cation exchange media composed of a macroporous highly crosslinked hydrophilic polymer matrix 55-75με/mL; Nuvia S, which has a UNOsphere base matrix with 90-150 με/mL ionic groups; Poros HS, which is a rigid polymeric bead with a backbone consisting of cross-linked poly[styrene-divinylbenzene]; Poros XS, which is a rigid polymetic bead with a backbone consisting of cross-linked poly[styrene divinylbenzene]; Toyo Pearl Giga Cap CM 650M, which is a polymeric base bead with 0.225 meq/mL ionic capacity; Toyo Pearl Giga Cap S 650M, which is a polymeric base bead; and Toyo Pearl MX TRP, which is a polymeric base bead. It is noted that CEX chromatography can be used with MM resins, described herein.
The protein load of a sample comprising a protein of interest is adjusted to a total protein load to the column of between about 5 g/L and about 150 g/L, or between about 10 g/L and about 100 g/L, between about 20 g/L and about 80 g/L, between about 30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. In certain embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material to be loaded onto the column of about 0.5 g/L and about 50 g/L, or between about 1 g/L and about 20 g/L.
Additives such as polyethylene glycol, detergents, amino acids, sugars, chaotropic agents can be added to enhance the performance of the separation so as to achieve better separation, recovery and/or product quality.
In certain embodiments, including those relating to aflibercept or anti-VEGF antibody or VEGF MiniTrap, the methods of the instant invention can be used to selectively remove, significantly reduce, or essentially remove all of the oxo-variants in a sample where the protein of interest will essentially be in the flowthrough of a CEX procedure while the oxo-variants will be substantially captured by the column media.
E. Mixed Mode Chromatography
Mixed mode (“MM”) chromatography may also be used to prepare the compositions of the invention. MM chromatography, also referred to herein as “multimodal chromatography”, is a chromatographic strategy that utilizes a support comprising a ligand that is capable of providing at least two different interactions with an analyte or protein of interest from a sample. One of these sites provides an attractive type of charge-charge interaction between the ligand and the protein of interest and the other site provides for electron acceptor-donor interaction and/or hydrophobic and/or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, etc.
The column resin employed for a mixed mode separation can be Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. Its base matrix is a highly cross-linked agarose with a ligand (N-benzyl-N-methyl ethanol amine) that exhibits different functionalities for interaction, such as ionic interaction, hydrogen bonding and hydrophobic interaction. In certain aspects, the resin employed for a mixed mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrices of PPA-HyperCel and HEA-HyperCel are high porosity cross-linked cellulose. Their ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine offer different selectivity and hydrophobicity options for protein separations. Additional mixed mode chromatographic supports include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain aspects, the mixed mode chromatography resin is comprised of ligands coupled to an organic or inorganic support, sometimes denoted by a base matrix, directly or via a spacer. The support may be in the form of particles, such as essentially spherical particles, a monolith, filter, membrane, surface, capillaries, and the like. In certain aspects, the support is prepared from a native polymer, such as cross-linked carbohydrate material, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate and the like. To obtain high adsorption capacities, the support can be porous, and ligands are then coupled to the external surfaces as well as to the pore surfaces. Such native polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teaching of which is incorporated herein by reference). Alternatively, the support can be prepared from a synthetic polymer, such as cross-linked synthetic polymers, for example, styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, and the like. Such synthetic polymers can be produced according to standard methods, see “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teaching of which is incorporated herein by reference). Porous native or synthetic polymer supports are also available from commercial sources, such as GE Healthcare, Uppsala, Sweden.
The protein load of a biological sample mixture comprising a protein of interest can be adjusted to a total protein load to the column of between about 25 g/L and about 750 g/L, or between about 75 g/L and about 500 g/L, or between about 100 g/L and about 300 g/L. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded to the column of about 1 g/L and about 50 g/L, or between about 9 g/L and about 25 g/L.
Additives such as polyethylene glycol, detergents, amino acids, sugars, chaotropic agents can be added to enhance the performance of the separation, so as to achieve better separation, recovery and/or product quality.
In certain embodiments, including those relating to aflibercept and/or MiniTrap, the methods of the instant invention can be used to selectively remove, significantly reduce, or essentially remove all of the PTMs, including oxo-variants.
The methods for producing the composition of the invention can also be implemented in a continuous chromatography mode. In this mode, at least two columns are employed (referred to as a “first” column and a “second” column). In certain embodiments, this continuous chromatography mode can be performed such that the eluted fractions and/or stripped fractions comprising PTMs, for example, oxo-variants, can then be loaded subsequently or concurrently onto the second column (with or without dilution).
In one embodiment, the media choice for continuous mode can be one of many chromatographic resins with pendant hydrophobic and anion exchange functional groups, monolithic media, membrane adsorbent media or depth filtration media.
F. Hydrophobic Interaction Chromatography
The compositions of the invention may also be prepared using hydrophobic interaction chromatography (HIC).
In performing the separation, a biological sample is contacted with a HIC material, for example, using a batch production technique or using a column or membrane chromatography. Prior to HIC processing it may be desirable to adjust the concentration of the salt buffer to achieve desired protein binding/interaction to the resin or the membrane.
Whereas ion exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography exploits the hydrophobic properties of proteins to achieve selective separation. Hydrophobic groups on or within a protein interact with hydrophobic groups of chromatography resin or a membrane. Typically, under suitable conditions, the more hydrophobic a protein is (or portions of a protein) the stronger it will interact with the column or the membrane. Thus, under suitable conditions, HIC can be used to facilitate the separation of process-related impurities (e.g., HCPs) as well as product-related substances (e.g., aggregates and fragments) from a protein of interest in a sample.
Like ion exchange chromatography, a HIC column or a HIC membrane device can also be operated in an elution mode, a flowthrough, or a hybrid mode wherein the product exhibits binding to or interacting with a chromatographic material yet can be washed from such material using a buffer that is the same or substantially similar to the loading buffer. (The details of these modes are outlined above in connection with AEX processing.) As hydrophobic interactions are strongest at high ionic strength, this form of separation is conveniently performed following a salt elution step such as those typically used in connection with ion exchange chromatography. Alternatively, salts can be added to a sample before employing a HIC step. Adsorption of a protein to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein of interest, salt type and the particular HIC ligand chosen. Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are ranked in terms of increasing salting out effect as Ba2+; Ca2+; Mg2+; Li+; Cs+, Na+; K+; Rb+; NH4+, while anions may be ranked in terms of increasing chaotropic effect as PO43−; SO42−; CH3CO3−; CI−; Br−; NO3−; ClO4−; I−; SCN−.
In general, Na+, K+ or NH4+ sulfates effectively promote ligand-protein interaction using HIC. Salts may be formulated that influence the strength of the interaction as given by the following relationship: (NH4)2SO4>Na2SO4>NaCl>NH4Cl>NaBr>NaSCN. In general, salt concentrations of between about 0.75 M and about 2 M ammonium sulfate or between about 1 M and about 4 M NaCl are useful.
HIC media normally comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. A suitable HIC media comprises an agarose resin or a membrane functionalized with phenyl groups (e.g., a Phenyl Sepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). Many HIC resins are available commercially. Examples include, but are not limited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or high substitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butyl columns (Bio-Rad, California); WP HI—Propyl (C3)™ (J. T. Baker, New Jersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, PA); ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen and Butyl FF HiScreen Octyl FF.
The protein load of a sample comprising a protein of interest is adjusted to a total protein load to the column of between about 50 g/L to about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/L and about 100 g/L, between about 20 g/L and about 80 g/L, between about 30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. In certain embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material to be loaded onto the column of about 0.5 g/L and about 50 g/L, or between about 1 g/L and about 20 g/L.
Because the pH selected for any particular production process must be compatible with protein stability and activity, particular pH conditions may be specific for each application. However, because at pH 5.0-8.5 particular pH values have very little significance on the final selectivity and resolution of a HIC separation, such conditions may be favored. An increase in pH weakens hydrophobic interactions and retention of proteins changes more drastically at pH values above 8.5 or below 5.0. In addition, changes in ionic strength, the presence of organic solvents, temperature and pH (especially at the isoelectric point, pI, when there is no net surface charge) can impact protein structure and solubility and, consequently, the interaction with other hydrophobic surfaces, such as those in HIC media and hence, in certain embodiments, the present invention incorporates production strategies wherein one or more of the foregoing are adjusted to achieve the desired reduction in process-related impurities and/or product-related substances.
In certain embodiments, spectroscopy methods such as UV, NIR, FTIR, Fluorescence, and Raman may be used to monitor the protein of interest and impurities in an on-line, at-line or in-line mode, which can then be used to control the level of aggregates in the pooled material collected from the HIC adsorbent effluent. In certain embodiments, on-line, at-line or in-line monitoring methods can be used either on the effluent line of the chromatography step or in the collection vessel, to enable achievement of the desired product quality/recovery. In certain embodiments, the UV signal can be used as a surrogate to achieve an appropriate product quality/recovery, wherein the UV signal can be processed appropriately, including, but not limited to, such processing techniques as integration, differentiation, and moving average, such that normal process variability can be addressed and the target product quality can be achieved. In certain embodiments, such measurements can be combined with in-line dilution methods such that ion concentration/conductivity of the load/wash can be controlled by feedback and hence facilitate product quality control.
G. Size Exclusion Chromatography
Size exclusion chromatography or gel filtration relies on the separation of components as a function of their molecular size. Separation depends on the amount of time that the substances spend in the porous stationary phase as compared to time in the fluid. The probability that a molecule will reside in a pore depends on the size of the molecule and the pore. In addition, the ability of a substance to permeate into pores is determined by the diffusion mobility of macromolecules which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all; and, for very small macromolecules the probability of penetration is close to unity. While components of larger molecular size move more quickly past the stationary phase, components of small molecular size have a longer path length through the pores of the stationary phase and are thus retained longer in the stationary, phase.
The chromatographic material can comprise a size exclusion material wherein the size exclusion material is a resin or membrane. The matrix used for size exclusion is preferably an inert gel medium which can be a composite of cross-linked polysaccharides, for example, cross-linked agarose and/or dextran in the form of spherical beads. The degree of cross-linking determines the size of pores that are present in the swollen gel beads. Molecules greater than a certain size do not enter the gel beads and thus move through the chromatographic bed the fastest. Smaller molecules, such as detergent, protein, DNA and the like, which enter the gel beads to varying extent depending on their size and shape, are retarded in their passage through the bed. Molecules are thus generally eluted in the order of decreasing molecular size.
Porous chromatographic resins appropriate for size-exclusion chromatography of viruses may be made of dextrose, agarose, polyacrylamide, or silica which have different physical characteristics. Polymer combinations can also be also used. Most commonly used are those under the tradename. “SEPHADEX” available from Amersham Biosciences.
Other size exclusion supports from different materials of construction are also appropriate, for example Toyopearl 55F (polymethacrylate, from Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, Calif.).
The protein load of a sample comprising a protein of interest can be adjusted to a total protein load to the column of between about 50 g/L and about 1000 g/L; about 5 g/L and about 150 g/L, between about 10 g/L and about 100 g/L, between about 20 g/L and about 80 g/L, between about 30 g/L and about 50 g/L, or between about 40 g/L and about 50 g/L. In certain embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material to be loaded onto the column of between about 0.5 g/L and about 50 g/L, or between about 1 g/L and about 20 g/L.
H. Viral Filtration
Viral filtration is a dedicated viral reduction step in a production process. This step is usually performed post chromatographic polishing. Viral reduction can be achieved via the use of suitable filters including, but not limited to, Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation. It will be apparent to one of ordinary skill in the art to select a suitable filter to obtain desired filtration performance.
I. Ultrafiltration/Diafiltration
Certain embodiments of the present invention employ ultrafiltration and diafiltration to further concentrate and formulate a protein of interest. Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9); the entire teachings of which are incorporated herein by reference. One filtration process is Tangential Flow Filtration as described in the Millipore catalogue entitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford, Mass., 1995/96), the entire teaching of which is incorporated herein by reference. Ultrafiltration is generally considered to mean filtration using filters with a pore size of smaller than 0.1μιη. By employing filters having such a small pore size, the volume of sample can be reduced through permeation of the sample buffer through the filter membrane pores while proteins are retained above the membrane surface.
One of ordinary skill in the art can select an appropriate membrane filter device for the UF/DF operation. Examples of membrane cassettes suitable for the present invention include, but not limited to, Pellicon 2 or Pellicon 3 cassettes with 10 kD, 30 kD or 50 kD membranes from EMD Millipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GE Healthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kD cassettes from Pall Corporation.
J. Exemplary Production Strategies
Primary recovery can proceed by sequentially employing pH reduction, centrifugation, and filtration to remove cells and cellular debris (including HCPs) from a production bioreactor harvest. The present invention is directed to subjecting a biological sample comprising a protein of interest from the primary recovery to one or more production steps, including (in no particular order) AEX, CEX, SEC, HIC and/or MM. Certain aspects of the present invention include further processing steps. Examples of additional processing procedures include ethanol precipitation, isoelectric focusing, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose™, further anion exchange chromatography and/or further cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g., using Protein A or G, an antibody, a specific substrate, ligand or antigen as the capture reagent). In certain aspects, the column temperature (as well as other parameters) can be independently varied to improve the separation efficiency and/or yield of any particular production step.
In certain embodiments, unbound flowthrough and wash fractions can be further fractionated and a combination of fractions providing a target product purity can be pooled.
Column loading and washing steps can be controlled by in-line, at-line or off-line measurement of the product related impurity/substance levels, either in the column effluent, or the collected pool or both, so as to achieve a particular target product quality and/or yield. In certain embodiments, the loading concentration can be dynamically controlled by in-line or batch or continuous dilutions with buffers or other solutions to achieve the partitioning necessary to improve the separation efficiency and/or yield.
Examples of such production procedures are depicted in FIGS. 5-8.
FIG. 5 represents one exemplary embodiment used for the production of aflibercept. Referring to FIG. 5, the method comprises: (a) expressing aflibercept in a host cell cultured in a CDM; (b) capturing aflibercept using a first chromatography support, which can include affinity capture resin; and (c) contacting at least a portion of aflibercept with a second chromatography support, which can include anion-exchange chromatography. Step (c) can further comprise washing an AEX column and collecting flowthrough fraction(s) of a sample comprising aflibercept. Optionally, step (c) can comprise stripping the second chromatographic support and collecting stripped fractions. The steps can be carried out by routine methodology in conjunction with methodology mentioned supra. It should be understood that one skilled in the art might opt to employ CEX rather than or in addition to AEX. In no particular order, additional chromatographic steps may be employed as well including, but not limited to, HIC and SEC.
In addition to the exemplary embodiment in FIG. 5, other additional exemplary embodiments can include (d) contacting at least a portion of said aflibercept of step (c) with a third chromatography support. In one aspect, the protocol can include (e) contacting at least a portion aflibercept of step (d) with a fourth chromatography support. In one aspect of this embodiment, the protocol can optionally comprise subjecting the sample comprising aflibercept of step (c) to a pH less than 5.5. In one aspect, the present method comprises a clarification step prior to step (a).
FIG. 6 represents one exemplary embodiment used for the production of VEGF MiniTrap. This method comprises: (a) expressing aflibercept in a host cell cultured in a CDM; (b) capturing aflibercept using a first chromatography support which can include affinity chromatography resin; (c) cleaving the aflibercept thereby removing the Fc domain and forming a sample comprising VEGF MiniTrap; (d) contacting the sample of step (c) with a second chromatographic support which can be affinity chromatography and (e) contacting the flowthrough of step (d) to a third chromatography support which can include an anion-exchange chromatography. Step (d) comprises the collection of flowthrough fraction(s) where due to the absence of an Fc domain, the MiniTrap should reside while the aflibercept or any other protein having an Fc domain should essentially interact with the affinity column of step (d). Optionally, step (d) can comprise stripping the third chromatographic support and collecting stripped fractions. The steps can be carried out by routine methodology in conjunction with methodology outlined above. In no particular order, additional chromatographic steps can be employed including, but not limited to, HIC and SEC.
FIG. 7 represents one exemplary embodiment for the production of aflibercept. This method comprises: (a) expressing aflibercept in a host cell cultured in a CDM; (b) capturing aflibercept using a first chromatography support, which can include cation exchange chromatography; and (c) contacting a flowthrough of step (b) to a second chromatography support which can include an anion-exchange chromatography. Optionally, step (c) can comprise stripping the second chromatographic support and collecting stripped fractions. The steps can be carried out by routine methodology in conjunction with protocols alluded to above. In no particular order, other chromatographic procedures may be employed including, but not limited to, HIC and SEC.
FIG. 8 represents one exemplary embodiment for producing VEGF MiniTrap. This method comprises: (a) expressing aflibercept in a host cell cultured in a CDM; (b) capturing aflibercept using a first chromatography support which can include an ion exchange chromatography; (c) subjecting a flowthrough fraction of (b) comprising aflibercept to affinity chromatography; eluting, wherein the elution comprises aflibercept; (d) subjecting the aflibercept of (c) to a cleavage activity, whereby the Fc domain is cleaved thus forming VEGF MiniTrap. In one aspect, the ion exchange of step (b) comprises AEX. Alternatively, step (b) may comprise CEX. In no particular order, additional chromatographic steps may be included such as further ion exchange chromatography steps following step (d), the addition of HIC and/or SEC.
VII. Pharmaceutical Formulations Comprising the Compositions
The invention also discloses formulations comprising anti-VEGF compositions (as described above). Suitable formulations for anti-VEGF proteins include, but are not limited to, formulations described in U.S. Pat. Nos. 7,608,261, 7,807,164, 8,092,803, 8,481,046, 8,802,107, 9,340,594, 9,914,763, 9,580,489, 10,400,025, 8,110,546, 8,404,638, 8,710,004, 8,921,316, 9,416,167, 9,511,140, 9,636,400, and 10,406,226, which are all incorporated herein by reference in their entirety.
The upstream process technologies (described in Section IV, supra) and downstream process technologies (described in Section V, supra) may be used alone or in combination with each other to effect formulation production.
The present invention discloses formulations comprising anti-VEGF compositions in association with one or more ingredients/excipients as well as methods of use thereof and methods of making such compositions. In an embodiment of the invention, a pharmaceutical formulation of the present invention has a pH of approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2.
To prepare pharmaceutical formulations for anti-VEGF compositions, an anti-VEGF composition is admixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.; the entire teachings of which are incorporated herein by reference. In an embodiment of the invention, the pharmaceutical formulation is sterile.
Pharmaceutical formulations of the present invention include an anti-VEGF composition and a pharmaceutically acceptable carrier including, for example, water, buffering agents, preservatives and/or detergents.
The present invention provides a pharmaceutical formulation comprising any of the anti-VEGF compositions set forth herein and a pharmaceutically acceptable carrier, for example, wherein the concentration of polypeptide is about 40 mg/mL, about 60 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 200 mg/mL or about 250 mg/mL.
The scope of the present invention includes desiccated, for example, freeze-dried, compositions comprising an anti-VEGF protein and a pharmaceutically acceptable carrier substantially (about 85% to about 99% or greater) lacking water.
In one embodiment, a further therapeutic agent that is administered to a subject in association with an anti-VEGF composition disclosed herein is administered to the subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002), the teaching of which is incorporated herein by reference).
The present invention provides a vessel (e.g., a plastic or glass vial with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-VEGF compositions or a pharmaceutical formulation comprising a pharmaceutically acceptable carrier described herein. The present invention also provides an injection device comprising the anti-VEGF composition or formulation set forth herein, for example, a syringe, a pre-filled syringe or an autoinjector. In one aspect, the vessel is tinted (e.g., brown) to block out light, natural or otherwise.
The present invention includes combinations including anti-VEGF compositions in association with one or more further therapeutic agents. The anti-VEGF composition and the further therapeutic agent can be in a single composition or in separate compositions. For example, the therapeutic agent is an Ang-2 inhibitor (e.g., nesvacumab), a Tie-2 receptor activator, an anti-PDGF antibody or antigen-binding fragment thereof, an anti-PDGF receptor or PDGF receptor beta antibody or antigen-binding fragment thereof and/or an additional VEGF antagonist such as aflibercept, conbercept, bevacizumab, ranibizumab, an anti-VEGF aptamer such as pegaptanib (e.g., pegaptanib sodium), a single chain (e.g., VL-VH) anti-VEGF antibody such as brolucizumab, an anti-VEGF DARPin such as the Abicipar Pegol DARPin, a bispecific anti-VEGF antibody, for example, which also binds to ANG2, such as RG7716, or a soluble form of human vascular endothelial growth factor receptor-3 (VEGFR-3) comprising extracellular domains 1-3, expressed as an Fc-fusion protein.
VIII. Methods of Treatment
The present invention provides methods for treating or preventing a cancer (e.g., whose growth and/or metastasis is mediated, at least in part, by VEGF, for example, VEGF-mediated angiogenesis) or an angiogenic eye disorder, in a subject, comprising administering a therapeutically effective amount of compositions as disclosed herein (Section III supra).
Upstream process technologies (Section IV supra), downstream process technologies (Sections V and VI supra) may be used alone or in combination with the each other to produce the compositions as described in Section III and/or the formulations as described in Section VII which can be used for treating or preventing a variety of disorders including ophthalmological and oncological disease.
The present invention also provides a method for administering compositions set forth herein (Section III and Section VII) to a subject (e.g., a human) comprising introducing the compositions with about 0.5 mg, 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg or 20 mg of the protein of interest (e.g., aflibercept or MiniTrap) in no more than about 100 μL, for example, about 50 μL, about 70 μL or about 100 μL, and optionally a further therapeutic agent, into the body of the subject by, for example, intraocular injection such as by intravitreal injection.
The present invention provides a method for treating cancer whose growth and/or metastasis is mediated, at least in part, by VEGF, for example, VEGF-mediated angiogenesis or an angiogenic eye disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the compositions set forth herein (Section III and Section VII above), for example, 2 mg, 4 mg, 6 mg, 8 mg or 10 mg of the protein of interest, in no more than about 100 μl, and optionally a further therapeutic agent, to a subject. In one embodiment of the invention, administration is done by intravitreal injection. Non-limiting examples of angiogenic eye disorders that are treatable or preventable using the methods herein, include:
age-related macular degeneration (e.g., wet or dry),
macular edema,
macular edema following retinal vein occlusion,
retinal vein occlusion (RVO),
central retinal vein occlusion (CRVO),
branch retinal vein occlusion (BRVO),
diabetic macular edema (DME),
choroidal neovascularization (CNV),
iris neovascularization,
neovascular glaucoma,
post-surgical fibrosis in glaucoma,
proliferative vitreoretinopathy (PVR),
optic disc neovascularization,
corneal neovascularization,
retinal neovascularization,
vitreal neovascularization,
pannus,
pterygium,
vascular retinopathy,
diabetic retinopathy in a subject with diabetic macular edema; and
diabetic retinopathies (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in a subject that does not suffer from DME).
The mode of administration of such compositions or formulations (Section III and Section VII) can vary and can be determined by a skilled practitioner. Routes of administration include parenteral, non-parenteral, oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, intraocular, intravitreal, transdermal or intra-arterial.
In one embodiment of the invention, intravitreal injection of a pharmaceutical formulation of the present invention (which includes a compositions or formulations of the present invention) includes the step of piercing the eye with a syringe and needle (e.g., 30-gauge injection needle) comprising the formulation and injecting the formulation (e.g., less than or equal to about 100 microliters; about 40, 50, 55, 56, 57, 57.1, 58, 60 or 70 microliters) into the vitreous of the eye with a sufficient volume as to deliver a therapeutically effective amount as set forth herein, for example, of about 2, 4, 6, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 10 or 20 mg of the protein of interest. Optionally, the method includes the steps of administering a local anesthetic (e.g., proparacaine, lidocaine or tetracaine), an antibiotic (e.g., a fluoroquinolone), antiseptic (e.g., povidone-iodine) and/or a pupil dilating agent to the eye being injected. In one aspect, a sterile field around the eye to be injected is established before the injection. Following intravitreal injection, the subject is monitored for elevations in intraocular pressure, inflammation and/or blood pressure.
An effective or therapeutically effective amount of protein of interest for an angiogenic eye disorder refers to the amount of the protein of interest sufficient to cause the regression, stabilization or elimination of the cancer or angiogenic eye disorder, for example, by regressing, stabilizing or eliminating one or more symptoms or indicia of the cancer or angiogenic eye disorder by any clinically measurable degree, for example, with regard to an angiogenic eye disorder, by causing a reduction in or maintenance of diabetic retinopathy severity score (DRSS), by improving or maintaining vision (e.g., in best corrected visual acuity as measured by an increase in ETDRS letters), increasing or maintaining visual field and/or reducing or maintaining central retinal thickness and, with respect to cancer, stopping or reversing the growth, survival and/or metastasis of cancer cells in the subject.
In one embodiment of the invention, an effective or therapeutically effective amount of a protein of interest such as aflibercept for treating or preventing an angiogenic eye disorder is about 0.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 7.25 mg, 7.7 mg, 7.9 mg, 8.0 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg, e.g., in no more than about 100 μL. The amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In certain exemplary embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the protein of interest in an amount that can be approximately the same or less or more than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.
It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
IX. Method of Assaying Protein Variants
The levels of protein variants in a chromatographic sample produced using the techniques described herein may be analyzed as described in the Examples below. In certain embodiments, a cIEF method is employed using an iCE3 analyzer (ProteinSimple) with a fluorocarbon coated capillary cartridge (100μπι××5 cm). The ampholyte solution consists of a mixture of 0.35% methyl cellulose (MC), 4% Pharmalyte 3-10 carrier ampholytes, 4% Pharmalyte 5-8 carrier ampholytes, 10 mM L-Arginine HCl, 24% formamide, and p1 markers 5.12 and 9.77 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted in purified water to 10 mg/mL. Samples were mixed with the ampholyte solution and then focused by introducing a potential of 1500 V for one minute, followed by a potential of 3000 V for 7 minutes. An image of the focused variants was obtained by passing 280 nm ultraviolet light through the capillary and into the lens of a charge coupled device digital camera. This image was then analyzed to determine the distribution of the various charge variants. Persons of skill in the art may vary the precise parameters while still achieving the desired outcome.
Various publications, including patents, patent applications, published patent applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited references is incorporated by reference, in its entirety.
The present invention will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the invention.
EXAMPLES
The MiniTraps (MT) 1-6 discussed in the Examples are as follows:
MT1: VEGF MiniTrap obtained by cleavage of aflibercept produced using CDM1.
MT2: VEGF MiniTrap obtained by cleavage of aflibercept produced using CDM2.
MT3: VEGF MiniTrap obtained by cleavage of aflibercept produced using CDM3.
MT4: VEGF MiniTrap obtained by cleavage of aflibercept produced using soy hydrolysate.
MT5: recombinant VEGF MiniTrap (dimer).
MT6: recombinant VEGF MiniTrap (scFv).
Characterization of MT1, MT5 and MT6 are described below in Example 8.
Color Assessment of Samples
The spectrophotometric assay method of measuring the b* value (CIELAB) was found suitable for performing color assessment.
The absorbance of a 1 mL protein sample was quantified over the visible light spectrum (380 to 780 nm) and the absorbance curve was transformed into the CIELAB color space using a set of matrix operations. The instrument can process approximately 6 samples per hour. The high throughput format of the assay used a CLARIOstar plate reader (BMG Labtech). Up to 96 samples can be analyzed using a 96-well plate requiring 0.3 mL of sample.
To convert the BY standards into the b* values, BY reference standards (BY1 to BY7) were quantified using the high throughput assay format.
The solutions were prepared as per the BY standards discussed above. The b* value for each of the standards are as shown in FIG. 9. This method provided a faster assay with a smaller sample requirement and shorter run times as shown in Table 3 below. For all the samples evaluated using this method, the protein concentration of the test samples was standardized to either 5 g/L or 10 g/L.
TABLE 3
Original
High-throughput
Amount/Sample
1 mL
0.3 mL
Measurement Format
cuvette (individual)
96-well-plate (bulk)
Run Time
6 samples per hour
96 samples per 5 minutes
Data Entry
manual
automated
Data Storage
Excel
LIMS
Example 1: Production of a Protein Using a Chemically Defined Medium
1.1 Cell Source and Harvest
An aflibercept producing cell line was employed in the present study. Aflibercept producing cell lines were cultured and harvested using chemically defined media (CDM).
1.2 Proteolytic Cleavage of Aflibercept
A column with an immobilized IdeS enzyme (FabRICATOR® obtained from Genovis (Cambridge, Mass.)) was used to generate MT1. Aflibercept obtained from a cell culture harvest (20 mg in 1.0 mL cleavage buffer) was added to the column and incubated for 30 min at 18° C. After 30 min, the column was washed with the cleavage buffer (1.0 mL). The digestion mixture and washing solutions were combined. The mixture was loaded onto and eluted from an analytical Protein A affinity column (Applied Biosystems™, POROS™ 20 μM Protein A Cartridge 2.1×30 mm, 0.1 mL (Cat #2-1001-00)). The processing was carried out according to Applied Biosystems™ protocol for POROS™ 20 μM Protein A Cartridge 2.1×30 mm, 0.1 mL (Cat #2-1001-00). The column height was 20±1.0 cm, residence time was 15 minutes and equilibration/wash was performed using 40 mM Tris, 54 mM Acetate pH 7.0±0.1.
Example 2. Anion Exchange Chromatography (AEX) for Color Minimization
(A) AEX was Employed to Reduce Color Formation
AEX chromatography was performed to remove the coloration obtained during production of aflibercept expressed using CDM1.
2.1 Design
Five AEX separations were performed for this study as detailed in Table 2-1 with the AEX protocol as described in Table 2-2. A 15.7 mL Q Sepharose Fast Flow column (19.5 cm bed height, 1.0 cm I.D.) and a 14.1 mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.) were integrated into an AKTA Avant benchtop liquid chromatography controller.
AEX load pH was adjusted to target ±0.05 pH units using 2 M Tris base or 2 M acetic acid. AEX load conductivity was adjusted to target ±0.1 mS/cm using 5 M sodium chloride or deionized water. All pool samples were analyzed for high molecular weight (HMW), color and yield.
TABLE 2-1
Summary of the Study Design for AEX Color Reduction
AEX Separation
Condition Evaluated
Resin
1
pH 8.30-8.50, 1.90-2.10 mS/cm
POROS 50 HQ
2
pH 7.90-8.10, 2.40-2.60 mS/cm
Q Sepharose FF
3
pH 7.90-8.10, 2.40-2.60 mS/cm
POROS 50 HQ
4
pH 7.70-7.90, 3.90-4.10 mS/cm
Q Sepharose FF
5
pH 7.70-7.90, 3.90-4.10 mS/cm
POROS 50 HQ
TABLE 2-2
AEX Protocol for Color Reduction
Column
Linear
Volumes
Velocity
Step
Description
Mobile Phase
(CVs)
(cm/h)
1
Pre-
2M Sodium Chloride (NaCl)
2
200
Equilibration
2
Equilibration
50 mM Tris, Variable mM
2
200
NaCl Variable pH and
Conductivity
3
Load
AEX Load
40 g/L-
200
Variable pH and Conductivity
resin
4
FT/Wash
50 mM Tris, Variable mM
2
200
NaCl Variable pH and
Conductivity
5
Strip 1
2M Sodium Chloride (NaCl)
2
200
6
Strip 2
1N Sodium Hydroxide (NaOH)
2
200
2.2 Results
Employing AEX separations for production exhibited a significant reduction in color. (Table 2-3). For example, as seen in Table 2-3, the color observed in the flowthrough (FT) and wash in AEX separation 1 (pH 8.30-8.50, 1.90-2.10 mS/cm) had a b* value of 1.05, as compared to the color of the Load for AEX (“AEX Load”) with a b* value of 3.06. The increase in b* value reflects the intensity of yellow-brown coloration of a sample.
Five AEX separations were performed to evaluate the impact of resin (Q Sepharose FF or POROS 50 HQ) and pH and conductivity setpoint (pH 8.40 and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm, or pH 7.80 and 4.00 mS/cm) on color reduction. For POROS 50 HQ, yields (64.4, 81.9, and 91.4%) and pool HMW levels (1.02, 1.29, and 1.83%) increased as the setpoint was changed to a lower pH and higher conductivity. Color (b* values) also increased (1.05, 1.33, and 1.55) as the setpoint was changed to a lower pH and higher conductivity. The higher pH levels and lower conductivities provided the most reduction in color over the AEX separation for POROS 50 HQ.
For Q Sepharose Fast Flow, yields (49.5 and 77.7%) and pool BMW levels (0.59 and 1.25%) also increased as the setpoint was changed to a lower pH and higher conductivity. Color (b* values) also increased (0.96 and 1.35) as the setpoint was changed to a lower pH and higher conductivity.
The use of AEX reduces yellow-brown coloration—see Table 2-3. Additionally, it was determined that Q Sepharose Fast Flow reduced color more than POROS 50 HQ for the two set points evaluated on both resins. At pH 8.00 and 2.50 mS/cm setpoint, POROS 50 HQ pool had a b* value of 1.33 while Q Sepharose Fast Flow pool had a b* value of 0.96. Similarly, at pH 7.80 and 4.00 mS/cm setpoint, POROS 50 HQ pool had a b* value of 1.55 while Q Sepharose Fast Flow pool had a b* value of 1.35 (Table 2-3).
TABLE 2-3
Summary of Experimental Results of the
AEX Color Reduction Study
AEX
Yield
HMW
Color
Color
Color
Separation
Fraction
(%)
(%)
(L*)
(a*)
(b*)
1
FT/wash
64.4
1.02
98.89
0.01
1.05
2
FT/wash
49.5
0.59
98.30
−0.03
0.96
3
FT/wash
81.9
1.29
99.07
−0.07
1.33
4
FT/wash
77.7
1.25
99.42
−0.04
1.35
5
FT/wash
91.4
1.83
99.19
−0.09
1.55
—
filtered pool
—
3.66-3.98
98.73
−0.21
3.06
(AEX Load)
AEX, anion exchange chromatography;
HMW, high molecular weight species;
N/A, not applicable
The fractions were adjusted to a protein concentration of 10 g/L for color measurements.
2.3 Conclusion
Use of AEX was found to reduce the yellow-brown coloration, see Table 2-3. Referring to Table 2-3, the AEX Load has a b* value of 3.06, but when subjected to AEX chromatography (AEX Separation 1-5), the b* value decreases indicating a decrease in yellow-brown coloration. Again, as the b* value decreases so does the coloration; as the b* value increases it is reflective of the yellow-brown color increasing in a given sample.
Color reduction was evaluated using two AEX resins (POROS 50 HQ and Q Sepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm, and pH 7.80 and 4.00 mS/cm). For both resins, color reduction was higher for the higher pH and lower conductivity set points. In addition, Q Sepharose Fast Flow provided more color reduction than POROS 50 HQ at the two set points evaluated on both resins (pH 8.00 and 2.50 mS/cm and pH 7.80 and 4.00 mS/cm). However, all the five AEX separation methods led to a significant color reduction when compared to the loading solution for AEX (“AEX Load”), demonstrating the importance of AEX production in the process of aflibercept production expressed using a CDM. The initial b* value of the AEX Load (at a concentration of 10 g/L) may range from about 0.5 to about 30, more particularly from about 1.0 to about 25.0, and even more particularly from about 2.0 to about 20.0. Following use of AEX, the b* value for the flowthrough (at a concentration of 10 g/L) may range from 0.5 to about 10.0, more particularly from about 0.5 to about 7.0, and even more particularly from about 0.5 to about 5.0.
2.4 Peptide Mapping
Sample preparation. Tryptic mapping of reduced and alkylated aflibercept samples obtained from AEX Load and flowthrough of the above experiment (Table 2-3) were performed to identify and quantify post-translational modification (PTM). An aliquot of each sample (Load and flowthrough) was denatured using 8.0 M Urea, 0.1 M Tris-HCl, pH 7.5, reduced with DTT and then alkylated with iodoacetamide. The denatured, reduced and alkylated sample was first digested with recombinant Lys-C(rLys-C) at an enzyme to substrate ratio of 1:100 (w/w) at 37° C. for 30 minutes, diluted with 0.1 M Tris-HCl, pH 7.5 such that a final urea concentration was 1.8 M, subsequently digested with trypsin at an enzyme to substance ratio of 1:20 (w/w) at 37° C. for 2 hours and then deglycosylated with PNGase F at an enzyme substrate ratio of 1:5 (w/w) for 37° C. for 1 hour. The digestion was stopped by bringing the pH below 2.0 using formic acid (FA).
LC-MS analysis. A 20 μg aliquot of resulting rLys-C/tryptic peptides from each sample was separated and analyzed by reverse-phase ultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLC CSH C18 column (130 Å, 1.7 μm, 2.1×150 mm) followed by on-line PDA detection (at wavelengths of 280 nm, 320 nm and 350 nm) and mass spectrometry analysis. Mobile phase A was 0.1% FA in water, and mobile phase B was 0.1% FA in acetonitrile. After sample injection, a gradient was initiated with a 5 minute hold at 0.1% B followed by a linear increase to 35% B over 75 minutes for optimum peptide separation. MS and MS/MS experiments were conducted using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with higher-energy collisional dissociation (HCD) employed for peptide fragmentation for MS/MS experiments. Peptide identity assignments were based on the experimentally determined accurate mass of a given peptide in the full MS spectrum as well as the b and y fragment ions in the corresponding HCD MS/MS spectrum. Extracted ion chromatograms of the peptides from the Load and flowthrough were generated (see FIG. 10). As seen in the extracted ion chromatogram in FIG. 10, the peptide fragments identified in “AEX Load” and “AEX FT/wash” from AEX separations 1-5 (as identified in Table 2-3) are shown. The relative abundance of some of these peptides identified in FIG. 10 from the peptide mapping analysis are shown in FIG. 11.
Referring to FIG. 11, this figure identifies various peptide fragments analyzed and their relative levels of oxidation. In particular, the third column identifies the amino acid residues (“Peptide Sequence”) of peptide fragments that were isolated and analyzed. Each Peptide Sequence has an amino acid residue that is underscored. The underscored amino acid residue identifies the amino acid in the Peptide Sequence that is oxidized. The oxidized amino acids correspond to either histidine (H) oxidation or tryptophan (W) oxidation. There is also depicted in this figure rows to the right of each of the Peptide Sequences showing the abundance of oxidized species. This shading in the rows indicates differences in the relative amount of oxidized residues in a particular sample using different AEX separations identified in the respective column headings. For example, referring to the second peptide, EIGLLTCEATVNGHLYK (SEQ ID NO.: 18) in FIG. 11, when read across along in a horizontal manner, the relative total population of this peptide in a particular sample (“aflibercept AEX Load”) that is oxidized is approximately 0.013% oxidized. As progression is made across the same row, there is a shift in the shading, indicating a change in the relative abundance of oxidized species. For example, using this same Peptide Sequence, the relative abundance of oxidized species for AEX separation are 0.006% to 0.010% when following different AEX separation protocols. Thus, it can be appreciated that AEX chromatography decreases the abundance of oxidized species.
(B) AEX was Employed to Reduce the Color Formation in MiniTrap Production
AEX chromatography was performed to remove the coloration obtained during production of MT1 which was obtained on performing cleavage of full-length aflibercept expressed using CDM1.
2.5 Design
Four AEX separations were performed for this study as described in Table 2-4. The AEX Load was obtained from a filtration sample of MT1 (“MT1 filtered pool”). A 15.7 mL Capto Q column (20.0 cm bed height, 1.0 cm I.D.), a 14.1 mL POROS 50 HQ column (18.0 cm bed height, 1.0 cm I.D.), and a 16.5 mL Q Sepharose FF column (21.0 cm bed height, 1.0 cm I.D.) were integrated into an AKTA Avant benchtop liquid chromatography controller for this experiment.
AEX load pH was adjusted to target ±0.05 pH units using 2 M tris base or 2 M acetic acid. AEX load conductivity was adjusted to target ±0.1 mS/cm using 5 M sodium chloride or deionized water. All pool samples were analyzed for HMW, color and yield.
TABLE 2-4
Summary of the Study Design for the AEX Color Reduction Study
AEX Separation
Resin
AEX Protocol
1
Capto Q
Table 2-6
2
POROS 50 HQ
Table 2-6
3
Q Sepharose FF
Table 2-6
4
POROS 50 HQ
Table 2-5
TABLE 2-5
Flowthrough AEX Protocol Used for the Color Reduction Study
Column
Linear
Volumes
Velocity
Step
Description
Mobile Phase
(CVs)
(cm/h)
1
Pre-
2M Sodium Chloride (NaCl)
2
200
Equilibration
2
Equilibration
50 mM Tris, 40 mM NaCl
2
200
pH 7.90-8.10, 6.50-7.50 mS/cm
3
Load
AEX Load
30 g/L-
200
pH 7.90-8.10, 6.50-7.50 mS/cm
resin
4
Wash
50 mM Tris, 40 mM NaCl
2
200
pH 7.90-8.10, 6.50-7.50 mS/cm
5
Strip 1
2M Sodium Chloride (NaCl)
2
200
6
Strip 2
1N Sodium Hydroxide (NaOH)
2
200
AEX, anion exchange chromatography; CV, column volume
TABLE 2-6
Bind and Elute AEX Protocol Used for the Color Reduction Study
Column
Linear
Volumes
Velocity
Step
Description
Mobile Phase
(CVs)
(cm/h)
1
Pre-
2M Sodium Chloride (NaCl)
2
200
Equil-
ibration
2
Equil-
50 mM Tris
2
200
ibration
pH 8.30-8.50, 1.90-2.10
mS/cm
3
Load
AEX Load
30 g/L-
200
pH 8.30-8.50, 1.90-2.10
resin
mS/cm
4
Wash
50 mM Tris
2
200
pH 8.30-8.50, 1.90-2.10
mS/cm
5
Elution
50 mM Tris, 70 mM NaCl
2
200
pH 8.30-8.50, 8.50-9.50
mS/cm
6
Strip 1
2M Sodium Chloride
2
200
(NaCl)
7
Strip 2
1 N Sodium Hydroxide
2
200
(NaOH)
AEX, anion exchange chromatography; CV, column volume
2.6 Results
All four AEX separations led to reduction in color as seen for coloration of the flowthrough and wash for AEX separations 1-4 (Table 2-7). While the first three AEX separations were evaluated in a bind and elute mode (Table 2-6), it was observed that the majority of the product was present in the load and wash blocks (62%-94%).
The first three separations evaluated the pH 8.4 and 2.0 mS/cm setpoint for Capto Q, POROS 50 HQ, and Q Sepharose FF resins. All three separations had a good yield (>80%). The POROS 50 HQ AEX pool showed the lowest yellow color in AEX pool (b* value of 2.09) followed by the Q Sepharose FF AEX pool (b* value of 2.22) and the Capto Q AEX pool (b* value of 2.55).
TABLE 2-7
Summary of Experimental Results of the AEX Color Reduction Study
AEX
Yield
HMW
Color
Color
Color
Separation
Fraction
(%)
(%)
(L*)
(a*)
(b*)
1
FT/wash
90.7
0.49
99.11
−0.27
2.55
2
FT/wash
93.8
0.33
99.20
−0.28
2.09
3
FT/wash
86.7
0.23
98.88
−0.23
2.22
4
FT/wash
99.5
1.13
98.90
−0.39
3.40
—
MT1 Filtered Pool
—
0.65
98.18
−0.37
4.17
(AEX Load)
AEX, anion exchange chromatography;
HMW, high molecular weight species.
The fractions were adjusted to a protein concentration of 5 g/L for color measurements.
2.7 Conclusion
As seen for aflibercept (see Section 2.3 above), use of AEX was found to reduce yellow-brown coloration (Table 2-7) for MiniTrap production. Referring to Table 2-7, the AEX Load has a b* value of 4.17, but when subjected to AEX chromatography (AEX Separation 1-4), the b* value decreases indicating a decrease in yellow-brown coloration. Again, as the b* value decreases so too does the coloration. The initial b* value of the AEX Load (at a concentration of 5 g/L) may range from about 0.5 to about 25, more particularly from about 1.0 to about 20.0, and even more particularly from about 1.5 to about 15.0. Following use of AEX, the b* value of the flowthrough (at a concentration of 5 g/L) may range from 0.5 to about 10.0, more particularly from about 0.5 to about 7.0, and even more particularly from about 0.5 to about 5.0.
Example 3. Oxidized Peptide Study
3.1 Peptide Mappings
Sample Preparation. Tryptic mapping of reduced and alkylated MiniTrap (MT1) and MT4 (MiniTrap similar to MT1 using a different full-length aflibercept one produced using soy hydrolysate cell culture) samples were performed to identify and quantify post-translational modification. An aliquot of sample was denatured using 8.0 M Urea in 0.1 M Tris-HCl, pH 7.5, reduced with DTT and then alkylated with iodoacetamide. The denatured, reduced and alkylated drug substance was first digested with recombinant Lys-C(rLys-C) at an enzyme to substrate ratio of 1:100 (w/w) at 37° C. for 30 minutes, diluted with 0.1 M Tris-HCl, pH 7.5 such that the final urea concentration was 1.8 M, subsequently digested with trypsin at an enzyme to substance ratio of 1:20 (w/w) at 37° C. for 2 hours and then deglycosylated with PNGase F at an enzyme substrate ratio of 1:5 (w/w) for 37° C. for 1 hour. The digestion was stopped by bringing the pH below 2.0 using formic acid (FA).
LC-MS Analysis. A 20 μg aliquot of resulting rLys-C/tryptic peptides from each sample was separated and analyzed by reverse-phase ultra-performance liquid chromatography (UPLC) using Waters ACQUITY UPLC CSH C18 column (130 Å, 1.7 μm, 2.1×150 mm) followed by on-line PDA detection (at wavelengths of 280 nm, 320 nm and 350 nm) and mass spectrometry analysis. Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. After sample injection, a gradient started with a 5 min hold at 0.1% B followed by a linear increase to 35% B over 75 minutes for optimum peptide separation. MS and MS/MS experiments were conducted on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with higher-energy collisional dissociation (HCD) employed for peptide fragmentation for MS/MS experiments. Peptide identity assignments were based on the experimentally determined accurate mass of a given peptide in the full MS spectrum as well as the b and y fragment ions in the corresponding HCD MS/MS spectrum. Extracted ion chromatograms of oxidized peptides and corresponding native peptide were generated with the peak areas integrated to calculate the site-specific percentage of oxidized amino acid residue(s) within the MT1 sample.
Peptide Fragments Linked to Increased Absorbance at 350 nm
The PTMs on MT1 were observed upon comparing the tryptic peptide maps for MT1 and MT4 (FIG. 12A shows the absorbance of peptides eluted from 20.0 to 75 minutes). The peptides with varying UV peaks are highlighted. The expanded view of the chromatogram is shown in FIG. 12B which shows the absorbance of peptides eluted from 16 to 30 minutes. The peptides with sharp contrast in UV absorbance between MT1 and MT4 were TNYLTH*R (SEQ ID NO.: 21), IIW(+4)DSR (SEQ ID NO.: 28) and IIIW(+132)DSR (SEQ ID NO.: 124) (* or underscoring represents oxidation of the residue). Further, the expanded view of the chromatogram is shown in FIG. 12C, which shows the absorbance of peptides eluted from 30 to 75 minutes. The peptides with sharp contrast in UV absorbance between MT1 and MT4 were DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), EIGLLTCEATVNGH*LYK (SEQ ID NO.: 18) and QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) (* represents oxidation of the residue). The peptide mapping revealed identity of peptides that are significantly different in abundance between the VEGF MiniTraps. The relative abundance of the peptides identified from the peptide mapping analysis is shown in Table 3-1. The amount of 2-oxo-histidines in MT1 (produced in a CDM) were higher than MT4 (produced in soy hydrolysate), suggesting that the media used to express aflibercept can have a significant effect on the relative abundance of peptides with oxidized histidines or oxidized tryptophans. For example, for the peptide QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), the percent relative abundance of the peptide in MT1 (CDM produced) was 0.015% compared to percent relative abundance of the peptide in MT4 (soy hydrolysate produced; which is about 15-fold less as compared to MT1).
TABLE 3-1
Peptide Modified
Fold change
Peptide
Sequence
MT1
MT4
MT1/MT4
EIGLLTCEATVNGH
EIGLLTC[+57]EATVN
0.011%
0.004%
2.75
LYK (SEQ ID NO.:
GH[+14]LYK (SEQ ID
57)
NO.: 18)
QTNTIIDVVLSPSH
QTNTIIDVVLSPSH[+14]
0.015%
0.001%
15.00
GIELSVGEK (SEQ
GIELSVGEK (SEQ
ID NO.: 58)
ID NO.: 19)
TELNVGIDFNWEYP
TELNVGIDFNWEYPS
0.204%
0.026%
7.85
SSKHQHK (SEQ ID
SKH[+14]QHK (SEQ ID
NO.: 59)
NO.: 20)
DKTHTCPPCPAPEL
DKTH[+14]TC[+57]PP
0.115%
0.018%
6.39
LG (SEQ ID NO.: 60)
C[+57]PAPELLG (SEQ
ID NO.: 17)
TNYLTHR (SEQ ID
TNYLTH[+14]R (SEQ
0.130%
0.020%
6.50
NO.: 61)
ID NO.: 21)
Color and 2-oxo-Histidine Quantitation. The percentage of 2-oxo-histidines in the oligopeptides that were generated by protease digestion, as measured by mass spectrometry, are also shown (Table 3-2). (Values were normalized against unmodified peptides.) Table 3-2 (I) shows the percent of oxidized histidines/tryptophans observed for AEX flowthrough: MT1 lot 1, AEX flowthrough for MT1 lot 2, and AEX flowthrough for MT1 lot 3. Table 3-2 (II) shows the percent of oxidized histidines/tryptophans observed for acidic fraction 1, acidic fraction 2, and main fraction obtained on performing CEX separation for MT1 lot 3. From this Table, it is clear that the acidic variants are comprised of oxidized species. From Table 3-2(I), it is clear that the % of 2-oxo-histidines and tryptophan dioxidation comprising peptides/protein is reduced in the AEX flowthrough compared to the AEX Strip. It is evident that stripping the AEX column enriches for the percentage of such modified peptides. For example, the % of the modified peptide “EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18)” in the AEX Flowthrough (MT1 lot 1) was 0.013% and in the “AEX Strip” was 0.080%. This also corroborates that the AEX column captures modified peptides, thus reducing the percentage of modified peptides in the AEX flowthrough.
TABLE 3-2 I
Percentage of 2-oxo-Histidines/Tryptophans
AEX Flowthrough
AEX Strip
MT1 lot 1
MT1 lot 2
MT1 lot 3
Intense
BY1, 110
≤BY3, 110
≤BY3, 110
Modified Peptides
yellow
mg/mL
mg/mL
mg/mL
EIGLLTC[+57]EATVNGH[+14]LYK
0.080%
0.013%
0.008%
0.006%
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK
0.054%
0.028%
0.023%
0.019%
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK
0.235%
0.085%
0.049%
0.049%
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57]PAPELLG
0.544%
0.092%
0.077%
0.057%
(SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21)
0.089%
0.022%
0.011%
0.010%
IIW[+32]DSR (SEQ ID NO.: 28)
0.738%
0.252%
0.198%
0.298%
TABLE 3-2 II
Percentage of 2-oxo-Histidines/Tryptophans
CEX flowthrough
Acidic fraction 1
Acidic fraction 2
Main fraction
from MT1 lot 3
from MT1 lot 3
from MT1 lot 3
Modified Peptides
Yellow
Yellow
No Color
EIGLLTC[+57]EATVNGH[+14]LYK
0.009%
0.008%
0.004%
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK
0.013%
0.015%
0.006%
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK
0.131%
0.151%
0.049%
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57]PAPELLG
0.117%
0.132%
0.068%
(SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21)
0.014%
0.008%
0.008%
IIW[+32]DSR (SEQ ID NO.: 28)
0.458%
0.269%
0.185%
In Table 3-2(11), [+57] represent alkylation of cysteine by iodoacetamide, which adds a carboxymethyl amine moiety on the cysteine, which is a net mass increase of about +57 over unmodified cysteine:
In Table 3-2(11), [+14] represent conversion from His to 2-oxo-His. One oxygen atom is added on carbon 2, but two hydrogen atoms are lost (one from Carbon 2, the other from nitrogen 3), which is a net mass increase of about +14 over unmodified histidine.
In Table 3-2(11), [+32] represents tryptophan dioxidation resulting in the formation of N-formylkynurenine, which is a net mass increase of about +32 over unmodified tryptophan (FIG. 4).
A second set of experiments were performed to evaluate the percentage of 2-oxo-histidines (and tryptophan dioxidation) in oligopeptides from protease digested FabRICATOR-cleaved aflibercept (MT4) which was processed by AEX chromatography (FIG. 13 and Table 3-3 below). The percent of 2-oxo-histidines and tryptophan dioxidation in AEX strip for oligopeptides from protease digested FabRICATOR-cleaved aflibercept (MT4) was significantly more than the percent of 2-oxo-histidines and tryptophan dioxidation in the AEX flowthrough (referring to “MT1” in Table 3-3 below).
TABLE 3-3
Percentage of 2-oxo-Histidines
fold change
Full length
AEX Strip
AEX Strip/
Modified peptides
aflibercept
MT1
from MT1
MT1
IIW[+32]DSR (SEQ ID NO.: 28)
0.22%
0.34%
0.81%
2.4
EIGLLTC[+57]EATVNGH[+14]LYK
0.00%
0.02%
0.08%
4.0
(SEQ ID NO.: 18)
QTNTIIDVVLSPSH[+14]GIELSVGEK
0.01%
0.04%
0.07%
1.8
(SEQ ID NO.: 19)
TELNVGIDFNWEYPSSKH[+14]QHK
0.01%
0.19%
0.42%
2.2
(SEQ ID NO.: 20)
DKTH[+14]TC[+57]PPC[+57]PAPELLG
0.01%a
0.11%
0.63%
5.7
(SEQ ID NO.: 17)
TNYLTH[+14]R (SEQ ID NO.: 21)
0.00%
0.03%
0.10%
3.3
avalue calculated using a different peptide for full-length aflibercept, as the C-terminal peptide is different from MiniTrap.
The percent of 2-oxo-histidines and tryptophan dioxidation in AEX strip was significantly more than the percent of 2-oxo-histidines and tryptophan dioxidation in the AEX flowthroughs during the MT1 productions (referring to “MT1” in Table 3-3 above). Compared to Table 3-2, Table 3-3 shows similar results that stripping the AEX column produced a sample with a significantly higher percent of 2-oxo-histidines and tryptophan dioxidation compared to the percent of 2-oxo-histidines and tryptophan dioxidation in AEX flowthrough, suggesting that the 2-oxo-histidines and tryptophan dioxidation species are bound to the AEX column during the separation and are removed upon stripping the AEX column. This is further evident in the extracted ion chromatogram as seen in FIG. 14.
Strong Cation Exchange Chromatogram (CEX)
A series of experiments were conducted in order to identify acidic species and other variants present in samples comprising anti-VEGF proteins.
Strong cation exchange chromatography was performed using a MonoS (10/100) GL column (GE Life Sciences, Marlborough, Mass.). For the sample separations, the mobile phases used were 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 (Mobile phase A) and 40 mM sodium phosphate, 100 mM sodium chloride pH 9.0 (Mobile phase B). A non-linear pH gradient was used to elute charge variants of MT1 with detection at 280 nm. Peaks that elute at a relative residence time earlier than the main peak are designated herein as acidic species.
A sample from the MT1 lot 2 (≤BY3), prior to any enrichment, was subjected to CEX using the method as depicted in FIG. 15. Desialylation was applied to the sample in order to reduce the complexity of variants of MT1. This was followed by preparative SEC processing (Superdex 200 prep grade XK26/100) using 1×DPBS, pH 7.2±0.2, as the mobile phase. The fractions obtained from the preparative SEC column comprising desialylated MiniTrap (dsMT1) were combined and further subjected to strong cation exchange (SCX) chromatography to enrich for charge variants of MT1 using a dual salt-pH gradient. The procedure resulted in a total of 7 fractions (F1-F7; MC represents the method control, FIG. 16 and FIG. 17).
On performing CEX, the acidic species elute earlier than the main peaks and basic species elute after the main peaks. As observed in FIG. 17, peaks 3-5 are the main peaks. Peaks 1 and 2 are eluted before elution of the main species of MT1 (peaks 3-5), and thus, comprise the acidic species. Peak 6 is eluted after the elution of the main species of MT1 (peaks 3-5), and thus, comprises the basic species. Table 3-4 shows the relative abundance of the peaks in MC (as identified in FIG. 16). For example, row two of Table 3-4 (labeled MC) shows that the total relative amount of acidic species in MC is about 19.8% (i.e., peak 1+peak 2). Table-3-4 also shows the relative abundance of the peaks for each individual fraction. While there are overlapping species in the different fractions (as reflected in FIG. 16 and FIG. 17), the majority of fractions F1 and F2 are acidic species (i.e., peak 1 and peak 2). For example, fraction F1 is comprised of 63.7% peak 1 and 19.2% peak 2 (for a total of 82.9% acidic species). Fraction F2 is comprised of 9.6% peak 1 and 75.9% peak 2 (for a total of 85.5% acidic species). The majority of fractions F3-F5 are the main species of MT1 (peaks 3-5). Lastly, the majority of fractions F6-F7 are the basic species (peak 6) but do include some portions of the main species (e.g., peaks 4 and 5).
It was also observed that fractions F1 and F2 (which comprises the acidic species) had an intense yellow-brown coloration compared to the fractions F3-F5 (which comprises the main species or “MT1”). All the fractions were inspected for color at concentrations ≥13 mg/mL. As evident from this Example, the presence of acidic species in the sample tracked with the appearance of yellow-brown coloration, removal (or minimization) of which can be accomplished by removing (or minimizing) the acidic species from MT1.
TABLE 3-4
Relative abundance of peaks based on analytical CEX
Peak Area (%)
Sample
Peak 1
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
MC
5.9
13.9
15.0
25.4
20.9
19.0
MT1 F1
63.7
19.2
17.1
ND
ND
ND
MT1 F2
9.6
75.9
10.6
2.2
1.6
ND
MT1 F3
ND
5.0
57.2
37.8
ND
ND
MT1 F4
ND
ND
16.3
56.3
27.4
ND
MT1 F5
ND
ND
ND
33.1
50.4
16.5
MT1 F6
ND
ND
ND
16.0
27.7
56.3
MT1 F7
2.8
7.7
8.0
16.1
16.5
48.9
ND: Not Detected
The 3D chromatograms for MT1 lot 2 and fractions F1-F7 are shown in FIGS. 18A-H. MT1 lot 2 did not exhibit any significant spectral features (FIG. 18A). Fractions 1 and 2 (comprising the acidic species) exhibited a spectral signature between 320-360 nm (see the circle in FIG. 18B). This feature was more prominent in fraction 1 compared to fraction 2 (FIGS. 18B and 18C) and was absent in fraction 3 and fractions 4-7 (main species, MT1) (FIGS. 18D and 18H), which did not exhibit yellow-brown coloration.
Thus, as observed above, CEX led to identification of acidic species/acidic fractions (fractions 1 and 2) which show an intense yellow-brown coloration as compared to the main species/fractions (fractions 3-6). This result was also observed in the form of a distinct spectral signature present in the 3D chromatograms of fractions F1-F2 and absent in fractions F3-F7.
Imaged Capillary Isoelectric Focusing (icIEF) Electropherograms
The distribution of variants in fractions F1-F7 and MC (from MT1—lot 2 after CEX) was further assessed by icIEF (FIG. 19).
The distribution of variants in fractions F1-F7 and MC (from MT1—lot 2 after CEX) was further assessed by icIEF using an iCE3 analyzer (ProteinSimple) with a fluorocarbon coated capillary cartridge (100 μm×5 cm). The ampholyte solution consisted of a mixture of 0.35% methyl cellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholytes, 4.2% Pharmalyte 8-10.5 carrier ampholytes, and 0.2% p1 marker 7.40 and 0.15% p1 marker 9.77 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted in purified water and sialidase A was added to each diluted sample at an enzyme to substrate ratio of 1:200 (units of sialidase A per milligram of MT1) followed by incubation at ambient temperature for approximately 16 hours. The sialidase A treated samples were mixed with the ampholyte solution and then focused by introducing a potential of 1500 V for one minute followed by a potential of 3000 V for 7 minutes. An image of the focused MT1 variants was obtained by passing 280 nm ultraviolet light through the capillary and into the lens of a charge coupled device digital camera. This image was then analyzed to determine the distribution of the various charge variants (FIG. 19). Referring to FIG. 19, fractions F1 and F2 (or the acidic fractions) showed an absence of the peak for MT1, which is clearly observed for MC and fractions F3-F7 (main species, MT1). Thus, icIEF electropherograms were considered able to detect and determine the distribution of the different charge variants of the protein under consideration, MT1 in this case. Thus, it was evident that acidic fractions on performing CEX analysis showed (a) increased relative abundance of percent of 2-oxo-histidine or dioxo-tryptophan (Table 3-2 (II)); (b) increased yellow-brown coloration (data not shown); and (c) presence of a spectral signature as seen on the 3D chromatograms for fractions 1 and 2 (FIG. 18B and FIG. 18C).
Example 4. Photo-Induction Study
In this Example, photo-induction of VEGF MiniTrap (MT), for example MT1, was performed by exposure of a protein sample to varying amounts of cool white (CW) fluorescent light or ultra-violet A (UVA) light. The color and oxidized amino acid content of the light exposed samples was determined. LCMS analysis was performed following exposure, as explained above. Exposure of MT to cool white light or UVA light produced an increase in oxidized amino acid residues, for example, histidine (Table 4-1, Table 4-2 and Table 4-3).
TABLE 4-1
Photo-Induction Study Design
Cumulative
0.2
0.5
0.8
1.0
2.0
Exposure
(xICH)
(xICH)
(xICH)
(xICH)H
(xICH)
CW fluorescent
0.24
0.6
0.96
1.2
2.4
exposure
million
million
million
million
million
(lux*hr)
lux*hr
lux*hr
lux*hr
lux*hr
lux*hr
Incubation time
30
75
100
150
300
with CW
hours
hours
hours
hours
hours
fluorescent light
(at 8 klux)
UVA exposure
40
100
160
200
400
(W*hr/m2)
Incubation time
4
10
16
20
40
with UVA
hours
hours
hours
hours
hours
(at 10 W/m2)
ICH refers to ICH Harmonised Tripartite Guideline: Stability Testing: Photostability Testing of New Drug Substances and Products Q1B which specifies photostability studies to be conducted with not less than 1.2 million lux*hours cool white fluorescent light and near ultraviolet energy of not less than 200 W*hr/m2.
Table 4-2 depicts the increase in coloration of the MT sample exposed to cool-white light and ultra-violet light. For example, b-value for sample (t=0) was 9.58. On exposing this sample to cool-white light at 2.4 million lux*hr, the b-value increases to 22.14. This increase in b-value indicates that the exposure of MT to cool-white light at 2.4 million lux*hr increases yellow-brown coloration of the sample as compared to sample (t=0). Similarly, on exposing MT sample (t=0) to ultra-violet light at 400 W*h/m2, the b-value increases to 10.72 from 9.58. This increase in b-value indicates that the exposure of MT sample to ultra-violet light at 400 W*h/m2 produces an increased yellow-brown coloration of the sample as compared to sample (t=0).
TABLE 4-2
Color of Samples Exposed to Cool White Light and Ultra-Violet Light
Photo exposure xICH
(lux*hr)
L*
a*
b*
BY Value
Cool White Light
T = 0
97.37
−1.12
9.58
4.0
0.2x (0.24 million lux*hr)
96.46
−0.72
11.75
3.7
0.5x (0.6 million lux*hr)
95.47
−0.4
11.3
3.7
0.8x (0.96 million lux*hr)
95.33
−0.38
11.96
3.6
1.0x (1.2 million lux*hr)
94.42
−0.2
13.72
3.3
2.0x (2.4 million lux*hr)
92.70
0.41
22.14
2.0
UVA
0.2x (40 W*h/m2)
97.26
−0.92
12.66
3.5
0.5x (100 W*h/m2)
100.39
−1.01
11.83
3.7
0.8x (160 W*h/m2)
79.69
−0.18
10.1
3.6
1.0x (200 W*h/m2)
97.48
−0.95
11.36
3.7
2.0x (400 W*h/m2)
97.76
−0.98
10.72
3.8
Sample colors are indicated using the CIELAB color space (L*, a* and b* variables) and relative to the EP BY color standard;
L* = white to black (L* is lightness);
a* = magenta to aqua;
b* = yellow to blue, the higher the b-value the more yellow.
TABLE 4-3 (I)
2-oxo-His Levels in Peptides from Ultra-Violet Light Stressed MiniTrap
Peptides
Site
t0
UV_4 h
UV_10 h
UV_16 h
UV_20 h
UV_40 h
DKTHTCPPCPAPEL
H209
0.056%
0.067%
0.081%
0.088%
0.077%
0.091%
LG (SEQ ID NO.: 17)
EIGLLTCEATVNGH
H86
0.010%
0.020%
0.034%
0.037%
0.033%
0.035%
LYK (SEQ ID NO.:
18)
QTNTIIDVVLSPSH
H110
0.024%
0.031%
0.028%
0.028%
0.027%
0.027%
GIELSVGEK (SEQ
ID NO.: 19)
TELNVGIDFNWEYP
H145
0.096%
0.147%
0.163%
0.173%
0.147%
0.125%
SSKHQHK (SEQ ID
NO.: 20)
TNYLTHR (SEQ ID
H95
0.014%
0.032%
0.044%
0.056%
0.058%
0.078%
SDTGRPFVEMYSEI
PEIIHMTEGR (SEQ
H19
0.007%
0.013%
0.021%
0.025%
0.024%
0.034%
ID NO.: 22)
VHEKDK (SEQ ID
H203
0.040%
0.105%
0.238%
0.255%
0.269%
0.324%
ID NO.: 23)
TABLE 4-3 (II)
2-oxo-His Levels in Peptides from Cool White Light Stressed MiniTrap
CW_
CW_
CW_
CW_
CW_
Peptides
Site
t0
30 h
75 h
100 h
150 h
300 h
DKTHTCPPCPAPELL
H209
0.056%
0.152%
0.220%
0.243%
0.258%
0.399%
G (SEQ ID NO.: 17)
EIGLLTCEATVNGHL
H86
0.010%
0.063%
0.110%
0.132%
0.170%
0.308%
YK (SEQ ID NO.: 18)
QTNTIIDVVLSPSHGI
H110
0.024%
0.085%
0.120%
0.128%
0.148%
0.180%
ELSVGEK (SEQ ID
NO.: 19)
TELNVGIDFNWEYP
H145
0.096%
0.423%
0.585%
0.634%
0.697%
0.748%
SSKHQHK (SEQ ID
NO.: 20)
TNYLTHR (SEQ ID
H95
0.014%
0.103%
0.175%
0.198%
0.267%
0.437%
NO.: 21)
SDTGRPFVEMYSEIP
H19
0.007%
0.025%
0.043%
0.049%
0.058%
0.115%
EIIHMTEGR (SEQ ID
NO.: 22)
VHEKDK (SEQ ID
H203
0.040%
0.426%
0.542%
0.622%
0.702%
1.309%
NO.: 23)
Exposure of aflibercept MT to cool white light or UVA light tracked with the appearance of oxidized histidines (2-oxo-his) (Table 4-3). Referring to Table 4-3, the peptide “SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.: 22)” with oxo-histidine was 0.007% in MT sample (t=0), whereas its abundance increased to 0.324% on exposure to ultra-violet light for 40 hours (Table 4-3(I)) and to 1.309% on exposure to cool-white light for 300 hours (Table 4-3(II)).
Two species of 2-oxo-histidine were observed, a 13.98 Da species (as shown in FIG. 2) and a 15.99 Da species (as shown in FIG. 3), with the 13.98 Da species being predominant in light stressed MiniTrap samples. The 15.99 Da species is known to be a product of a copper metal-catalyzed process (Schoneich, J. Pharm. Biomed Anal., 21:1093-1097 (2000)). Moreover, the 13.98 Da species is a product of a light-driven process (Liu et al., Anal. Chem., 86, 10, 4940-4948 (2014)).
Similar to the increased abundance of oxidized histidines in samples exposed to cool white light and UVA light, exposure of MT to cool white light or UVA light also induced formation of other PTMs (Table 4-4 and Table 4-5).
TABLE 4-4 (I)
Other PTMs in Peptides from Ultra-Violet Light Stressed MiniTrap
UV_
UV_
UV _
UV_
UV_
Peptides
Site
t0
40 h
10 h
16 h
20 h
40 h
Deamidation
EIGLLTCEATVNGHLYK (SEQ
N84
20.8%
21.7%
21.5%
21.5%
22.7%
22.4%
ID NO.: 62)
QTNTIIDVVLSPSHGIELSVGE
N99
5.3%
5.4%
5.5%
5.4%
5.5%
5.6%
K (SEQ ID NO.: 63)
Oxidation
SD T GRPF VEMYSEIPEIIRMTE
M10
4.5%
8.2%
11.1%
13.3%
13.8%
19.3%
GR (SEQ ID NO.: 64)
SDTGRPFVEMYSEIPEIIHMTE
M20
1.1%
2.0%
2.8%
3.4%
3.4%
4.6%
GR (SEQ ID NO.: 65)
TQSGSEMK (SEQ ID NO.: 66)
M163
2.0%
2.7%
4.1%
4.6%
7.9%
8.7%
SDQGLYTCAASSGLMTK
M192
5.4%
8.1%
10.9%
12.1%
12.5%
18.3%
(SEQ ID NO.: 67)
3-deoxygluconasone
SDTGRPFVEMYSEIPEIIRMTE
R5
9.9%
10.0%
9.9%
9.7%
9.8%
9.3%
GR (SEQ ID NO.: 68)
TABLE 4-4 (II)
Other PTMs in Peptides from Cool White Light Stressed MiniTrap
CW_
CW_
CW_
CW _
CW _
Peptides
Site
t0
30 h
75 h
100 h
150 h
300 h
Deamidation
EIGLLTCEATVNGHLYK (SEQ
N84
20.8%
22.0%
22.9%
20.3%
21.8%
21.3%
ID NO.: 62)
QTNTIIDVVLSPSHGIELSVGEK
N99
5.3%
5.6%
5.2%
5.6%
5.5%
5.8%
(SEQ ID NO.: 63)
Oxidation
SDTGRPFVEMYSEIPEIIHMTEG
M10
4.5%
11.7%
17.3%
19.9%
25.1%
39.7%
R (SEQ ID NO.: 64)
SDTGRPFVEMYSEIPEIIHMTEG
M20
1.1%
3.1%
4.3%
5.1%
6.1%
8.2%
R (SEQ ID NO.: 65)
TQSGSEMK (SEQ ID NO.: 66)
M163
2.0%
3.3%
15.7%
11.7%
26.4%
20.5%
SDQGLYTCAASSGLMTK (SEQ
M192
5.4%
10.7%
15.3%
18.7%
22.8%
37.6%
ID NO.: 67)
3-deoxygluconasone
SDTGRPFVEMYSEIPEIIHMTEG
R5
9.9%
9.9%
9.6%
9.3%
9.3%
9.0%
R (SEQ ID NO.: 68)
TABLE 4-5 (I)
Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides from
Ultra-Violet Light Stressed MiniTrap
Mod-
UV_
UV_
UV _
UV_
UV_
Peptides
ification
Site
t0
4 h
10 h
16 h
20 h
40 h
SDQGLYTCAASSGLM
+4
W58
0.016%
0.049%
0.089%
0.119%
0.132%
0.221%
TK (SEQ ID NO.: 67)
+16
0.047%
0.109%
0.177%
0.225%
0.242%
0.514%
+32
0.200%
0.487%
0.415%
0.481%
0.423%
0.498%
+48
0.000%
0.000%
0.000%
0.001%
0.001%
0.001%
TELNVGIDFNWEYPSS
+4
W138
0.435%
0.462%
0.550%
0.557%
0.502%
0.512%
K (SEQ ID NO.: 29)
+16
0.083%
0.100%
0.161%
0.206%
0.239%
0.448%
+32
0.009%
0.018%
0.027%
0.039%
0.044%
0.115%
+48
0.284%
0.278%
0.270%
0.302%
0.343%
0.275%
GFIISNATYK (SEQ ID
+16
Y64
0.032%
0.041%
0.046%
0.053%
0.054%
0.073%
NO.: 69)
KFPLDTLIPDGK (SEQ
+16
F44
0.068%
0.077%
0.087%
0.084%
0.070%
0.096%
ID NO.: 70)
FLSTLTIDGVTR (SEQ
+16
F166
0.066%
0.075%
0.085%
0.089%
0.089%
0.124%
ID NO.: 71)
TABLE 4-5 (II)
Oxidation Levels of Tryptophan/Tyrosine/Phenylalanine in Peptides from
Cool White Light Stressed MiniTrap
Mod-
CW_
CW_
CW_
CW_
CW_
Peptides
ification
Site
t0
30 h
7 5h
100 h
150 h
300 h
SDQGLYTCAASSG
+4
W58
0.016%
0.063%
0.124%
0.161%
0.228%
0.526%
LMTK (SEQ ID NO.:
+16
0.047%
0.129%
0.227%
0.283%
0.377%
0.795%
67)
+32
0.200%
1.601%
2.706%
3.139%
3.925%
6.974%
+48
0.000%
0.001%
0.002%
0.002%
0.003%
0.005%
TELNVGIDFNWEY
+4
W138
0.435%
0.555%
0.481%
0.490%
0.429%
0.522%
PSSK (SEQ ID NO.:
+16
0.083%
0.109%
0.364%
0.251%
0.399%
0.753%
29)
+32
0.009%
0.019%
0.027%
0.033%
0.048%
0.135%
+48
0.284%
0.284%
0.330%
0.308%
0.347%
0.316%
GFIISNATYK (SEQ
+16
Y64
0.032%
0.043%
0.057%
0.063%
0.078%
0.127%
ID NO.: 69)
KFPLDTLIPDGK
+16
F44
0.068%
0.087%
0.072%
0.088%
0.079%
0.144%
(SEQ ID NO.: 70)
FLSTLTIDGVTR
+16
F166
0.066%
0.091%
0.088%
0.101%
0.112%
0.168%
(SEQ ID NO.: 71)
Thus, exposure of MT to cool white light or UVA light tracked with the appearance of oxidized residues (such as histidines/tryptophans (oxo-Trp)). Four species of oxo-trp were observed: +4 Da, +16 Da, +32 Da and +48 Da. The +4 Da species is explained by formation of kynurenine (FIG. 4), whereas the +16 Da, +32 Da and +48 Da are the mono-oxidation, di-oxidation and tri-oxidation of tryptophan residues. Peptide mapping of tryptic digests of MT samples monitored at 320 nm is shown in FIG. 20. The relative presence of oxidized residues comprising peptides can be compared in FIG. 20. For example, for the peptide IIW(+4)DSRK (SEQ ID NO.: 114), a significant difference in its presence can be seen for MT sample at t=0, and MT1 sample exposed to UVA for 40 hour and MT sample exposed to CWL for 300 hours.
Exposure of MT to cool white light or UVA light was also evaluated for the presence of HMW/low molecular weight (LMW) species (Table 4-6).
TABLE 4-6
HMW/LMW Species Were Generated After Extended
UVA and CWL Stress Sample: MT1, 80mg/mL,911 5.8
Light
Dark
Light
Dark
Light
Dark
exposed
control
exposed
control
exposed
control
Samples
samples
Samples
samples
Samples
samples
% HMW
% Native
% LMW
Cumulative UVA exposure (×ICH)
t = 0
2.1
NA
96.7
NA
1.2
NA
0.2 × ICH (40
2.1
2.1
96.7
96.7
1.2
1.3
W * h/m2)
0.5 × ICH (100
11.6
2.2
86.5
96.6
1.9
1.2
W * h/m2)
0. × ICH (160
14.5
2.2
83.4
96.6
2.1
1.2
W * h/m2)
1.0 × ICH (200
15.8
2.2
81.9
96.6
2.3
1.3
W * h/m2)
2.0 × ICH (400
22.7
2.3
74.5
96.7
2.8
1.0
W * h/m2)
Cumulative CWL exposure (×ICH)
0.2 × ICH (0.24
12.1
2.2
86.6
96.6
1.4
1.2
million lux * h)
0.5 × ICH (0.6
20.4
2.3
77.9
96.4
1.6
1.3
million lux * hr)
0.8 × ICH
23.2
2.4
75.1
96.2
1.7
1.4
(0.96 million
lux * hr)
1.0 × ICH (1.2
30.1
2.6
68.1
96.2
1.9
1.3
million lux * hr)
2.0 × ICH (2.4
45.0
2.9
52.6
95.8
2.4
1.4
million lux * hr)
To track the coloration with respect to HMW/LMW species for each sample, analytical size-exclusion chromatography with full-spectrum PDA detection (SEC-PDA) was performed as shown above on all the stressed samples (CWL and UVA). SEC-PDA analysis of CWL-stressed MT reveals significant increases in absorbance at ˜350 nm for all size variants except the LMW species (FIG. 21), whereas SEC-PDA on UVA-stressed MT reveals no increases in absorbance at ˜350 nm (FIG. 22). Unlike CWL-treated stress samples, UVA-treated stress samples did not produce any significantly quantifiable yellow-brown color.
A similar result was obtained after studying absorbance ratios at 320 nm and 280 nm for the samples stressed by UVA and CWL. The A320/A280 ratios, analyzed by either raw intensity or total peak area, tracked with increasing intensity of yellow color in CWL-stressed samples (FIG. 23), whereas the A320/A280 ratios did not track with increasing intensity of yellow color in UVA-stressed samples (FIG. 24). This corroborates the previous observation that MT1 samples subjected to UVA stress does not result in the same yellow-brown color observed following CWL stress.
Example 5. Upstream Methods for Reducing Coloration
5.1 Chemically Defined Medium Incubation Study
The effect of various constituents spiked into fresh chemically defined media (CDM) comprising aflibercept with respect to coloration was examined.
One or more 50 mL vent-capped shaker tubes with 10 mL working volume (fresh CDM1) were incubated for 7 days, taking samples on day 0 and day 7. Aflibercept samples (aflibercept recombinant protein in an aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5 mM sodium citrate and 100 mM sodium chloride) were spiked into shaker tubes at a concentration of 6 g/L.
Components added to reach a cumulative concentration:
Cysteine: 16.6 mM
Iron: 0.23 mM
Copper: 0.0071 mM
Zinc: 0.54 mM
The scaled effect of each constituent added on the b* value (CIE L*, a*, b* color space) is set forth in FIG. 25A and plot of actual b* value against predicted b* value is set forth in FIG. 25B. Addition of cysteine resulted in the largest yellow-brown color increase. Iron and zinc also generated color. Folic acid and B vitamin group (including thiamine, niacinamide, D-pantothenic acid, D-biotin, and pyridoxine) increased the yellow-brown color. Riboflavin and Vitamin B12 did not statistically impact color.
5.2 Effect of Decreasing Cysteine and Metals on b* value
Bioreactors (e.g., 2 L) were inoculated from a seed culture of an aflibercept producing cell line. The inoculated cultures were grown at a temperature of 35.5° C., pH of 7.1±0.25, and air sparge set point of 22 ccm. Glucose, antifoam, and basal feeds were supplemented to the bioreactors as needed. The effect of lowering the concentration of cysteine and of metals on color when aflibercept is expressed was evaluated in CDM1.
Medium at day 0=CDM1, including 1.48 mM of cysteine
Nutrient Feeds:
Day 2=Chemically defined feed (CDF)+1.3-2.1 mM cysteine
Day 4=CDF+1.6-1.7 mM cysteine
Day 6=CDF+1.6-1.7 mM cysteine
Day 8=CDF+1.6-1.7 mM cysteine
The bioreactor conditions were as follows:
Cysteine was added at a cumulative concentration of about 6-7 millimoles per L of culture, 8-9 millimoles per L of culture, or 10-11 millimoles per L of culture.
Metals in CDM1 (0.5x, lx, or 1.5×CDM1 levels) at 1× levels are listed below (where the concentrations are prior to inoculum addition):
Fe=68-83 micromoles per liter of culture
Zn=6-7 micromoles per liter of culture
Cu=0.1-0.2 micromoles per liter of culture
Ni=0.5-1 micromoles per liter of culture
Decreasing cumulative cysteine levels to 6-7 millimoles/L reduced yellow-brown color with no significant impact to titer. Decreasing metal concentrations to 0.5× in the medium reduced color with significant increase in titer. There was a minimal impact to titer, VCC (viable cell concentration), viability, ammonia or osmolality (See FIG. 26A-E). The predicted scale effect of metal content and cysteine on b* value and titer is set forth in FIG. 27.
5.3 Evaluation of the Effect of Antioxidants on b* Value
The effect of the antioxidants taurine, hypotaurine, thioctic acid, glutathione, glycine and vitamin C on color when spiked into spent CDM comprising aflibercept was evaluated. One or more 50 mL vent-capped shaker tubes with 10 mL working volume (CDM1) were incubated for 7 days, taking samples on day 0 and day 7.
The conditions for component additions to spent CDM1 were as follows:
Aflibercept sample (aflibercept recombinant protein in an aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5 mM sodium citrate and 100 mM sodium chloride) spiked into shaker tubes at 6 g/L concentration
Antioxidants added to spent CDM1 at the following concentrations:
Taurine=10 mM of culture
Hypotaurine=10 mM of culture
Glycine=10 mM of culture
Thioctic Acid=0.0024 mM of culture
Glutathione, reduced=2 mM of culture
Hydrocortisone=0.0014 mM of culture
Vitamin C (ascorbic acid)=0.028 mM of culture
Multiple antioxidants decreased color formation in spent medium: a combination of hypotaurine, taurine and glycine; thioctic acid; and vitamin C. Glutathione increased b* value.
TABLE 5-1
Summary of Antioxidant Effect on Color Formation of MiniTrap
Condition
b* value
Spent Medium Day 0
0.37
Spent Medium Day 7 Control
1.47
Spent Medium Day 7 + Antioxidants*
1.02
*Antioxidants that significantly decreased b* value: Hypotaurine/Taurine/Glycine, Thioctic Acid, Vitamin C.
A summary of the predicted effect of various antioxidants on b* value (CIE L*, a*, b* color space) is set forth in FIG. 28 (A-C).
The effect of the further addition to the antioxidants on color, when spiked into spent CDM comprising aflibercept, was evaluated. One or more 50 mL vent-capped shaker tubes with 10 mL working volume (CDM1) were incubated for 7 days, taking samples on day 0 and day 7.
The conditions for component additions to spent CDM1 were as follows:
Aflibercept sample (aflibercept recombinant protein in an aqueous buffered solution, pH 6.2, comprising 5 mM sodium phosphate, 5 mM sodium citrate and 100 mM sodium chloride) spiked into shaker tubes at 6 g/L concentration
Two DOE experiments were run:
(i) Antioxidants added to spent CDM1 at the following concentrations:
Taurine=10 mM of culture
Hypotaurine=10 mM of culture
Glycine=10 mM of culture
Thioctic Acid=0.0024 mM of culture
Vitamin C (ascorbic acid)=0.028 mM of culture
(ii) Antioxidants added to reach the following cumulative concentrations:
ATA=2.5-5 μM
Deferoxamine mesylate (DFO)=5-10 μM
Catalase=101.5 mg/L
S-carboxymethyl-L-Cysteine=10 mM
Hypotaurine was found to decrease the color formation in spent medium (FIG. 28D). DFO also significantly decreased the color formation in spent medium (FIG. 28D). The other antioxidants did not have a statistical impact on the color formation.
TABLE 5-2
Summary of Antioxidant Effect on Color Formation of MiniTrap
Condition
b* value
Spent Medium Day 0
0.44
Spent Medium Day 7 Control
1.73
Spent Medium Day 7 + Hypotaurine
1.32
Spent Medium Day 7 + DFO
0.92
Shake-Flask Antioxidant Study:
Taurine, hypotaurine, glycine, thioctic acid and vitamin C were evaluated individually and in combination for their ability to decrease the color formation in cell culture (Table 5-3).
250 mL shake flasks were inoculated from a seed culture of an aflibercept producing cell line. The inoculated cells were grown at 35.5° C. in incubators with 5% CO2 control. Glucose and basal feeds were supplemented to the shake flasks as needed. The process described above was used wherein metals were present at 0.5× concentration in CDM1 and cysteine was added at a cumulative concentration of 6-7 mM.
TABLE 5-3
Level 1
Level 1
Level 1
Antioxidant
0x
0.5x
1x
Taurine
0
3.75
mM
7.5
mM
Hypotaurine
0
3.75
mM
7.5
mM
Glycine
2.0
mM
5.75
mM
9.5
mM
Thioctic acid
1.0
μM
1.9
μM
2.8
μM
Vitamin C
0
11.0
μM
21.0
μM
FIG. 28E shows the predicted effect of the antioxidants in Table 5-3 on b* value (CIE L*, a*, b* color space) and final titer. Taurine, hypotaurine, and glycine significantly reduced b* value without negatively impacting titer.
Example 6. Glycosylation and Viability Studies for Aflibercept Production Using CDM
Bioreactors (e.g., 2 L) were inoculated from a seed culture of an aflibercept producing cell line. The inoculated cultures were grown at a temperature of 35.5° C., pH of 7.1±0.25, and air sparge set point of 22 ccm. Glucose, antifoam, and basal feeds were supplemented to the bioreactors as needed. Production of aflibercept protein was carried out using CDM1 (proprietary). Production of a host cell line expressing aflibercept fusion protein was carried out using CDM1 (proprietary), CDM2 (commercially obtained), and CDM3 (commercially obtained). A set of experiments was carried out using CDM 1, 2, and 3 with no additional media components. Another set of experiments was performed using CDMs 1-3 to which manganese (manganese chloride tetrahydrate, Sigma, 3.2 mg/L), galactose (Sigma, 8 g/L), and uridine (Sigma, 6 g/L) were added to the feeds to modify the galactosylation profile. Lastly, a set of experiments was performed using CDMs 1-3 to which manganese (manganese chloride tetrahydrate, Sigma, 3.2 mg/L), galactose (Sigma, 8 g/L), and uridine (Sigma, 6 g/L) were added to the feeds to modify the galactosylation profile and dexamethasone (Sigma, 12 mg/L) was added to the feeds to modify the sialyation profile of the composition. A clarified harvest using each of the CDM was prepared by centrifugation followed by 0.45 μm filtration.
Samples were processed by Protein A prior to N-glycan analysis.
Titer Measurements
Throughout these examples, unless stated otherwise, aflibercept titers were measured daily using an Agilent (Santa Clara, Calif.) 1200 Series HPLC, or equivalent, operating with a low pH, and step elution gradient with detection at 280 nm. Concentrations were assigned with respect to a reference standard calibration curve.
Viable Cell Density (VCD) and Cell Viability Values
Throughout these examples, unless stated otherwise, viable cell density (VCD) and cell viability values were measured through trypan blue exclusion via Nova BioProfile Flex automated cell counters (Nova Biomedical, Waltham, Mass.). Glucose, lactate, offline pH, dissolved oxygen (DO), pCO2 measurements, and osmolality were measured with the Nova BioProfile Flex (Nova Biomedical, Waltham, Mass.).
N-Glycan Oligosaccharide Profiling
Approximately 15 μg of Protein A processed samples from CDM 1-3 harvests were prepared for N-glycan analysis in accordance with the Waters GlycoWorks protocol using the GlycoWorks Rapid Deglycosylation and GlycoWorks RapiFluor-MS Label kits (Waters part numbers 186008939 and 186008091, respectively). N-glycans were removed from the aflibercept protein by treating the samples with PNGase-F at 50.5° C. for 5 minutes, followed by a cool down at 25° C. for 5 minutes. The released glycans were labeled with RapiFluor-MS fluorescent dye through reaction at room temperature for 5 minutes. The protein was precipitated by adding acetonitrile to the reaction mixture and pelletized to the bottom of the well through centrifugation at 2,204×g for 10 minutes. The supernatant comprising the labeled glycans was collected and analyzed on an UPLC using hydrophilic interaction liquid chromatography (Waters BEH Amide column) with post-column fluorescence detection. After binding to the column, the labeled glycans were separated and eluted using a binary mobile phase gradient comprised of acetonitrile and aqueous 50 mM ammonium formate (pH 4.4). The labeled glycans were detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm. Using the relative area percentages of the N-glycan peaks in the resultant chromatograms, the N-glycan distribution is reported as the total percentage of N-glycans (1) containing a core fucose residue (Total Fucosylation, Table 6-1), (2) containing at least one sialic acid residue (Total Sialylation, Table 6-2), (3) identified as Mannose-5 (Mannose-5, Table 6-3), (4) containing at least one galactose residue (Total Galactosylation, Table 6-4), and (5) of known identity (Total Identified Peaks, Table 6-5).
Results
The viable cell count (VCC), viability, and harvest titer results are shown in FIGS. 29-31 for CDMs 1-3 with and without additional components.
Amongst the nine cultures, the CDM1 culture comprising uridine, manganese, and galactose showed the highest titer at 12 days (5.5 g/L). CDM1 without additional components also showed a high titer at 12 days (about 4.25 g/L) compared to the other seven cultures (FIG. 29).
Cell viability results were similar across the various conditions up to process day 6. After process day 7, the CDM2 and CDM3 cultures with or without additional media components showed more than about 90% viability (FIG. 30).
CDM1 culture with uridine, manganese and galactose showed the highest VCC around day 6 (FIG. 31).
The influence of cultures and supplements had a significant impact on the overall N-glycan distribution (Tables 6-1 to 6-5). The glycan levels were compared using Protein A processed aflibercept (two samples were evaluated) made using soy hydrolysate. The total identified peaks are listed in Table 6-5.
TABLE 6-1
Total Fucosylation (%)
Condition
Day 6
Day 10
Day 12
CDM1
48.75
—
46.26
CDM1 + UMG
49.21
—
44.38
CDM1 + UMG + Dex
48.88
—
46.23
CDM2
—
45.68
45.14
CDM2 + UMG
—
46.36
45.27
CDM2 + UMG + Dex
—
46.92
—
CDM3
49.24
—
45.59
CDM3 + UMG
48.71
—
42.61
CDM3 + UMG + Dex
49.36
—
44.56
Soy hydrolysate 1
51.37
Soy hydrolysate 2
52.43
U is uridine, M is manganese, G is galactose, Dex is dexamethasone
TABLE 6-2
Total Sialylation (%)
Condition
Day 6
Day 10
Day 12
CDM1
44.06
—
39.14
CDM1 + UMG
43.72
—
35.8
CDM1 + UMG + Dex
43.2
—
36.72
CDM2
—
37.62
36.67
CDM2 + UMG
—
37.57
36.29
CDM2 + UMG + Dex
—
38.06
—
CDM3
44
—
31.21
CDM3 + UMG
42.48
—
30.84
CDM3 + UMG + Dex
43.82
—
32.74
Soy hydrolysate 1
58.24
Soy hydrolysate 2
59.23
U is uridine, M is mananese, G is alactose, Dex is dexamethasone
TABLE 6-3
Mannose-5 (%)
Condition
Day 6
Day 10
Day 12
CDM1
6.76
—
10.1
CDM1 + UMG
6.9
—
13.17
CDM1 + UMG + Dex
6.23
—
8.86
CDM2
—
9.71
11.96
CDM2 + UMG
—
9.44
10.93
CDM2 + UMG + Dex
—
8.21
—
CDM3
2.31
—
12.63
CDM3 + UMG
2.71
—
13.38
CDM3 + UMG + Dex
2.05
—
11.98
Soy hydrolysate 1
5.19
Soy hydrolysate 2
5.24
U is uridine, M is manganese, G is galactose, Dex is dexamethasone
TABLE 6-4
Total Galactosylation (%)
Condition
Day 6
Day 10
Day 12
CDM1
68.44
—
62.9
CDM1 + UMG
69.25
—
59.02
CDM1 + UMG + Dex
69.05
—
63.26
CDM2
—
65.33
63.68
CDM2 + UMG
—
68.13
66
CDM2 + UMG + Dex
—
69.35
—
CDM3
74.57
—
62.28
CDM3 + UMG
74.82
—
62.2
CDM3 + UMG + Dex
76.48
—
65.18
Soy hydrolysate 1
79.64
Soy hydrolysate 2
80.55
U is uridine, M is manganese, G is galactose, Dex is dexamethasone
TABLE 6-5
Total Identified Peaks (%)
Condition
Day 6
Day 10
Day 12
CDM1
87.28
—
84.67
CDM1 + UMG
88.43
—
83.82
CDM1 + UMG + Dex
87.36
—
83.44
CDM2
—
86.23
86.67
CDM2 + UMG
—
87.81
86.87
CDM2 + UMG + Dex
—
87.53
—
CDM3
86.38
—
86.31
CDM3 + UMG
87.07
—
86.13
CDM3 + UMG + Dex
87.18
—
87.43
Soy hydrolysate 1
93.93
Soy hydrolysate 2
94.74
U is uridine, M is manganese, G is galactose, Dex is dexamethasone
The total fucosylation, total sialylation, total galactosylation and mannose-5 observed on day 12 of the cultures of the various CDMs was 42.61% to 46.26%, 30.84% to 39.14%, 59.02 to 66% and 8.86% to 13.38%, respectively. These values for glycosylation differ from the glycosylation values obtained using soy hydrolysate.
Lastly, color measurements were carried out for the clarified harvests obtained from cells expressing aflibercept in CDM1, CDM2, and CDM3 supplemented with uridine, manganese, and galactose. The operating parameters for the bioreactor study steps will be known to one of ordinary skill in the art.
Example 7. Affinity Production of Anti-VEGF Proteins
7.1 Expression of VEGF MiniTrap
The coding regions of recombinant VEGF MiniTrap (e.g., MT5, SEQ ID NO.: 46) were operably linked to a signal sequence, cloned into a mammalian expression vector and transfected into Chinese hamster ovary (CHO-K1) cells; the stably transfected pools were isolated after selection with 400 μg/mL hygromycin for 12 days. The stable CHO cell pools, grown in chemically defined protein-free medium, were used to produce proteins for testing. The recombinant polypeptides were secreted from the cells into the growth medium.
Sequences of constituent domains of the VEGF MiniTrap
Human Flt1 (accession #NP 001153392.1)
Human Flk1 (accession #NP 002244.1)
Human Fc (IGHG1, accession #P01857-1)
The recombinant VEGF MiniTrap (MT5) was obtained from this process and was further processed.
7.2 Preparation of Affinity Chromatography Columns
Five distinct proteins capable of binding to the VEGF MiniTrap (MT5) were evaluated. The proteins used include VEGF165 (SEQ ID NO.: 72), mAb1 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 73 is a heavy chain and SEQ ID NO.: 74 is a light chain); mAb2 (a mouse anti-VEGFR1 mAb human IgG1 where SEQ ID NO.: 75 is a heavy chain and SEQ ID NO.: 76 is a light chain); mAb3 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 77 is a heavy chain and SEQ ID NO.: 78 is a light chain) and mAb4 (a mouse anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain).
The column was activated by washing the columns with 6 column volumes (CV) of 1 mM ice-cold hydrochloric acid at a flow rate not exceeding 1 mL/min. Ten mg of each of the proteins were loaded onto three HiTrap NETS-Activated HP affinity columns (1 mL, GE Healthcare, Cat #17-0716-01) and the columns were closed to allow coupling to take place for 30 minutes at room temperature. The columns were washed with 18 column volumes of 0.5 M sodium acetate, 0.5 M NaCl, pH 4.0 and the open sites were blocked with 18 column volumes of 0.5 M Tris-HCl, 0.5 M NaCl pH 8.3 (the wash was carried out in the following order: 6 column volumes of 0.5 M Tris-HCl, 0.5 M NaCl, pH 8.3; 6 column volumes of 0.5 M sodium acetate (sodium acetate: JT Baker, Cat #3470-01), 0.5 M NaCl, pH 4.0; 6 column volumes of 0.5 M Tris, pH 8.3; incubate the column for 30 minutes at room temperature; 6 column volumes of 0.5 M sodium acetate buffer, 0.5 M NaCl pH 4.0; 6 column volumes of 0.5 M Tris-HCl, 0.5 NaCl, pH 8.3 and 6 column volumes of 0.5 M sodium acetate buffer, 0.5 M NaCl pH 4.0). The columns were stored in DPBS, pH 7.5. The five columns evaluated are designated as column 1 (comprising VEGF165), column 2 (comprising mAb1), column 3 (comprising mAb2), column 4 (comprising mAb3) and column 5 (comprising mAb4).
7.3 Production of MiniTrap Using Affinity Chromatography
Sample Preparation. Two different production processes for the MiniTrap were performed. In one case, material comprising a MiniTrap sample was produced using each of the affinity columns where the parent material (MiniTrap) was diluted in 1×DPBS buffer to 20 mg/mL and was applied to the column and included at RT for 30 minutes. Using the affinity column, the MiniTrap was isolated from 7000 ppm of HCP.
Alternatively, harvested culture supernatant was used which comprised 0.4 mg/mL of protein in the supernatant and loaded onto the different affinity columns (1-5) separately. No further dilution was performed. The affinity columns were then washed with 9 CV of 1×DPBS buffer followed by eluting the proteins with IgG elution buffer, pH 2.8 (Thermo, Cat #21009).
MiniTrap material obtained as described above was then filtered through a 0.45 μm filter or centrifuged before loading onto the columns prepared as described in Section 7.2 above. Twenty-five mL of loading solution comprising approximately 0.4 mg/mL protein was loaded onto each of the columns and incubated for 20 minutes. Each column was washed with 9 CV of DPBS (Invitrogen, Cat #14190-144) before elution for equilibration. The amount of MT5 in the wash fractions is shown in Table 7-1. The washes were followed by elution using 6 CV of pH 2.8 (Commercial Elution Buffer, (Thermo, Cat #21009)) and 100 mM glycine buffer pH 2.5 and fractions were quickly neutralized with the addition of 1 M Tris, pH 7.5 (Invitrogen, Cat #15567-027). The amount of MiniTrap in the eluted fractions is also shown in Table 7-1.
The MiniTrap (MT5) was successfully produced from all five affinity columns. The yield from the column with VEGF165 was higher than compared to mAb1 and mAb2 columns. The mAb3 and mAb4 comprising humanized anti-VEGFR1 mAb also showed successful production of MT5 with similar yield to mAb1 and mAb2. In Table 7-1, the expected yield was calculated based on 100% conjugation efficiency and 1:1 molar ratio of affinity-captured protein to MT5.
TABLE 7-1
Affinity
Column 1
Column 2
Column 3
Column 4
Column 5
Column
VEGF165
mAb1
mAb2
mAb3
mAb4
Conjugation
10
10
10
10
10
Amount (mg)
Load (mg)
21.2
21.2
21.2
20.1
20.1
Expected (mg)
~12
~3.2
~3.2
~3.2
~3.2
Wash (mg)
14.9
13.2
12.6
15.2
14.7
Eluate (mg)
4.8
1.6
1.8
1.5
1.6
7.4 Column Stability Study
Multiple runs were carried out using columns 1 and 2 following the method discussed in Section 7.3 (Table 7-2 for column 1 and Table 7-3 for column 2).
TABLE 7-2
Production Yield
Run #
1
2
3
4
5
6
7
Load (mg)
21.2
19.7
19.7
19.7
19.6
19.6
19.6
Wash (mg)
14.9
13.5
15.0
15.0
14.6
14.6
14.3
Eluate (mg)
4.8
5.4
5.2
4.8
5.2
4.6
4.8
TABLE 7-3
Production Yield
Run #
1
2
3
4
5
6
7
Load (mg)
21.2
19.7
19.7
19.7
19.6
19.6
19.6
Wash (mg)
13.2
16.0
16.4
16.4
17.1
17.2
17.2
Eluate (mg)
1.6
1.8
1.8
1.8
2.0
2.0
2.0
The columns were stored at 4° C. for about 5 weeks. A similar amount of MT5 was eluted from each production demonstrating good column stability.
7.5 Stability Study of the Produced VEGF MiniTrap
SDS-PAGE analysis of the eluted fractions from the three columns (column 1, column 2, and column 3) was performed. The samples were prepared in non-reducing and reducing SDS-PAGE sample buffer and run on a 4-12% gradient NuPage bis-Tris gel using 1×IVIES (Cat. No. NP0322, Invitrogen, Carlsbad, Calif.).
The wells were loaded with (1) molecular weight standard, (2) loading solution, (3) column wash from column 1, (4) eluted fraction from column 2, (5) eluted fraction from column 1, (6) eluted fraction from column 3, (7) MT5 stored at pH 2.8 for 1 min, (8) MT5 stored at pH 2.8 for 30 min, and (9) molecular weight standard (FIG. 33 and FIG. 34). The analysis demonstrated that fractions obtained from the eluted fractions from all the three affinity columns (columns 1-3) showed similar size profiles and the use of the affinity columns did not destabilize the MiniTrap.
7.6 Host Cell Protein Level Calculations
A standard curve of concentration of host cell proteins was obtained using CHO HCP ELISA Kit, 3G (F550) (Cygus Technologies) (FIG. 32 and Table 7-4). The amount of HCPs in the loading solutions and the eluted fractions was calculated using the standard curve as depicted in FIG. 32 and curve formula listed in Table 7-4.
TABLE 7-4
Low
EC50
High
Curve Formula
Asymptote
Slope
(ng/mL)
Asymptote
R2
Y = (A-D)/
0.2
1.9
32.9
2.3
1
(1 + (X/C){circumflex over ( )}B) + D
The total HCPs were calculated using the standard curve and the chart with the total amount of host cell proteins is shown in FIG. 35A. FIG. 35B also shows total amount of host cell proteins in the load compared to the washes and eluted fractions from columns 1, 2, 4 and 5. Multiple runs were carried out using the columns and the (#) in FIG. 35B represent the run from which the fraction was evaluated.
The use of affinity capture using proteins capable of binding to MiniTrap showed an efficient reduction of HCPs from about 7000 ppm to about 25-50 ppm. As observed for the yield, the column with VEGF165 showed higher purity of MiniTrap from HCPs than shown by mAb1 and mAb2 columns.
7.7 SEC Profiles of VEGF MiniTrap Before and After Affinity Production
SEC profiles of the eluted fractions from three columns (columns 1-3) were compared to the SEC profile of MiniTrap in the loading solution. As seen in FIG. 36 and Table 7-5, the SEC profiles of the MT5 before or after affinity production were highly similar.
TABLE 7-5
Peak
%
%
%
%
No. as
Retention
Peak
Retention
Peak
Retention
Peak
Retention
Peak
in FIG.
Time
Area
Time
Area
Time
Area
Time
Area
36
Loading solution
Column 1 Eluate
Column 2 Eluate
Column 3 Eluate
1
6.8
1.8
6.8
1.2
6.9
1.1
7.0
1.2
2
7.8
94.6
7.9
97.2
7.9
97.3
7.9
98.3
3
9.4
3.6
10.2
1.7
11.4
1.6
11.3
0.5
7.8 Kinetics of VEGF MiniTrap Pre and Post Column Samples Binding to mAb1, mAb2 and VEGF165
Kinetic studies were performed using a Biacore T200 instrument.
Equilibrium dissociation constants (KD values) for VEGF165 binding to MiniTrap in the eluates from columns 1 and 2 and loading solution were determined using a real-time surface plasmon resonance biosensor using a Biacore T200 instrument. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. The Biacore sensor surface was first derivitized by amine coupling with a mAb 1 to capture MT5. A cartoon representation of the binding study is shown in FIG. 37.
Briefly, the eluates from the columns and loading solution were diluted into an HBS-EP (Biacore) buffer and injected across the immobilized protein matrices at a capture level of ˜70 RUs. The VEGF165 was then injected at a flow rate of 50 μL/min. Equivalent concentration of analyte was simultaneously injected over an untreated reference surface to serve as blank sensorgrams for subtraction of bulk refractive index background. The sensor chip surface was regenerated between cycles with two 5-min injections of 10 mM Glycine, at 25 μL/min. The resultant experimental binding sensorgrams were then evaluated using the BIA evaluation 4.0.1 software to determine kinetic rate parameters. Datasets for each sample were fit to the 1:1 Langmuir model. For these studies, binding and dissociation data were analyzed under the Global Fit Analysis protocol while selecting fit locally for maximum analyte binding capacity (RU) or Rmax attribute. In this case, the software calculated a single dissociation constant (kd), association constant (ka), and affinity constant (Kd). The equilibrium dissociation constant is KD=kd/ka. The kinetic on-rate, the kinetic off rate, and the overall affinities were determined by using different VEGF165 concentrations in the range of 0.03-2 nM (Table 7-6). The dissociative half-lives (t1/2) were calculated from the kinetic rate constants as: t1/2=ln(2)/60*Kd. Binding kinetic parameters for MT5 to VEGF165 obtained from before and after the affinity chromatography production at 25° C. are shown in Table 7-6.
The affinity (KD), on rate (ka, M-1s-1) and off rate (kd) for MT5 produced by affinity chromatography compared with loading solution to assess the effect(s) of affinity chromatography step showed no change in the kinetics of MT5 from different samples. The SPR sensorgrams of the VEGF MiniTrap constructs are shown in FIG. 38.
TABLE 7-6
VEGF MiniTrap
ka
kd
KD
t½
Chi2
Rmax
samples
(106 M−1s−1)
(10−5 s−1)
(10−12 M)
(min)
(RU2)
(RU)
Loading solution
9.44
1.74
1.84
664
0.10
20
column 2 eluate
8.83
1.49
1.69
775
0.17
28
column 1 eluate
12.18
1.80
1.48
641
0.18
19
7.9 Multiple Production Cycles
Chromatographic production of harvest as obtained by step 7.1 was carried out using column 1 (hVEGF165) and column 2 (mAb1) as shown in 7.3. The columns were used for multiple chromatographic cycles. The yields in the columns did not vary significantly due to additional runs, suggesting that the columns retained binding capacity (Table 7-7).
TABLE 7-7
Production Yield
Affinity Column
Column 1
Column 2
Run #
1
2
3
4
1
2
3
4
Load (mg)
21.2
19.7
19.7
19.7
21.2
19.7
19.7
19.7
Wash (mg)
14.9
13.5
15.0
15.0
13.2
16.0
16.4
16.4
Eluate (mg)
4.8
5.4
5.2
4.8
1.6
1.8
1.8
1.8
HCP calculations in the loading solutions, wash fractions and eluted fractions for columns 1 and 2 were obtained using the method described in 7.4 (FIG. 39). The total HCPs calculated showed that repeated use of the columns did not reduce the ability of the columns to bind to MiniTrap.
7.10 Optimizing the Affinity Chromatographic Columns
The chromatographic production of harvest material as obtained in Section 7.1 was performed using column 1 (VEGF165) and column 2 (mAb1). For the optimization studies, 14 mg or 45 mg instead of 10 mg of the VEGF165 or the anti-VEGFR1 mAb were loaded onto two HiTrap NETS-Activated HP affinity columns (1 mL, GE Healthcare) and the columns were closed to allow coupling to take place for 30 minutes at room temperature. The column preparation and production of the harvest including the MiniTrap was carried out as discussed in 7.2 and 7.3 above. The amount of MT5 in the wash and eluted fractions is shown in Table 7-8. The comparison of affinity column with 14 mg or 45 mg (VEGF165 or anti-VEGFR1 mAb (mAb1)) conjugation amount instead of 10 mg shows an increased yield of MiniTrap from both columns. Thus, the column yield using the outlined method can be improved by optimizing the protein to column ratio or by increasing the conjugation efficiency by changing the pH, incubation time, incubation temperature, etc.
TABLE 7-8
Affinity Column
(hVEGF165)
(mouse anti-VEGFR1 mAb)
MW (kDa)
~40 (Dimer)
145
Conjugation
10
14
10
45
Amount (mg)
Load (mg)
21.2
45.5
21.2
45.5
Wash (mg)
14.9
36.8
13.2
29.2
Eluate (mg)
~5.0
7.6
~1.8
5.5
7.11 Use of CEX with the Affinity Chromatography
A cell culture sample from MT5 expression was produced using column 1 as discussed in Section 7.3 above. The eluate obtained was subjected to cation exchange chromatography (CEX) column (HiTrap Capto S, 1 mL). The operating conditions of the column are shown in Table 7-9.
TABLE 7-9
Steps
Affinity
Cation Exchange (CEX)
Column
Affinity Column, 1 mL
HiTrap Capto S, 1 mL
Load
MT5 CM2926
20 mM Acetate, pH 5.0 (Load/
Wash1)
Wash
1X DPBS pH 7.2
10 mM Phosphate, pH 7.0
Elution
Pierce ™ IgG Elution
50 mM Tris, 62.5 mM (NH4)2SO4,
Buffer
pH 8.5
Regeneration/
10 mM Glycine pH 2.5
50 mM Tris, 1M (NH4)2SO4,
Strip
pH 8.5
The total HCP in the original/starting cell culture sample, the affinity chromatography column 1 eluate and CEX eluate was about 230,000 ng/mL, about 9,000 ng/mL and about 850 ng/mL, respectively. The HCP amounts were quantitated determined using the Cygnus CHO HCP ELISA Kit, 3G, as mentioned above.
7.12 Use of Affinity Chromatography to Produce Other Anti-VEGF Proteins
Column 1 was evaluated to study its ability to produce other anti-VEGF proteins. Aflibercept and a scFv fragment with VEGF binding potential were used for this study. The production processes were carried out as discussed in Section 7.3. Table 7-10 demonstrates that column 1 was successful in binding and eluting other anti-VEGF proteins.
TABLE 7-10
Affinity Column 1
scFv
Aflibercept
Load (mg)
10
20
Wash (mg)
4.5
10.6
Eluate (mg)
3.6
10.2
Example 8. Mass Spectrometry-Based Characterization of VEGF MiniTrap Constructs Materials.
VEGF MiniTrap (MT1) was produced from aflibercept as described in Example 1. VEGF MiniTrap 5 (MT5) was produced as described in Example 7. VEGF MiniTrap (MT6) was produced by the following method: the coding regions of recombinant VEGF MiniTrap (MT5) were operably linked to a signal sequence and cloned into a mammalian expression vector, transfected into Chinese hamster ovary (CHO-K1) cells and stably transfected pools were isolated after selection with 400 μg/mL hygromycin for 12 days. The stable CHO cell pools, grown in CDM, were used to produce proteins for analysis.
8.1 Deglycosylation of Glycoproteins.
Samples from clarified harvest of MT1, MT5 and MT6 were diluted or reconstituted to a concentration of 0.52 mg/mL into a 28.8 μL solution of 1% (w/v) RG surfactant (RapiGest SF, Waters, Milford, Mass.) and 50 mM HEPES (pH 7.9). These solutions were heated to approximately 95° C. over 2 min, allowed to cool to 50° C., and mixed with 1.2 μL of PNGase F solution (GlycoWorks Rapid PNGase F, Waters, Milford, Mass.). Deglycosylation was completed by incubating the samples at 50° C. for 5 min.
8.2 HILIC-Fluorescence-ESI-MS (MS/MS) Analysis.
MT1 was analyzed via HILIC separation combined with fluorescence and mass spectrometric detection. MT5 and MT6 were analyzed using only HILIC. Chromatography was performed using a Waters 2D Acquity UPLC equipped with photodiode array and fluorescence (FLR) detectors and interfaced with a Waters Synapt G2-S mass spectrometer (MS conditions). A hydrophilic interaction chromatography (HILIC) mode of separation was used with a Waters UPLC Glycan BEH Amide column, 150×2.1 mm, 1.7 μm. The column temperature was set to 60° C. and the autosampler temperature was set to 5° C. The injection volume was 50 μL. The photodiode array scan range was 190-700 nm. The FLR was set to excitation 265 nm, emission 425 nm for RapiFluor-labeled glycans and excitation 274 nm, and emission 303 nm for tyrosine present in the glycopeptides. The initial flow rate was 0.4 mL/min with mobile phase A comprising of 100 mM ammonium formate (pH 4.4) and mobile phase B being acetonitrile.
8.3 MS Conditions
Liquid chromatography/mass spectrometry (LC/MS) experiments were conducted using a Waters Synapt G2-S mass spectrometer. The scan range was mass-to-charge ratio 100-2400 for positive and negative ion mode analyses. Scan time was is, and glu-fibrinopeptide B was constantly infused (2 μL/min) as a calibrant (“lock mass”). The capillary voltage was set to 2.5 kV, with a source temperature of 120° C. and desolvation temperature of 500° C. The nitrogen nebulizer gas flow was set to 7001/h.
8.4 Native SEC-MS
ACQUITY UPLC I class system (Waters, Milford, Mass.) was coupled to Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) for all online SEC-MS analyses. ACQUITY UPLC Protein BEH SEC Column (200 Å, 1.7 μm, 4.6×300 mm) was set at 30° C. and used for protein separation. The mobile phase was 100 mM ammonium acetate at pH 6.8. Each separation was 30 minutes with a flow rate of 0.3 mL/min, and the injection amount was set to 40 μg. The following MS parameters were used for online SEC-nano-ESI-MS data acquisition. Each acquisition was 25 minutes beginning immediately after sample injection. The deglycosylated samples were ionized in positive mode with 3 kV spray voltage, 200° C. capillary temperature, and 70 S-lens RF level. In-source CID was set at 75 eV. Full MS scans were acquired at 15 K resolving power with mass range between m/z 2000-8000. A maximum injection time of 100 ms, automatic gain control target value of 3e6, and 10 microscans were used for full MS scans.
8.5 Peptide Mapping
Sample preparation for peptide mapping. Reduction was achieved by the addition of 500 mmol/L dithiothreitol (DTT) to a final concentration of 5 mmol/L followed by incubation at 4° C. for 60 min. Alkylation was performed by adding 500 mmol/L iodoacetamide (IAM) to a final concentration of 10 mmol/L and incubating at 4° C. for 60 min in the dark. The denaturing buffer was exchanged for digestion buffer (1 mol/L urea in 0.1 mol/L Tris, pH 7.8) using Zeba™ Spin 7 K MWCO size-exclusion desalting columns (P/N 89882) (Thermo Scientific, Waltham, Mass.) according to the manufacturer's instructions. Recombinant porcine trypsin (purchased from Sigma, Cat #03708985001) was added at a 1:18 (enzyme: sample) mass ratio (based on VEGF MiniTrap protein concentration as measured by UV-Vis spectrophotometry after buffer exchange), the concentration of VEGF MiniTrap proteins was adjusted to 0.5 μg/μL and digestion allowed to proceed during a 4 h incubation at room temperature. When the digestion was complete, 0.1% formic acid in LC-MS grade water was added at a 1:1 volume ratio. Digests were stored at −80° C. until analysis.
LC-MS/MS analysis of tryptic digests. One or more 2.5 μg (10 μL) of peptide digests were loaded via autosampler onto a C18 column enclosed in a thermostatted column oven set to 40° C. Samples were held at 7° C. while queued for injection. The chromatographic method was initiated with 98% Mobile Phase A (0.1% volume fraction of formic acid in water) and 2% Mobile Phase B (0.1% volume fraction of formic acid in acetonitrile) with the flow rate set at a constant 0.200 mL/min. After a 10 min wash, peptides were eluted over a 110 min gradient in which Mobile Phase B content rose at a rate of 0.39% per min to reach a final composition comprising 45% Mobile Phase B. Prior to the next sample injection, the column was washed for 15 min with 97% Mobile Phase B, then equilibrated at 98% Mobile Phase A for 25 min. The eluate was diverted to waste for the first 1.5 minutes and final 5 minutes of the run. Peptides eluting from the chromatography column were analyzed by UV absorption at 214 nm followed by mass spectrometry on the LTQ Orbitrap Elite or Discovery XL. Replicate peptide mapping data were collected for PS 8670 and RM 8671 samples to include three tandem MS (MS/MS) analyses and one MS-only analysis each. The MS/MS analyses were performed for peptide identification in data-dependent mode in which one cycle of experiments consisted of one full MS scan of 300 m/z to 2000 m/z followed by five sequential MS/MS events performed on the first through fifth most intense ions detected at a minimum threshold count of 500 in the MS scan initiating that cycle. The sequential mass spectrometry (MS″) AGC target was set to 1E4 with microscans=3. The ion trap was used in centroid mode at normal scan rate to analyze MS/MS fragments. Full MS scans were collected in profile mode using the high resolution FTMS analyzer (R=30,000) with a full scan AGC target of 1E6 and microscans=1. Ions were selected for MS/MS using an isolation width of 2 Da, then fragmented by collision induced dissociation (CID) with helium gas using a normalized CID energy of 35, an activation Q of 0.25 and an activation time of 10 msec. A default charge state was set at z=2. Data dependent masses were placed on the exclusion list for 45s if the precursor ion triggered an event twice within 30s; the exclusion mass width was set at ±1 Da. Charge state rejection was enabled for unassigned charge states. A rejection mass list included common contaminants at 122.08 m/z, 185.94 m/z, 355.00 m/z, 371.00 m/z, 391.00 m/z, 413.30 m/z, 803.10 m/z, 1222.10 m/z, 1322.10 m/z, 1422.10 m/z, 1522.10 m/z, 1622.10 m/z, 1722.10 m/z, 1822.10 m/z, and 1922.10 m/z. MS-only analyses were performed for the generation of the TIC non-reduced peptide map and reduced maps.
8.6 Results
Structure of VEGF MiniTrap constructs. Structure of VEGF MiniTraps MT1, MT5 and MT6 are depicted in FIG. 40, FIG. 41, FIG. 43 and FIG. 44.
Initial mass analysis using SEC-MS confirmed the identities of all three molecules at intact protein level after deglycosylation (FIG. 42). The Total Ion Chromatogram (TIC) of the native SEC-MS analysis demonstrates detection of an intact VEGF MiniTrap molecules at around 12-13 minutes. The expansion of the low molecular weight (LMW) region of the TIC showed presence of LMW impurities in all the three protein samples.
The deconvoluted mass spectra for the VEGF MiniTraps further confirmed their identity and provided data for elucidation of the major PTMs present in the samples comprising MT1 and MT5 (FIG. 43), which are dimers and MT6 (FIG. 44) which is a single chain protein.
Analysis of MT1 sample. The LMW species identified from the TIC of the SEC-MS analysis of the samples comprising MT1 was extracted to examine three distinct LMW impurities—LMW1, LMW2, and LMW3 (FIG. 45A and FIG. 45B). LMW1 species comprised a truncated species of aflibercept. LMW2 species comprised the Fc impurity present in the sample form the cleavage of aflibercept which was performed to produce MiniTrap. LMW3 species comprised a monomer possibly cleaved from the MT1 (dimer) molecule.
MT1 sample did not show presence of FabRICATOR enzyme, which had been used to cleave aflibercept to form a MiniTrap protein. The enzyme, if present, is detected at about 11.5 and 12.5 minutes. No such peak was detected during the SEC-MS analysis of the MT1 sample (FIG. 46).
Analysis of MT5 sample. The LMW species identified from the TIC of the SEC-MS analysis of the samples comprising MT5 was extracted to examine the presence of two distinct LMW impurities—LMW1 and LMW2 (FIG. 47).
Analysis of MT6 sample. The LMW species identified from the TIC of the SEC-MS analysis of the samples comprising MT1 was extracted to examine the presence of three distinct LMW impurities—LMW1, LMW2, and LMW3 (FIG. 48). LMW2 species comprised a fragment of the MT6 wherein the cleavage produced the fragment of VEGF MiniTrap with the G45 linker (SEQ ID NO.: 111). LMW5 species comprised a fragment of the MT6 wherein the cleavage occurred right before or after the G45 linker (SEQ ID NO.: 111).
The glycans in the MT6 sample were identified by their mass and elution order in the HILIC chromatography method using the glucose unit value pioneered by Waters and the National Institute for Bioprocessing Research and Training (Dublin, Ireland) (FIG. 49A and FIG. 49B).
Free thiol Quantification. Cysteine residues of the VEGF MiniTrap constructs may be involved in the formation of intra- and inter-molecular disulfide bond(s) or they may exist as free thiols. The presence of sulfide bonds in peptides and proteins has been shown to impose conformational rigidity on a protein. Thiols can be detected by a variety of reagents and separation techniques. The analysis of the three VEGF MiniTrap constructs for a very low level of free thiols is shown in Table 8-1.
TABLE 8-1
Location
Peptide (site of free cysteine)
MT1
MT5
MT6
VEGFR1
ELVIPCR (SEQ ID NO.: 81)
<0.1%
<0.1%
<0.1%
VEGFR2
LVLNCTAR (SEQ ID NO.: 82)
0.3%
0.3%
0.3%
Fc Hinge
THTCPPCPAPELLG
0.0%
0.0%
N/A
(SEQ ID NO.: 83)
Trisulfide Quantification. Similar to free thiols in Cys residues of the VEGF MiniTrap constructs, trisulfide bonds can influence the structure of the protein. The analysis of the three VEGF MiniTrap constructs under conditions with very low level of free thiols is shown in Table 8-2.
TABLE 8-2
Location
Peptide
MT1
MT5
MT6
VEGFR1
ELVIPCR -
0.1%
<0.1%
0.1%
EIGLLTCEATVNGHLYK
(SEQ ID NO.: 84)
VEGFR2
LVLNCTAR -
<0.1%
<0.1%
<0.1%
SDQGLYTCAASSGLMTK(K)
(SEQ ID NO.: 85)
Fc Hinge
THTCPPCPAPELLG -
1.5%
3.7%
N/A
THTCPPCPAPELL(G)
(SEQ ID NO.: 86)
Intra-chain disulfide in the Hinge region. Mispaired disulfide bonds in the hinge region can have implications on the structure, function and stability of the VEGF MiniTrap constructs. The analysis of the three VEGF MiniTrap constructs for a very low or no intra-chain disulfide binds in the hinge region of the VEGF MiniTrap constructs [THTC*PPC*PAPELLG, C* shows where intra-chain sulfide bond can be formed] (SEQ ID NO.: 83) is shown in Table 8-3.
TABLE 8-3
Peptide
MT1
MT5
MT6
Disulfide
<0.1%
<0.1%
N/A
Trisulfide
<0.1%
<0.1%
N/A
Cross and parallel disulfide linkage isomer quantification. For MT1 and MT5, which are dimers connected by parallel disulfide bonds in the hinge regions, there is a possibility of isomers wherein the disulfide bonds in the hinge region can be crossed (FIG. 50).
The quantification of types of disulfide bond, parallel versus cross, showed that MT5 recombinantly expressed protein had a slightly higher level of cross disulfide bridge in the Fc hinge region compared to the MT1—which is a FabRICATOR digested molecule (Table 8-4).
TABLE 8-4
Disulfide
MT1
MT5
MT6
Cross
0.2%
3.9%
N/A
Parallel
99.8%
96.1%
N/A
Post-Translational Modifications (PTMs).
TABLE 8-5
PTM
Site
Modified Peptide
MT1
MT5
MT6
Deamidation
Asn84
EIGLLTCEATVNGHLYK
Succinimide
3.1%
3.2%
3.2%
(Asn319)
(SEQ ID NO.: 87)
Asp/iso Asp
21.9%
18.9%
20.9%
Asn99
QTNTIIDVVLSPSHGIELSVGEK
Succinimide
4.6%
4.6%
4.0%
(Asn334)
(SEQ ID NO.: 88)
Asp/iso Asp
0.7%
0.5%
0.6%
Oxidation
Met10
SDTGRPFVEMYSEIPEIIHMTEGR
1.8%
2.1%
2.1%
(SEQ ID NO.: 89)
Met20
SDTGRPFVEMYSEIPEIIHMTEGR
2.9%
3.0%
2.7%
(SEQ ID NO.: 90)
Met245
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS
—
—
1.4%
DTGRPFVEMYSEIPEIIHMTEGR
(SEQ ID NO.: 91)
Met255
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSS
—
—
2.7%
DTGRPFVEMYSEIPEIIHMTEGR
(SEQ ID NO.: 92)
Met163
TQSGSEMK
4.3%
4.3%
3.8%
(Met398)
(SEQ ID NO.: 93)
Met192
SDQGLYTCAASSGLMTK
5.0%
5.0%
4.2%
(Met427)
(SEQ ID NO.: 94)
C-term
Gly211
THTCPPCPAPELLG
0.1%
2.0%
—
Glycine loss
(SEQ ID NO.: 95)
Evaluation of PTMs in all the three VEGF MiniTrap constructs showed comparable levels of PTMs (Table 8-5). The deamidation observed at Asn84 to form succinimide was in the range of about 3.1-3.2% and to form aspartic acid/iso aspartic acid was 18.9-21.9%. Oxidation of several methionine residues (e.g., Met10, Met 20m Met163 and Met192) was observed in the range of about 0.7-6.8% for all the three VEGF MiniTrap constructs. MT6, which, in contrast to MT1 and MT5, comprises a linker, showed additional oxidation of methionine residues on the linker (e.g., Met245 and Met255). About 0.1% and 2.0% of the C-terminal glycine (Gly211) in MT1 and MT5 showed a glycine loss. This was not observed for MT6, which lacks a C-terminal glycine.
Advanced glycation end-product modifications related to lysine and arginine glycation. Glycation of the VEGF MiniTrap constructs can alter their structure and function, leading to impaired anti-VEGF activity.
TABLE 8-6
Site
PTM
MT1
MT5
MT6
Arg5
3-Deoxyglucosone
8.0%
8.1%
9.2%
Glycation
0.1%
0.1%
0.1%
Carboxymethylation
1.5%
1.4%
1.4%
Arg153
3-Deoxyglucosone
<0.1%
<0.1%
<0.1%
Arg96
3-Deoxyglucosone
<0.1%
<0.1%
<0.1%
Lys62
Glycation
1.1%
1.1%
1.3%
Carboxymethylation
<0.1%
<0.1%
<0.1%
Lys68
Glycation
0.4%
0.3%
0.5%
Lys149
Glycation
0.6%
0.5%
0.6%
Carboxymethylation
<0.1%
<0.1%
<0.1%
Lys185
Glycation
<0.1%
<0.1%
<0.1%
Evaluation of modifications in all three VEGF MiniTrap constructs showed comparable levels (Table 8-6).
Modified sites. The modified sites on the VEGF MiniTrap constructs, as elucidated by the intact mass analysis as per Section 8.4, were confirmed and quantified using reduced peptide mapping as illustrated in Section 8.5 (Table 8-7). The site T90N91 for peptide sequence TNYLTHR (SEQ ID NO.: 21), the ** represents that asparagine was converted to aspartic acid after truncation, whereas for site N99T100 the peptide sequence QTNTIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 19), the * represents a high level of no-specific cleavage by trypsin. These two truncation sites were found to form LMW species impurities during evaluation of MT1 and MT5. The truncation at M245Y246 was found only on MT6 which had the unique linker and was responsible for the LMW2 species impurity during the MT6 preparation.
TABLE 8-7
Site
Peptide Sequence
MT1
MT5
MT6
N99T100
QTNTIIDVVLSPSHGIELSVGEK*
12.6%
13.2%
13.6%
(SEQ ID NO.: 96)
T90N91
TNYLTHR** (SEQ ID NO.: 97)
0.5%
0.1%
0.3%
M245Y246
GGGGSGGGGSGGGGSGGGGSG
—
—
1.8%
GGGSGGGGSSDTGRPFVEMYSE
IPEIIHMTEGR (SEQ ID NO.: 98)
M10Y11
SDTGRPFVEMYSEIPEIIHMTEGR
0.2%
1.5%
1.7%
(SEQ ID NO.: 99)
Glycosites occupancy quantification. N-glycosylation is a common PTM. Characterizing the site-specific N-glycosylation including N-glycan macroheterogeneity (glycosylation site occupancy) and microheterogeneity (site-specific glycan structure) is important for the understanding of glycoprotein biosynthesis and function. The extent of glycosylation can change depending on how the protein is expressed. The levels of glycosylation at N36 were similar for all the three VEGF MiniTraps (Table 8-8 and FIG. 51). Similarly, the levels of glycosylation at N68 were also similar for all the three VEGF MiniTraps (Table 8-8 and FIG. 52). The levels of glycosylation at N123 were also similar for all the three VEGF MiniTraps (Table 8-8 and FIG. 53), but mannose-5 was found to be elevated in the MT1 preparation. For the VEGF MiniTrap constructs, glycosylation at Asn196 was lower for MT5 and MT6, compared to MT1 (Table 8-8 and FIG. 54). Additionally, the mannose-5 was also elevated for the MT1 preparation than MT5 and MT6 preparations.
TABLE 8-8
Site
Peptide
MT1
MT5
MT6
N36
(R)VTSPNITVTLK (SEQ ID NO.: 100)
98.3%
98.1%
99.4%
N68
(K)GFIISNATYK (SEQ ID NO.: 101)
51.9%
55.4%
64.9%
N123
(K)LVLNCTAR (SEQ ID NO.: 102)
99.9%
99.4%
98.4%
N196
(K)NSTFVR (SEQ ID NO.: 103)
98.6%
44.5%
55.1%
Analysis of N-glycans. The glycosylation at N36 is shown in Table 8-9. G2F, G2FS, G2FS2 were the major N-glycans found in all the three VEGF MiniTraps. For glycosylation at N68 shown in Table 8-10, G2F and G2FS were the major N-glycans found in all the three VEGF MiniTraps. For glycosylation at N123 is shown in Table 8-11, G2F and G2S were the major N-glycans found in all the three VEGF MiniTraps and Mannose-5 was detected at high levels in MT1 compared to MT5 and MT6. For glycosylation at N196 shown in Table 8-12, G2, G2S, G2S2 were the major N-glycans found in all the three VEGF MiniTraps and Mannose-5 was detected at high levels in MT1 compared to MT5 and MT6.
TABLE 8-9
Glycans at N36
MT1
MT5
MT6
G0F-GlcNAc
2.0%
1.8%
1.8%
G1F
3.2%
1.0%
1.4%
G1F-GlcNAc
4.8%
4.6%
4.9%
G1FS-GlcNAc
3.1%
3.8%
3.1%
G2F
17.4%
15.1%
19.8%
G2F2S
1.7%
2.0%
2.2%
G2FS
34.2%
31.5%
31.9%
G2FS2
20.4%
25.8%
19.0%
G3FS
2.3%
4.0%
5.5%
G3FS2
2.6%
4.7%
5.0%
G3FS3
1.1%
2.4%
1.9%
G1_Man5 + Phos
1.2%
0.3%
0.2%
Man6 + Phos
5.7%
2.5%
2.8%
TABLE 8-10
Glycans at N68
MT1
MT5
MT6
G0F-GlcNAc
1.2%
1.1%
1.1%
G1F
5.1%
1.4%
1.7%
G1F-GlcNAc
3.9%
3.9%
4.0%
G1FS
1.2%
0.4%
0.4%
G1FS1-GlcNAc
1.2%
1.6%
1.4%
G2F
27.4%
23.6%
28.6%
G2F2S
2.2%
3.0%
3.4%
G2FS
52.4%
55.2%
50.2%
G2FS2
3.9%
6.9%
5.8%
G3FS
0.5%
1.2%
1.6%
G3FS2
0.4%
1.1%
1.2%
TABLE 8-11
Glycans at N123
MT1
MT5
MT6
G0-GlcNAc
3.5%
3.7%
3.5%
G1-GlcNAc
6.2%
6.8%
6.4%
G1S-GlcNAc
4.1%
3.5%
2.8%
G2
10.6%
16.7%
17.1%
G2F
1.5%
7.2%
7.0%
G2FS
2.1%
13.6%
14.2%
G2S
12.7%
26.1%
25.5%
G2S2
1.3%
5.0%
6.6%
G1_Man4
3.8%
1.3%
1.4%
G1S_Man4
3.9%
2.1%
1.8%
G1_Man5
4.0%
1.2%
1.1%
G1S_Man5
3.2%
1.4%
1.4%
Man4
2.6%
1.9%
1.8%
Man5
35.5%
4.3%
3.1%
Man6
1.1%
0.1%
0.1%
Man7
2.8%
0.1%
0.1%
TABLE 8-12
Glycans at N196
MT1
MT5
MT6
G0-GlcNAc
1.9%
1.8%
1.9%
G1
4.1%
3.6%
4.2%
G1-GlcNAc
1.9%
2.5%
2.4%
G1S-GlcNAc
2.9%
2.6%
1.8%
G2
20.7%
28.2%
32.1%
G2F
2.0%
5.1%
6.0%
G2FS
2.0%
6.1%
6.2%
G2FS2
0.5%
1.6%
1.3%
G2S
17.7%
31.2%
29.9%
G2S2
4.4%
9.7%
6.7%
G3S
0.1%
0.7%
1.0%
G1S_Man4
1.0%
0.3%
0.3%
G1_Man5
2.3%
0.5%
0.5%
Man3
3.1%
0.7%
0.6%
Man4
2.7%
0.8%
0.6%
Man5
30.4%
3.6%
3.4%
O-glycans at the linker for MT6. The GS linker for MT6 was evaluated to study O-glycans on MT6. O-xylosylation was found to on serine residues located on the GS linker of MT6 (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGR, underlined serine residues were glycosylated) (SEQ ID NO.: 98). The composition of the O-glycans is shown in Table 8-13.
TABLE 8-13
Composition
Mass
Annotation
Number
Level
Xylosylation
+132.0
Tri
<0.1%
di
1.5%
mono
15%
Xylose + Galactose
+294.1
mono
0.9%
Xylose + Galactose +
+585.2
mono
0.7%
Sialic Acid
HILIC-FLR-MS analysis. HILIC-FLR-MS analysis was performed for all the VEGF MiniTrap proteins as described in Section 8.2. The analysis showed that the N-linked glycans for MT5 and MT6 were similar but were different than the ones obtained for MT1 (FIG. 55 shows the full scale and stacked chromatograms, FIG. 56 shows full scale and overlaid chromatograms and FIG. 57 shows the full scale, stacked and normalized chromatograms).
Finally, the percent glycosylation and detailed glycan identification and quantification for all three VEGF MiniTrap proteins is listed in Table 8-14 and FIG. 58A-C, respectively. As observed in all the glycan analysis, the glycosylation profile and mannose levels for MT5 and MT6 are similar, but different from MT1.
TABLE 8-14
MT1
MT5
MT6
% Fucosylation
42.9%
57.8%
57.2%
% Galactosylation
71.6%
92.9%
93.7%
% Sialylation
33.1%
47.6%
44.8%
% High Mannose
17.6%
2.6%
2.3%
% Bisecting
1.9%
0.4%
0.4%
Example 9. Production and Color Quantification Using Upstream Medium and Feed Process Optimization
(A) Un-optimized CDM (Control Bioreactor)
The manufacture of MiniTrap described in Example 5 was employed.
The operating parameters for the study steps are as known to one of ordinary skill in the art.
Medium at day 0=CDM1 and included the following nutrients, antioxidants and metals:
Cysteine was added at a cumulative concentration of 8-9 mM
Metals in Starting Medium are listed below at 1× concentration (where the concentrations are prior to inoculum addition):
Fe=68-83 micromoles per liter of culture
Zn=6-7 micromoles per liter of culture
Cu=0.1-0.2 micromoles per liter of culture
Ni=0.5-1 micromoles per liter of culture
On harvesting MT1, the production procedure as shown in FIG. 59 was followed. The operating parameters for the chromatography are known to one of ordinary skill in the art. The operating parameters for the affinity capture (step 3 of FIG. 59), affinity Flowthrough (step 5 of FIG. 59), AEX (step 8 of FIG. 59), and HIC (step 9 of FIG. 59) are outlined in Table 9-1. The proteolytic cleavage of aflibercept following affinity capture and filtration step was carried out using the procedure as outlined in Example 1.2.
TABLE 9-1
Affinity
Affinity
Steps
Capture
flowthrough
AEX
HIC
Resin
MabSelect
MabSelect
POROS 50
Capto Phenyl
SuRe
SuRe
HQ
HS
Load
30 g/L resin
30 g/L resin
40 g/L resin
100 g/L resin
pH 6.80-7.20
pH 8.30-8.50,
pH 4.40-4.60
1.90-2.10
7.50-10.50
mS/cm
mS/cm
Equilibra-
20 mM
26 mM Tris, 16
50 mM Tris
40 mM Tris, 30
tion
Sodium
mM Sodium
pH 8.30-8.50,
mM Sodium
Phosphate
Phosphate, 18
1.90-2.10
Citrate, 74 mM
pH
mM Acetate pH
mS/cm
Acetate pH
7.10-7.30,
6.90-7.10,
4.40-4.60,
2.60-3.20
2.00-4.00
7.50-10.50
mS/cm
mS/cm
mS/cm
Wash 1
10 mM
26 mM Tris, 16
50 mM Tris
40 mM Tris, 30
Sodium
mM Sodium
pH 8.30-8.50,
mM Sodium
Phosphate,
Phosphate, 18
1.90-2.10
Citrate, 74 mM
500 mM
mM Acetate pH
mS/cm
Acetate pH
NaCl pH
6.90-7.10,
4.40-4.60,
7.10-7.30,
2.00-4.00
7.50-10.50
40-50
mS/cm
mS/cm
mS/cm
Wash 2
20 mM
N/A
N/A
N/A
Sodium
Phosphate
pH
7.10-7.30,
2.60-3.20
mS/cm
Elution
40 mM
40 mM Acetic
N/A
N/A
Acetic
Acid pH 2.80-
Acid pH
3.20, 0.28-
2.80-3.20,
0.36 mS/cm
0.28-0.36
mS/cm
Regener-
500 mM
500 mM Acetic
2M Sodium
Proprietary
ation/Strip
Acetic
Acid, pH 2.25-
Chloride
buffer
1
Acid, pH
2.65, 0.90-
(NaCl)
2.25-2.65,
1.25 mS/cm
0.90-1.25
mS/cm
Regener-
N/A
N/A
1N Sodium
N/A
ation/Strip
Hydroxide
2
(NaOH)
Table 9-2 shows the color quantification of the pools obtained on performing various chromatographic steps. The color quantification was carried using samples from the pool having a protein concentration of 5 g/L.
Affinity Capture Pool refers to the eluate collected on performing the affinity capture step (step 3 of FIG. 59). Enzymatic Pool refers to the flowthrough collected on performing the enzymatic cleavage step (step 4 of FIG. 59). Affinity flowthrough Pool refers to the flowthrough collected on performing the affinity flowthrough step (step 5 of FIG. 59) and Affinity flowthrough Eluate refers to the eluate collected on performing the affinity flowthrough step (step 5 of FIG. 59). AEX Pool and AEX Strip refer to the flowthrough and stripped fractions obtained on performing anion exchange chromatography step (step 8 of FIG. 59). HIC Pool refers to the flowthrough collected on performing the hydrophobic interaction chromatography step (step 9 of FIG. 59).
Each step as seen in Table 9-2 shows a reduction in coloration (as observed from the reduction in b* values of the pools). For example, on performing affinity flowthrough chromatography, the flowthrough fraction has a b* value of 2.16 (reduced from a b* value of 2.52 for the flowthrough collected from the affinity capture step). The flowthrough and wash following the AEX separation further reduced the coloration, as observed by reduction in the b* value from 2.16 to 0.74. As expected, stripping the AEX column led to a sample with a yellow-brown color which was significantly more intense than the coloration from the flowthrough and wash following the AEX separation as seen from the b* values (8.10 versus 0.74). Lastly, a HIC step afforded a further reduction in color (the b* value can be normalized for 5 g/L protein concentration from the b* value obtained for HIC pool at 28.5 g/L protein concentration).
TABLE 9-2
Color Quantification of Samples at Various Production Steps
Sample
Conc. (g/L)
L*
a*
b*
Affinity Capture Pool
5.0 ± 0.1
98.75
−0.12
2.52
Enzymatic Cleavage Pool
5.0 ± 0.1
99.03
−0.07
1.61
Affinity flowthrough Pool
5.0 ± 0.1
98.95
−0.08
2.16
Affinity flowthrough Eluate
5.0 ± 0.1
98.92
−0.01
0.83
AEX Pool
5.0 ± 0.1
99.72
−0.03
0.74
AEX 2M NaCl Strip
5.0 ± 0.1
96.25
−0.42
8.10
HIC Pool
28.5
98.78
−0.28
3.11
(B) Optimized CDM (Low Cysteine, Low Metals and Increased Antioxidants Bioreactor)
The effect of lowering the concentration of cysteine, lowering the concentration of metals, and increasing antioxidants on coloration was evaluated using the following protocols:
Medium at day 0=CDM1
Cysteine was added at a cumulative concentration of 5-6 mM
Antioxidants were added to CDM1 to reach the following cumulative concentrations (where the concentrations are prior to inoculum addition):
Taurine=10 mM of culture
Glycine=10 mM of culture
Thioctic Acid=0.0024 mM of culture
Vitamin C (ascorbic acid)=0.028 mM of culture
Metals in Starting Medium are listed below for the 1× level.
Fe=68-83 micromoles per liter of culture
Zn=6-7 micromoles per liter of culture
Cu=0.1-0.2 micromoles per liter of culture
Ni=0.5-1 micromoles per liter of culture.
The reduction of all the metals included using 0.25× the concentrations noted above for the medium.
Upon harvesting of the MT1 sample, the production procedure as shown in FIG. 59 was followed. The operating parameters for the chromatography are known to one of ordinary skill in the art. The operating parameters for the affinity capture, affinity flowthrough, and HIC are outlined in Table 9-1. The proteolytic cleavage of aflibercept following affinity capture and filtration step was carried out using the procedure as outlined in Example 1.2.
Table 9-3 shows the color quantification of the pools obtained on performing the various chromatographic steps. The color quantification was carried using samples from the pool having a protein concentration of 5 g/L. The steps as seen in Table 9-3 afforded a similar production as seen for steps in Table 9-2.
TABLE 9-3
Color Quantification of Samples at Various Production Steps of MiniTrap
Sample
Conc. (g/L)
L*
a*
b*
Affinity Capture Pool
5.0 ± 0.1
99.18
−0.09
1.77
Enzymatic Cleavage Pool
5.0 ± 0.1
99.44
−0.06
1.17
Affinity flowthrough Pool
5.0 ± 0.1
99.32
−0.10
1.58
Affinity flowthrough Eluate
5.0 ± 0.1
99.74
−0.05
0.60
AEX Pool
5.0 ± 0.1
99.63
−0.07
0.50
AEX 2M NaCl Strip
5.0 ± 0.1
97.63
−0.49
6.10
HIC Pool
27.6
99.07
−0.29
2.32
Comparing Table 9-2 and Table 9-3, it is evident that the “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition” had lower color in affinity capture pool (b* value of 1.77) compared to the “Control Bioreactor Condition” (b* value 2.52).
An MT sample with a concentration of 160 g/L, where the MT is formed using the steps listed in Table 9-2 and Table 9-3, is predicted to have a b* value of 13.45 for the “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition” and a b* value of 17.45 for the “Control Bioreactor Condition.” A 23% reduction in color is achieved through optimization of the upstream media and feeds. Similarly, an MT sample with a concentration of 110 g/L, where the MT is formed using the steps listed in Table 9-2 and Table 9-3, is predicted to have a b* value of 9.25 for the “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition” and a b* value of 12 for the “Control Bioreactor Condition.”
To understand how each production unit operation contributes to color reduction, the b* value for each production process intermediate as a percentage of the color of affinity capture pool was calculated (Table 9-4).
TABLE 9-4
b* as
% of
Affinity
Conc.
Capture
Sample
(g/L)
b*
Δb*
Pool
Control
Affinity Capture Pool
5.0 ± 0.1
2.52
N/A
100.0
Bioreactor
Enzymatic Cleavage
5.0 ± 0.1
1.61
−0.91
63.8
Pool
Affinity flowthrough
5.0 ± 0.1
2.16
0.55
85.7
Pool
AEX Pool
5.0 ± 0.1
0.74
−1.42
29.4
HIC Pool
5.0 ± 0.1
0.55
−0.19
21.8
Low Cysteine,
Affinity Capture Pool
5.0 ± 0.1
1.77
N/A
100.0
Low Metals,
Enzymatic Cleavage
5.0 ± 0.1
1.17
−0.60
66.1
and Increased
Pool
Antioxidants
Affinity flowthrough
5.0 ± 0.1
1.58
0.41
89.2
Bioreactor
Pool
AEX Pool
5.0 ± 0.1
0.50
−1.08
28.2
HIC Pool
5.0 ± 0.1
0.42
−0.08
23.7
The AEX unit operation provides the most color reduction (1.08 to 1.42 change in b*) while the HIC unit operation provides some additional color reduction (0.08 to 0.19 change in b*). The unit operations evaluated overall remove 76.3%-78.2% of the color present in affinity capture pool.
The color of various production process intermediates for “Control Bioreactor Condition” and “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition” were also studied for the percentage of 2-oxo-histidines and percentage of oxo-tryptophans in the oligopeptides that were generated by protease digestion, as measured by mass spectrometry as shown in Table 9-5 and Table 9-6, respectively. The peptide mapping was performed as discussed in Example 3.
Referring to Table 9-5, on comparing the histidine oxidation levels in the pools in different production steps, it is evident that relative abundance of the percentage of histidine oxidation levels for MT formed reduces in the pool as the production process progresses. For example, for H209 in the “Control Bioreactor Condition”, the percent histidine oxidation level was 0.062 for the enzymatic cleavage pool and this was reduced to 0.029 for AEX flowthrough and further reduced to 0.020 for the HIC pool. Similarly, for H209 in the “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition”, the percent histidine oxidation level was 0.039 for the enzymatic cleavage pool and this was reduced to 0.023 for AEX flowthrough and further reduced to 0.016 for the HIC pool. Thus, the production strategy led to a reduction in percentage of histidine oxidation levels in MT. As the coloration reduced, presence of some of the oxidized residues in the sample also reduced. Similar to histidine oxidation, tryptophan oxidation levels were also tracked for the pools in different production steps for both the “Control Bioreactor Condition” and “Low Cysteine, Low Metals, and Increased Antioxidants Bioreactor Condition” (Table 9-6).
TABLE 9-5
Histidine Oxidation Levels (%)
Color
H19
H86
H95
H110
H145
H209
Fraction
(b*)
(+14)
(+14)
(+14)
(+14)
(+14)
(+14)
Control
Enzymatic
1.61
0.023
0.018
0.011
0.014
0.007
0.062
Bioreactor
Cleavage Pool
Condition
Affinity
2.16
0.030
0.027
0.018
0.015
0.011
0.067
flowthrough
Pool (AEX
Load)
Affinity
0.83
0.030
0.022
0.000
0.018
0.004
0.046
flowthrough
Eluate
AEX
0.74
0.026
0.025
0.013
0.016
0.010
0.029
flowthrough
AEX 2M
8.10
0.024
0.063
0.033
0.019
0.012
0.063
NaCl Strip
HIC Pool
0.55
0.018
0.009
0.002
0.021
0.005
0.020
Low
Enzymatic
1.17
0.019
0.017
0.009
0.014
0.008
0.039
Cysteine,
Cleavage Pool
Low Metals,
Affinity
1.58
0.026
0.025
0.013
0.014
0.010
0.043
and
flowthrough
Increased
Pool (AEX
Antioxidants
Load)
Bioreactor
Affinity
0.60
0.031
0.017
0.007
0.020
0.003
0.016
Condition
flowthrough
Eluate
AEX
0.50
0.020
0.022
0.009
0.014
0.010
0.023
flowthrough
AEX 2M
NaCl Strip
6.10
0.020
0.055
0.025
0.016
0.011
0.042
HIC Pool
0.42
0.013
0.009
0.002
0.017
0.003
0.016
TABLE 9-6
Tryptophan Oxidation Levels (%)
Color
W58
W58
W58
W58
W138
W138
W138
Fraction
(b*)
(+4)
(+16)
(+32)
(+48)
(+4)
(+16)
(+32)
Control
Enzymatic
1.61
0.006
0.032
0.289
0.000
0.020
1.093
0.106
Bioreactor
Cleavage
Condition
Pool
Affinity
2.16
0.016
0.055
0.327
0.000
0.017
0.771
0.111
flowthrough
Pool (AEX
Load)
Affinity
0.83
0.009
0.031
0.453
0.000
0.025
1.039
0.132
flowthrough
Eluate
AEX
0.74
0.014
0.038
0.283
0.000
0.023
0.720
0.120
flowthrough
AEX 2M
8.10
0.043
0.089
0.462
0.000
0.031
0.620
0.175
NaCl Strip
HIC Pool
0.55
0.037
0.126
0.413
0.000
0.020
0.656
0.274
Low
Enzymatic
1.17
0.009
0.027
0.239
0.001
0.027
1.026
0.136
Cysteine,
Cleavage
Low Metals,
Pool
and
Affinity
1.58
0.013
0.045
0.284
0.000
0.021
0.628
0.107
Increased
flowthrough
Antioxidants
Pool (AEX
Bioreactor
Load)
Condition
Affinity
0.60
0.003
0.026
0.421
0.021
0.025
1.032
0.132
flowthrough
Eluate
AEX
0.50
0.011
0.031
0.235
0.000
0.022
0.676
0.102
flowthrough
AEX 2M
6.10
0.034
0.073
0.478
0.000
0.032
0.635
0.169
NaCl Strip
HIC Pool
0.42
0.029
0.122
0.355
0.000
0.022
0.800
0.236
|
16996007
|
regeneron pharmaceuticals, inc.
|
USA
|
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 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Mar 1st, 2022 12:00AM
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Apr 6th, 2016 12:00AM
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https://www.uspto.gov?id=US11259510-20220301
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Humanized T cell mediated immune responses in non-human animals
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Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express a humanized T cell co-receptor (e.g., humanized CD4 and/or CD8 (e.g., CD8α and/or CD8β)), a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment and/or a human or humanized major histocompatibility complex that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and/or MHC II β chains) and/or MHC I (e.g., MHC I α) respectively, and optionally human or humanized β 2 microglobulin). Also provided are embryos, tissues, and cells expressing the same. Methods for making a genetically engineered animal that expresses at least one humanized T cell co-receptor (e.g., humanized CD4 and/or CD8), at least one humanized MHC that associates with the humanized T cell co-receptor (e.g., humanized MHC II and/or MHC I, respectively) and/or the humanized TCR are also provided. Methods for using the genetically engineered animals that mount a substantially humanized T cell immune response for developing human therapeutics are also provided.
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11259510
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1. A genetically modified mouse comprising in its genome
(a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide;
(b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide,
wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide,
wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide;
(c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II α polypeptide and a second nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptide,
wherein the chimeric human/mouse MHC II α polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide,
wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide;
(d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide,
wherein the chimeric human/mouse MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide; and
(e) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one human Vα segment and at least one human Jα segment, wherein the unrearranged T cell receptor (TCR) α variable region sequence is operably linked to a mouse TCRα constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unrearranged TCRβ variable region sequence is operably linked to a mouse TCRβ constant region sequence,
wherein the mouse expresses:
(A) the chimeric human/mouse CD4 co-receptor,
(B) a chimeric human/mouse CD8 co-receptor comprising the chimeric human/mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide,
(C) a chimeric human/mouse MHC II complex comprising the chimeric human/mouse MHC II_α polypeptide and the chimeric human/mouse MHC II β polypeptide, wherein the chimeric human/mouse MHC II complex is capable of binding the chimeric human/mouse CD4 co-receptor,
(D) the chimeric human/mouse MHC I polypeptide, wherein the chimeric human/mouse MHC I polypeptide is capable of binding the chimeric human/mouse CD8 co-receptor, and
(E) a T cell receptor comprising a humanized TCRα chain and a humanized TCRβ chain,
wherein the humanized TCRα chain is encoded by a rearranged human Vα/Jα sequence operably linked to the mouse TCRα constant region sequence, wherein the rearranged human Vα/Jα sequence is formed by rearrangement of the at least one human Vα segment and the at least one human Jα segment,
wherein the humanized TCRβ chain is encoded by a rearranged human Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ constant region sequence, wherein the rearranged human Vβ/Dβ/Jβ sequence is formed by rearrangement of the at least one human Vβ segment, the at least one Dβ segment, and the at least one human Jβ segment.
2. The genetically modified mouse of claim 1, comprising in its germline genome
(a) the first nucleotide sequence encoding the chimeric human/mouse CD4 co-receptor;
(b) the second nucleotide sequence encoding the chimeric human/mouse CD8α polypeptide and the third nucleotide sequence encoding the chimeric human/mouse CD8β polypeptide;
(c) the first nucleic acid sequence encoding the chimeric human/mouse MHC II α polypeptide and the second nucleic acid sequence encoding the chimeric human/mouse MHC II β polypeptide;
(d) the third nucleic acid sequence encoding the chimeric human/mouse MHC I polypeptide; and
(e) the unrearranged TCR α variable region sequence operably linked to the mouse TCRα constant region sequence and the unrearranged TCRβ variable region sequence operably linked to the mouse TCRβ constant region sequence.
3. The genetically modified mouse of claim 1, wherein
(a) the first nucleotide sequence is present at an endogenous CD4 locus;
(b) the second nucleotide sequence is present at an endogenous CD8α locus and the third nucleotide sequence is present at an endogenous CD8β locus;
(c) the first nucleic acid sequence is present at an endogenous MHC II α locus and the second nucleic acid sequence is present at an endogenous MHC II β locus;
(d) the third nucleic acid sequence is present at an endogenous MHC I locus;
(e) the unrearranged TCRα variable region sequence is present at an endogenous TCRα variable region locus and the unrearranged TCRβ variable region sequence is present at an endogenous TCRβ variable region locus, or
(f) any combination of (a)-(e).
4. The genetically modified mouse of claim 3, wherein
(a) the first nucleotide sequence is present at the endogenous CD4 locus and expressed under regulatory control of mouse CD4 promoter and regulatory elements;
(b) the second nucleotide sequence is present at the endogenous CD8α locus and expressed under regulatory control of mouse CD8α promoter and regulatory elements, and the third nucleotide sequence is present at an endogenous CD8β locus and expressed under regulatory control of mouse CD8β promoter and regulatory elements;
(c) the first nucleic acid sequence is present at the endogenous MHC II α locus and expressed under regulatory control of mouse MHC II α promoter and regulatory elements, and the second nucleic acid sequence is present at an endogenous MHC II β locus and expressed under regulatory control of mouse MHC II β promoter and regulatory elements;
(d) the third nucleic acid sequence is present at the endogenous MHC I locus and expressed under regulatory control of mouse MHC I promoter and regulatory elements; or
(e) any combination of (a)-(d).
5. The genetically modified mouse of claim 4, wherein
the chimeric human/mouse CD4 co-receptor comprises D1, D2 and D3 domains of the human CD4 polypeptide operably linked to D4, transmembrane, and cytoplasmic domains of the mouse CD4 polypeptide.
6. The genetically modified mouse of claim 1, wherein
(a) the human HLA class II α polypeptide is selected from the group consisting of any α chain of HLA-DR, HLA-DQ, and HLA-DP;
(b) the human HLA class II β polypeptide is selected from the group consisting of any β chain of HLA-DR, HLA-DQ, and HLA-DP;
(c) the human HLA class I polypeptide is selected from the group consisting of HLA-A, HLA B, and HLA-C gene; or
(d) any combination of (a)-(c).
7. The genetically modified mouse of claim 6, wherein the human HLA class II α polypeptide is the α chain of HLA-DR and the human HLA class II β polypeptide is the β chain of HLA-DR, and
wherein the human HLA class I polypeptide is HLA-A.
8. The genetically modified mouse of claim 7, wherein the chimeric human/mouse MHC II complex comprises α1, α2, β1, and β2 domains of a human HLA-DR2 protein.
9. The genetically modified mouse of claim 7, wherein the chimeric human/mouse MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA-A2 protein.
10. The genetically modified mouse of claim 1, wherein
(a) the unrearranged TCRα variable region sequence comprises a complete repertoire of human Vα gene segments and a complete repertoire of human Jα gene segments,
(b) the unrearranged TCRβ variable region sequence comprises a complete repertoire of human Vβ gene segments, a complete repertoire of human Dβ gene segments, and a complete repertoire of human Jβ gene segments, or
(c) the unrearranged TCRα variable region sequence comprises a complete repertoire of human Vα gene segments and a complete repertoire of human Jα gene segments and the unrearranged TCRβ variable region sequence comprises a complete repertoire of human Vβ gene segments, a complete repertoire of human Dβ gene segments, and a complete repertoire of human Jβ gene segments.
11. The genetically modified mouse of claim 1, wherein:
(i) an endogenous TCRα variable region locus (a) lacks all or substantially all functional endogenous Vα gene segments, (b) lacks all or substantially all functional endogenous Jα gene segments, or (c) lacks all or substantially all functional endogenous Vα gene segments and lacks all or substantially all functional endogenous Jα gene segment;
(ii) an endogenous TCRβ variable region locus (a) lacks all or substantially all functional endogenous Vβ gene segments, (b) lacks all or substantially all functional endogenous Dβ gene segments, (c) lacks all or substantially all functional endogenous Jβ gene segments, or (d) lacks all or substantially all functional endogenous Vβ gene segments, lacks all or substantially all functional Dβ gene segments, and lacks all or substantially all functional Jβ gene segments; or
(iii) the endogenous TCRα variable region locus lacks all or substantially all functional endogenous Vα gene segments and lacks all or substantially all functional endogenous Jα gene segment, and the endogenous TCRβ variable region locus lacks all or substantially all functional endogenous Vβ gene segments, lacks all or substantially all functional Dβ gene segments, and lacks all or substantially all functional Jβ gene segments.
12. The genetically modified mouse of claim 1, wherein
(a) the first nucleotide sequence comprises a sequence encoding D1, D2 and D3 domains of the human CD4 polypeptide that, at an endogenous CD4 locus, (i) replaces a sequence encoding D1, D2 and D3 domains of a mouse CD4 polypeptide and (ii) is operably linked to mouse CD4 D4, transmembrane and cytoplasmic domains encoding sequences;
(b) the second nucleotide sequence comprises a sequence encoding the IgV-like domain of the human CD8α polypeptide that, at an endogenous CD8α locus, (i) replaces a sequence encoding the IgV-like domain of a mouse CD8α polypeptide and (ii) is operably linked to mouse CD8α transmembrane and cytoplasmic domain encoding sequences, and
the third nucleotide sequence comprises a sequence encoding the IgV-like domain of the human CD8β polypeptide that, at an endogenous CD8β locus, (i) replaces a sequence encoding the IgV-like domain of a mouse CD8β polypeptide and (ii) is operably linked to mouse CD8β transmembrane and cytoplasmic domain encoding sequences;
(c) the first nucleic acid sequence comprises a sequence encoding α1 and α2 domains of the human HLA class II α polypeptide that, at an endogenous MHC II α locus, (i) replaces a sequence encoding α1 and α2 domains of a mouse MHC II α polypeptide and (ii) is operably linked to mouse MHC II α polypeptide transmembrane and cytoplasmic domain encoding sequences, and
the second nucleic acid comprises a sequence encoding β1 and β2 domains of the human HLA class II β polypeptide that, at an endogenous MHC II β locus, (i) replaces a sequence encoding β1 and β2 domains of a mouse MHC II β polypeptide and (ii) is operably linked to mouse MHC II β polypeptide transmembrane and cytoplasmic domain encoding sequences;
(d) the third nucleic acid sequence comprises a sequence encoding α1, α2, and α3 domains of the human HLA class I polypeptide that, at an endogenous MHC I locus, (i) replaces a sequence encoding α1, α2, and α3 domains of a mouse MHC I polypeptide and (ii) is operably linked to mouse MHC I polypeptide transmembrane and cytoplasmic domain encoding sequences;
(e) the unrearranged TCRα variable region sequence replaces one or more endogenous Vα and/or Jα gene segments at an endogenous TCRα variable region locus and the unrearranged TCRβ variable region sequence replaces one or more endogenous Vβ, Dβ and/or Jβ gene segments at an endogenous TCRβ variable region locus; or
(f) any combination of (a)-(e).
13. The genetically modified mouse of claim 1, wherein the mouse does not express:
(a) a functional mouse CD4 co-receptor from an endogenous CD4 locus;
(b) a functional mouse CD8 co-receptor from an endogenous CD8 locus;
(c) a mouse TCRα variable domain from an endogenous TCRα locus;
(d) a mouse TCRβ variable domain from an endogenous TCRβ locus;
(e) on a cell surface, an extracellular domain of a classical MHC class I polypeptide from an endogenous MHC I locus;
(f) on a cell surface, an extracellular domain of a classical MHC class II polypeptide from an endogenous MHC II locus; or
(e) any combination of (a)-(f).
14. The genetically modified mouse of claim 1, further comprising a β2 microglobulin locus comprising a sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence, wherein the mouse expresses a human or humanized β2 microglobulin polypeptide.
15. The genetically modified mouse of claim 14, wherein the mouse does not express a functional endogenous mouse β2 microglobulin polypeptide from an endogenous mouse β2 microglobulin locus.
16. The genetically modified mouse of claim 14, wherein the sequence encoding the polypeptide comprising the human β2 microglobulin amino acid sequence is operably linked to mouse β2 microglobulin regulatory elements.
17. The genetically modified mouse of claim 14, wherein the β2 microglobulin locus comprises a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of a human β2 microglobulin gene.
18. The genetically modified mouse of claim 17, wherein the β2 microglobulin locus further comprises a nucleotide sequence set forth in exon 1 of a mouse β2 microglobulin gene.
19. The genetically modified mouse of claim 1, wherein the mouse expresses:
(a) at least 50% of all functional human TCRVα gene segments;
(b) at least 50% of all functional human TCRVβ gene segments; or
(c) at least 50% of all functional human TCRVα gene segments and at least 50% of all functional human TCRVβ gene segments.
20. A method of making the genetically modified mouse of claim 1 comprising modifying the genome of the mouse to comprise:
(a) the first nucleotide sequence encoding the chimeric human/mouse CD4 co-receptor;
(b) the second nucleotide sequence encoding the chimeric human/mouse CD8α polypeptide and the third nucleotide sequence encoding the chimeric human/mouse CD8β polypeptide;
(c) the first nucleic acid sequence encoding the chimeric human/mouse MHC II α polypeptide and the second nucleic acid sequence encoding the chimeric human/mouse MHC II β polypeptide;
(d) the third nucleic acid sequence encoding the chimeric human/mouse MHC I polypeptide; and
(e) the unrearranged TCR α variable region sequence operably linked to the mouse TCRα constant region sequence and the unrearranged TCRβ variable region sequence operably linked to the mouse TCRβ constant region sequence; and
(f) optionally, a β2 microglobulin locus comprising a sequence encoding a human or humanized β2 microglobulin polypeptide.
21. The method of claim 20, wherein modifying the genome comprises homologous recombination in one or more mouse ES cell(s) such that the first, second, and third nucleotide sequences; the unrearranged TCRα variable region sequence and unrearranged TCRβ variable region sequence; the first, second, and third nucleic acid sequences; and optionally the β2 microglobulin locus; are added, in any order, into the genome of the one or more mouse ES cell(s).
22. The method of claim 21, further comprising generating a mouse from the one or more mouse ES cell(s).
23. A method of obtaining any one of: (1) a TCR protein that is specific for an antigen and comprises a human TCR variable domain, (2) the human TCR variable domain and (3) a nucleic acid sequence encoding the human TCR variable domain,
the method comprising
isolating from a mouse according to claim 1 any one of:
(1) a T cell expressing a TCR protein that is specific for an antigen and comprises both a human TCR α variable domain and a human TCR β variable domain,
(2) either or both (i) the human TCR α variable domain and (ii) the human TCR β variable domain, and
(3) either or both (i) a nucleic acid sequence encoding the human TCR α variable domain and (ii) a nucleic acid sequence encoding the human TCR β variable domain.
24. The method of claim 23, wherein the method comprises isolating from the mouse a nucleic acid sequence encoding the human TCR α variable domain and a nucleic acid sequence encoding the human TCR β variable domain, the method further comprising
culturing a host cell in sufficient conditions for expressing (i) the nucleic acid sequence encoding the human TCR α variable domain in operable linkage with a human TCR α constant region and (ii) the nucleic acid sequence encoding the human TCR β variable domain in operable linkage with a human TCR β constant region,
wherein the nucleic acid sequences encoding the human TCR α variable domain and the human TCR β variable domain are on the same or different expression vectors.
25. The method of claim 23, wherein the antigen is a tumor antigen, a viral antigen, or a bacterial antigen.
26. A genetically modified mouse embryonic stem cell comprising in its genome
(a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide,
(b) a second nucleotide sequence and a third nucleotide sequence respectively encoding a chimeric human/mouse CD8α polypeptide and a chimeric human/mouse CD8β polypeptide,
wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide,
wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide, and
(c) a first nucleic acid sequence and a second nucleic acid sequence respectively encoding a chimeric human/mouse MHC II α polypeptide and a chimeric human/mouse MHC II β polypeptide,
wherein the chimeric human/mouse MHC II α polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide,
wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide,
(d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide,
wherein the chimeric human/mouse MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide, and
(e) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one human Vα segment and at least one human Jα segment, wherein the unrearranged TCR α variable region sequence is operably linked to a mouse TCRα constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unrearranged TCRβ variable region sequence is operably linked to a mouse TCRβ constant region sequence.
27. The genetically modified mouse embryonic stem cell of claim 26, wherein
(a) the first nucleotide sequence is present at an endogenous CD4 locus;
(b) the second nucleotide sequence is present at an endogenous CD8α locus, and the third nucleotide sequence is present at an endogenous CD8β locus;
(c) the first nucleic acid sequence is present at an endogenous MHC II α locus and the second nucleic acid sequence is present at an endogenous MHC II β locus;
(d) the third nucleic acid sequence is present at an endogenous MHC I locus; and/or
(e) the unrearranged TCRα variable region sequence is present at an endogenous TCRα variable region locus and the unrearranged TCRβ variable region sequence is present at an endogenous TCRβ variable region locus.
28. The genetically modified mouse embryonic stem cell of claim 27, wherein
(a) the first nucleotide sequence is present at the endogenous CD4 locus and operably linked to mouse CD4 promoter and regulatory elements;
(b) the second nucleotide sequence is present at the endogenous CD8α locus and operably linked to mouse CD8α promoter and regulatory elements, and the third nucleotide sequence is present at the endogenous CD8β locus and operably linked to mouse CD8β promoter and regulatory elements;
(c) the first nucleic acid sequence is present at the endogenous MHC II α locus and operably linked to mouse MHC II α promoter and regulatory elements and the second nucleic acid sequence is present at the endogenous MHC II β locus and operably linked to mouse MHC II β promoter and regulatory elements; and/or
(d) the third nucleic acid sequence is present at the endogenous MHC I locus and operably linked to mouse MHC I promoter and regulatory elements.
29. The genetically modified mouse embryonic stem cell of claim 26, wherein
(a) the chimeric human/mouse CD4 co-receptor comprises D1, D2 and D3 domains of the human CD4 polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD4 polypeptide, and/or
(b) the chimeric human/mouse CD8α polypeptide comprises an extracellular portion of the human CD8α polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide comprises an extracellular portion of the human CD8β polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8β polypeptide.
30. A mouse embryonic stem (ES) cell made by a method comprising the steps of
adding into the genome of the ES cell by homologous recombination in any order
(a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide,
(b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide,
wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide,
wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide,
(c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II α polypeptide and a second nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptide,
wherein the chimeric human/mouse MHC II α polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide,
wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide,
(d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide,
wherein the chimeric human/mouse MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide, and
(e) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one human Vα segment and at least one human Jα segment, wherein the unrearranged TCR α variable region sequence is operably linked to a mouse TCRα constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unrearranged TCR β variable region sequence is operably linked to a mouse TCRβ constant region sequence.
31. The genetically modified mouse of claim 4, wherein the chimeric human/mouse CD8α polypeptide comprises an extracellular portion of the human CD8α polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide comprises an extracellular portion of the human CD8β polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8β polypeptide.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application filed under 35 U.S.C. § 371 of PCT Application No. PCT/US2016/026260 (filed Apr. 6, 2016), which claims priority to U.S. Provisional Application Ser. Nos. 62/143,687 (filed Apr. 6, 2015), 62/158,804 (filed May 8, 2015), and 62/186,935 (filed Jun. 30, 2015), each applications of which are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 2016-04-06-10145WO01-SEQ-LIST_ ST25.txt, created on Apr. 6, 2016, and having a size of 56.7 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
TECHNICAL FIELD
The present invention relates to a non-human animals (e.g., rodents, e.g., mice or rats) capable of mounting substantially human(ized) T cell mediated immune responses and expressing (i) one or more human(ized) T cell co-receptor(s) (e.g., CD4 and/or CD8 (e.g., CD8α, and/or CD8β)), (ii) one or more human(ized) major histocompatibility complex(es) that associates with the one or more human(ized) T cell co-receptor(s) (e.g., MHC II (e.g., MHC α and/or MHC II β) and/or MHC I (e.g., MHC I α and/or β2 microglobulin)) and/or (iii) a human(ized) T cell receptor (TCR) (e.g., TCRα and/or TCRβ); embryos, tissues, cells and/or nucleic acids isolated from the non-human animals; methods of making the non-human animals; and methods of using the non-human animals for the development of human therapeutics.
BACKGROUND OF THE INVENTION
In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptor also referred to as TCR). These foreign antigens are presented on the surface of cells as peptide fragments by specialized proteins, generically referred to as major histocompatibility complex (MHC) molecules, and specifically referred to as human leukocyte antigen (HLA) in humans. During a T cell-mediated response, antigens presented by MHC molecules are recognized by a T cell receptor. However, more than T cell receptor recognition of MHC-antigen complex is required for an effective immune response. The binding of a T cell co-receptor molecule (e.g., CD4 or CD8) to an invariant portion of MHC is also required.
T cells come in several varieties, including helper T cells and cytotoxic T cells. Helper T cells express co-receptor CD4 and recognize antigens bound to MHC II molecules. CD4+ T cells activate other effector cells in the immune system, e.g., MHC II expressing B cells to produce antibody, MHC II expressing macrophages to destroy pathogens, etc. The binding of CD4 and T cell receptor to the same MHC II-presented foreign antigen makes a T cell significantly more sensitive to that antigen.
In contrast, cytotoxic T cells (CTLs) express co-receptor CD8 and recognize foreign antigens bound to MHC I molecules. CTLs are specialized to kill any cell that bears an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell displays peptides derived from cellular proteins not normally present (e.g., of viral, tumor, or other non-self origin), such peptides are recognized by CTLs, which become activated and kill the cell displaying the peptide. Similar to CD4, engagement of CD8 makes CTLs more sensitive to MHC I-presented antigen.
Not all antigens will provoke T cell activation due to tolerance mechanisms. However, in some diseases (e.g., cancer, autoimmune diseases) peptides derived from self-proteins become the target of the cellular component of the immune system, which results in destruction of cells presenting such peptides. There has been significant advancement in recognizing antigens that are clinically significant (e.g., antigens associated with various types of cancer) and/or TCR sequences that bind the clinically significant antigens. However, in order to improve identification and selection of clinically significant peptides that will provoke a suitable response in a human T cell and/or of TCR capable of binding the clinically significant antigens (e.g., for adoptive immunotherapy of cancer, T cell vaccination for autoimmunity, etc.), there remains a need for in vivo and in vitro systems that mimic aspects of human immune system. Thus, there is a need for biological systems (e.g., genetically modified non-human animals and cells) that can display components of a human immune system, particularly components of the T cell immune response.
SUMMARY OF THE INVENTION
As disclosed herein, the thymus of genetically modified non-human animals comprising a substantially humanized T cell immune system has similar absolute numbers of thymocytes and CD3+ T cells as control animals. Additionally, these cells show comparable development into single positive T cells to control animals and are capable of generating a robust human cellular response against antigen, e.g., a viral antigen. The human cellular response of the non-human animals generally comprises activated non-human T cells expressing human or humanized T cell receptor (TCR) variable domains that recognize antigen presented in the peptide binding cleft formed by human leukocyte antigen (HLA) extracellular domains, which may be expressed on the surface of non-human antigen presenting cells. In some embodiments, the substantially humanized T cell immune system comprises
(A) a non-human T cell that expresses
(i) a T cell co-receptor polypeptide comprising a part or all of the extracellular portion of a human T cell co-receptor, e.g., a T cell co-receptor polypeptide comprising one or more human T cell co-receptor extracellular domains such that the T cell co-receptor polypeptide is capable of associating with and/or associates with
(a) one or more extracellular domains of a human or humanized HLA molecule (e.g., a first human HLA extracellular domain that is a binding site for the T cell co-receptor polypeptide and/or a second human HLA extracellular domain that forms a peptide binding cleft, e.g., with a third human HLA extracellular domain),
(b) an extracellular domain of a human or humanized TCR variable domain (e.g., a human or humanized TCRα variable domain and/or a human or humanized TCRβ variable domain that is respectively encoded by at least one human TCRα and/or TCRβ variable region gene segment), and/or
(c) an extracellular domain of a human TCR constant domain, and
(ii) a T cell receptor (TCR) comprising at least a human TCR variable domain; and optionally
(B) a non-human antigen presenting cell that presents antigen in the context of human HLA, e.g., a non-human antigen presenting cell that expresses on its cell surface at least one MHC molecule that comprises a peptide binding cleft formed by two human HLA extracellular domains, and is capable of activating and/or activates the non-human T cell.
In one aspect, the non-human T cell and the non-human antigen presenting cell are found in or isolated from the same non-human animal.
Accordingly, provided herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express
(A) a human or humanized T cell co-receptor (e.g., human or humanized CD4 and/or human or humanized CD8 (e.g., human or humanized CD8α and/or human or humanized CD8β)),
(B) a human or humanized major histocompatibility complex that associates with the human or humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., human or humanized MHC II α and/or human or humanized MHC IIβ) that binds the human or humanized CD4 and/or human or humanized MHC I (e.g., human or humanized MHC Iα, and optionally human or humanized β2 microglobulin) that binds the human or humanized CD8), and/or
(C) a human or humanized T cell receptor (TCR);
as well as embryos, tissues, and cells expressing the same, and nucleic acids encoding the same. Also provided are methods of making and using the disclosed non-human animals.
In one aspect, provided is a genetically modified non-human animal, comprising
(A) a humanized CD4 co-receptor and/or a humanized CD8 co-receptor comprising a humanized CD8α polypeptide and a humanized CD8β polypeptide (e.g., the non-human animal comprises, e.g., in its germline genome, first nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, and/or a second nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide),
wherein each humanized T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, e.g., wherein the humanized CD4 co-receptor comprises at least transmembrane and cytoplasmic domains of a non-human CD4 co-receptor and/or the humanized CD8 co-receptor comprises at least transmembrane and cytoplasmic domains of non-human CD8α and non-human CD8β polypeptides,
wherein each chimeric T cell co-receptor polypeptide comprises part or all of an extracellular portion of a human T cell co-receptor, e.g., one or more extracellular domains of a human T cell co-receptor, e.g., at least an extracellular domain of a human T cell co-receptor that associates with an HLA molecule, e.g., wherein the humanized CD4 co-receptor comprises the extracellular portion (or parts thereof, e.g., extracellular domain(s)) of human CD4 that is responsible for interacting with MHC II, T cell receptor variable domains, T cell receptor constant domains, or a combination thereof, and/or e.g., wherein the humanized CD8 co-receptor comprises the extracellular portions (or parts thereof, e.g., extracellular domains) of human CD8α and human CD8β that is responsible for interacting with MHC I, T cell receptor variable domains, T cell receptor constant domains, or a combination thereof;
(B) a human(ized) TCR (e.g., the non-human animal comprises, e.g., in its germline genome, an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence); and optionally,
(C) a human(ized) MHC II complex that associates with the humanized CD4 co-receptor and/or a human(ized) MHC I complex that associates with the humanized CD8 co-receptor (e.g., the non-human animal comprises, e.g., in its germline genome, first nucleic acid sequence encoding a chimeric human/non-human MHC IIα polypeptide and a second nucleic acid sequence encoding a chimeric human/non-human MHC IIβ polypeptide, and/or a third nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide),
wherein each chimeric MHC polypeptide comprises at least an extracellular portion (or part thereof) of a human MHC polypeptide (e.g., HLA polypeptide) that, either alone (e.g., MHC I) or when complexed with another chimeric MHC polypeptide (e.g., MHC II α and MHC II β) is respectively capable of associating with the human(ized) CD8 co-receptor or human(ized)CD4 co-receptor and presenting peptide in the context of HLA, e.g., wherein a humanized MHC II complex comprises (i) a chimeric human/non-human MHC II α polypeptide comprising α1 and β2 domains of a human HLA class II α polypeptide and the transmembrane and cytoplasmic domains of a non-human HLA class II α polypeptide and (ii) a chimeric human/non-human MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide the transmembrane and cytoplasmic domains of a non-human HLA class II β polypeptide and/or wherein a humanized MHC I complex comprises α1, α2, and α3 domains of a human MHC I polypeptide, and optionally a human(ized) β2 microglobulin.
In some embodiments, the non-human animal comprises
(A) a humanized CD4 co-receptor and a humanized CD8 co-receptor comprising a humanized CD8α polypeptide and a humanized CD8β polypeptide (e.g., the non-human animal comprises, e.g., in its germline genome, first nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, a second nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide),
wherein each humanized T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, e.g., wherein the humanized CD4 co-receptor comprises at least transmembrane and cytoplasmic domains of a non-human CD4 co-receptor and the humanized CD8 co-receptor comprises at least transmembrane and cytoplasmic domains of non-human CD8α and non-human CD8β polypeptides,
wherein each chimeric T cell co-receptor polypeptide comprises part or all of an extracellular portion of a human T cell co-receptor, e.g., one or more extracellular domains of a human T cell co-receptor, e.g., at least an extracellular domain of a human T cell co-receptor that associates with an HLA molecule, e.g., wherein the humanized CD4 co-receptor comprises the extracellular portion (or parts thereof, e.g., extracellular domain(s)) of human CD4 that is responsible for interacting with MHC II, T cell receptor variable domains, T cell receptor constant domains, or a combination thereof, and/or e.g., wherein the humanized CD8 co-receptor comprises the extracellular portions (or parts thereof, e.g., extracellular domains) of human CD8α and human CD8β that is responsible for interacting with MHC I, T cell receptor variable domains, T cell receptor constant domains, or a combination thereof;
(B) a humanized TCR (e.g., the non-human animal comprises, e.g., in its germline genome, an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence); and
(C) a humanized MHC II complex that associates with the humanized CD4 co-receptor and a humanized MHC I complex that associates with the humanized CD8 co-receptor (e.g., the non-human animal comprises, e.g., in its germline genome, first nucleic acid sequence encoding a chimeric human/non-human MHC IIα polypeptide, a second nucleic acid sequence encoding a chimeric human/non-human MHC IIβ polypeptide and a third nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide),
wherein each chimeric MHC polypeptide comprises at least an extracellular portion (or part thereof) of a human MHC polypeptide (e.g., HLA polypeptide) that, either alone (e.g., MHC I) or when complexed with another chimeric MHC polypeptide (e.g., MHC II α and MHC II β) is respectively capable of associating with the humanized CD8 co-receptor or humanized CD4 co-receptor and presenting peptide in the context of HLA, e.g., wherein a humanized MHC II complex comprises (i) a chimeric human/non-human MHC II α polypeptide comprising α1 and β2 domains of a human HLA class II α polypeptide and the transmembrane and cytoplasmic domains of a non-human HLA class II α polypeptide and (ii) a chimeric human/non-human MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide the transmembrane and cytoplasmic domains of a non-human HLA class II β polypeptide and (iii) a humanized MHC I complex comprises α1, α2, and α3 domains of a human MHC I polypeptide, and optionally a human(ized) β2 microglobulin (e.g., the non-human animal further comprises a β2 microglobulin locus encoding a polypeptide comprising a human β2 microglobulin amino acid sequence, or a portion thereof).
In some embodiments, the first nucleotide sequence encoding a chimeric T cell CD4 co-receptor polypeptide is present at an endogenous CD4 T cell co-receptor locus, and/or the second nucleotide sequence encoding a chimeric T cell CD8α co-receptor polypeptide is present at an endogenous CD8α T cell co-receptor locus and the third nucleotide sequence encoding a chimeric T cell CD8β co-receptor polypeptide is present at an endogenous CD8β T cell co-receptor locus. Additional embodiments include a chimeric human/non-human CD4 polypeptide encoded by the gene set forth in FIG. 5A (e.g., wherein the human portion of the resulting chimeric human/non-human CD4 T cell co-receptor polypeptide comprises at least human Ig1, human Ig2 and human Ig3 domains, otherwise respectively referred to as D1, D2 and D3 domains) and/or a chimeric CD8 co-receptor encoded by the genes set forth in FIG. 5B (e.g., wherein the human portion of the chimeric CD8 co-receptor comprises all or substantially all of the extracellular portion of a human CD8 polypeptide (e.g., CD8α and/or CD8β), including human immunoglobulin V (IgV)-like α and β domains. In some embodiments, the human portion of the chimeric CD4 T cell co-receptor polypeptide comprises one or more extracellular domains of a human CD4 polypeptide (e.g., D1, D2, D3, D4, or any combination thereof) and the non-human portion of the chimeric CD4 T cell co-receptor polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD4 T cell co-receptor, the human portion of the chimeric CD8α polypeptide comprises an extracellular domain (e.g., an IgV-like domain) of a human CD8α polypeptide and the non-human portion of the chimeric CD8α polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD8α polypeptide, and/or the human portion of the CD8β polypeptide comprises an extracellular domain (e.g., an IgV-like domain) of the human CD8β polypeptide and the non-human portion of the chimeric CD8β T cell co-receptor polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD8β polypeptide.
In some embodiments, the first nucleic acid sequence encoding the human(ized) MHC II α is present at an endogenous non-human MHC II α locus and the second nucleic acid sequence encoding the human(ized) MHC II β is present at an endogenous non-human MHC II β locus, and/or the third nucleic acid sequence encoding the human(ized) MHC I is present at an endogenous non-human MHC I locus. In one aspect, the human(ized) MHC IIα polypeptide comprises the extracellular portion (or part thereof) of a human MHD IIα polypeptide (e.g., an HLA class IIα polypeptide), the human(ized) MHC IIβ polypeptide comprises the extracellular portion (or part thereof) of a human MHC IIβ polypeptide (e.g., an HLA class Iβ polypeptide) and/or the human(ized) MHC I polypeptide comprises the extracellular portion (or part thereof) of a human MHC I polypeptide (e.g., an HLA class I polypeptide). In some embodiments, the humanized MHC II α polypeptide comprises human MHC II α 1 and α2 domains, the humanized MHC II β polypeptide comprises human MHC II β1 and β2 domains and/or the humanized MHC I polypeptide comprises human MHC I α1, α2, and α3 domains. In some embodiments, the first nucleic acid sequence encoding the chimeric human/non-human MHC II α polypeptide is expressed under regulatory control of endogenous non-human MHC II α promoter and regulatory elements, the second nucleic acid sequence encoding the chimeric human/non-human MHC II β polypeptide is expressed under regulatory control of endogenous non-human MHC II β promoter and regulatory elements, and/or the third nucleic acid sequence encoding the chimeric human/non-human MHC I polypeptide is expressed under regulatory control of an endogenous non-human MHC I promoter and regulatory elements. In additional embodiments, a non-human portion of the chimeric human/non-human MHC II α polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II α polypeptide, a non-human portion of the chimeric human/non-human MHC II β polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II β polypeptide and/or a non-human portion of the chimeric human/non-human MHC I polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide. Embodiments include non-human animals wherein the human portion of the proteins of chimeric human/non-human MHC II complex is derived from corresponding human HLA class II proteins selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP and/or wherein the human portion of the third chimeric human/non-human MHC I polypeptide is derived from human HLA-A, human HLA-B, or human HLA-C. As non-limiting examples, in some embodiments, the chimeric MHC II α polypeptide comprises the extracellular portion, or a part thereof, of a HLA-DRα protein, a HLA-DQ α protein, or a HLA-DP α protein, the chimeric MHC II β polypeptide comprises the extracellular portion, or a part thereof, of a HLA-DRβ protein, a HLA-DQ β protein, or a HLA-DP β protein, and/or the chimeric MHC I polypeptide comprises the extracellular portion, or a part thereof, of a human HLA-A protein, a human HLA-B protein, or a human HLA-C protein. Non-human animals are also provided, wherein the human portion of the chimeric human/non-human MHC II proteins derived from corresponding human HLA-DR proteins, e.g., the human portion of the human/non-human MHC II α polypeptide comprises α1 and β2 domains of the α chain of HLA-DR2 and the human portion of the human/non-human MHC II β polypeptide comprises β1 and β2 domains of the β chain of HLA-DR2 and/or wherein the human portion of the MHC I polypeptide is derived from a human HLA-A polypeptide, e.g., the human portion of the human/non-human MHC I polypeptide comprises the α1, α2, and α3 domains of a human HLA-A2 polypeptide, e.g., the α1, α2, and α3 domains of a human HLA-A2.1 polypeptide. Non-human animals wherein the non-human portions of the MHC II complex are derived from a murine H-2E encoding sequence and/or wherein the non-human portions of the MHC I polypeptide is derived from a murine H-2K encoding sequence are also provided. For example, the chimeric MHC II α polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2E α polypeptide, the chimeric MHC II β polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2E β polypeptide, and the chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2K polypeptide.
In some embodiments, the unrearranged TCRα variable gene locus is present at an endogenous TCRα variable gene locus and the unrearranged TCRβ variable gene locus is present at an endogenous TCRβ variable gene locus. In some aspects, the unrearranged TCRα variable gene locus comprises a complete repertoire of human unrearranged Vα gene segments and a complete repertoire of human unrearranged Jα gene segments and/or the unrearranged TCRβ variable gene locus comprises a complete repertoire of human unrearranged Vβ gene segments, a complete repertoire of human unrearranged Dβ gene segments and a complete repertoire of human unrearranged Jβ gene segments. In some embodiments, the human unrearranged Vα and Jα gene segments rearrange to form a rearranged human Vα/Jα sequence and/or the human unrearranged Vβ Dβ and Jβ gene segment rearrange to form a rearranged human Vβ/Dβ/Jβ sequence. In some embodiments, a non-human animal as disclosed herein expresses a T cell receptor comprising a human TCRα variable region and/or a human TCRβ variable region on the surface of a T cell. In some embodiments, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence and/or endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence, e.g., the animal may lack a functional endogenous non-human TCRα variable locus and/or the animal may lack a functional endogenous non-human TCRβ variable locus, e.g., the animal comprises (a) a deletion of all or substantially all functional endogenous Vα gene segments, (b) a deletion of all or substantially all functional endogenous Jα gene segments, (c) a deletion of all or substantially all functional endogenous Vβ gene segments, (d) a deletion of all or substantially all functional endogenous Dβ gene segments, (e) a deletion of all or substantially all functional endogenous Jβ gene segments, and/or (f) a combination thereof. In some embodiments, the endogenous non-human TCRα variable locus lacks all or substantially all functional endogenous Vα gene segments and/or lacks all or substantially all functional endogenous Jα gene segments; and/or the endogenous non-human TCRβ variable locus (a) lacks all or substantially all functional endogenous Vβ gene segments, (b) lacks all or substantially all functional endogenous Dβ gene segments, (c) lacks all or substantially all functional endogenous Jβ gene segments, or (d) any combination of (a), (b), and (c)
In some embodiments, the first, second and/or third nucleotide sequence(s) respectively encoding the chimeric T cell CD4, CD8α and/or CD8 β co-receptor polypeptide(s) is present at endogenous T cell co-receptor loci, e.g., endogenous CD4, CD8α and/or CD8 β co-receptor loci respectively; the unrearranged TCRα variable gene locus is present at an endogenous TCRα variable gene locus; the unrearranged TCRβ variable gene locus is present at an endogenous TCRβ variable gene locus; and/or the first, second and/or third nucleic acid sequence(s) respectively encoding the chimeric MHC II α, MHC II β, and/or MHC I polypeptide(s) is present at endogenous MHC loci; e.g., MHC II α, MHC II β, and/or MHC I loci, respectively. In some embodiments, the nucleotide sequence(s) encoding the chimeric T cell co-receptor(s), the unrearranged TCRα variable gene locus, the unrearranged TCRβ variable gene locus and/or the nucleic acid sequence(s) encoding the chimeric MHC molecule(s) may be operably linked to non-human promoter and regulatory sequences. For example, the first nucleotide sequence may be expressed under regulatory control of endogenous non-human CD4 promoter and regulatory elements, the second nucleotide sequence may be expressed under regulatory control of endogenous non-human CD8α promoter and regulatory elements, and and/or the third nucleotide sequence may expressed under regulatory control of endogenous non-human CD8β promoter and regulatory elements; the unrearranged TCRα variable gene locus may be expressed under regulatory control of endogenous TCRα (variable) regulatory and promoter elements and the unrearranged TCRβ variable gene locus may be expressed under regulatory control of endogenous TCRβ (variable) regulatory and promoter elements; the first nucleic acid sequence may be expressed under regulatory control of endogenous non-human MHC II α promoter and regulatory elements, the second nucleic acid sequence may be expressed under regulatory control of endogenous non-human MHC II β promoter and regulatory elements, and the third nucleic acid sequence may expressed under regulatory control of an endogenous non-human MHC I promoter and regulatory elements.
In some embodiments, a nucleotide sequence encoding the extracellular portion (or parts thereof, e.g., D1, D2, D3 and/or D4) of the human CD4 polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., D1, D2, D3 and/or D4) of an endogenous non-human (mouse) CD4 co-receptor polypeptide, and may be operably linked to endogenous non-human (mouse) CD4 transmembrane and cytoplasmic domain encoding sequences, at the endogenous non-human (mouse) CD4 co-receptor locus; a nucleotide sequence encoding all or part of the extracellular portion of a human CD8α polypeptide replaces a sequence encoding all or part of an extracellular portion of an endogenous non-human (mouse) T cell CD8α polypeptide, and may be operably linked to endogenous non-human (mouse) CD8α transmembrane and cytoplasmic domain encoding sequences, at the endogenous non-human (mouse) CD8α locus; a nucleotide sequence encoding all or part of the extracellular domain of a human CD8β polypeptide replaces a sequence encoding all or part of an extracellular domain of an endogenous non-human (mouse) T cell CD8β polypeptide and may be operably linked to endogenous non-human CD8β transmembrane and cytoplasmic domain encoding sequences, at the endogenous CD8β locus; an unrearranged TCRα variable gene locus replaces one or more endogenous Vα and/or Jα gene segments at an endogenous non-human (mouse) TCRα variable gene locus; an unrearranged TCRβ variable gene locus replaces one or more endogenous Vβ, Dβ and/or Jα gene segments at an endogenous non-human (mouse) TCRβ variable gene locus; a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., α1 and β2 domains) of a human MHC II α polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., α1 and β2 domains) of an endogenous non-human (mouse) MHC II α polypeptide, and may be operably linked to endogenous non-human (mouse) MHC II α transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC II α locus; a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., β1 and β2 domains) of a human MHC II β polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., β1 and β2 domains) of an endogenous non-human (mouse) MHC II β polypeptide, and may be operably linked to endogenous non-human (mouse) MHC II β transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC II β locus; and/or a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., α1, α2 and/or α3 domains) of a human MHC I polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., α1, α2 and/or α3 domains) of an endogenous non-human (mouse) MHC I polypeptide, and may be operably linked to endogenous non-human (mouse) MHC I transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC I locus.
In some embodiments, a genetically modified non-human animal as disclosed herein does not express a functional endogenous non-human T cell CD4 co-receptor from its endogenous locus, does not express a functional endogenous non-human T cell CD8 co-receptor from its endogenous CD8 locus, does not express a functional TCRα variable domain from an endogenous TCRα variable locus, does not express a function TCRβ variable domain from an endogenous TCRβ variable locus, does not express an extracellular domain of an endogenous MHC II complex from an endogenous MHC II locus (e.g., on a cell surface) and/or does not express an extracellular domain of an endogenous MHC I polypeptide from an endogenous MHC I locus (e.g., on a cell surface).
Any non-human animal disclosed herein may further comprise a β2 microglobulin locus encoding a polypeptide comprising a human or humanized β2 microglobulin amino acid sequence, wherein the non-human animal expresses the human or humanized β2 microglobulin polypeptide. In some embodiments, the non-human animal does not express a functional endogenous non-human animal β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In some embodiments, the β2 microglobulin locus is operably linked to endogenous non-human β2 microglobulin regulatory elements. In one embodiment, the β2 microglobulin locus comprises a nucleotide sequence set forth in exon 2, exon 3, and exon 4 (e.g., exon 2 to exon 4) of a human β2 microglobulin gene, and optionally, the β2 microglobulin locus further comprises a nucleotide sequence set forth in exon 1 of a non-human, e.g., rodent, β2 microglobulin gene.
Non-human animals as provided herein may be a rodent, e.g., a mouse or a rat.
Also provided herein is a mouse that expresses chimeric human/murine T cell CD4, CD8α, and CD8β co-receptor polypeptides each respectively comprising murine CD4, CD8α, and CD8β transmembrane and cytoplasmic domains; a T cell receptor comprising a human TCRα variable region and a human TCRβ variable region on the surface of a T cell; chimeric human/murine MHD IIα, MHD IIβ, and MHC I polypeptides each respectively comprising extracellular domains of a human MHC II α (e.g., human HLA class II α1 and β2 domains), MHC II β (human HLA class II β1 and β2 domains), and MHC I polypeptide (e.g., human HLA class I α1, α2, and α3 domains); and optionally a human or humanized β2 microglobulin polypeptide. In one embodiment, provided herein are non-human animals, e.g., mice, wherein the first nucleic acid sequence encodes an a chain of a chimeric human/murine HLA-DR/H-2E polypeptide, the second nucleotide sequence encodes a β chain of a chimeric HLA-DR/H-2E polypeptide, and the third nucleic acid sequence encodes a chimeric human/murine HLA-A/H-2K polypeptide, and wherein the mouse expresses HLA-A/H-2K and HLA-DR/H-2E proteins.
Also provided herein is a non-human animal comprising a substantially humanized T cell immune system, e.g., wherein the substantially humanized T cell immune system mounts a substantially humanized T cell immune response against an antigen. In some embodiments, the substantially humanized T cell immune response comprises activated T cells expressing human T cell receptor (TCR) variable domains that recognize antigen presented in the context of human leukocyte antigen (HLA) extracellular domains and/or antigen presenting cells that present antigen in the context of HLA extracellular domains. In some embodiments, the substantially humanized T cell immune system comprises: (a) a non-human T cell that expresses a T cell co-receptor polypeptide comprising a human T cell co receptor domain that binds to a human HLA molecule and/or a T cell receptor (TCR) comprising a TCR variable domain that is encoded by at least one human TCR variable region gene segment; and (b) a non-human antigen presenting cell that presents antigen in the context of human HLA and activates the non-human T cell.
Also provided are methods of making and using the non-human animals disclosed herein. Generally, methods of making a genetically modified non-human animal as disclosed herein comprise (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide (e.g., a chimeric CD4 polypeptide), and/or a second nucleotide sequence encoding a second chimeric human/non-human T cell co-receptor polypeptide (e.g., a chimeric CD8α polypeptide) and a third nucleotide sequence encoding a third chimeric human/non-human T cell co-receptor polypeptide (e.g., a CD8β polypeptide), wherein a non-human portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, and wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof, e.g., one or more domains) of a human T cell co-receptor; (b) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR constant gene sequence; and optionally (c) placing into the genome a first nucleic acid sequence encoding a first chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC IIα polypeptide), a second nucleic acid sequence encoding a second chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC IIβ polypeptide) and/or a third nucleic acid sequence encoding a third chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC I polypeptide) and/or (d) adding into the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the first nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD4 operably linked to at least transmembrane and cytoplasmic domains of a non-human CD4 co-receptor, the second nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD8α and at least the transmembrane and cytoplasmic domains of a non-human CD8α, the third nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD8β and at least the transmembrane and cytoplasmic domains of non-human CD8β, the first nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class II α polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC II α polypeptide, the second nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class II β polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC II β polypeptide, the third nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class I polypeptide and the transmembrane and cytoplasmic domains of a non-human MHC I polypeptide, and the β2 microglobulin locus comprises a nucleotide sequence set forth in exons 2 to 4 of the human β2 microglobulin gene, e.g., nucleotide sequences set forth in exons 2, 3, and 4 of the human β2 microglobulin gene.
Methods of making non-human animals include embodiments wherein (a) introducing the first, second and/or third nucleotide sequence(s) encoding the chimeric T cell co-receptor polypeptide(s) into the genome of the non-human animal comprises replacing at an endogenous CD4 locus a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, and/or replacing at an endogenous CD8α locus a nucleotide sequence encoding an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and replacing at an endogenous CD8β locus a nucleotide sequence encoding an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide; (b) inserting the unrearranged TCRα locus and/or unrearranged TCR locus into the genome of the animal comprises replacing an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region and/or replacing an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region; (c) placing the first, second and/or third nucleic acid sequence(s) encoding chimeric MHC polypeptide(s) into the genome of the non-human animal comprises replacing at an endogenous non-human MHC II locus a nucleotide sequence encoding a non-human MHC II complex with a nucleotide sequence encoding a chimeric human/non-human MHC II complex and replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and/or (d) adding the β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide into the genome of a non-human animal comprises replacing at the endogenous non-human β2 microglobulin locus a nucleotide sequence encoding a non-human β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.
In some embodiments, (a) introducing the first, second and/or third nucleotide sequence into the genome of the non-human animal respectively comprises (i) replacing at an endogenous CD4 locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD4 polypeptide in operable linkage with sequences encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains, (ii) replacing at an endogenous CD8α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8α polypeptide in operable linkage with sequences encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains and/or (iii) replacing at an endogenous CD8β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8β polypeptide in operable linkage with sequences encoding the endogenous non-human CD8β transmembrane and cytoplasmic domains; (b) inserting the unrearranged TCRα locus and/or unrearranged TCRβ locus into the genome of the animal respectively comprises (i) replacing an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region and/or (ii) replacing an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region; (c) placing the first, second and/or third nucleic acid sequence into the genome of the non-human animal respectively comprises (i) replacing at an endogenous non-human MHC II α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class II α polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II α transmembrane and cytoplasmic domains, (ii) replacing at an endogenous non-human MHC II β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class II β polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II β transmembrane and cytoplasmic domains and/or (iii) replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class I polypeptide in operable linkage with sequences encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains; and/or replacing at an endogenous β2 microglobulin locus a nucleotide sequence set forth in exon 2-exon 4 with a nucleotide sequence comprising exons 2, 3, and 4 of a human β2 microglobulin gene.
In one embodiment, the introducing step comprises replacing in a first non-human animal at an endogenous CD4 locus a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, replacing in a second non-human animal at an endogenous CD8α locus a nucleotide sequence encoding an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and replacing at an endogenous CD8β locus a nucleotide sequence encoding an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide. In some embodiments, the introducing step comprises replacing in a first non-human animal at an endogenous CD4 locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD4 polypeptide in operable linkage with sequences encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains, replacing in a second non-human animal at an endogenous CD8α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8α polypeptide in operable linkage with sequences encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains and replacing at an endogenous CD8β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8β polypeptide in operable linkage with sequences encoding the endogenous non-human CD8β transmembrane and cytoplasmic domains. In some embodiments, the replacing steps are performed simultaneously or in any order.
In some embodiments, the inserting step comprises replacing in a third non-human animal an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region; replacing in a fourth non-human animal an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region. In some embodiments, the replacing steps are performed simultaneously or in any order.
In some embodiments, the placing step comprises, in no particular order, replacing in a fifth non-human animal at an endogenous non-human MHC II locus one or more nucleotide sequence encoding a non-human MHC II complex with one or more nucleotide sequence encoding a chimeric human/non-human MHC II complex; and replacing in the fifth non-human animal at an endogenous non-human MHC I locus a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide. In some embodiments, the placing step comprises replacing in a fifth non-human animal at an endogenous non-human MHC II α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC II α polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II α transmembrane and cytoplasmic domains and replacing at an endogenous non-human MHC II β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC II β polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II β transmembrane and cytoplasmic domains; and replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC I polypeptide in operable linkage with sequences encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains in the fifth non-human animal. In some embodiments, the replacing steps are performed simultaneously or in any order.
In some embodiments, the adding step comprises replacing in a sixth non-human animal at the endogenous non-human β2 microglobulin locus a nucleotide sequence encoding a non-human β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the human or humanized β2 microglobulin polypeptide is encoded by the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.
Methods disclosed herein include embodiments wherein a first, second, and/or third nucleotide sequence(s) encoding chimeric T cell co receptor polypeptide(s) is introduced; the TCRα locus and/or unrearranged TCR locus is inserted; first, second and/or third nucleic acid sequence(s) encoding chimeric MHC polypeptide(s) is placed; and/or the β2 microglobulin locus is added by breeding a non-human animal comprising one or more of the genetic modifications as described herein to another (or more) non-human animal(s) of the same species comprising the remaining genetic modifications. A non-limiting embodiment includes breeding, in any order, the first, second, third, fourth, fifth and sixth non-human animals as described above.
Methods disclosed herein may comprise homologous recombination in non-human embryonic stem (ES) cells. Methods disclosed herein may be used to generate mice as disclosed herein. Non-human animals expressing chimeric human/non-human CD4, CD8α and/or CD8β T cell co-receptor polypeptides, human(ized) TCR α/β proteins, and chimeric MHC II complex and MHC I (with human or humanized β2 microglobulin) may be generated by (a) first introducing each individual human(ized) gene by homologous recombination in individual ES cells respectively and generating each individual non-human animal from such ES cells, and subsequent breeding of each generated non-human animal in any order, (b) introducing all human(ized) genes by sequential homologous recombination in a single ES cell and then generating a non-human animal from such ES cell, or (c) a combination of sequential homologous recombination at some loci in ES cells and breeding. Animals as disclosed herein may also be generated by breeding the progeny of the initial breeding with other animals as appropriate. Breeding and/or homologous recombination may be accomplished in any preferred order.
Also provided are methods of isolating human TCR variable domains specific for an antigen from a non-human animal comprising isolating from a non-human animal provided herein or made according to a method disclosed herein a T cell or TCR protein that binds to the antigen. In some embodiments, the methods may further comprise identifying a first and/or second nucleic acid encoding the TCRα and/or TCRβ variable domains that binds to the antigen and/or culturing a cell comprising one or more vectors in sufficient conditions for expression of the vector(s), wherein the vector(s) comprises a third and/or fourth nucleic acid respectively identical to or substantially identical to the first and/or second nucleic acids, and wherein the third and/or fourth nucleic acid is cloned in-frame with, e.g., a human TCR constant region gene, e.g., a TCRα constant region gene and/or TCR constant region gene, respectively. Tissues and cells comprising the genetic modifications as disclosed herein (which may include rearranged human TCRα and/or TCRβ variable region genes), and nucleic acids encoding such human TCR variable domains expressed by such tissues or cells isolated from a non-human animal modified as described herein are also provided. Also included are (1) recombinant nucleic acids, e.g., expression vectors, comprising the nucleic acid sequences encoding a human TCR variable domain as disclosed herein, e.g., a human rearranged TCRα or human rearranged TCRβ variable region gene, cloned in-frame to an appropriate human TCR constant region gene, e.g., a TCRα constant region gene or TCR constant region gene, respectively, (2) host cells comprising such nucleic acids (e.g., expression vectors) and (3) the TCR expressed by the host cells. In some embodiments, recombinant nucleic acids provided herein comprise a human rearranged TCRδ variable region gene or a TCRγ variable region gene, e.g., derived from a non-human animal genetically modified as disclosed herein or a tissue isolated therefrom, cloned in-frame with a human TCRδ constant region gene or a TCRγ constant region gene, respectively.
A method of generating a humanized T cell response in a non-human animal is also provided, the method generally comprising immunizing a non-human animal a non-human animal genetically modified or having a substantially humanized T cell immune system as described herein with an antigen, e.g., a human antigen, e.g., a human tumor antigen, a human bacterial pathogen, a human viral pathogen, etc. In some embodiments, the non-human animal immunized expresses at least 50% of all functional human TCRVα gene segments and/or at least 50% of all functional human TCRVβ gene segments and/or comprises all or substantially all functional human TCRVα gene segments and/or all or substantially all functional human TCRVβ gene segments.
Also provided are in vitro methods of isolating human TCR specific for an antigen, which generally comprise detecting activation of a first cell of a non-human animal after (a) contact with a second cell of a non-human animal and (b) incubation with the antigen; wherein the first cell expresses a chimeric human/non-human T cell co-receptor and either or both (i) a chimeric human/non-human TCRα chain and (ii) a chimeric human/non-human TCRβ chain, and wherein the second cell expresses a chimeric human/non-human MHC polypeptide. The methods may further comprise isolating a TCR from the first cell, or nucleic acids encoding same.
In the in vitro methods disclosed herein, the antigen may be tumor antigen, a viral antigen, an autoantigen, or a bacterial antigen. In some embodiments, the non-human animal is a rodent, e.g., a rat or a mouse. Also provided herein is tissue, a T cell, a TCR (e.g., a soluble TCR), or a nucleic acid encoding all or part of the TCR that is isolated from a non-human animal genetically modified or having a substantially humanized T cell immune system as described herein, a hybridoma or quadroma derived from such a T cell.
Also provided are compositions, e.g., comprising a first and second cell of a non-human animal; wherein the first cell expresses a chimeric human/non-human T cell co-receptor and optionally, either or both (i) a chimeric human/non-human TCRα chain and (ii) a chimeric human/non-human TCRβ chain, and wherein the second cell expresses a chimeric human/non-human MHC polypeptide that associates with the chimeric human/non-human T cell co-receptor. In some embodiments, the first cell is a non-human T cell. In other embodiments, the second cell is a non-human antigen presenting cell.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation (not to scale) of humanized T cell receptor complex comprising humanized TCR alpha and beta proteins, humanized MHC Class I complexed with humanized β2 microglobulin, and humanized CD8 heterodimer (left panel); as well as T cell receptor complex comprising humanized TCR alpha and beta proteins, humanized MHC Class II heterodimer, and humanized CD4 (right panel). Antigen presented by humanized MHC is depicted as a circle. Mouse regions are depicted as filled shapes while human regions are depicted as striped shapes.
FIGS. 2A-C provide a schematic representation (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., chimeric HLA-A2/H-2K locus (FIG. 2A), chimeric HLA-DR2/H-2E locus (FIG. 2B), and humanized β2M locus (FIG. 2C). Unless otherwise indicated, human sequences are depicted as empty shapes and mouse sequences are depicted as filled shapes. The striped shape represents exon 1 of H-2E derived from a different mouse strain than the endogenous locus (see Example 1.3 and FIG. 3B). Floxed neomycin phosphotransferase cassette(s) are depicted with arrows labeled accordingly.
FIGS. 3A-C depicts a strategy for generating a humanized MHC locus comprising humanized MHC I and MHC II genes. In the particular embodiment depicted in FIG. 3A, the MHC locus of the generated mouse comprises chimeric HLA-A2/H-2K and HLA-DR2/H-2E sequences (H2-K+/1666 MHC-II+/6112) and lacks H2-D sequence (H2-D+/delete) and H-2A sequence (the genetic engineering scheme also results in a deletion of H-2A, see Example 1.2). Large Targeting Vectors (LTVECs) or Cre recombinase construct introduced into ES cells at each stage of humanization are depicted to the right of the arrows. MAID or 4 digit numbers refer to modified allele ID number. FIG. 3B is a schematic diagram (not to scale) of an exemplary HLA-DR2/H-2E large targeting vector. Unless otherwise indicated, human sequences are depicted as empty shapes and mouse sequences are depicted as filled shapes. The striped shape represents exon 1 of H-2E derived from a different mouse strain than the endogenous locus (see Example 1.3). A floxed hygromycin cassette is depicted as an arrow labeled accordingly. FIG. 3C is a schematic representation (not to scale) of exemplary genotypes of chimeric human/mouse MHC loci (** represents H-2L gene that is not present in all mouse strains, e.g., is not present in C57BL/6 or 129 mouse strains), where endogenous mouse H-2K and H-2E loci are respectively replaced by chimeric human/mouse HLA-A2/H-2K and HLA-DR2/H-2E loci (striped shapes), H-2A and H-2D loci were deleted (empty shapes outlined with dotted lines), and remaining loci are endogenous mouse genes (solid shapes outlined with solid lines).
FIG. 4A depicts (not to scale) a progressive strategy for humanization of the mouse TCRα locus, wherein TCRα variable region gene segments are sequentially added upstream of an initial humanization of a deleted mouse locus (MAID1540). Mouse sequence is indicated by filled shapes; human sequence is indicated by empty shapes. MAID refers to modified allele ID number. TRAV=TCR Vα segment, TRAJ=TCR Jα segment (hTRAJ=human TRAJ), TRAC=TCR Cα domain, TCRD=TCRδ. FIG. 4B depicts (not to scale) a progressive strategy for humanization of the mouse TCRβ locus, wherein TCRβ variable region gene segments are sequentially added to a deleted mouse TCRβ variable locus. Mouse sequence is indicated by filled shapes; human sequence is indicated by empty shapes. MAID refers to modified allele ID number. TRBV or TCRBV=TCRβ V segment.
FIG. 5A depicts a schematic representation (not to scale) of the chimeric CD4 locus. Human coding exons are presented by striped shapes, mouse coding exons are presented by filled shapes, and non-coding exons are presented by empty shapes. Immunoglobulin-like domains (Ig), transmembrane (TM), cytoplasmic (CYT) and signal peptide (Signal) coding exons, as well as 3′ untranslated regions (UTR), are indicated. A floxed (loxP) neomycin phosphotransferase (Pgk-neo) cassette is depicted with arrows labeled accordingly. FIG. 5B depicts a schematic representation (not to scale) of the chimeric CD8α and CD8b loci. Human coding exons are presented by striped shapes, mouse coding exons are presented by filled shapes, and non-coding exons are presented by empty shapes. Immunoglobulin-like domains (IgV), transmembrane (TM), cytoplasmic (CYT) and signal peptide (Signal) coding exons, as well as 3′ untranslated regions (UTR), are indicated. Floxed (loxP) hygromycin (Hyg) and neomycin phosphotransferase (Pgk-neo) cassettes are depicted with arrows labeled accordingly.
FIGS. 6A-C are FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on singlets, and stained with (FIG. 6A) anti-mouse CD19 and anti-mouse CD3 antibodies, (FIG. 6B) anti-mouse CD19 and anti-mouse F4/80 antibodies, or (FIG. 6C) anti-mouse CD8α and anti-mouse CD4 antibodies (left panel) or anti-human CD8α and anti-human CD4 antibodies (right panel).
FIGS. 7A-G are FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on CD19+ cells, F4/80+ cells or CD3+ cells, and stained with (FIGS. 7A, 7B) anti-human B2M or anti-mouse H-2D antibodies; (FIGS. 7C, 7D) anti-HLA-A2 or anti-HLA-DR antibodies; (FIGS. 7E, 7F) anti-H-2D and anti-IAIE antibodies; or (FIG. 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom).
FIG. 8 provides FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8), gated on CD3+CD4+ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies
FIGS. 9A-E are FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on singlets, CD3+ cells, CD4+ T cells, or CD8+ T cells, and stained with (FIG. 9A) anti-mouse CD19 and anti-mouse CD3, (FIG. 9B) anti-mouse CD19 and anti-mouse F4/80 antibodies, (FIG. 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (FIGS. 9D, 9E) anti-mouse CD44 and anti-mouse CD62L antibodies.
FIGS. 10A-G are FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on CD19+ cells, F4/80+ cells, or CD3+ cells, and stained with (FIGS. 10A, 10B) anti-human B2M or anti-mouse H-2D antibodies, (FIGS. 10C, 10D) anti-HLA-A2 or anti-HLA-DR antibodies, (FIGS. 10E, 10F) anti-H-2D and anti-IAIE antibodies, or (FIG. 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom).
FIG. 11 provides FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8), gated on CD3+CD4+ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies.
FIG. 12 provides the number of splenic cells (spots per well (Mean+SD); y-axis) that produce IFN-γ in an enzyme-linked immunosorbent spot assay after isolation from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci and incubation in the absence of peptide (200 k cells only; x-axis) or presence of 10 μg/ml or 1 μg/ml MAGE-A3 peptide (x-axis).
FIG. 13A depicts progression of acute Armstrong strain viral infection in either control or mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci; the timeline for the experiment is depicted at the top of the figure, and measurement of viral titers on various days post-infection for both mouse strains is depicted in the bottom graph. FIG. 13B depicts progression of chronic Clone 13 strain viral infection in either control or mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci; the timeline for the experiment is depicted at the top of the figure, and the measurement of viral titers on Day 21 post-infection for both mouse strains is depicted in the bottom graph. T cells from uninfected or chronically infected TM I/II B C4/8 or control B6 mice were stained with anti-PD1, anti-Lag3, and anti-Tim3 antibodies (FIG. 13C; x-axis); the figure provides a quantification of cells staining positive (% positive cells; y-axis).
FIG. 14 depicts progression of chronic Clone 13 strain viral infection in either control or TM I/II B C4/8 mice after prior acute Armstrong strain infection; the timeline for the experiment is depicted at the top of the figure, and measurement of viral titers on Day 31 post-infection is depicted in the bottom graph. Mock infected mice were included in the experiment as an additional control.
FIGS. 15A-B depicts the number of CD8+ cells (y-axis; IFN-γ Positive Cells) that produced IFN-γ in response to LCMV peptides that are HLA-A2 restricted (GPC10-18; N69-77; Z49-58), H2Db restricted (GP33-41), ovalbumin, or incubation alone and were isolated from either control animals (FIG. 15A) or mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci (FIG. 15B), each of which received a mock infection (mock; n=1 each group) or an acute Armstrong strain infection (Arm; n=3 each group). The % of IFNγ+CD8+ lymphocytes (y-axis) after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447 or Z49) during a time course of infection (days post infection; x-axis) in mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci or control B6 animals are shown in FIGS. 15C and 15D, respectively.
DETAILED DESCRIPTION
Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express a humanized T cell co-receptor (e.g., humanized CD4 and/or CD8 (e.g., CD8α and/or CD8β)), a human or humanized major histocompatibility complex (MHC) that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and/or MHC II β chains) and/or MHC I (e.g., MHC Iα), and optionally human or humanized β2 microglobulin) and/or a human or humanized T cell receptor (TCR), as well as embryos, tissues, and cells expressing the same. The development of the cellular arm of the immune system of the non-human animals disclosed herein is comparable to control animals, e.g., the thymus and spleen comprises similar absolute numbers of thymocytes and CD3+ cells. This is in stark contrast to other non-human animals modified to comprise both human TCR (α and β) and a chimeric human/mouse MHC I molecule, see, e.g., Li (2010) Nature Medicine 16:1029-1035 and supplementary materials. Such animals showed a decrease in T cell populations compared not only to wildtype control animals, but also animals modified with only human TCR, and animals modified with only the chimeric human/mouse MHC I molecule, id. Accordingly, provided herein are non-human animals engineered to co-express a humanized CD4 co-receptor and a humanized MHC II and/or a humanized CD8 co-receptor and a humanized MHC I, and optionally a humanized TCR. Methods for making a genetically engineered animal that expresses at least one humanized T cell co-receptor (e.g., humanized CD4 and/or CD8), at least one humanized MHC that associates with the humanized T cell co-receptor (e.g., humanized MHC II and/or MHC I that associate with humanized CD4 and/or CD8, respectively) and/or the humanized TCR are also provided. Methods for using the genetically engineered animals that mount a substantially humanized T cell immune response for developing human therapeutics are also provided.
Substantially Humanized T Cell Immune Responses
Disclosed herein are non-human animals that are genetically modified to mount substantially humanized T cell immune responses. The mice disclosed herein express at least one human or humanized T cell co-receptor, at least one human or humanized major histocompatibility complex (MHC) capable of associating with the at least one human or humanized T cell co-receptor, and/or a human or humanized T cell receptor (TCR), which is preferably capable of recognizing an antigen presented in the context of human or humanized MHC in association with a human or humanized T cell co-receptor and providing activation signals to the non-human cell, e.g., non-human T cell, expressing the human or humanized TCR. The human or humanized T cell co-receptor, human or humanized TCR and/or human or humanized MHC may be encoded by the genome of the non-human animal. In preferred embodiments, upon immunization with an antigen, the non-human animals present HLA restricted epitopes of the antigen to TCR derived from human TCR gene segments, e.g., a human TCRα V segment, a human TCRα J segment, a human TCRβ V segment, human TCRβ D segment and/or a human TCRβ J segment.
Accordingly, encompassed by the invention is a genetically modified non-human animal whose genome comprises (e.g., at an endogenous locus) a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), wherein the chimeric T cell co-receptor polypeptide comprises conservative amino acid substitutions of the amino acid sequence(s) described herein and/or a nucleic acid sequence encoding a humanized MHC polypeptide that associates with the humanized T cell co-receptor polypeptide, wherein the humanized MHC polypeptide comprises conservative amino acid substitutions of the amino acid sequence(s) described herein.
A conservative amino acid substitution includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of CD4 or CD8 to associate with, e.g., bind to MHC II or MHC I, respectively, and may, e.g., increase sensitivity of TCR to MHC-presented antigen. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
One skilled in the art would understand that in addition to the nucleic acid residues encoding humanized T cell co-receptor polypeptides, humanized MHC polypeptides, and/or TCR variable regions described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptides of the invention. Therefore, in addition to a genetically modified non-human animal that comprises in its genome a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), an unrearranged T cell receptor variable gene locus (e.g., TCRα and/or TCRβ) comprising human unrearranged gene segments, and/or a nucleic acid sequence encoding a humanized MHC polypeptide capable of associating with the humanized T cell co-receptor polypeptide with conservative amino acid substitutions, also provided is a non-human animal whose genome comprises a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), an unrearranged T cell receptor variable gene locus (e.g., TCRα and/or TCRβ) comprising human unrearranged gene segments, and/or a nucleic acid sequence encoding a humanized MHC polypeptide capable of associating with the humanized T cell co-receptor polypeptide, which differs from that described herein due to the degeneracy of the genetic code.
The identity of a sequence may be determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular sequences. In various embodiments, identity is determined by comparing the sequence of a mature protein from its N-terminal to its C-terminal. In various embodiments when comparing a chimeric human/non-human sequence to a human sequence, the human portion of the chimeric human/non-human sequence (but not the non-human portion) is used in making a comparison for the purpose of ascertaining a level of identity between a human sequence and a human portion of a chimeric human/non-human sequence (e.g., comparing a human ectodomain of a chimeric human/mouse protein to a human ectodomain of a human protein).
The terms “homology” or “homologous” in reference to sequences, e.g., nucleotide or amino acid sequences, means two sequences which, upon optimal alignment and comparison, are identical in, e.g., at least about 75% of nucleotides or amino acids, e.g., at least about 80% of nucleotides or amino acids, e.g., at least about 90-95% nucleotides or amino acids, e.g., greater than 97% nucleotides or amino acids. One skilled in the art would understand that, for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.
The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In addition, various portions of the chimeric or humanized protein of the invention may be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless stated otherwise, various domains of the chimeric or humanized proteins of the invention are operably linked to each other.
The term “replacement” in reference to gene replacement refers to placing exogenous genetic material at an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the Examples below, in one embodiment, nucleic acid sequences of endogenous loci encoding portions of mouse CD4 or CD8 (CD8α and/or CD8β) polypeptides were replaced by nucleotide sequences encoding portions of human CD4 or CD8 (CD8α and/or CD8β) polypeptides, respectively.
“Functional” as used herein, e.g., in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non-human CD4 or CD8 locus) results in a locus that fails to express a functional endogenous polypeptide.
Humanized T Cell Co-Receptor(s)
Disclosed herein are non-human animals that express at least one human or humanized T cell co-receptor, e.g., CD4, CD8α and/or CD8β). Accordingly, a non-human animal as disclosed herein comprises at least one of a first, second, and/or third nucleotide sequence, each of which encodes a different human or chimeric human/non-human T cell co-receptor polypeptide selected from a human or humanized CD4 polypeptide, a human or humanized CD8α polypeptide, and a human or humanized CD8β polypeptide. Use of the first, second, third designations herein is not to be construed as limiting the non-human animals disclosed herein as requiring all three nucleotide sequences or the presence of any of the co-receptor nucleotide sequences in any order. Accordingly, a non-human animal as disclosed herein may comprise a nucleic acid sequence or nucleic acid sequences encoding a human or humanized CD4 and/or a human or humanized CD8 (e.g., human or humanized CD8α and/or CD8β) polypeptide(s).
In one embodiment, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD4 polypeptide. In another embodiment, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD8α polypeptide and a second nucleotide sequence encoding a human or humanized CD8β polypeptide. In another embodiment, a non-human animal as disclosed herein comprises first and second nucleotide sequences encoding human or humanized CD8α and CD8β polypeptides and further comprises a third nucleotide sequence encoding a human or humanized CD4 polypeptide.
Human or Humanized CD4
In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at an endogenous CD4 locus, a nucleotide sequence encoding a human or humanized CD4 polypeptide; thus, the animals express a human or humanized CD4 polypeptide.
Human CD4 gene is localized to chromosome 12, and is thought to contain 10 exons. CD4 gene encodes a protein with amino-terminal hydrophobic signal sequence, encoded by exons 2 and 3 of the gene. The protein comprises four extracellular immunoglobulin-like domains, Ig1-Ig4, also commonly and respectively referred to as D1-D4 domains. Maddon et al. (1987) Structure and expression of the human and mouse T4 genes, Proc. Natl. Acad. Sci. USA 84:9155-59. D1 domain is believed to be encoded by exon 3 (sequence downstream of signal peptide) and exon 4, while D2, D3, and D4 are encoded by a separate exon each—exons 5, 6, and 7, respectively (see FIG. 5A: D1, D2, D3 and D4 domains are encoded by sequences designated as Ig1, Ig2, Ig3 and Ig4, respectively). Littman (1987) The Structure of the CD4 and CD8 Genes, Ann. Rev. Immunol. 5:561-84; Hanna et al. (1994) Specific Expression of the Human CD4 Gene in Mature CD4+CD8− and Immature CD4+CD8+ T cells and in Macrophages of Transgenic Mice, Mol. Cell. Biol. 14(2):1084-94; Maddon et al., supra. At areas of high protein concentration, such as the area of contact between T cell and antigen-presenting cell, the molecule tends to homodimerize through interactions between opposing D4 domains. Zamoyska (1998) CD4 and CD8: modulators of T cell receptor recognition of antigen and of immune responses? Curr. Opin. Immunol. 10:82-87; Wu et al. (1997) Dimeric association and segmental variability in the structure of human CD4, Nature 387:527; Moldovan et al. (2002) CD4 Dimers Constitute the Functional Component Required for T Cell Activation, J. Immunol. 169:6261-68.
D1 domain of CD4 resembles immunoglobulin variable (V) domain, and, together with a portion of D2 domain, is believed to bind (associate with) MHC II, e.g., at an MHC II co-receptor binding site. Huang et al. (1997) Analysis of the contact sites on the CD4 Molecule with Class II MHC Molecule, J. Immunol. 158:216-25. In turn, MHC II interacts with T cell co-receptor CD4 at the hydrophobic crevice at the junction between MHC II α 2 and β2 domains. Wang and Reinherz (2002) Structural Basis of T Cell Recognition of Peptides Bound to MHC Molecules, Molecular Immunology, 38:1039-49.
Domains D3 and D4 of the CD4 co-receptor are believed to interact with the TCR-CD3 complex as the substitution of these two domains abrogated the ability of CD4 to bind to TCR. Vignali et al. (1996) The Two Membrane Proximal Domains of CD4 Interact with the T Cell Receptor, J. Exp. Med. 183:2097-2107. CD4 molecule exists as a dimer, and residues in the D4 domain of the molecule are believed to be responsible for CD4 dimerization. Moldovan et al. (2002) CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. 169:6261-68.
Exon 8 of the CD4 gene encodes the transmembrane domain, while the remainder of the gene encodes the cytoplasmic domain. CD4 cytoplasmic domain possesses many distinct functions. For example, the cytoplasmic domain of CD4 recruits a tyrosine kinase Lck. Lck is a Src family kinase that is associated with CD4 and CD8 cytoplasmic domains and simultaneous binding of the co-receptors and TCRs to the same MHC leads to increased tyrosine phosphorylation of CD3 and ζ chain of the TCR complex, which in turn leads to recruitment of other factors that play a role in T cell activation. Itano and colleagues have proposed that cytoplasmic tail of CD4 also promotes differentiation of CD4+CD8+ T cells into CD4+ lineage by designing and testing expression of hybrid protein comprising CD8 extracellular domain and CD4 cytoplasmic tail in transgenic mice. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4 Lineage T Cells, J. Exp. Med. 183:731-41. The expression of the hybrid protein led to the development of MHC I-specific, CD4 lineage T cells. Id.
CD4 co-receptor appears to be the primary receptor for HIV virus, with the CD4+ T cell depletion being an indicator of disease progression. The cytoplasmic tail of CD4 appears to be essential for delivering apoptotic signal to CD4+ T cells in HIV-induced apoptosis. Specifically, the interaction of CD4 and Lck was shown to potentiate HIV-induced apoptosis in these cells. Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and Is Accelerated by Interaction of CD4 with p56lck, J. Exp. Med. 183:39-48.
T cells develop in the thymus progressing from immature CD4−/CD8− (double negative or DN) thymocytes to CD4+/CD8+ (double positive or DP) thymocytes, which eventually undergo positive selection to become either CD4+ or CD8+ (single positive or SP) T cells. DP thymocytes that receive signals through MHC I-restricted TCR differentiate into CD8+ T cells, while DP thymocytes that receive signals through MHC II-restricted TCR differentiate into CD4+ T cells. The cues received by the DP cell that lead to its differentiation into either CD4+ of CD8+ T cell have been a subject of much research. Various models for CD4/CD8 lineage choice have been proposed and are reviewed in Singer et al. (2008) Lineage fate and intense debate: myths, models and mechanisms of CD4− versus CD8− lineage choice, Nat. Rev. Immunol. 8:788-801.
Deactivation of a specific T cell co-receptor as a result of positive selection is a product of transcriptional regulation. For CD4, it has been shown that an enhancer located 13 kb upstream of exon 1 of CD4 upregulates CD4 expression in CD4+ and CD8+ T cells. Killeen et al. (1993) Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4, EMBO J. 12:1547-53. A cis-acting transcriptional silencer located within the first intron of murine CD4 gene functions to silence expression of CD4 in cells other than CD4+ T cells. Siu et al. (1994) A transcriptional silencer control the developmental expression of the CD4 gene, EMBO J. 13:3570-3579.
Because important transcriptional regulators (e.g., promoters, enhancers, silencers, etc.) that control CD4 lineage choice were missing in several strains of previously developed transgenic mice expressing human CD4, these mice were not able to recapitulate normal T cell lineage development, and produced immune cells other than CD4+ T cells that expressed CD4. See, e.g., Law et al. (1994) Human CD4 Restores Normal T Cell Development and Function in Mice Deficient in CD4, J. Exp. Med. 179:1233-42 (CD4 expression in CD8+ T cells and B cells); Fugger et al. (1994) Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region β-chain T-cell repertoire and mediates an HLA-D-restricted immune response, Proc. Natl. Acad. Sci. USA, 91:6151-55 (CD4 expressed on all CD3+ thymocytes and B cells). Thus, in one embodiment, there may be a benefit in developing a genetically modified animal that retains endogenous mouse promoter and other regulatory elements in order for the animal to produce T cells that are capable of undergoing T cell development and lineage choice.
Thus, in various embodiments, the invention provides a genetically modified non-human animal, comprising, e.g., at its endogenous T cell co-receptor locus (e.g., CD4 locus), a nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide. In one embodiment, a human portion of the chimeric polypeptide comprises all or substantially all of an extracellular portion (or part thereof, e.g., one or more extracellular domains, e.g., at least two consecutive extracellular domains) of a human T cell co-receptor. In one embodiment, a non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a non-human T cell co-receptor. In one embodiment, the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. Thus, in one aspect, the invention provides a genetically modified non-human animal comprising at its endogenous CD4 locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, wherein a human portion of the chimeric polypeptide comprises all or substantially all of an extracellular portion of a human CD4, wherein a non-human portion comprises at least transmembrane and cytoplasmic domains of a non-human CD4, and wherein the animal expresses a functional chimeric CD4 polypeptide. In one aspect, the non-human animal only expresses the humanized CD4 polypeptide, i.e., chimeric human/non-human CD4 polypeptide, and does not express a functional endogenous non-human CD4 protein from its endogenous CD4 locus.
In one embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of a human CD4 polypeptide. In another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises at least all or substantially all of the MHC II binding domain of the human CD4 polypeptide (e.g., a substantial portion of human D1 and D2 domains); in one embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of D1, D2, and D3 domains of the human CD4 polypeptide; in yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of immunoglobulin-like domains of CD4, e.g., domains termed D1, D2, D3, and D4. In yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises in its human portion all or substantially all of the human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of the human CD4 that is responsible for interacting with MHC II and/or the variable domain of a T cell receptor. Therefore, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises all or substantially all of the coding sequence of domains D1-D2 of the human CD4 (e.g., a portion of exon 3 and exons 4-5 of the human CD4 gene); in another embodiment, it comprises all or substantially all of the coding sequence of D1-D3 of the human CD4 (e.g., portion of exon 3 and exons 4-6 of the human CD4). Thus, in one embodiment, the nucleotide sequence encoding chimeric human/non-human CD4 comprises nucleotide sequences encoding all or substantially all D1-D3 domains of the human CD4. In another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the coding sequence of D1-D4 domains of the human CD4 gene. In another embodiment, the nucleotide sequence may comprise the nucleotide sequence encoding mouse CD4 signal peptide, e.g., region encoded by portions of exons 2-3 of the mouse gene. In another embodiment, the nucleotide sequence may comprise the nucleotide sequence encoding a human CD4 signal peptide. In one embodiment, the chimeric human/non-human CD4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:78, and the human portion of the chimeric polypeptide spans about amino acids 27-319 of SEQ ID NO:78 (set forth separately in SEQ ID NO:79).
In one embodiment, the non-human animal expresses a chimeric human/non-human CD4 polypeptide sequence. In one embodiment, a human portion of the chimeric CD4 sequence comprises one or more conservative or non-conservative modifications.
In one aspect, a non-human animal that expresses a human CD4 sequence is provided, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human CD4 sequence. In a specific embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence described in the Examples. In one embodiment, the human CD4 sequence comprises one or more conservative substitutions. In one embodiment, the human CD4 sequence comprises one or more non-conservative substitutions.
In some embodiments, a portion, e.g., a human portion of the chimeric CD4, may comprise substantially all of the sequence indicated herein (e.g., substantially all of a protein domain indicated herein). Substantially all sequence generally includes 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). One skilled in the art would understand that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction methods used.
In one aspect, the non-human portion of the chimeric human/non-human CD4 polypeptide comprises at least transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Due to the important functions served by CD4 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in genetically engineered animals ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. Although a specific mouse CD4 sequence is described in the Examples, any suitable sequence derived therefrom, e.g., sequence comprising conservative/non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human portion of the chimeric CD4 co-receptor comprises any sequence of the endogenous CD4 that has not been humanized.
The non-human animal described herein may comprise at its endogenous locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide. In one aspect, this results in a replacement of a portion of an endogenous CD4 gene with a nucleotide sequence encoding a portion of a human CD4 polypeptide. In one embodiment, such replacement is a replacement of endogenous nucleotide sequence encoding, e.g., all or substantially all of the extracellular domain of a non-human CD4, e.g., a sequence encoding at least all or substantially all of the first immunoglobulin-like domain (i.e., D1) of a non-human CD4 (e.g., a sequence encoding all or substantially all of domains D1-D2 of a non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D3 of a non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D4 of a non-human CD4), with a human nucleotide sequence encoding the same. In one embodiment, the replacement results in a chimeric protein comprising human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In yet another embodiment, the replacement results in a chimeric protein comprising human CD4 sequence that is responsible for interacting with MHC II and/or variable domain of a T cell receptor. In one embodiment, the replacement does not comprise a replacement of a CD4 sequence encoding at least transmembrane and cytoplasmic domains of a non-human CD4 polypeptide. Thus, in one aspect, the non-human animal expresses a chimeric human/non-human CD4 polypeptide from the endogenous non-human CD4 locus. In yet another embodiment, the replacement results in a protein comprising a polypeptide sequence set forth in SEQ ID NO:78.
In one embodiment, the nucleotide sequence of the chimeric human/non-human CD4 locus (e.g., chimeric human/rodent CD4 locus, e.g., chimeric human/mouse CD4 locus) described herein is provided. In one aspect, because the chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) CD4 sequence is placed at the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus, it retains the CD4 enhancer element located upstream of the first CD4 exon. In one embodiment, the replacement at the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus comprises a replacement of, e.g., a portion of exon 3 encoding D1, and exons 4-6 encoding the rest of D1 and D2-D3 of CD4 polypeptide; thus, in one aspect, the chimeric CD4 locus retains the cis-acting silencer located in intron 1 of the non-human (e.g., mouse) CD4 gene. Thus, in one embodiment, the chimeric locus retains endogenous non-human (e.g., rodent, e.g., mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus may contain human promoter and regulatory elements to the extent those allow proper CD4 expression, CD4+ T cell development, CD4 lineage choice, and co-receptor function. Thus, in some aspects, the animals of the invention comprise a genetic modification that does not alter proper lineage choice and development of T cells. In one aspect, the animals (e.g., rodents, e.g., mice) of the invention do not express chimeric CD4 polypeptide on immune cells other than cells that normally express CD4. In one aspect, animals do not express CD4 on B cells or mature CD8+ T cells. In one embodiment, the replacement results in retention of elements that allow proper spatial and temporal regulation of CD4 expression.
In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD4 protein from a chimeric CD4 locus as described herein displays the chimeric protein on a cell surface, e.g., T cell surface. In one embodiment, the non-human animal expresses the chimeric CD4 protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the CD4 protein of the invention is capable of interacting with an MHC II protein expressed on the surface of a second cell, e.g., an antigen presenting cell (APC).
Human or Humanized CD8
In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8 polypeptide; thus, the animals express a human or humanized CD8 polypeptide. In various embodiments, the invention provides non-human animals that comprise in their genome, e.g., at an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8α polypeptide and/or a nucleotide sequence encoding a human or humanized CD8β polypeptide. Thus, the genetically modified non-human animal of the invention expresses a human or humanized CD8α and/or a human or humanized CD8β polypeptide(s).
Human CD8 protein is typically expressed on cell surface as heterodimer of two polypeptides, CD8α and CD8β, although disulfide-linked homodimers and homomultimers have also been detected (e.g., in NK cells and intestinal γδ T cells, which express CD8αα). The genes encoding human CD8α and CD8β are located in close proximity to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human CD8 β-chain genes, J. Immunol. 148:1919-27. CD8α protein contains a leader peptide, an immunoglobulin V-like region, a hinge region, a transmembrane domain and a cytoplasmic tail. Norment et al. (1989) Alternatively Spliced mRNA Encodes a Secreted Form of Human CD8α. Characterization of the Human CD8α gene, J. Immunol. 142:3312-19. The exons/introns of the CD8α gene are depicted schematically in FIG. 5B.
Human CD8β gene lies upstream of the CD8α gene on chromosome 2. Multiple isoforms generated by alternative splicing of CD8β gene have been reported, with one isoform predicted to lack a transmembrane domain and generate a secreted protein. Norment et al. (1988) A second subunit of CD8 is expressed in human T cells, EMBO J. 7:3433-39. The exons/introns of CD8β gene are also depicted schematically in FIG. 5B.
The membrane-bound CD8β protein contains an N-terminal signal sequence, followed by immunoglobulin V-like domain, a short extracellular hinge region, a transmembrane domain, and a cytoplasmic tail. See, Littman (1987) The structure of the CD4 and CD8 genes, Ann Rev. Immunol. 5:561-84. The hinge region is a site of extensive glycosylation, which is thought to maintain its conformation and protect the protein from cleavage by proteases. Leahy (1995) A structural view of CD4 and CD8, FASEB J. 9:17-25.
CD8 protein is commonly expressed on cytotoxic T cells, and interacts with MHC I molecules. The interaction is mediated through CD8 binding to the α3 domain of MHC I. Although binding of MHC class I to CD8 is about 100-fold weaker than binding of TCR to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366.
CD8 binding to MHC class I molecules is species-specific; the mouse homolog of CD8, Lyt-2, was shown to bind H-2Dd molecules at the α3 domain, but it did not bind HLA-A molecules. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341. Differential binding was presumably due to CDR-like determinants (CDR1- and CDR2-like) on CD8 that were not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174:371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8αα and HLA-A2, Nature 387:630-634. It has been reported that CD8 binds HLA-A2 in a conserved region of the α3 domain (at position 223-229). A single substitution (V245A) in HLA-A reduced binding of CD8 to HLA-A, with a concomitant large reduction in T cell-mediated lysis. Salter et al. (1989), Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphism in the α3 domain of HLA-A molecules also affected binding to CD8. Id. In mice, amino acid substitution at residue 227 in H-2Dd affected the binding of mouse Lyt-2 to H-2Dd, and cells transfected with a mutant H-2Dd were not lysed by CD8+ T cells. Potter et al. (1989) Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75. Thus, expression of human or humanized CD8 may be beneficial for studying T cell responses to antigen presented by human or humanized MHC I.
Similarly to CD4, the cytoplasmic domain of CD8 interacts with tyrosine kinase Lck, which in turn leads to T cell activation. Although Lck seems to interact with the cytoplasmic domain of CD8α, it appears that this interaction is regulated by the presence of the cytoplasmic domain of CD8β because mutations or deletion of CD8β cytoplasmic domain resulted in reduced CD8α-associated Lck activity. Irie et al. (1998) The cytoplasmic domain of CD8β Regulates Lck Kinase Activation and CD8 T cell Development, J. Immunol. 161:183-91. The reduction in Lck activity was associated with impairment in T cell development. Id.
Expression of CD8 on appropriate cells, e.g., cytotoxic T cells, is tightly regulated by a variety of enhancer elements located throughout the CD8 locus. For instance, at least 4 regions of DNAse I-hypersensitivity, regions often associated with regulator binding, have been identified at the CD8 locus. Hosert et al. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. 158:4270-81. Since the discovery of these DNAse I-hypersensitive regions at CD8 locus, at least 5 enhancer elements have been identified, spread throughout the CD8 locus, that regulate expression of CD8α and/or β in T cells of various lineages, including DP, CD8 SP T cells, or cells expressing γδTCR. See, e.g., Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8B gene expression during thymic differentiation, Nature Rev. 2:909-919 and Online Erratum; Ellmeier et al. (1998) Multiple Development Stage-Specific Enhancers Regulate CD8 Expression in Developing Thymocytes and in Thymus-Independent T cells, Immunity 9:485-96.
Thus, similarly to the benefit derived from retaining endogenous CD4 promoter and regulatory elements for human or humanized CD4 genetically modified animals, in some embodiments, there may be a benefit in developing a genetically modified non-human animal that retains endogenous mouse promoter and regulatory elements that would control expression of human or humanized CD8. There may be a particular benefit in creating genetically modified animals comprising a replacement of endogenous non-human sequences encoding CD8α and/or β proteins with those encoding human or humanized CD8α and/or β proteins, as described herein.
In various embodiments, the invention provides a genetically modified non-human animal comprising in its genome, e.g., at its endogenous CD8 locus, at least one nucleotide sequence encoding a chimeric human/non-human CD8 polypeptide (e.g., CD8α and/or β polypeptide), wherein a human portion of the polypeptide comprises all or substantially all of an extracellular portion (or a part thereof, e.g., an extracellular domain) of a human CD8 polypeptide (e.g., CD8α and/or β), wherein a non-human portion comprises at least transmembrane and cytoplasmic domains of a non-human CD8 (e.g., CD8α and/or β), and wherein the animal expresses the chimeric CD8 polypeptide (e.g., CD8α and/or β polypeptide). Thus, in one embodiment, the invention provides a genetically modified non-human animal comprising at its endogenous non-human CD8 locus a first nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and a second nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide, wherein the first nucleotide sequence comprises a sequence that encodes all or substantially all of the extracellular portion of a human CD8α polypeptide and at least transmembrane and cytoplasmic domains of a non-human CD8α polypeptide, and wherein the second nucleotide sequence comprises a sequence that encodes all or substantially all of the extracellular portion of a human CD8β polypeptide and at least transmembrane and cytoplasmic domains of a non-human CDβ polypeptide, wherein the animal expresses a functional chimeric human/non-human CD8 protein. In one aspect, the non-human animal only expresses a humanized CD8 polypeptide (e.g., chimeric human/non-human CD8α and/or β polypeptide), and does not express a corresponding functional non-human CD8 polypeptide(s) from the endogenous CD8 locus.
In one embodiment, the chimeric human/non-human CD8α polypeptide comprises in its human portion all or substantially all of the extracellular portion of a human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises at least the MHC I binding domain of the human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises the sequence of at least all or substantially all of the immunoglobulin V-like domain of the human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8α polypeptide comprises at least the exons that encode an extracellular portion of the human CD8α polypeptide. In one embodiment, the nucleotide sequence comprises at least the exons that encode the Ig V-like domains. In one embodiment, the extracellular portion of a human CD8α polypeptide is a region encompassing the portion of the polypeptide that is not transmembrane or cytoplasmic domain. In one embodiment, the nucleotide sequence encoding the chimeric human/non-human CD8α polypeptide comprises the sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8α signal peptide. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8α signal sequence. In one embodiment, the chimeric human/non-human CD8α polypeptide comprises an amino acid sequence set forth in SEQ ID NO:88, and the human portion of the chimeric polypeptide is set forth at amino acids 28-179 of SEQ ID NO:88 (represented separately in SEQ ID NO:89).
Similarly, in one embodiment, the chimeric human/non-human CD8β polypeptide comprises in its human portion all or substantially all of the extracellular portion of a human CD8β polypeptide. In one embodiment, the human portion of the chimeric CD8β polypeptide comprises the sequence of all or substantially all of the immunoglobulin V-like domain of human CD8β. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8β polypeptide comprises at least the exons that encode the extracellular portion of the human CD8β polypeptide. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric human/non-human CD8β polypeptide comprises at least the exons that encode the IgG V-like domain of human CD8β. In one embodiment, the nucleotide sequence encoding the chimeric human/non-human CD8β polypeptide comprises the sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8β signal peptide. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8β signal sequence. In one embodiment, the chimeric human/non-human CD8β polypeptide comprises an amino acid sequence set forth in SEQ ID NO:83, and the human portion of the chimeric polypeptide is set forth at amino acids 15-165 of SEQ ID NO:83 (represented separately in SEQ ID NO:84).
In one embodiment, the non-human animal expresses a chimeric human/non-human CD8α and/or CD8β polypeptides. In some embodiments, the human portion of the chimeric human/non-human CD8α and/or β polypeptide comprises one or more conservative or nonconservative modification(s).
In one aspect, a non-human animal that expresses a human CD8α and/or β polypeptide sequence is provided, wherein the human CD8α and/or β polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human CD8α and/or β polypeptide sequence, respectively. In a specific embodiment, the human CD8α and/or β polypeptide sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the respective human CD8α and/or β polypeptide sequence described in the Examples. In one embodiment, the human CD8α and/or β polypeptide sequence comprises one or more conservative substitutions. In one embodiment, the human CD8α and/or β polypeptide sequence comprises one or more non-conservative substitutions.
In some embodiments, a portion, e.g., a human portion of the chimeric CD8, may comprise substantially all of the sequence indicated herein (e.g., substantially all of a protein domain indicated herein). Substantially all sequence generally includes 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). One skilled in the art would understand that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction methods used.
In one aspect, the non-human portion of the chimeric human/non-human CD8α and/or β polypeptide comprises at least transmembrane and/or cytoplasmic domain of the non-human CD8α and/or β polypeptide, respectively. Due to the important functions served by CD8 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in genetically engineered animals ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD8α and/or β polypeptide is a mouse CD8α and/or β polypeptide, respectively. Although specific mouse CD8α and β sequences are described in the Examples, any suitable sequence derived therefrom, e.g., sequence comprising conservative/non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) retains any endogenous sequence that has not been humanized.
The non-human animal described herein may comprise at its endogenous locus a nucleotide sequence encoding a chimeric human/non-human CD8α and/or β polypeptide. In one aspect, this results in a replacement of a portion of an endogenous CD8α gene with a nucleotide sequence encoding a portion of a human CD8α polypeptide, and/or a replacement of a portion of an endogenous CD8β gene with a nucleotide sequence encoding a portion of a human CD8β polypeptide. In one embodiment, such replacement is a replacement of endogenous nucleotide sequence encoding all or substantially all of extracellular portion of a non-human CD8α and/or β with a human nucleotide with a human nucleotide sequence encoding the same. In one embodiment, such replacement is a replacement of a sequence encoding at least all or substantially all of the immunoglobulin V-like domain of a non-human CD8α and/or β with a human nucleotide sequence encoding the same. In one embodiment, the replacement does not comprise a replacement of a CD8α and/or β sequence encoding transmembrane and cytoplasmic domain of a non-human CD8α and/or β polypeptide. Thus, the non-human animal expresses a chimeric human/non-human CD8α and/or β polypeptide from the endogenous non-human CD8 locus. In yet another embodiment, the replacement results in a CD8α and/or β protein comprising a polypeptide sequence set forth in SEQ ID NO:88 and/or 84, respectively.
In one embodiment, the nucleotide sequence of the chimeric human/non-human CD8 locus (e.g., chimeric rodent CD8 locus, e.g., chimeric mouse CD8 locus) is provided. In one aspect, because the chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) CD8α and/or β sequence is placed at respective endogenous non-human (e.g., rodent, e.g., mouse) CD8α and/or β locus, it retains endogenous CD8α and/or β promoter and regulatory elements. In another embodiment, the chimeric locus may contain human CD8α and/or β promoter and regulatory elements to the extent those allow proper CD8α and/or β expression (proper spatial and temporal protein expression), CD8+ T cell development, CD8 lineage choice, and co-receptor function. Thus, in one aspect, the animals of the invention comprise a genetic modification that does not alter proper lineage choice and development of T cells. In one aspect, the animals (e.g., rodents, e.g., mice) of the invention do not express chimeric CD8 protein on immune cells other than cells that normally express CD8, e.g., animals do not express CD8 on B cells or mature CD4+ T cells. In one embodiment, the replacement results in retention of elements that allow proper spatial and temporal regulation of CD8α and/or β expression.
In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD8 protein (e.g., CD8α β or CD8αα) from a chimeric CD8 locus as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expresses the chimeric CD8 protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the CD8 protein of the invention is capable of interacting with an MHC I protein expressed on the surface of a second cell.
Human or Humanized T Cell Receptor
Disclosed herein are genetically modified non-human animals comprising a substantially humanized T cell immune system. In some embodiment a non-human animal as disclosed herein comprises, e.g., in its genome, (a) a nucleotide sequence encoding a chimeric human/non-human T cell co-receptor, wherein the human portion of the chimeric T cell co-receptor polypeptide is encoded by a sequence encoding an extracellular domain of a human T cell co-receptor, and wherein the sequence encoding the extracellular domain of a human T cell co-receptor is operably linked to a nucleotide comprising a sequence encoding a non-human T cell co-receptor transmembrane and/or cytoplasmic domain; (b) an unrearranged T cell receptor (TCR) variable gene region comprising at least one human V segment, optionally at least on human D segment, and at least one human J segment, wherein the unrearranged V, optionally D, and J segments of the TCR variable region gene can recombine to form a rearranged gene operably linked to a non-human TCR constant gene sequence; and (c) a nucleic acid sequence encoding a chimeric human/non-human MHC polypeptide, wherein a human portion of the chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide that associates with the human portion of the chimeric T cell co-receptor polypeptide. Optionally, the non-human animal also comprises a human or humanized β2 microglobulin polypeptide.
Accordingly, in various embodiments, the invention generally provides genetically modified non-human animals wherein the non-human animals comprise in the genome unrearranged humanized TCR variable gene loci, e.g., an unrearranged human TCR variable gene region comprising human TCR variable segments capable of recombining to form a rearranged TCR variable gene sequence. TCR locus or TCR gene locus (e.g., TCRα locus or TCR locus), as used herein, refer to the genomic DNA comprising the TCR coding region, including the entire TCR coding region, including unrearranged V(D)J sequences, enhancer sequence, constant sequence(s), and any upstream or downstream (UTR, regulatory regions, etc.), or intervening DNA sequence (introns, etc.). TCR variable locus, TCR variable region, or TCR variable gene locus (e.g., TCRα variable gene locus or TCRβ variable gene locus), refers to genomic DNA that includes TCR variable region segments (V(D)J region) but excludes TCR constant sequences and, in various embodiments, enhancer sequences. Other sequences may be included in the TCR variable gene locus for the purposes of genetic manipulation (e.g., selection cassettes, restriction sites, etc.), and these are encompassed herein.
T cells bind epitopes on small antigenic determinants on the surface of antigen-presenting cells that are associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex. T cells bind these epitopes through a T cell receptor (TCR) complex on the surface of the T cell. T cell receptors are heterodimeric structures composed of two types of chains: an α (alpha) and β (beta) chain, or a γ (gamma) and δ (delta) chain. The a chain is encoded by the nucleic acid sequence located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus, and the β chain is encoded by the nucleic acid sequence located within the β locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells has an αβ TCR; while a minority of T cells bears a γδ TCR. Interactions of TCRs with MHC class I (presenting to CD8+ T cells) and MHC class II (presenting to CD4+ T cells) molecules are shown in FIG. 1 (closed symbols represent non-human sequences; striped symbols represent human sequences, showing one particular embodiment of the TCR protein of the present invention).
T cell receptor α and β polypeptides (and similarly γ and δ polypeptides) are linked to each other via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail also being a part of the constant region). The variable region of the TCR determines its antigen specificity, and similar to immunoglobulins, comprises three complementary determining regions (CDRs). Also similar to immunoglobulin genes, T cell receptor variable gene loci (e.g., TCRα and TCRβ loci) contain a number of unrearranged V(D)J segments (variable (V), joining (J), and in TCRβ and δ, diversity (D) segments). During T cell development in the thymus, TCRα variable gene locus undergoes rearrangement, such that the resultant TCR α chain is encoded by a specific combination of VJ segments (Vα/Jα sequence); and TCRβ variable gene locus undergoes rearrangement, such that the resultant TCR β chain is encoded by a specific combination of VDJ segments (vβ/Dβ/Jβ sequence).
Interactions with thymic stroma trigger thymocytes to undergo several developmental stages, characterized by expression of various cell surface markers. A summary of characteristic cell surface markers at various developmental stages in the thymus is presented in Table 1. Rearrangement at the TCRβ variable gene locus begins at the DN2 stage and ends during the DN4 stage, while rearrangement of the TCRα variable gene locus occurs at the DP stage. After the completion of TCRβ locus rearrangement, the cells express TCRβ chain at the cell surface together with the surrogate α chain, pTα. See, Janeway's Immunobiology, Chapter 7, 7th Ed., Murphy et al. eds., Garland Science, 2008.
TABLE 1
Developmental Stages of T cells in the Thymus
Developmental Stage
DN1
DN2
DN3
DN4
DP
SP
Marker(s)
CD44+/
CD44+/
CD44low/
CD44−/
CD4+/
CD4+ or
CD25−
CD25+
CD25+
CD25−
CD8+
CD8+
Naive CD4+ and CD8+ T cells exit the thymus and enter the peripheral lymphoid organs (e.g., spleen) where they are exposed to antigens and are activated to clonally expand and differentiate into a number of effector T cells (Teff), e.g., cytotoxic T cells, TREG cells, TH17 cells, TH1 cells, TH2 cells, etc. Subsequent to infection, a number of T cells persist as memory T cells, and are classified as either central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and colleagues proposed that, after initial infection, Tem cells represent a readily available pool of antigen-primed memory T cells in the peripheral tissues with effector functions, while Tcm cells represent antigen-primed memory T cells in the peripheral lymphoid organs that upon secondary challenge can become new effector T cells. While all memory T cells express CD45RO isoform of CD45 (naïve T cells express CD45RA isoform), Tcm are characterized by expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling in the peripheral lymphoid organs and lymph nodes. Id. Thus, all T cells found in the peripheral lymphoid organs (e.g., naïve T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a number of different cell surface markers, e.g., CD44. For summary of various cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, supra.
While TCR variable domain functions primarily in antigen recognition, the extracellular portion of the constant domain, as well as transmembrane, and cytoplasmic domains of the TCR also serve important functions. A complete TCR receptor complex requires more than the α and β or γ and δ polypeptides; additional molecules required include CD3γ, CD3δ, and CD3ε, as well as the ζ chain homodimer (ζζ). At the completion of TCRβ rearrangement, when the cells express TCRβ/pTα, this pre-TCR complex exists together with CD3 on the cell surface. TCRα (or pTα) on the cell surface has two basic residues in its transmembrane domain, one of which recruits a CD3δε heterodimer, and another recruits ζζ via their respective acidic residues. TCRβ has an additional basic residue in its transmembrane domain that is believed to recruit CD3δε heterodimer. See, e.g., Kuhns et al. (2006) Deconstructing the Form and Function of the TCR/CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:α005140. The assembled complex, comprising TCRαβ heterodimer, CD3γε, CD3δε, and ζζ, is expressed on the T cell surface. The polar residues in the transmembrane domain have been suggested to serve as quality control for exiting endoplasmic reticulum; it has been demonstrated that in the absence of CD3 subunits, TCR chains are retained in the ER and targeted for degradation. See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. Immunol. 23:101-25.
CD3 and ζ chains of the assembled complex provide components for TCR signaling as TCRαβ heterodimer (or TCRγδ heterodimer) by itself lacks signal transducing activity. The CD3 chains possess one Immune-Receptor-Tyrosine-based-Activation-Motif (ITAM) each, while the ζ chain contains three tandem ITAMs. ITAMs contain tyrosine residues capable of being phosphorylated by associated kinases. Thus, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, e.g., Love and Hayes (2010) ITAM-Mediated Signaling by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 2:e002485. Following TCR engagement, ITAM motifs are phosphorylated by Src family tyrosine kinases, Lck and Fyn, which initiates a signaling cascade, resulting in Ras activation, calcium mobilization, actin cytoskeleton rearrangements, and activation of transcription factors, all ultimately leading to T cell differentiation, proliferation, and effector actions. Id., see also, Janeway's Immunobiology, supra; both incorporated herein by reference.
Additionally, TCRβ transmembrane and cytoplasmic domains are thought to have a role in mitochondrial targeting and induction of apoptosis; in fact, naturally occurring N-terminally truncated TCRβ molecules exist in thymocytes. Shani et al. (2009) Incomplete T-cell receptor-β peptides target the mitochondrion and induce apoptosis, Blood 113:3530-41. Thus, several important functions are served by the TCR constant region (which, in various embodiments, comprises a portion of extracellular as well as transmembrane and cytoplasmic domains); and in various embodiments the structure of this region should be taken into consideration when designing humanized TCRs or genetically modified non-human animals expressing the same.
Mice transgenic for rearranged T cell receptor sequences are known in the art. The present invention relates to genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise unrearranged human or humanized T cell variable gene loci that are capable of rearranging to form nucleic acid sequences that encode human T cell receptor variable domains, including animals that comprise T cells that comprise rearranged human variable domains and non-human (e.g., mouse or rat) constant regions. The present invention also provides non-human animals (e.g., rodents, e.g., rats, mice) that are capable of generating a diverse repertoire of human T cell receptor variable region sequences; thus, the present invention provides non-human animals that express TCRs with fully human variable domains in response to an antigen of interest and that bind an epitope of the antigen of interest. In some embodiments, provided are non-human animals that generate a diverse T cell receptor repertoire capable of reacting with various antigens, including but not limited to antigens presented by APCs.
In one embodiment, the invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR variable region segments (V(D)J segments), wherein the unrearranged human TCR variable region segments replace, at an endogenous non-human (e.g., rodent) TCR variable gene locus (e.g., TCRα, β, δ, and/or γ variable gene locus), endogenous non-human TCR variable region segments. In one embodiment, unrearranged human TCR variable gene locus replaces endogenous non-human TCR variable gene locus.
In another embodiment, the invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR variable region segments (V(D)J segments), wherein the unrearranged human TCR variable region segments are operably linked to a non-human TCR constant region gene sequence resulting in a humanized TCR locus, wherein the humanized TCR locus is at a site in the genome other than the endogenous non-human TCR locus. Thus, in one embodiment, a non-human animal (e.g., rodent, e.g., mouse, rat) comprising a transgene that comprises unrearranged human TCR variable region segments operably linked to non-human TCR constant region gene sequence is also provided.
In one aspect, the genetically modified non-human animals of the invention comprise in their genome human TCR variable region segments, while retaining non-human (e.g., rodent, e.g., mouse, rat) TCR constant gene sequence(s) that encode TCR constant domains. In various embodiments, a TCR constant domain includes the transmembrane domain and the cytoplasmic tail of the TCR. Thus, in various embodiments of the present invention, the genetically modified non-human animals retain endogenous non-human TCR transmembrane domain and cytoplasmic tail. In other embodiments, non-human animals comprise non-human non-endogenous TCR constant gene sequences, e.g., encoding non-human non-endogenous TCR transmembrane domain and cytoplasmic tail. As indicated above, the constant domain of the TCR participates in a signaling cascade initiated during antigen-primed T cell activation; thus, endogenous TCR constant domain interacts with a variety of non-human anchor and signaling proteins in the T cell. Thus, in one aspect, the genetically modified non-human animals of the invention express humanized T cell receptors that retain the ability to recruit a variety of endogenous non-human anchor or signaling molecules, e.g., CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), the ζ chain, Lck, Fyn, ZAP-70, etc. A nonlimiting list of molecules that are recruited to the TCR complex is described in Janeway's Immunobiology, supra. It is believed that the ability of T cell development and T cell differentiation processes in the non-human animals to proceed and allow for a robust immune response may be due, at least in part, to the placement of variable regions at the endogenous mouse loci and the maintenance of mouse constant domains.
In some embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRα variable region segments, wherein the unrearranged human TCRα variable region segments are operably linked to a non-human TCRα constant region gene sequence resulting in a humanized TCRα locus. In one embodiment, the humanized TCRα locus is at a site in the genome other than the endogenous non-human TCRα locus. In another embodiment, the unrearranged human TCRα variable region segments replace endogenous non-human TCRα variable region segments while retaining endogenous non-human TCRα constant region gene sequence(s). In one embodiment, the unrearranged human TCRα variable gene locus replaces endogenous non-human TCRα variable gene locus. In some embodiments, replacement of an endogenous non-human TCRα variable region gene locus with the unrearranged human TCRα variable gene locus comprises a deletion or inactivation of a TCRδ variable gene locus. In other embodiments, replacement of an endogenous non-human TCRα variable region gene with the unrearranged human TCRα gene locus comprises a replacement of an endogenous TCRδ variable gene locus with unrearranged human TCRδ variable region segments. In some embodiments, the animal retains endogenous non-human TCRβ variable region and constant region gene sequence(s). Thus, the animal expresses a TCR that comprises a chimeric human/non-human (i.e., humanized) TCRα chain and a non-human TCR chain.
In some embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRδ variable region segments, wherein the unrearranged human TCRδ variable region segments are operably linked to a non-human TCRδ constant region gene sequence resulting in a humanized TCRδ locus. In one embodiment, the humanized TCRδ locus is at a site in the genome other than the endogenous non-human TCRδ locus. In another embodiment, the unrearranged human TCRδ variable region segments replace endogenous non-human TCRδ variable region segments while retaining endogenous non-human TCRδ constant region gene sequence(s). In one embodiment, the unrearranged human TCRδ variable gene locus replaces endogenous non-human TCRδ variable gene locus.
In other embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRβ variable region segments, wherein the unrearranged human TCRβ variable region segments are operably linked to a non-human TCR constant region gene sequence resulting in a humanized TCR locus. In one embodiment, the humanized TCR locus is at a site in the genome other than the endogenous non-human TCRβ locus. In another embodiment, the unrearranged human TCRβ variable region segments replace endogenous non-human TCRβ variable region segments while retaining endogenous non-human TCRβ constant region gene sequence(s). In one embodiment, the unrearranged human TCRβ variable gene locus replaces endogenous non-human TCRβ variable gene locus. In some embodiments, the animal retains endogenous non-human TCRα variable region and constant region gene sequence(s). Thus, the animal expresses a TCR that comprises a chimeric human/non-human (i.e., humanized) TCRβ chain and a non-human TCRα chain.
In some specific embodiments, the invention provides a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that comprises in its genome (a) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRα constant gene sequence(s), (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCR constant region gene sequence(s) and/or (c) an unrearranged TCRδ variable gene locus comprising at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRδ constant region gene sequence. Another non-human animal as provided herein comprises in its genome (a) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRα constant gene sequence(s), (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRβ constant gene sequence(s), (c) an unrearranged TCRδ variable gene locus comprising at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRδ constant region gene sequence(s) and/or (d) an unrearranged TCRγ variable gene locus comprising at least one human Vγ segment, and at least one human Jγ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRγ constant region gene sequence.
In various embodiments of the invention, the unrearranged human or humanized TCR variable gene locus (e.g., TCRα TCR and/or TCRδ variable gene locus) is comprised in the germline of the non-human animal (e.g., rodent, e.g., mouse or rat). In various embodiments, the replacements of TCR V(D)J segments by unrearranged human TCR V(D)J segments (e.g., Vα and Jα; Vβ and Dβ and Jβ; Vδ and Dδ and Jδ; Vγ and Jγ segments) are at an endogenous non-human TCR variable locus (or loci), wherein the unrearranged human V and J and/or V and D and J segments are operably linked to non-human TCR constant region gene sequences.
In some embodiments of the invention, the non-human animal comprises two copies of the unrearranged human or humanized TCRα variable gene locus, two copies of the unrearranged human or humanized TCRβ variable gene locus and/or two copies of the unrearranged human or humanized TCRδ variable gene locus. Thus, the non-human animal is homozygous for one or more unrearranged human or humanized TCRα, TCRβ and/or TCRδ variable gene loci. In some embodiments of the invention, the non-human animal comprises one copy of the unrearranged human or humanized TCRα variable gene locus one copy of the unrearranged human or humanized TCRβ variable gene locus and/or one copy of the unrearranged human or humanized TCRδ variable gene locus. Thus, the non-human animal is heterozygous for unrearranged human or humanized TCRα, TCRβ and/or TCRδ variable gene locus. In other embodiment, a non-human animal is heterozygous or homozygous for unrearranged human or humanized TCRγ variable gene locus.
In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments (e.g., human Vα and Jα segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments replaces endogenous TCRα variable gene locus. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCRα variable gene locus are capable of rearranging to form a rearranged human Vα/Jα sequence.
Similarly, in one embodiment, the unrearranged TCRβ variable gene locus comprising human variable region segments (e.g., human Vβ, Dβ, and Jβ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRβ variable gene locus comprising human variable region segments replaces endogenous TCRβ variable gene locus. In one aspect, endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence. Thus, in one aspect, the human Vβ, Dβ, and Jβ segments in the unrearranged TCR variable gene locus are capable of rearranging to form a rearranged human Vβ/Dβ/Jβ sequence.
In one embodiment, the unrearranged TCRδ variable gene locus comprising human variable region segments (e.g., human Vδ, Dδ, and Jδ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRδ variable gene locus comprising human variable region segments replaces endogenous TCRδ variable gene locus. In one aspect, endogenous non-human Vδ, Dδ, and Jδ segments are incapable of rearranging to form a rearranged Vδ/Dδ/Jδ sequence. Thus, in one aspect, the human Vδ, Dδ, and Jδ segments in the unrearranged TCRδ variable gene locus are capable of rearranging to form a rearranged human Vδ/Dδ/Jδ sequence.
In one embodiment, the unrearranged TCRγ variable gene locus comprising human variable region segments (e.g., human Vγ and Jγ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRγ variable gene locus comprising human variable region segments replaces endogenous TCRγ variable gene locus. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vγ/Jγ sequence. Thus, in one aspect, the human Vγ and Jγ segments in the unrearranged TCRγ variable gene locus are capable of rearranging to form a rearranged human Vγ/Jγ sequence.
In yet another embodiment, both the unrearranged TCRα, β, δ and/or γ variable gene loci comprising human variable region segments replace respective endogenous TCRα, β, δ, and γ variable gene loci. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence, endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence, endogenous Vδ, Dδ, and Jδ segments are incapable of rearranged to form a rearranged Vδ/Dδ/Jδ sequence and/or endogenous non-human Vγ and Jγ segments are incapable of rearranging to form a rearranged Vγ/Jγ sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCRα variable gene locus are capable of rearranging to form a rearranged human Vα/Jα sequence, the human Vβ, Dβ, and Jβ segments in the unrearranged TCRβ variable gene locus are capable of rearranging to form a rearranged human Vβ/Dβ/Jβ sequence, the human Vδ, Dδ, and Jδ segments in the unrearranged TCRδ variable gene locus are capable of rearranged to form a rearranged human Vδ/Dδ/Jδ sequence and/or the human Vγ and Jγ segments in the unrearranged TCRα variable gene locus are capable of rearranging to form a rearranged human Vγ/Jγ sequence.
In some aspects of the invention, the non-human animal comprising a humanized TCRα, TCR and/or TCR δ gene locus (comprising an unrearranged human TCRα, TCRβ and/or TCR δ variable gene locus) retains an endogenous non-human TCRα TCRβ and/or TCRδ variable gene locus. In one embodiment, the endogenous non-human TCRα, TCRβ and/or TCRδ variable gene locus is a non-functional locus. In one embodiment, the non-functional locus is an inactivated locus, e.g., an inverted locus (e.g., the coding nucleic acid sequence of the variable gene locus is in inverted orientation with respect to the constant region sequence, such that no successful rearrangements are possible utilizing variable region segments from the inverted locus). In one embodiment, the humanized TCRα, TCRβ and/or TCR δ variable gene locus is positioned between the endogenous non-human TCRα, TCRβ and/or TCRδ variable gene locus and the endogenous non-human TCRα, TCRβ and/or TCRδ constant gene locus, respectively. Similar chromosomal arrangements may be made for placing human or humanized TCRγ into the genome of a non-human animal, e.g., at a TCRγ locus.
The number, nomenclature, position, as well as other aspects of V and J and/or V, D, and J segments of the human and mouse TCR loci may be ascertained using the IMGT database, available at the website of the International Immunogenetics Information System (IMGT). The mouse TCRα variable locus is approximately 1.5 megabases and comprises a total of 110Vα and 60 Jα segments. The human TCRα variable locus is approximately 1 megabase and comprises a total of 54Vα and 61Jα segments, with 45Vα and 50Jα believed to be functional. Unless stated otherwise, the numbers of human V(D)J segments referred to throughout the specification refers to the total number of V(D)J segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) comprises at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vα segments. In some embodiments, the humanized TCRα locus comprises 2, 8, 23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, the humanized TCRα locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vα; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or 100% of human Vα.
In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human Vα40 to Vα41 (Vα segment is also referred to as “TRAV” or “TCRAV”) and a DNA fragment comprising a contiguous human sequence of 61 human Jα segments (Jα segment is also referred to as “TRAJ” or “TCRAJ”). In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV22 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV1-1 to TRAV 41 and 61 human TRAJs. In various embodiments, the DNA fragments comprising contiguous human sequences of human TCRα variable region segments also comprise restriction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with proper functioning (e.g., rearrangement, splicing, etc.) of various genes at the TCRα locus.
In one embodiment, the humanized TCRα locus comprises 61 human Jα segments, or 100% of human Jα segments. In a particular embodiment, humanized TCRα locus comprises 8 human Vα segments and 61 human Jα segments; in another particular embodiment, humanized TCRα locus comprises 23 human Vα segments and 61 human Jα segments. In another particular embodiment, the humanized TCRα locus comprises a complete repertoire of human Vα and Jα segments, i.e., all human variable a region gene segments encoded by the α locus, or 54 human Vα and 61 human Jα segments. In various embodiments, the non-human animal does not comprise any endogenous non-human Vα or Jα segments at the TCRα locus.
The mouse TCRβ variable locus is approximately 0.6 megabases and comprises a total of 33 Vβ, 2 Dβ, and 14 Jβ segments. The human TCRβ variable locus is approximately 0.6 megabases and comprises a total of 67 Vβ, 2 Dβ, and 14 Jβ segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) comprises at least one human Vβ, at least one human Dβ, and at least one human Jα segment. In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to human 67 Vβ segments. In some embodiments, the humanized TCRβ locus comprises 8, 14, 40, 66, or human 67 Vβ segments. Thus, in some embodiments, the humanized TCRβ locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vβ; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98%, or 100% of human V.
In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human Vβ18 to Vβ29-1 (Vβ segment is also referred to as “TRBV” or “TCRBV”). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV6-5 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., human Dβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7 segments). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV6-5 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., human Dβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7 segments). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV1 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2. In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV1 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2, and a separate DNA fragment comprising the sequence of human TRBV30. In various embodiments, the DNA fragments comprising contiguous human sequences of human TCRβ variable region segments also comprise restriction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with proper functioning (e.g., rearrangement, splicing, etc.) of various genes at the TCR locus.
In one embodiment, the humanized TCR locus comprises 14 human Jβ segments, or 100% of human Jβ segments, and 2 human Dβ segments or 100% of human Jβ segments. In another embodiment, the humanized TCR locus comprises at least one human Vβ segment, e.g., 14 human Vβ segments, and all mouse Dβ and Jβ segments. In a particular embodiment, humanized TCR locus comprises 14 human Vβ segments, 2 human Dβ segments, and 14 human Jβ segments. In another particular embodiment, the humanized TCR locus comprises a complete repertoire of human Vβ, Dβ, and Jβ segments, i.e., all human variable β region gene segments encoded by the β locus or 67 human Vβ, 2 human Dβ, and 14 human Jβ segments. In one embodiment, the non-human animal comprises one (e.g., 5′) non-human Vβ segment at the humanized TCR locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCR locus.
In various embodiments, wherein the non-human animal (e.g., rodent) comprises a repertoire of human TCRα and TCRβ (and optionally human TCRδ and TCRγ) variable region segments (e.g., a complete repertoire of variable region segments), the repertoire of various segments (e.g., the complete repertoire of various segments) is utilized by the animal to generate a diverse repertoire of TCR molecules to various antigens.
In various aspects, the non-human animals comprise contiguous portions of the human genomic TCR variable loci that comprise V, D, and J, or D and J, or V and J, or V segments arranged as in an unrearranged human genomic variable locus, e.g., comprising promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., arranged as in a human genomic TCR variable locus. In other aspects, the various segments are arranged as in an unrearranged non-human genomic TCR variable locus. In various embodiments of the humanized TCR α, β, δ and/or γ locus, the humanized locus can comprise two or more human genomic segments that do not appear in a human genome juxtaposed, e.g., a fragment of V segments of the human variable locus located in a human genome proximal to the constant region, juxtaposed with a fragment of V segments of the human variable locus located in a human genome at the upstream end of the human variable locus.
In both mouse and human, the TCRδ gene segments are located with the TCRα locus (see FIG. 4A, top, TCRD region boxed). TCRδ J and D segments are located between Vα and Jα segments, while TCRδ V segments are interspersed throughout the TCRα locus, with the majority located among various Vα segments. The number and locations of various TCRδ segments can be determined from the IMGT database. Due to the genomic arrangement of TCRδ gene segments within the TCRα locus, successful rearrangement at the TCRα locus may delete or inactivate the TCRδ gene segments.
In some embodiments of the invention, a non-human animal comprising an unrearranged human TCRα variable gene locus also comprises at least one human Vδ segment, e.g., up to complete repertoire of human Vδ segments. Thus, in some embodiments, the replacement of endogenous TCRα variable gene locus results in a replacement of at least one non-human Vδ segment with a human Vδ segment. In other embodiments, the non-human animal of the invention comprises a complete repertoire of human Vδ, Dδ, and Jδ segments at the unrearranged humanized TCRα locus; in yet other embodiments, the non-human animal comprises a complete unrearranged human TCRδ locus at the unrearranged humanized TCRα locus (i.e., a TCRδ locus including human variable region segments, as well as human enhancer and constant region). An exemplary embodiment for constructing an unrearranged humanized TCRα locus comprising complete unrearranged TCRδ locus is depicted in U.S. Pat. No. 9,113,616, incorporated herein by reference.
In yet another embodiment, the non-human animal of the invention further comprises an unrearranged humanized TCRγ locus, e.g., a TCRγ locus comprising at least one human Vγ and at least one human Jγ segments (e.g., a complete repertoire of human Vγ and human Jγ variable region segments). The human TCRγ locus is on human chromosome 7, while the mouse TCRγ locus is on mouse chromosome 13. See the IMGT database for more detail on the TCRγ locus.
In one aspect, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising humanized TCRα and β variable gene loci (and, optionally humanized TCRδ/γ variable gene loci) described herein expresses a humanized T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell. In some aspects, the non-human animal is capable or expressing a diverse repertoire of humanized T cell receptors that recognize a variety of presented antigens.
In various embodiments of the invention, the humanized T cell receptor polypeptides described herein comprise human leader sequences. In alternative embodiments, the humanized TCR receptor nucleic acid sequences are engineered such that the humanized TCR polypeptides comprise non-human leader sequences.
The humanized TCR polypeptides described herein may be expressed under control of endogenous non-human regulatory elements (e.g., rodent regulatory elements), e.g., promoter, silencer, enhancer, etc. The humanized TCR polypeptides described herein may alternatively be expressed under control of human regulatory elements. In various embodiments, the non-human animals described herein further comprise all regulatory and other sequences normally found in situ in the human genome.
In various embodiments, the human variable region of the humanized TCR protein is capable of interacting with various proteins on the surface of the same cell or another cell. In one embodiment, the human variable region of the humanized TCR interacts with MHC proteins (e.g., MHC class I or II proteins) presenting antigens on the surface of the second cell, e.g., an antigen presenting cell (APC). In some embodiments, the MHC I or II protein is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC I or II protein is a human(ized) protein. In one aspect, the second cell, e.g., the APC, is an endogenous non-human cell expressing a human or humanized MHC molecule. In a different embodiment, the second cell is a human cell expressing a human MHC molecule.
In one aspect, the non-human animal expresses a humanized T cell receptor with a non-human constant region on the surface of a T cell, wherein the receptor is capable of interacting with non-human molecules, e.g., anchor or signaling molecules expressed in the T cell (e.g., CD3 molecules, the chain, or other proteins anchored to the TCR through the CD3 molecules or the ζ chain). Thus, in one aspect, a cellular complex is provided, comprising (a) a non-human T-cell that expresses (i) a TCR that comprises a humanized TCRα chain as described herein and humanized TCRβ chain as described herein and (ii) a chimeric co-receptor as described herein and (b) a non-human antigen-presenting cell comprising an antigen bound to a chimeric MHC I and/or chimeric MHC II α s described herein. In one embodiment, the non-human constant TCRα and TCRβ chains are complexed with a non-human zeta (ζ) chain homodimer and CD3 heterodimers. In one embodiment, the cellular complex is an in vivo cellular complex. In one embodiment, the cellular complex is an in vitro cellular complex.
In various embodiments, the non-human animals (e.g., rodents, e.g., mice or rats) described herein produce T cells that are capable of undergoing thymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP and to CD4 or CD8 SP T cells. Such T cells of the non-human animal of the invention express cell surface molecules typically produced by a T cell during a particular stage of thymic development (e.g., CD25, CD44, Kit, CD3, pTα, etc.). Thus, in one embodiment, the non-human animals described herein may express pTα complexed with TCRβ at the DN3 stage of thymic development. The non-human animals described herein express T cells capable of undergoing thymic development to produce CD4+ and CD8+ T cells.
In various embodiments, the non-human animals described herein produce T cells that are capable of undergoing T cell differentiation in the periphery. In some embodiments, the non-human animals described herein are capable of producing a repertoire of effector T cells, e.g., CTL (cytotoxic T lymphocytes), TH1, TH2, TREG, TH17, etc. Thus, in these embodiments, the non-human animals described herein generate effector T cells that fulfill different functions typical of the particular T cell type, e.g., recognize, bind, and respond to foreign antigens. In various embodiments, the non-human animals described herein produce effector T cells that kill cells displaying peptide fragments of cytosolic pathogens expressed in the context of MHC I molecules; recognize peptides derived from antigens degraded in intracellular vesicles and presented by MHC II molecules on the surface of macrophages and induce macrophages to kill microorganisms; produce cytokines that drive B cell differentiation; activate B cells to produce opsonizing antibodies; induce epithelial cells to produce chemokines that recruit neutrophils to infection sites; etc.
In additional embodiments, the non-human animals described herein comprise CD3+ T cells in the periphery, e.g., in the spleen. In other aspects, the non-human animals described herein are capable of generating a population of memory T cells in response an antigen of interest. For example, the non-human animals generate both central memory T cells (Tcm) and effector memory T cells (Tem) to an antigen, e.g., antigen of interest (e.g., antigen being tested for vaccine development, etc.).
DN1 and DN2 cells that do not receive sufficient signals (e.g., Notch signals) may develop into B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. See, e.g., Yashiro-Ohtani et al. (2010) Notch regulation of early thymocyte development, Seminars in Immunology 22:261-69. In some embodiments, the non-human animals described herein develop B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. In some embodiments, the non-human animals described herein develop a dendritic cell population in the thymus.
The predominant type of T cell receptors expressed on the surface of T cells is TCRα/β, with the minority of the cells expressing TCRδ/γ. In some embodiments of the invention, the T cells of the non-human animals comprising humanized TCRα and/or β loci exhibit utilization of TCRα/β and TCRδ/γ loci, e.g., utilization of TCRα/β and TCRδ/γ loci that is similar to the wild type animal (e.g., the T cells of the non-human animals described herein express TCRα/β and TCRδ/γ proteins in comparable proportions to that expressed by wild type animals). Thus, in some embodiments, the non-human animals comprising humanized TCRα/p and endogenous non-human TCRδ/γ loci exhibit utilization of all loci.
Human or Humanized MHC Molecules
In various embodiments, provided herein are genetically modified non-human animals that co-express at least one humanized T cell co-receptor, at least one humanized MHC that associates with the humanized T cell co-receptor, and optionally, a humanized TCR, which upon recognizing and binding peptide presented by the humanized MHC, and in conjunction with the humanized co-receptor, provides activation signals to the cell expressing the humanized TCR and chimeric T cell co-receptor polypeptides. Accordingly, a non-human animal as disclosed herein comprises at least one of a first, second, and/or third nucleic acid sequence, each of which encodes a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC II α polypeptide, a human or humanized MHC II β polypeptide, and a human or humanized MHC I α polypeptide; the non-human animal also optionally comprises a human or humanized β2 microglobulin. Use of the first, second, and third designations herein is not to be construed as limiting the non-human animals disclosed herein as requiring all three nucleic acid sequences or the presence of any of the human or humanized MHC polypeptides in any specific order.
Accordingly, in some embodiments, a non-human animal as disclosed herein may comprise, e.g., a first and second nucleotide sequence encoding e.g., a human or chimeric CD8α polypeptide and a human or chimeric CD8β polypeptide, an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR constant gene sequence, and optionally a first and second nucleic acid sequence encoding, e.g., a human or humanized MHC I α polypeptide and a human or humanized β2-microglobulin polypeptide. In other embodiments, a non-human animal as disclosed herein may comprise, e.g., a first nucleotide sequence encoding, e.g., a chimeric CD4 polypeptide; an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCR variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCR constant gene sequence; and optionally a first and second nucleic acid sequence encoding, e.g., a human or humanized MHC II α polypeptide and a human or humanized MHC II β polypeptide. In some embodiment, a non-human animal as disclosed herein may comprise, e.g., a first, second and third nucleotide sequence encoding e.g., a chimeric CD4 polypeptide, a chimeric CD8α polypeptide, and a chimeric CD8β polypeptide; an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence; and optionally a first, second, third and fourth nucleic acid sequence encoding, e.g., a human or humanized MHC II α polypeptide, a human or humanized MHC II β polypeptide, a human or humanized MHC I α polypeptide, and a human or humanized a β2-microglobulin polypeptide.
In various embodiments, provided herein is a genetically modified non-human animal, e.g., rodent (e.g., mouse or rat) comprising in its genome a nucleic acid sequence encoding a human or humanized MHC I polypeptide and/or a nucleic acid sequence encoding human or humanized MHC II protein. The MHC I nucleic acid sequence may encode an MHC I polypeptide that is partially human and partially non-human, e.g., chimeric human/non-human MHC I polypeptide, and the MHC II nucleic acid sequence may encode an MHC II protein that is partially human and partially non-human, e.g., chimeric human/non-human MHC II protein (e.g., comprising chimeric human/non-human MHC II α and β polypeptides). In some aspects, the animal does not express endogenous MHC I and/or endogenous MHC II polypeptides, e.g., functional endogenous MHC I and/or MHC II polypeptides on a cell surface. In some embodiments, the only MHC I and/or MHC II molecules expressed on a cell surface of the animal are chimeric MHC I and/or MHC II molecules.
A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide is disclosed in U.S. Patent Publication Nos. 20130111617 and 20130185819, which publications are incorporated herein by reference in their entireties. A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding humanized, e.g., chimeric human/non-human MHC II polypeptides is disclosed in U.S. Pat. No. 8,847,005 and in U.S. Patent Publication No 20130185820, each of which are incorporated herein by reference in their entireties. A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide and comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding humanized, e.g., chimeric human/non-human MHC II polypeptides, is disclosed in U.S. Patent Publication No. 20140245467, which is incorporated herein by reference in its entirety.
In various embodiments provided herein is a genetically modified non-human animal comprising in its genome, e.g., at one or more endogenous MHC loci, a first nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric MHC I polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC I polypeptide; a second nucleic acid sequence encoding a chimeric human/non-human MHC II α polypeptide, wherein a human portion of the chimeric MHC II α polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC II α polypeptide; and/or a third nucleic acid sequence encoding a chimeric human/non-human MHC II β polypeptide, wherein a human portion of the chimeric MHC II β polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC II β polypeptide; wherein the non-human animal expresses functional chimeric human/non-human MHC I and MHC II proteins from its endogenous non-human MHC locus. In one embodiment, the first, second, and/or third nucleic acid sequences are respectively located the endogenous non-human MHC I, MHC II α and MHC II β loci. In one embodiment, wherein the non-human animal is a mouse, the first, second, and/or third nucleic acid sequences are located at the endogenous mouse MHC locus on mouse chromosome 17. In one embodiment, the first nucleic acid sequence is located at the endogenous non-human MHC I locus. In one embodiment, the second nucleic acid sequence is located at the endogenous non-human MHC II α locus. In one embodiment, the third nucleic acid sequence is located at the endogenous non-human MHC II β locus.
In one embodiment, the non-human animal only expresses the chimeric human/non-human MHC I, MHC II α and/or MHC β11 polypeptides and does not express endogenous non-human MHC polypeptides (e.g., functional endogenous MHC I, II α and/or II β polypeptides) from the endogenous non-human MHC locus. In one embodiment, the animal described herein expresses a functional chimeric MHC I and a functional chimeric MHC II on the surface of its cells, e.g., antigen presenting cells, etc. In one embodiment, the only MHC I and MHC II expressed by the animal on a cell surface are chimeric MHC I and chimeric MHC II, and the animal does not express any endogenous MHC I and MHC II on a cell surface.
In one embodiment, the chimeric human/non-human MHC I polypeptide comprises in its human portion a peptide binding cleft, e.g., of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular portion of a human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises an extracellular domain of an a chain of a human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises α1 and β2 domains of a human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises α1, α2, and α3 domains of a human MHC I.
In one aspect, a human portion of the chimeric MHC II α polypeptide and/or a human portion of the chimeric MHC II β polypeptide comprises a peptide-binding domain of a human MHC II α polypeptide and/or human MHC II β polypeptide, respectively. In one aspect, a human portion of the chimeric MHC II α and/or β polypeptide comprises an extracellular portion of a human MHC II α and/or β polypeptide, respectively. In one embodiment, a human portion of the chimeric MHC II α polypeptide comprises α1 domain of a human MHC II α polypeptide; in another embodiment, a human portion of the chimeric MHC II α polypeptide comprises α1 and β2 domains of a human MHC II α polypeptide. In an additional embodiment, a human portion of the chimeric MHC II β polypeptide comprises β1 domain of a human MHC II β polypeptide; in another embodiment, a human portion of the chimeric MHC II β polypeptide comprises β1 and β2 domains of a human MHC II β polypeptide.
In some embodiments, the human or humanized MHC I polypeptide may be derived from a functional human HLA molecule encoded by any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. The human or humanized MHC II polypeptide may be derived from a functional human HLA molecule encoded by an of HLA-DP, -DQ, and -DR loci. A list of commonly used HLA antigens and alleles is described in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), incorporated herein by reference. Shankarkumar et al. also present a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75:291-455, both incorporated herein by reference. In some embodiments, the MHC I or MHC II polypeptides may be derived from any functional human HLA-A, B, C, DR, or DQ molecules. Thus, the human or humanized MHC I and/or II polypeptides may be derived from any functional human HLA molecules described therein. In some embodiments, all MHC I and MHC II polypeptides expressed on a cell surface comprise a portion derived from human HLA molecules.
Of particular interest are human HLA molecules, specific polymorphic HLA alleles, known to be associated with a number of human diseases, e.g., human autoimmune diseases. In fact, specific polymorphisms in HLA loci have been identified that correlate with development of rheumatoid arthritis, type I diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune disorders. See, e.g., Wong and Wen (2004) What can the HLA transgenic mouse tell us about autoimmune diabetes?, Diabetologia 47:1476-87; Taneja and David (1998) HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity, J. Clin. Invest. 101:921-26; Bakker et al. (2006), A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information; and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106:18680-85. Thus, the human or humanized MHC I and/or II polypeptides may be derived from a human HLA molecule known to be associated with a particular disease, e.g., autoimmune disease.
In one specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., and HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is commonly used amongst the North American population. Although the present Examples describe this particular HLA sequence, any suitable HLA-A sequence is encompassed herein, e.g., polymorphic variants of HLA-A2 exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequence described herein due to the degeneracy of genetic code, etc.
In another specific aspect, the human portion of the chimeric MHC I polypeptide is derived from human MHC I selected from HLA-B and HLA-C. In one aspect, it is derived from HLA-B, e.g., HLA-B27. In another aspect, it is derived from HLA-A3, -B7, -Cw6, etc.
In one specific aspect, the human portions of the humanized MHC II α and β polypeptides described herein are derived from human HLA-DR, e.g., HLA-DR2. Typically, HLA-DR α chains are monomorphic, e.g., the α chain of HLA-DR complex is encoded by HLA-DRA gene (e.g., HLA-DRα*01 gene). On the other hand, the HLA-DR β chain is polymorphic. Thus, HLA-DR2 comprises an a chain encoded by HLA-DRA gene and a β chain encoded by HLA-DR1β *1501 gene. Although the present Examples describe these particular HLA sequences; any suitable HLA-DR sequences are encompassed herein, e.g., polymorphic variants exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequences described herein due to the degeneracy of genetic code, etc.
The human portions of the chimeric MHC II α and/or β polypeptide may be encoded by nucleic acid sequences of HLA alleles known to be associated with common human diseases. Such HLA alleles include, but are not limited to, HLA-DRB1*0401, -DRB1*0301, -DQA1*0501, -DQB1*0201, DRB1*1501, -DRB1*1502, -DQB1*0602, -DQA1*0102, -DQA1*0201, -DQB1*0202, -DQA1*0501, and combinations thereof. For a summary of HLA allele/disease associations, see Bakker et al. (2006), supra, incorporated herein by reference.
In one aspect, the non-human portion of a chimeric human/non-human MHC I, MHC II α and/or MHC II β polypeptide(s) comprises transmembrane and/or cytoplasmic domains of an endogenous non-human (e.g., rodent, e.g., mouse, rat, etc.) MHC I, MHC II α and/or MHC II β polypeptide(s), respectively. Thus, the non-human portion of the chimeric human/non-human MHC I polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC I polypeptide. The non-human portion of a chimeric MHC II α polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC II α polypeptide. The non-human portion of a chimeric human/non-human MHC II β polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC II β polypeptide. In one aspect, the non-human animal is mouse, and a non-human portion of the chimeric MHC I polypeptide is derived from a mouse H-2K protein. In one aspect, the animal is a mouse, and non-human portions of the chimeric MHC II α and β polypeptides are derived from a mouse H-2E protein. Thus, a non-human portion of the chimeric MHC I polypeptide may comprise transmembrane and cytoplasmic domains derived from a mouse H-2K, and non-human portions of the chimeric MHC II α and β polypeptides may comprise transmembrane and cytoplasmic domains derived from a mouse H-2E protein. Although specific H-2K and H-2E sequences are contemplated in the Examples, any suitable sequences, e.g., polymorphic variants, conservative/non-conservative amino acid substitutions, etc., are encompassed herein. In one aspect, the non-human animal is a mouse, and the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In other aspects, the mouse does not express any functional endogenous mouse MHC I and MHC II on a cell surface.
A chimeric human/non-human polypeptide may be such that it comprises a human or a non-human leader (signal) sequence. In one embodiment, the chimeric MHC I polypeptide comprises a non-human leader sequence of an endogenous MHC I polypeptide. In one embodiment, the chimeric MHC II α polypeptide comprises a non-human leader sequence of an endogenous MHC II α polypeptide. In one embodiment, the chimeric MHC II β polypeptide comprises a non-human leader sequence of an endogenous MHC II β polypeptide. In an alternative embodiment, the chimeric MHC I, MHC II α and/or MHC II β polypeptide(s) comprises a non-human leader sequence of MHC I, MHC II α and/or MHC II β polypeptide(s), respectively, from another non-human animal, e.g., another rodent or another mouse strain. Thus, the nucleic acid sequence encoding the chimeric MHC I, MHC II α and/or MHC II β polypeptide may be operably linked to a nucleic acid sequence encoding a non-human MHC I, MHC II α and/or MHC II β leader sequence, respectively. In yet another embodiment, the chimeric MHC I, MHC II α and/or MHC II β polypeptide(s) comprises a human leader sequence of human MHC I, human MHC II α and/or human MHC II β polypeptide, respectively (e.g., a leader sequence of human HLA-A2, human HLA-DRα and/or human HLA-DRβ1*1501, respectively).
A chimeric human/non-human MHC I, MHC II α and/or MHC II β polypeptide may comprise in its human portion a complete or substantially complete extracellular domain of a human MHC I, human MHC II α and/or human MHC II β polypeptide, respectively. Thus, a human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more of the amino acids encoding an extracellular domain of a human MHC I, human MHC II α and/or human MHC II β polypeptide (e.g., human HLA-A2, human HLA-DRα and/or human HLA-DRβ1*1501). In one example, substantially complete extracellular domain of the human MHC I, human MHC II α and/or human MHC II β polypeptide lacks a human leader sequence. In another example, the chimeric human/non-human MHC I, chimeric human/non-human MHC II α and/or the chimeric human/non-human MHC II β polypeptide comprises a human leader sequence.
Moreover, the chimeric MHC I, MHC II α and/or MHC II β polypeptide may be operably linked to (e.g., be expressed under the regulatory control of) endogenous non-human promoter and regulatory elements, e.g., mouse MHC I, MHC II α and/or MHC II β regulatory elements, respectively. Such arrangement will facilitate proper expression of the chimeric MHC I and/or MHC II polypeptides in the non-human animal, e.g., during immune response in the non-human animal.
In a further embodiment, a non-human animal of the invention, e.g., a rodent, e.g., a mouse, comprises (e.g., at an endogenous β2 microglobulin locus) a nucleic acid sequence encoding a human or humanized β2 microglobulin. β2 microglobulin or the light chain of the MHC class I complex (also abbreviated “β2M”) is a small (12 kDa) non-glycosylated protein, that functions primarily to stabilize the MHC I α chain. Generation of human or humanized β2 microglobulin animals is described in detail in U.S. Patent Publication No. 20130111617, and is incorporated herein by reference.
The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin protein (i.e., amino acid residues corresponding to the mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin protein, for example, amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin protein. The nucleic and amino acid sequences of human β2 microglobulin are described in Gussow et al., supra, incorporated herein by reference.
Thus, the human or humanized β2 microglobulin polypeptide may comprise amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.
In some embodiments, the nucleotide sequence encoding a human or humanized β2 microglobulin comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to allow for normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises about 2.8 kb of a human β2 microglobulin gene.
Thus, the human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin, e.g., nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In a specific embodiment, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8 kb of a human β2 microglobulin gene. As exon 4 of the β2 microglobulin gene contains the 5′ untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.
It would be understood by those of ordinary skill in the art that although specific nucleic acid and amino acid sequences to generate genetically engineered animals are described herein, sequences of one or more conservative or non-conservative amino acid substitutions, or sequences differing from those described herein due to the degeneracy of the genetic code, are also provided.
Therefore, a non-human animal that expresses a human β2 microglobulin sequence is provided, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human β2 microglobulin sequence. In a specific embodiment, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence described herein. In one embodiment, the human β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence comprises one or more non-conservative substitutions.
In addition, provided are non-human animals wherein the nucleotide sequence encoding a human or humanized β2 microglobulin protein also comprises a nucleotide sequence set forth in exon 1 of a non-human β2 microglobulin gene. Thus, in a specific embodiment, the non-human animal comprises in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin wherein the nucleotide sequence comprises exon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of a non-human β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2 and 3 of a human β2 microglobulin gene).
In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) of the invention, in addition to a nucleotide sequence encoding a chimeric CD8 protein, further comprises a nucleic acid sequence encoding a human or humanized MHC I protein, such that the chimeric CD8 protein expressed on the surface of a T cell of the animal is capable of associating, binding and/or interacting with a human or humanized MHC I expressed on a surface of a second cell, e.g., an antigen presenting cell. In one embodiment, the MHC I protein comprises an extracellular domain of a human MHC I polypeptide. In one embodiment, the animal further comprises a human or humanized β2 microglobulin polypeptide. Exemplary genetically modified animals expressing a human or humanized MHC I polypeptide and/or β2 microglobulin polypeptide are described in U.S. Patent Publication Nos. 20130111617 and 20130185819, both incorporated herein by reference in their entireties. Thus, in one embodiment, the animal comprising chimeric CD8 protein described herein may further comprise a humanized MHC I complex, wherein the humanized MHC I complex comprises: (1) a humanized MHC I polypeptide, e.g., wherein the humanized MHC I polypeptide comprises a human MHC I extracellular domain and transmembrane and cytoplasmic domains of an endogenous (e.g., mouse) MHC I, e.g., wherein the humanized MHC I comprises al, α2, and α3 domains of a human MHC I polypeptide, and (2) a human or humanized β2 microglobulin polypeptide (e.g., the animal comprises in its genome a nucleotide sequence set forth in exons 2, 3, and 4 of a human β2 microglobulin). In one aspect, both humanized MHC I and human or humanized β2 microglobulin polypeptides are encoded by nucleotide sequences located at endogenous MHC I and β2 microglobulin loci, respectively; in one aspect, the animal does not express functional endogenous MHC I and β2 microglobulin polypeptides. Thus, the MHC I expressed by the animals may be a chimeric human/non-human, e.g., human/rodent (e.g., human/mouse) MHC I polypeptide. A human portion of the chimeric MHC I polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in a human population. In the embodiment, wherein the animal is a mouse, a non-human (i.e., a mouse) portion of the chimeric MHC I polypeptide may be derived from a mouse MHC I protein selected from H-2D, H-2K and H-2L.
In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) of the invention further comprises a nucleotide sequence encoding a human or humanized MHC II protein, such that the chimeric CD4 protein expressed on the surface of a T cell of the animal is capable of interacting with a human or humanized MHC II expressed on a surface of a second cell, e.g., an antigen presenting cell. In one embodiment, the MHC II protein comprises an extracellular domain of a human MHC II α polypeptide and an extracellular domain of a human MHC II β polypeptide. Exemplary genetically modified animals expressing a human or humanized MHC II polypeptide are described in U.S. Pat. No. 8,847,005, issued Sep. 30, 2014, and U.S. Patent Publication No. 20130185820, incorporated herein by reference in their entireties. Thus, in one embodiment, the animal comprising chimeric CD4 protein described herein may further comprise a humanized MHC II protein, wherein the humanized MHC II protein comprises: (1) a humanized MHC II α polypeptide comprising a human MHC II α extracellular domain and transmembrane and cytoplasmic domains of an endogenous, e.g., mouse, MHC II, wherein the human MHC II α extracellular domain comprises α1 and β2 domains of a human MHC II α and (2) a humanized MHC II β polypeptide comprising a human MHC II β extracellular domain and transmembrane and cytoplasmic domains of an endogenous, e.g., mouse, MHC II, wherein the human MHC II β extracellular domain comprises β1 and β2 domains of a human MHC II β. In one aspect, both humanized MHC II α and β polypeptides are encoded by nucleic acid sequences located at endogenous MHC II α and β loci, respectively; in one aspect, the animal does not express functional endogenous MHC II α and β polypeptides. Thus, the MHC II expressed by the animals may be a chimeric human/non-human, e.g., human/rodent (e.g., human/mouse) MHC II protein. A human portion of the chimeric MHC II protein may be derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g., HLA-DR4, HLA-DR2, HLA-DQ2.5, HLA-DQ8, or any other HLA class II molecule present in a human population. In the embodiment, wherein the animal is a mouse, a non-human (i.e., a mouse) portion of the chimeric MHC II polypeptide may be derived from a mouse MHC II protein selected from H-2E and H-2A.
Various other embodiments of a genetically modified non-human animal, e.g. rodent, e.g., rat or mouse, would be evident to one skilled in the art from the present disclosure and from the disclosure of U.S. Patent Publication Nos. 20130111617, 20130185819 and 20130185820, and U.S. Pat. No. 8,847,005, incorporated herein by reference.
In various embodiments, the genetically modified non-human animals described herein make cells, e.g., APCs, with human or humanized MHC I and II on the cell surface and, as a result, present peptides as epitopes for T cells in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in vaccine development; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC expression.
Non-Human Animals, Tissues and Cells
The genetically modified non-human animal of the invention may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.
In a specific embodiment, the non-human animal is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In an embodiment, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and another aforementioned strain. Non-human animals as provided herein may be a mouse derived from any combination of the aforementioned strains.
In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
Thus, in one embodiment of the invention, a genetically modified mouse is provided, wherein the mouse comprises, e.g., in its genome, e.g., in its germline genome, (a) a first nucleotide sequence encoding a first chimeric human/murine T cell co-receptor polypeptide (e.g., CD4), a second nucleotide sequence encoding a second chimeric human/murine T cell co-receptor polypeptide (e.g., CD8α), and/or a third nucleotide sequence encoding a third chimeric human/murine T cell co-receptor polypeptide (e.g., CD8β), wherein a murine portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a murine T cell co-receptor, wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human T cell co-receptor, and wherein the mouse expresses the first, second and/or third chimeric T cell co-receptor polypeptide; (b) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a murine TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a murine TCRβ constant gene sequence; and optionally, (c) a first nucleic acid sequence encoding a first chimeric human/murine MHC polypeptide (e.g., MHC II α), a second nucleic acid sequence encoding a second chimeric human/murine MHC polypeptide (e.g., MHC II β) and/or a third nucleic acid sequence encoding a third chimeric human/murine MHC polypeptide (e.g., MHC I) and a β2 microglobulin locus encoding a human or humanized β2 microglobulin, wherein a human portion of each chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide that associates with the first, second and/or third chimeric T cell co-receptor polypeptide (e.g., wherein a human portion of a chimeric MHC II complex (e.g., humanized MHC II α and β polypeptides) associates with the chimeric CD4 polypeptide and/or a human portion of the chimeric MHC I polypeptide (or MHC I complex, e.g., humanized MHC Iα and human(ized) β2 microglobulin) associates with the chimeric CD8 co-receptor (e.g., humanized CD8 α and β polypeptides).
A genetically modified mouse is provided herein comprising in its genome, e.g., at its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human/mouse CD4 polypeptide, wherein a mouse portion of the chimeric polypeptide comprises at least transmembrane and cytoplasmic domains of a mouse CD4 polypeptide, and wherein the mouse expresses a chimeric human/mouse CD4. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of the extracellular domain of a human CD4 polypeptide. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of the D1 domain of a human CD4 protein. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of D1-D2 domains of a human CD4 protein, e.g., at least all or substantially all of D1-D3 domains of a human CD4 protein, e.g., all or substantially all of D1-D4 domains of a human CD4 protein. Thus, in one embodiment, the mouse comprises at the endogenous CD4 locus a nucleotide sequence comprising at least all or substantially all of exons 4, 5, and 6 of the human CD4 gene, e.g., the sequence of exon 3 of the human CD4 gene encoding a portion of the D1 domain of human CD4 and exons 4-6 of the human CD4 gene. In one embodiment, the mouse comprises at the endogenous CD4 locus a chimeric human/mouse CD4 that comprises a human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In another embodiment, the mouse comprises at the endogenous CD4 locus a chimeric human/mouse CD4 that comprises a human CD4 sequence that is responsible for interacting with MHC II and/or variable domain of a T cell receptor. In one embodiment, the nucleotide sequence comprises the sequence encoding mouse CD4 signal peptide. In one embodiment, the mouse comprises a replacement of the nucleotide sequence encoding a mouse CD4 extracellular domain with a nucleotide sequence encoding a human CD4 extracellular domain. In another embodiment, the mouse comprises a replacement of the nucleotide sequence encoding at least all or substantially all of mouse CD4 D1 domain, e.g., a nucleotide sequence encoding at least all or substantially all of mouse CD4 D1-D2 domains, e.g., a nucleotide sequence encoding at least all or substantially all of mouse CD4 D1-D3 domains, with human nucleotide sequence encoding the same. In one embodiment, the domains of chimeric CD4 polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5A.
In one embodiment, the mouse does not express a functional endogenous mouse CD4 from it endogenous mouse CD4 locus. In one embodiment, the mouse described herein comprises the chimeric human/mouse CD4 nucleotide sequence in the germline of the mouse.
In one embodiment, the mouse retains any endogenous sequences that have not been humanized, e.g., in the embodiment wherein the mouse comprises a replacement of the nucleotide sequence encoding all or substantially all of D1-D3 domains, the mouse retains endogenous nucleotide sequence encoding mouse CD4 D4 domain as well a nucleotide sequence encoding transmembrane and cytoplasmic domains of mouse CD4.
In one aspect, the mouse expressing chimeric human/mouse CD4 protein retains mouse CD4 promoter and regulatory sequences, e.g., the nucleotide sequence in the mouse encoding chimeric human/mouse CD4 is operably linked to endogenous mouse CD4 promoter and regulatory sequences. In one aspect, these mouse regulatory sequences retained in the genetically engineered animal of the invention include the sequences that regulate expression of the chimeric protein at proper stages during T cell development. Thus, in one aspect, the mouse does not express chimeric CD4 on B cells or mature CD8+ T cells. In one aspect, the mouse also does not express chimeric CD4 on any cell type, e.g., any immune cell type, that normally does not express endogenous CD4.
A genetically modified mouse disclosed herein may comprise in its genome, e.g., at its endogenous CD8 locus, a first nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide. In one embodiment, the first nucleotide sequence comprises a sequence that encodes all or substantially all of an extracellular portion of a human CD8α polypeptide and at least transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence that encodes all or substantially all of an extracellular portion of a human CD8β polypeptide and at least transmembrane and cytoplasmic domains of a mouse CD8β polypeptide, and wherein the mouse expresses a functional chimeric human/mouse CD8 protein. In one embodiment, the first nucleotide sequence comprises a sequence that encodes at least the immunoglobulin V-like domain of the human CD8α polypeptide and the remaining sequences of a mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence that encodes at least the immunoglobulin V-like domain of the human CD8β polypeptide and the remaining sequences of a mouse CD8β polypeptide. In one embodiment, first nucleotide sequence comprises at least the MHC I-binding domain of a human CD8α polypeptide. In one embodiment, the first and the second nucleotide sequences comprise at least the exons that encode the extracellular portion of a human CD8α polypeptide and/or CD8β polypeptide, respectively. In one embodiment, the extracellular portion of a human CD8α polypeptide and/or CD8β polypeptide is a region encompassing the portion of the human CD8α polypeptide and/or CD8β polypeptide that is not transmembrane or cytoplasmic domain. In one embodiment, the domains of a chimeric CD8α polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5B. In one embodiment, the domains of a chimeric CD8β polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5B. In one embodiment, the nucleotide sequence encoding the chimeric human/mouse CD8α polypeptide and/or CD8β polypeptide comprises the sequence encoding a mouse CD8α and/or CD8β signal peptide, respectively. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8α and/or CD8β signal sequence. In one embodiment, the mouse comprises a replacement of a nucleotide sequence encoding all or substantially all of the mouse CD8α and/or CD8β extracellular domain with a nucleotide sequence encoding all or substantially all of the human CD8α and/or CD8β extracellular domain, respectively.
In one embodiment, the mouse does not express a functional endogenous mouse CD8α and/or CD8β polypeptide from its endogenous CD8 locus. In one embodiment, the mouse as described herein comprises the chimeric human/mouse CD8 sequence in its germline.
In one aspect, the mouse expressing chimeric human/mouse CD8α and/or CD8β polypeptide retains mouse CD8α and/or CD8β promoter and regulatory sequences, e.g., the nucleotide sequence in the mouse encoding chimeric human/mouse CD8 is operably linked to endogenous mouse CD8 promoter and regulatory sequences. In one aspect, these regulatory sequences retained in the mouse include the sequences regulating CD8 protein expression at proper stages of T cell development. In one aspect, the genetically modified mouse does not express chimeric CD8 on B cells or mature CD4+ T cells, or any cell, e.g., immune cell, that does not normally express endogenous CD8.
The invention also provides a genetically modified mouse comprising in its genome an unrearranged human or humanized TCR variable gene locus, e.g., TCRα, TCRβ, TCRδ, and/or TCRγ variable gene locus. In some embodiments, the unrearranged human or humanized TCR variable gene locus replaces endogenous mouse TCR variable gene locus. In other embodiments, unrearranged human or humanized TCR variable gene locus is at a site in the genome other than the corresponding endogenous mouse TCR locus. In some embodiments, human or humanized unrearranged TCR variable gene locus is operably linked to mouse TCR constant region.
In one embodiment, a genetically modified mouse is provided, wherein the mouse comprises in its genome an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and least one human Jα segment, operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a mouse TCR constant gene sequence. In one specific embodiment, the mouse comprises in its genome an unrearranged TCRα variable gene locus comprising a complete repertoire of human Vα segments and a complete repertoire of human Jα segments operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus comprising a complete repertoire of human Vβ segments, a complete repertoire of human Dβ segments, and a complete repertoire of human Jβ segments operably linked to a mouse TCR constant gene sequence.
In some embodiments, the unrearranged TCRα variable gene locus comprising human TCRα variable region segments replaces endogenous mouse TCRα variable gene locus, and the unrearranged TCRβ variable gene locus comprising human TCRβ variable region segments replaces the endogenous mouse TCRβ variable gene locus. In some embodiments, the endogenous mouse Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence, and the endogenous mouse Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence. In some embodiments, the human Vα and Jα segments rearrange to form a rearranged human Vα/Jα sequence, and the human Vβ, Dβ, and Jβ segments rearrange to form a rearranged human Vβ/Dβ/Jβ sequence.
The invention also relates to a genetically modified mouse that comprises in its genome a nucleic acid sequence encoding a chimeric MHC polypeptide, wherein the human portion of the chimeric MHC polypeptide associates with a human extracellular domain of a chimeric T cell co-receptor as disclosed herein. Genetically modified mice as disclosed herein may comprise a first nucleic acid sequence encoding a chimeric human/mouse MHC I, a second nucleic acid sequence encoding a chimeric human/mouse MHC II α, and/or a third nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptides. A human portion of the chimeric MHC I, MHC II α, and/or MHC II β may comprise an extracellular domain of a human MHC I, MHC II α, and MHC II β, respectively. In one embodiment, the mouse expresses functional chimeric human/mouse MHC I, MHC II α, and MHC II β polypeptides from its endogenous mouse MHC locus. In one embodiment, the mouse does not express functional mouse MHC polypeptides, e.g., functional mouse MHC I, MHC II α, and MHC II β polypeptides, from its endogenous mouse MHC locus. In other embodiments, the only MHC I and MHC II expressed by the mouse on a cell surface are chimeric MHC I and II.
In one embodiment, a human portion of the chimeric human/mouse MHC I polypeptide comprises a peptide binding domain or an extracellular domain of a human MHC I (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does not express a peptide binding or an extracellular domain of an endogenous mouse MHC I polypeptide from its endogenous mouse MHC I locus. The peptide binding domain of the human MHC I may comprise α1 and β2 domains. Alternatively, the peptide binding domain of the human MHC I may comprise al, α2, and α3 domains. In one aspect, the extracellular domain of the human MHC I comprises an extracellular domain of a human MHC I α chain. In one embodiment, the endogenous mouse MHC I locus is an H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric MHC I polypeptide comprises transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. Thus, in one embodiment, the mouse of the invention comprises at its endogenous mouse MHC I locus a nucleic acid sequence encoding a chimeric human/mouse MHC I, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide, and a mouse expresses a chimeric human/mouse HLA-A2/H-2K protein. In other embodiment, the mouse portion of the chimeric MHC I polypeptide may be derived from other mouse MHC I, e.g., H-2D, H-2L, etc.; and the human portion of the chimeric MHC I polypeptide may be derived from other human MHC I, e.g., HLA-B, HLA-C, etc. In one aspect, the mouse does not express a functional endogenous H-2K polypeptide from its endogenous mouse H-2K locus. In one embodiment, the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In other embodiments, the only MHC I polypeptides expressed by the mouse on a cell surface are chimeric human/mouse MHC I polypeptides.
In one embodiment, a human portion of the chimeric human/mouse MHC II α polypeptide comprises a human MHC II α peptide binding or extracellular domain and a human portion of the chimeric human/mouse MHC II β polypeptide comprises a human MHC II β peptide binding or extracellular domain. In some embodiments, the mouse does not express a peptide binding or an extracellular domain of endogenous mouse a and/or β polypeptide from an endogenous mouse locus (e.g., H-2A and/or H-2E locus). In some embodiments, the mouse comprises a genome that lacks a gene that encodes a functional MHC class II molecule comprising an H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, H-2Ea, and a combination thereof. In some embodiments, the only MHC II polypeptides expressed by the mouse on a cell surface are chimeric human/mouse MHC II polypeptides. The peptide-binding domain of the human MHC II α polypeptide may comprise α1 domain and the peptide-binding domain of the human MHC II β polypeptide may comprise a β1 domain; thus, the peptide-binding domain of the chimeric MHC II complex may comprise human al and β1 domains. The extracellular domain of the human MHC II α polypeptide may comprise α1 and β2 domains and the extracellular domain of the human MHC II β polypeptide may comprise β1 and β2 domains; thus, the extracellular domain of the chimeric MHC II complex may comprise human α1, α2, β1 and β2 domains. In one embodiment, the mouse portion of the chimeric MHC II complex comprises transmembrane and cytosolic domains of mouse MHC II, e.g. mouse H-2E (e.g., transmembrane and cytosolic domains of mouse H-2E α and β chains). Thus, in one embodiment, the mouse of the invention comprises at its endogenous mouse MHC II locus a nucleic acid sequence encoding a chimeric human/mouse MHC II α, wherein a human portion of the chimeric MHC II α polypeptide comprises an extracellular domain derived from an a chain of a human MHC II (e.g., a chain of HLA-DR2) and a mouse portion comprises transmembrane and cytoplasmic domains derived from an a chain of a mouse MHC II (e.g., H-2E); and a mouse comprises at its endogenous mouse MHC II locus a nucleic acid sequence encoding a chimeric human/mouse MHC II β, wherein a human portion of the chimeric MHC II β polypeptide comprises an extracellular domain derived from a β chain of a human MHC II (e.g., β chain of HLA-DR2) and a mouse portion comprises transmembrane and cytoplasmic domains derived from a β chain of a mouse MHC II (e.g., H-2E); e.g., wherein the mouse expresses a chimeric human/mouse HLA-DR2/H-2E protein. In other embodiment, the mouse portion of the chimeric MHC II protein may be derived from other mouse MHC II, e.g., H-2A, etc.; and the human portion of the chimeric MHC II protein may be derived from other human MHC II, e.g., HLA-DQ, etc. In one aspect, the mouse does not express functional endogenous H-2A and H-2E polypeptides from their endogenous mouse loci (e.g., the mouse does not express H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea polypeptides). In some embodiments, the mouse lacks expression of any endogenous MHC I or MHC II molecule on a cell surface.
In various aspects, the human or humanized β2 microglobulin expressed by a genetically modified non-human animal, or cells, embryos, or tissues derived from a non-human animal, preserves all the functional aspects of the endogenous and/or human β2 microglobulin. For example, it is preferred that the human or humanized β2 microglobulin binds the α chain of MHC I polypeptide (e.g., endogenous non-human or human MHC I polypeptide). The human or humanized β2 microglobulin polypeptide may bind, recruit or otherwise associate with any other molecules, e.g., receptor, anchor or signaling molecules that associate with endogenous non-human and/or human β2 microglobulin (e.g., HFE, etc.).
In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), also provided is a tissue or cell, wherein the tissue or cell is derived from a non-human animal as described herein, and comprises a heterologous β2 microglobulin gene or β2 microglobulin sequence, i.e., nucleotide and/or amino acid sequence. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or human or humanized β2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express MHC I complex, e.g., an antigen presenting cell. The human or humanized β2 microglobulin polypeptide expressed by said cell may interact with endogenous non-human MHC I (e.g., rodent MHC I), to form a functional MHC I complex. The resultant MHC I complex may be capable of interacting with a T cell, e.g., a cytotoxic T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a T cell.
Also provided are non-human cells that comprise human or humanized β2 microglobulin gene or sequence, and an additional human or humanized sequence, e.g., chimeric MHC I polypeptide presently disclosed. In such an instance, the human or humanized β2 microglobulin polypeptide may interact with, e.g., a chimeric human/non-human MHC I polypeptide, and a functional MHC I complex may be formed. In some aspects, such complex is capable of interacting with a TCR on a T cell, e.g., a human or a non-human T cell. Thus, also provided in an in vitro complex of a cell from a non-human animal as described herein and a human or a non-human T cell.
Another aspect of the disclosure is a rodent embryo (e.g., a mouse or a rat embryo) comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence as described herein. In one embodiment, the embryo comprises an ES donor cell that comprises the heterologous β2 microglobulin gene or β2 microglobulin sequence, and host embryo cells. The heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or β2 microglobulin sequence.
This invention also encompasses a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein (e.g., wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide). The non-human cell may comprise a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.
In one aspect, a non-human induced pluripotent cell comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized gene or sequence.
In some embodiments of the invention, the mouse described herein expresses chimeric human/mouse MHC II only on professional antigen presenting cells, e.g., B cell, monocytes/macrophages, and/or dendritic cells of the mouse. In some embodiments, a mouse described herein elicits an immune response, e.g., a cellular immune response, to one or more human antigens. In some embodiments, a mouse described herein elicits a humanized T cell response to one or more human antigens.
In addition to a genetically engineered non-human animal, a non-human embryo (e.g., a rodent, e.g., a mouse or a rat embryo) is also provided, wherein the embryo comprises a donor ES cell that is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein. In one aspect, the embryo comprises an ES donor cell that comprises the chimeric CD4 gene, the chimeric CD8 (e.g., CD8α and/or CD8β) gene, a humanized MHC I (e.g., MHC I α) nucleic acid sequence, a humanized MHC II (e.g., MHC II α and/or MHC II β) nucleic acid sequence, an unrearranged humanized TCR (e.g., TCRα and/or TCRβ, or TCRδ, and/or TCRγ) locus and/or human or humanized β2 microglobulin gene sequence and host embryo cells.
Also provided is a tissue, wherein the tissue is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein, and expresses the chimeric CD4 protein, the chimeric CD8 protein (e.g., chimeric CD8α and/or CD8β protein), a humanized TCR polypeptide (e.g., TCRα and/or TCRβ, or TCRδ, and/or TCRγ polypeptide), a humanized MHC I polypeptide (e.g., MHC I α), a humanized MHC II polypeptide (e.g., MHC II α and/or MHC II β polypeptide) and/or a human or humanized β2 microglobulin.
In one aspect, a method for making a chimeric human/non-human CD4 molecule is provided, comprising expressing in a single cell a chimeric CD4 protein from a nucleotide construct as described herein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
In one aspect, a cell that expresses a chimeric CD4 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD4 sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
A chimeric CD4 molecule made by a non-human animal as described herein is also provided, wherein, in one embodiment, the chimeric CD4 molecule comprises an amino acid sequence of all or substantially all of an extracellular domain of a human CD4 protein, and at least transmembrane and cytoplasmic domains from a non-human CD4 protein, e.g., mouse CD4 protein. In another embodiment, a chimeric CD4 molecule made by a non-human animal as described herein is provided, wherein the chimeric CD4 molecule comprises an amino acid sequence of at least all or substantially all D1 domain of a human CD4, e.g., at least all or substantially all D1-D2 domains of a human CD4, e.g., at least all or substantially all D1-D3 domains of a human CD4, e.g., an amino acid sequence of human CD4 that is responsible for binding MHC II and/or extracellular domain of a TCR, e.g., an amino acid sequence of human CD4 that is responsible for binding MHC II and/or a variable domain of a TCR; and wherein the remainder of the protein (e.g., transmembrane domain, cytoplasmic domain, any portion of extracellular domain that has not been humanized) is derived from the endogenous non-human protein sequence. An exemplary chimeric human/non-human CD4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:78, and the human portion of the chimeric polypeptide spans about amino acids 27-319 of SEQ ID NO:78 (set forth separately in SEQ ID NO:79).
In one aspect, a method for making a chimeric human/non-human CD8 molecule (e.g., CD8α and/or CD8β) is provided, comprising expressing in a single cell a chimeric CD8 polypeptide(s) from a nucleotide construct(s) as described herein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
In one aspect, a cell that expresses a chimeric CD8 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD8 sequence(s) as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
A chimeric CD8 molecule made by a non-human animal as described herein is also provided, wherein the chimeric CD8 molecule comprises all or substantially all of the extracellular domain from a human CD8 protein (e.g., CD8α and/or CD8β), and at least transmembrane and cytoplasmic domains from a non-human CD8 protein, e.g., mouse CD8 protein. Exemplary chimeric CD8α polypeptide is set forth in SEQ ID NO:88, and exemplary chimeric CD8β protein is set forth in SEQ ID NO:83.
A humanized TCR protein made by a non-human animal (e.g., rodent, e.g., mouse or rat) as described herein is also provided, wherein the humanized TCR protein comprises a human variable region and a non-human constant region. Thus, the humanized TCR protein comprises human complementary determining regions (i.e., human CDR1, 2, and 3) in its variable domain and a non-human constant region. Also provided are nucleic acids that encode the human TCR variable domains generated by a non-human animal described herein.
In addition, a non-human cell isolated from a non-human animal as described herein is provided. In one embodiment, the cell is an ES cell. In one embodiment, the cell is a T cell, e.g., a CD4+ T cell. In one embodiment, the cell is a helper T cell (TH cell). In one embodiment, the TH cell is an effector TH cell, e.g., TH1 cell or TH2 cell. In one embodiment, the cell is CD8+ T cell. In one embodiment, the cell is a cytotoxic T cell. Also provided is a non-human cell that expresses a TCR protein comprising a human variable region and a non-human constant region. The TCR protein may comprise TCRα, TCRδ, or a combination thereof. In one embodiment, the cell is a T cell, e.g., a CD4+ or a CD8+ T cell. Additionally, non-human T cells as provided herein may express on its cell surface (a) a chimeric human/non-human T cell co-receptor, e.g., a chimeric CD4 polypeptide or a chimeric CD8 polypeptide, comprising a human T cell co-receptor extracellular domain operably linked to a non-human T cell co-receptor transmembrane and/or intracellular domain; and (b) a TCR protein comprising a human variable region and a non-human constant region.
In another embodiment, the cell is an antigen presenting cell. In one embodiment, the antigen presenting cell presents antigen on humanized MHC I molecules. In another embodiment, the antigen presenting cell is a professional antigen presenting cell, e.g., a B cell, a dendritic cell, and a macrophage. In another embodiment, the antigen presenting cell presents antigen on humanized MHC I and/or humanized MHC II molecules.
In one aspect, a cell that expresses a chimeric human/non-human MHC I and MHC II proteins (e.g., HLA-A2/H-2K and HLA-DR2/H-2E proteins) is provided. In one aspect, the cell is a mouse cell that does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the cell is a mouse cell engineered to lack all or a portion of an endogenous H-2D locus. In some embodiments, the cell is a mouse cell that does not express any functional endogenous MHC I and MHC II polypeptide on its surface. In one embodiment, the cell comprises an expression vector comprising a chimeric MHC class I sequence and chimeric MHC class II sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
A chimeric MHC II complex comprising an extracellular domain of HLA-DR2 described herein may be detected by anti-HLA-DR antibodies. Thus, a cell displaying chimeric human/non-human MHC II polypeptide may be detected and/or selected using anti-HLA-DR antibody. The chimeric MHC I complex comprising an extracellular domain of HLA-A2 described herein may be detected using anti-HLA-A, e.g., anti-HLA-A2 antibodies. Thus, a cell displaying a chimeric human/non-human MHC I polypeptide may be detected and/or selected using anti-HLA-A antibody. Antibodies that recognize other HLA alleles are commercially available or can be generated, and may be used for detection/selection.
Although the Examples that follow describe a genetically engineered animal whose genome comprises a replacement of a nucleic acid sequence encoding mouse H-2K, and H-2A and H-2E proteins with a nucleic acid sequence encoding a chimeric human/mouse HLA-A2/H-2K and HLA-DR2/H-2E protein, respectively, one skilled in the art would understand that a similar strategy may be used to introduce chimeras comprising other human MHC I and II genes (other HLA-A, HLA-B, and HLA-C; and other HLA-DR, HLA-DP and HLA-DQ genes). Such animals comprising multiple chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) MHC I and MHC II genes at endogenous MHC loci are also provided. Examples of such chimeric MHC I and MHC II proteins are described in U.S. Publication Nos. 20130111617, 20130185819, 20130185820 and 20140245467 and U.S. Pat. No. 8,847,005, each of which are incorporated herein by reference.
Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.
In one aspect, a non-human induced pluripotent cell comprising a gene encoding a chimeric CD4 polypeptide, a gene encoding a chimeric CD8 polypeptide (e.g., CD8α and/or CD8β polypeptide), a gene encoding a humanized MHC I polypeptide (e.g., MHC I α and/or β2 microglobulin), a gene encoding a humanized MHC II polypeptide (e.g., MHC II α and/or MHC II β) and/or an unrearranged humanized TCR locus encoding a humanized TCRα and/or TCRβ polypeptide as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.
In one aspect, a hybridoma or quadroma is provided, derived from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.
Making Genetically Modified Non-Human Animals that Mount Substantially Humanized T Cell Immune Responses
Also provided is a method for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) described herein. Generally, the methods comprise (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide, a second nucleotide sequence encoding a second chimeric human/non-human T cell co-receptor polypeptide, and/or a third nucleotide sequence encoding a third chimeric human/non-human T cell co-receptor polypeptide, wherein a non-human portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, and wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof) of a human T cell co-receptor; (b) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence; and optionally (c) placing into the genome a first nucleic acid sequence encoding a first chimeric human/non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human/non-human MHC polypeptide and/or a third nucleic acid sequence encoding a third chimeric human/non-human MHC polypeptide and/or (d) adding into the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the steps of introducing, inserting and/or placing comprise targeting sequences encoding the extracellular domain(s) of the T cell co-receptor, the variable domain(s) of the TCR, the extracellular domain(s) of the MHC polypeptide, or a portion of the β2 microglobulin and replacing them with sequences encoding human T cell co-receptor extracellular domain(s), human TCR variable domains, human MHC extracellular domain(s), and/or a human portion of the β2 microglobulin, respectively.
In other embodiments, introducing, inserting, placing and/or adding may comprise breeding, e.g., mating, animals of the same species. In other embodiments, introducing, inserting, placing and/or adding comprises sequential homologous recombination in ES cells. In some embodiments, the ES cells are derived from non-human animals genetically modified to comprise one or more, but not all, of the genetic modifications desired, and homologous recombination in such ES cells completes the genetic modification. In other embodiments, introducing, inserting, placing and/or adding may comprise a combination of breeding and homologous recombination in ES cells, e.g., breeding an animal to another (or more) animal of the same species, wherein some or all of the animals may be generated from ES cells genetically modified via a single homologous recombination or sequential homologous recombination events, and wherein some ES cell may be isolated from a non-human animal comprising one or more of the genetic modifications disclosed herein.
In some embodiments, the method utilizes a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. Targeting construct may comprise 5′ and/or 3′ homology arms that target the endogenous sequence to be replaced, an insert sequence (that replaces the endogenous sequence) and one or more selection cassettes. A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Commonly, a selection cassette enables positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). In addition, a selection cassette may be flanked by recombination sites, which allow deletion of the selection cassette upon treatment with recombinase enzymes. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, but others are known in the art. A selection cassette may be located anywhere in the construct outside the coding region. In one embodiment, the selection cassette is located at the 5′ end the human DNA fragment. In another embodiment, the selection cassette is located at the 3′ end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragment.
In one embodiment, the method for making a genetically engineered non-human animal results in the animal that comprises at an endogenous CD4 locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide. In one embodiment, the invention comprises a method of modifying a CD4 locus of a non-human animal to express a chimeric human/non-human CD4 polypeptide described herein. In one embodiment, the invention provides a method of modifying a CD4 locus of a mouse to express a chimeric human/mouse CD4 polypeptide comprising introducing, e.g., replacing at an endogenous CD4 locus of a non-human animal, e.g., a mouse, a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/mouse CD4 polypeptide. In one aspect of the method, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of an extracellular domain of a human CD4 polypeptide and at least transmembrane and cytoplasmic domains of an endogenous mouse CD4 polypeptide. In another aspect of the method, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of D1-D2 domains of a human CD4 polypeptide. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of D1-D3 domains of a human CD4 polypeptide. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of amino acid sequence of human CD4 that is responsible for interacting with MHC II and/or an extracellular domain of a T cell receptor. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of amino acid sequence of human CD4 that is responsible for interacting with MHC II and/or a variable domain of a T cell receptor.
Thus, a nucleotide construct for generating genetically modified animals comprising chimeric human/non-human CD4 is provided. In one aspect, the nucleotide sequence comprises 5′ and 3′ homology arms, a DNA fragment comprising human CD4 gene sequence (e.g., human CD4 extracellular domain gene sequence, e.g., gene sequence of all or substantially all of domains D1-D2 of human CD4, e.g., gene sequence of all or substantially all of domains D1-D3 and/or D2-D3 of human CD4, e.g., gene sequence of all or substantially all of domains D1-D4 of human CD4), and a selection cassette flanked by recombination sites. In one embodiment, human CD4 gene sequence is a genomic sequence that comprises introns and exons of human CD4. In one embodiment, homology arms are homologous to non-human (e.g., mouse) CD4 genomic sequence. An exemplary construct of the invention is depicted in FIG. 5A.
In some embodiments, the method results in an animal that comprises at an endogenous CD8 locus a nucleotide sequence(s) encoding a chimeric human/non-human CD8α and/or CD8β polypeptide. In one embodiment, the invention provides a method of modifying a CD8 locus of a non-human animal to express a chimeric human/non-human CD8 polypeptide described herein. In one aspect, provided is a method of modifying a CD8 locus of a mouse to express a chimeric human/mouse CD8 polypeptide comprising introducing, e.g., replacing, at an endogenous CD8 locus of a non-human animal, e.g., a mouse, a nucleotide sequence encoding an endogenous non-human CD8 polypeptide with a nucleotide sequence encoding a chimeric human/mouse CD8 polypeptide. The CD8 polypeptide may be selected from CD8α, CD8β, and combination thereof. In one aspect, the chimeric polypeptide comprises all or substantially all of an extracellular domain of a human CD8 polypeptide and at least transmembrane and cytoplasmic domains of an endogenous mouse CD8 polypeptide.
Thus, a nucleotide construct for generating genetically modified animals comprising human/non-human CD8 is also provided. In one aspect, the sequence of the nucleotide construct comprises 5′ and 3′ homology arms, a DNA fragment comprising human CD8α or CD8β sequence, and a selection cassette flanked by recombination sites. In some embodiments, the human sequence comprises introns and exons of human CD8α or CD8β, e.g., exons encoding the extracellular domain of the human CD8α or CD8β, respectively. In one embodiment, homology arms are homologous to non-human CD8α or CD8β sequence. Exemplary constructs for CD8α and CD8β are depicted in FIG. 5B.
Because of close chromosomal localization of the genes encoding CD8α and CD8β, sequential targeting of the two genes improves the chances of successful humanization. In one embodiment, the targeting strategy comprises introducing chimeric CD8β construct described herein into ES cells, generating a mouse from the targeted ES cells, deriving genetically modified ES cells from said mouse, and introducing chimeric CD8α construct described herein into said genetically modified ES cells. In another embodiment, the targeting strategy comprises introducing a chimeric CD8β construct described herein into ES cells, selecting the cells that have incorporated the chimeric CD8β construct, introducing a chimeric CD8α construct described herein into ES cells that have incorporated and are harboring the chimeric CD8β construct, and selecting the cells that have incorporated both chimeric CD8β and CD8α. In one aspect of this embodiment, the steps of selecting are performed utilizing different selection markers. In alternative embodiments, CD8α humanization can be accomplished first. Upon completion of gene targeting, ES cells of genetically modified non-human animals can be screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide by a variety of methods known in the art (e.g., modification of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659).
In some embodiments, the method for making a genetically modified non-human animal results in the animal whose genome comprises a humanized unrearranged TCR locus (e.g., a humanized unrearranged TCRα, TCRβ, TCRδ, and/or TCRγ locus). In one embodiment, a method for making a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that expresses a T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell is provided, wherein the method comprises inserting, e.g., replacing, in a first non-human animal an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the humanized TCRα variable gene locus is operably linked to endogenous TCRα constant region; inserting, e.g., replacing in a second non-human animal an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, one human Dβ segment, and one human Jβ segment, wherein the humanized TCRβ variable gene locus is operably linked to endogenous TCR constant region; and breeding the first and the second non-human animal to obtain a non-human animal that expresses a T cell receptor comprising a human variable region and a non-human constant region. In other embodiments, the invention provides methods of making a genetically modified non-human animal whose genome comprises a humanized unrearranged TCRα locus, or a non-human animal whose genome comprises a humanized unrearranged TCR locus. In various embodiments, the replacements are made at the endogenous loci. In various embodiments, the method comprises progressive humanization strategy, wherein a construct comprising additional variable region segments is introduced into ES cells at each subsequent step of humanization, ultimately resulting in a mouse comprising a complete repertoire of human variable region segments (see, e.g., FIGS. 4A and 4B).
The disclosure also provides a method of modifying a TCR variable gene locus (e.g., TCRα, TCRβ, TCRδ, and/or TCRγ gene locus) of a non-human animal to express a humanized TCR protein described herein. In one embodiment, the invention provides a method of modifying a TCR variable gene locus to express a humanized TCR protein on a surface of a T cell wherein the method comprises inserting, e.g., replacing, in a non-human animal an endogenous non-human TCR variable gene locus with an unrearranged humanized TCR variable gene locus. In one embodiment wherein the TCR variable gene locus is a TCRα variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Vα segment and at least one human Jα segment. In one embodiment wherein the TCR variable gene locus is a TCRβ variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment. In various aspects, the unrearranged humanized TCR variable gene locus is operably linked to the corresponding endogenous non-human TCR constant region.
Thus, nucleotide constructs for generating genetically modified animals comprising humanized TCR variable region genes are also provided. In one aspect, the nucleotide construct comprises: 5′ and 3′ homology arms, a human DNA fragment comprising human TCR variable region gene segment(s), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a TCRα gene fragment and it comprises at least one human TCRα variable region segment. In another embodiment, the human DNA fragment is a TCRβ fragment and it comprises at least one human TCRβ variable region gene segment. In one aspect, at least one homology arm is a non-human homology arm and it is homologous to non-human TCR locus (e.g., non-human TCRα or TCRβ locus).
In various aspects of the invention, the sequence(s) encoding a chimeric human/non-human MHC I and MHC II polypeptides are located at an endogenous non-human MHC locus (e.g., mouse H-2K and/or H-2E locus). In one embodiment, this results in placement, e.g., replacement, of an endogenous MHC gene(s) or a portion thereof with a nucleic acid sequence(s) encoding a human or humanized MHC I polypeptides. Since the nucleic acid sequences encoding MHC I, MHC II α and MHC II β polypeptides are located in proximity to one another on the chromosome, in order to achieve the greatest success in humanization of both MHC I and MHC II in one animal, the MHC I and MHC II loci should be targeted sequentially. Thus, also provided herein are methods of generating a genetically modified non-human animal comprising nucleic acid sequences encoding chimeric human/non-human MHC I, MHC II α and MHC II β polypeptides as described herein.
Thus, a nucleotide construct for generating genetically modified animals comprising chimeric human/non-human MHC is provided. In one aspect, the nucleic acid construct comprises: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human MHC gene sequences (e.g., human HLA-A2 or human HLA-DRs gene sequences), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human MHC gene (e.g., human HLA-A2 or HLA-DR2 gene). In one embodiment, the non-human homology arms are homologous to a non-human MHC locus (e.g., MHC I or MHC II locus).
In one embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence at 5′ and 3′ locations, respectively, of an endogenous non-human (e.g., murine) MHC class I or class II gene locus (e.g., 5′ of the first leader sequence and 3′ of the α3 exon of the mouse MHC I gene, or upstream of mouse H-2Ab1 gene and downstream of mouse H-2Ea gene). In one embodiment, the endogenous MHC class I locus is selected from mouse H-2K, H-2D and H-2L. In a specific embodiment, the endogenous MHC class I locus is mouse H-2K. In one embodiment, the endogenous MHC II locus is selected from mouse H-2E and H-2A. In one embodiment, the engineered MHC II construct allows replacement of both mouse H-2E and H-2A genes. In one embodiment, the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In another embodiment, the mouse does not express any functional endogenous MHC I and MHC II polypeptides on a cell surface. In one embodiment, the only MHC I and MHC II expressed by the mouse on a cell surface are chimeric human/mouse MHC I and MHC II.
The disclosure also provides methods for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or a rat) whose genome comprises a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the methods result in a genetically engineered rodent, e.g., mouse, whose genome comprises at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some instances, the mouse does not express a functional mouse β2 microglobulin from an endogenous mouse β2 microglobulin locus. In some aspects, the methods utilize a targeting construct, e.g., made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo, e.g., using VELOCIMOUSE® technology, as described in herein.
Also provided is a nucleotide construct used for generating genetically engineered non-human animals. The nucleotide construct may comprise: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human β2 microglobulin sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human β2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to a non-human β2 microglobulin locus. The genomic fragment may comprise exons 2, 3, and 4 of the human β2 microglobulin gene. In one instance, the genomic fragment comprises, from 5′ to 3′: exon 2, intron, exon 3, intron, and exon 4, all of human β2 microglobulin sequence. The selection cassette may be located anywhere in the construct outside the β2 microglobulin coding region, e.g., it may be located 3′ of exon 4 of the human β2 microglobulin. The 5′ and 3′ non-human homology arms may comprise genomic sequence 5′ and 3′ of endogenous non-human β2 microglobulin gene, respectively. In another embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence 5′ of exon 2 and 3′ of exon 4 of endogenous non-human gene, respectively.
Another aspect of the invention relates to a method of modifying a β2 microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide described herein. One method of modifying a β2 microglobulin locus of a non-human animal, e.g., mouse, to express a human or humanized β2 microglobulin polypeptide, comprises replacing at an endogenous β2 microglobulin locus a nucleotide sequence encoding a mouse β2 microglobulin with a nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide. In one embodiment of such method, the non-human animal, e.g., mouse does not express a functional β2 microglobulin polypeptide from an endogenous non-human, e.g., mouse β2 microglobulin locus. In some specific embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequence set forth in exons 2 to 4 of the human β2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequences set forth in exons 2, 3, and 4 of the human β2 microglobulin gene.
Various exemplary embodiments of the humanized loci described herein are presented in FIGS. 2-5.
Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) bearing the genetic modification of interest can be identified by screening for loss of mouse allele and/or gain of human allele using a modification of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.
In some embodiments, animals are generated herein by breeding.
In one non-limiting aspect, for example, a non-human animal comprising the chimeric human/non-human CD8 described herein and the humanized MHC I and/or β2 microglobulin may be generated by breeding an animal comprising a chimeric CD8 locus (e.g., chimeric CD8 α and/or β locus) as described herein with an animal comprising a humanized MHC I and/or β2 microglobulin locus. The animal may also be generated by introducing into ES cells of an animal comprising humanized MHC I and/or β2 microglobulin locus a nucleotide sequence encoding chimeric CD8 (e.g., chimeric CD8 α and/or β), e.g., for replacement at the endogenous CD8 locus (e.g., chimeric CD8 α and/or β locus); or introducing into ES cells of an animal comprising a chimeric CD8 locus (e.g., chimeric CD8 α and/or β locus) a nucleotide sequence(s) encoding humanized MHC I and/or β2 microglobulin.
In some embodiments, the animal comprising a chimeric CD8 locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized CD8 and TCR variable region loci, which may then be bred with an animal comprising humanized MHC I and/or β2 microglobulin loci to generate an animal comprising humanized MHC I, TCR variable gene and/or β2 microglobulin loci.
Alternatively, the animal comprising a humanized MHC I and/or β2 microglobulin loci may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized MHC I and TCR variable region loci, which may then be bred with an animal comprising a chimeric CD8 locus generate an animal comprising humanized MHC I, TCR variable gene and/or β2 microglobulin loci, respectively.
In one aspect, the non-human animal comprising a chimeric human/non-human CD4 and the humanized MHC II may be generated by breeding an animal comprising a chimeric CD4 locus as described herein with an animal comprising a humanized MHC II locus. The animal may also be generated by introducing into ES cells of an animal comprising humanized MHC II locus a nucleotide sequence encoding chimeric CD4, e.g., for replacement at the endogenous CD4 locus; or introducing into ES cells of an animal comprising a chimeric CD4 locus a nucleotide sequence encoding humanized MHC II.
In some embodiments, the animal comprising a chimeric CD4 locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized CD4 and TCR variable region loci, which may then be bred with an animal comprising a humanized MHC II locus to generate an animal comprising humanized CD4, MHC II and TCR variable gene loci. Alternatively, the animal comprising a comprising humanized MHC II locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized MHC II and TCR variable region loci, which may then be bred with an animal comprising a chimeric CD4 locus generate an animal comprising humanized MHC II, TCR variable gene and/or β2 microglobulin loci, respectively.
In some embodiments, a non-human animal comprising the chimeric human/non-human CD8 described herein and the humanized MHC I and/or β2 microglobulin is bred with an animal comprising a chimeric CD4 locus as described herein and an animal comprising a humanized MHC II locus to generate a non-human animal comprising chimeric CD4 and CD8 polypeptides and humanized MHC I (and/or β2 microglobulin) and MHC II molecules. In some embodiments, the animal comprising chimeric human/non-human CD4 and CD8 polypeptides and humanized MHC I and MHC II molecules is bred with an animal comprising a humanized TCR variable domain to generate an animal comprising a substantially humanized T cell immune system, e.g., chimeric human/non-human CD4 and CD8 polypeptides, humanized MHC I (and/or β2 microglobulin) and MHC II molecules and humanized TCR variable domains.
Any of the genetically modified no-human animal (e.g., mouse) described herein may comprise one or two copies of the genes encoding chimeric human/non-human CD8 (e.g., CD8α and/or CD8β); chimeric human/non-human CD4; human or humanized MHC I; human or humanized β2 microglobulin; human or humanized MHC II (e.g., MHC IIα and/or MHC IIβ); and human or humanized TCR (e.g., TCR α and/or TCRβ). Accordingly, the animal may be heterozygous or homozygous for any or all of these genes.
Using Genetically Modified Non-Human Animals that Mount Substantially Humanized T Cell Immune Responses
The genetically modified non-human animals, e.g., rodents, e.g., mice or rats, comprising either humanized CD4 and MHC II or humanized CD8 and MHC I (and β2 microglobulin), or both, present peptides to T cells (CD4+ or CD8+ T cells, respectively) in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in vaccine development; for identification of high affinity T cells to human pathogens or cancer antigens (i.e., T cells that bind to antigen in the context of human MHC I complex with high avidity), e.g., for use in adaptive T cell therapy; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC and CD4/CD8 expression.
Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the capacity of an antigen to initiate an immune response in a human, and for generating a diversity of antigens and identifying a specific antigen that may be used in human vaccine development.
In one aspect, a method for determining whether a peptide will provoke a cellular immune response in a human is provided, comprising exposing a genetically modified non-human animal as described herein to the peptide, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell (e.g., a CD8+ or CD4+ T cell, comprising a human CD8 or CD4, respectively) that binds a sequence of the peptide presented by a chimeric human/non-human MHC I or II molecule as described herein. In one embodiment, the non-human animal following exposure comprises an MHC class I-restricted CD8+ cytotoxic T lymphocyte (CTL) that binds the peptide. In another embodiment, the non-human animal following exposure comprises an MHC II-restricted CD4+ T cell that binds the peptide.
In one aspect, a method for identifying a human T cell epitope is provided, comprising exposing a non-human animal as described herein to an antigen comprising a putative T cell epitope, allowing the non-human animal to mount an immune response, isolating from the non-human animal an MHC class I- or MHC class II-restricted T cell that binds the epitope, and identifying the epitope bound by said T cell.
In one aspect, a method is provided for identifying an antigen that generates a T cell response in a human, comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying the antigen bound by the HLA class I- or class II-restricted molecule.
In one aspect, a method is provided for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA class I- or class II-restricted immune response, comprising exposing a mouse as described herein to the putative antigen and measuring an antigen-specific HLA class I- or HLA class II-restricted immune response in the mouse.
In addition, the genetically engineered non-human animals described herein may be useful for identification of T cell receptors, e.g., high-avidity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. The method may comprise: exposing the non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, isolating from the non-human animal a T cell comprising a T cell receptor that binds the antigen presented by a human or humanized MHC I or MHC II, and determining the sequence of said T cell receptor.
Non-human animals expressing a diverse repertoire of functional human TCR V(D)J gene segments may be useful for the study of human diseases. Accordingly, in one embodiment, the genetically engineered non-human animals described herein may express a TCR repertoire substantially similar to a TCR repertoire expressed in a human, e.g., the TCR repertoire of a non-human animal disclosed herein may be derived from at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR α, TCR β, TCRγ and/or TCRδ gene segments. In some embodiments, a non-human animal as disclosed expresses a TCR repertoire derived from
(i) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Vα gene segments;
(ii) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Jα gene segments;
(iii) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Vβ gene segments;
(iv) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Dβ gene segments; and/or
(v) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Jβ gene segments.
In one embodiment, the mouse produces a T cell repertoire comprising all or substantially all functional human TCR Vα gene segments, and comprising all or substantially all functional human TCR Vβ gene segments. In one embodiment, the mouse provided herein utilizes human TCR Vα and/or Vβ genes with a frequency similar to the frequency of human TCR Vα and/or Vβ genes, respectively, utilized by human T cells in a human. Methods of detecting the gene segments expressed in the TCR repertoire of the non-human animal include, e.g., flow cytometric and/or sequencing methods (e.g., real time PCR, Next Generation Sequencing, etc.).
In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic, comprising exposing a genetically modified animal as described herein to a putative human therapeutic (or e.g., exposing a human or humanized MHC II- or MHC I-expressing cell of such an animal to a peptide sequence of the putative therapeutic), exposing a cell of the genetically modified animal that displays a human or humanized MHC/peptide complex to a T cell comprising a chimeric human/non-human (e.g., human/mouse) CD4 or CD8 capable of binding the cell of the genetically modified animal, and measuring activation of the T cell that is induced by the peptide-displaying cell of the genetically modified animal.
In addition to the ability to identify antigens and antigen epitopes from human pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. Also, the genetically modified animals of the invention can be used to study various aspects of human autoimmune disease, and may be utilized as autoimmune disease models.
In various embodiments, the genetically modified non-human animals of the invention make T cells with humanized TCR molecules on their surface, and as a result, would recognize peptides presented to them by MHC complexes in a human-like manner. The genetically modified non-human animals described herein may be used to study the development and function of human T cells and the processes of immunological tolerance; to test human vaccine candidates; to generate TCRs with certain specificities for TCR gene therapy; to generate TCR libraries to disease associated antigens (e.g., tumor associated antigens (TAAs); etc.
There is a growing interest in T cell therapy in the art, as T cells (e.g., cytotoxic T cells) can be directed to attack and lead to destruction of antigen of interest, e.g., viral antigen, bacterial antigen, tumor antigen, etc., or cells that present it. Initial studies in cancer T cell therapy aimed at isolation of tumor infiltrating lymphocytes (TILs; lymphocyte populations in the tumor mass that presumably comprise T cells reactive against tumor antigens) from tumor cell mass, expanding them in vitro using T cell growth factors, and transferring them back to the patient in a process called adoptive T cell transfer. See, e.g., Restifo et al. (2012) Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews 12:269-81; Linnermann et al. (2011) T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131:1806-16. However, success of these therapies have thus far been limited to melanoma and renal cell carcinoma; and the TIL adoptive transfer is not specifically directed to defined tumor associated antigens (TAAs). Linnermann et al., supra.
Attempts have been made to initiate TCR gene therapy where T cells are either selected or programmed to target an antigen of interest, e.g., a TAA. Current TCR gene therapy relies on identification of sequences of TCRs that are directed to specific antigens, e.g., tumor associated antigens. For example, Rosenberg and colleagues have published several studies in which they transduced peripheral blood lymphocytes derived from a melanoma patient with genes encoding TCRα and β chains specific for melanoma-associated antigen MART-1 epitopes, and used resulting expanded lymphocytes for adoptive T cell therapy. Johnson et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114:535-46; Morgan et al. (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314:126-29. The MART-1 specific TCRs were isolated from patients that experienced tumor regression following TIL therapy. However, identification of such TCRs, particularly high-avidity TCRs (which are most likely to be therapeutically useful), is complicated by the fact that most tumor antigens are self antigens, and TCRs targeting these antigens are often either deleted or possess suboptimal affinity, due primarily to immunological tolerance.
In various embodiments, the present invention solves this problem by providing genetically engineered non-human animals comprising in their genome an unrearranged human TCR variable gene locus. The non-human animal described herein is capable of generating T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human animals described herein may be a source of a diverse repertoire of humanized T cell receptors, e.g., high-avidity humanized T cell receptors for use in adoptive T cell transfer.
Thus, in one embodiment, the present invention provides a method of generating a T cell receptor to a human antigen comprising immunizing a non-human animal (e.g., a rodent, e.g., a mouse or a rat) described herein with an antigen of interest, allowing the animal to mount an immune response, isolating from the animal an activated T cell with specificity for the antigen of interest, and determining the nucleic acid sequence of the T cell receptor expressed by the antigen-specific T cell.
In one embodiment, the invention provides a method of producing a human T cell receptor specific for an antigen of interest (e.g., a disease-associated antigen) comprising immunizing a non-human animal described herein with the antigen of interest; allowing the animal to mount an immune response; isolating from the animal a T cell reactive to the antigen of interest; determining a nucleic acid sequence of a human TCR variable region expressed by the T cell; cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region; and expressing from the construct a human T cell receptor specific for the antigen of interest. In one embodiment, the steps of isolating a T cell, determining a nucleic acid sequence of a human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region, and expressing a human T cell receptor are performed using standard techniques known to those of skill the art.
In one embodiment, the nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell. In one embodiment, the cell expressing the TCR is selected from a CHO, COS, 293, HeLa, PERC.6™ cell, etc.
The antigen of interest may be any antigen that is known to cause or be associated with a disease or condition, e.g., a tumor associated antigen; an antigen of viral, bacterial or other pathogenic origin; etc. Many tumor associated antigens are known in the art. A selection of tumor associated antigens is presented in Cancer Immunity (A Journal of the Cancer Research Institute) Peptide Database (archive.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). In some embodiments of the invention, the antigen of interest is a human antigen, e.g., a human tumor associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and a T cell receptor is used to kill a cell expressing the antigen.
In one embodiment, provided herein is a method of identifying a T cell with specificity against an antigen of interest, e.g., a tumor associated antigen, comprising immunizing a non-human animal described herein with the antigen of interest, allowing the animal to mount an immune response, and isolating from the non-human animal a T cell with specificity for the antigen.
The present invention provides new methods for adoptive T cell therapy. Thus, provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject (e.g., a mammalian subject, e.g., a human subject) comprising immunizing a non-human animal described herein with an antigen associated with the disease or condition, allowing the animal to mount an immune response, isolating from the animal a population of antigen-specific T cells, and infusing isolated antigen-specific T cells into the subject. In one embodiment, the invention provides a method of treating or ameliorating a disease or condition in a human subject, comprising immunizing the non-human animal described herein with an antigen of interest (e.g., a disease- or condition-associated antigen, e.g., a tumor associated antigen), allowing the animal to mount an immune response, isolating from the animal a population of antigen-specific T cells, determining the nucleic acid sequence of a T cell receptor, (e.g., a first and/or second nucleic acid sequence encoding the human rearranged TCRα and/or human rearranged TCRβ variable region gene); a third and/or fourth nucleic acid sequence encoding the human rearranged TCRδ variable region gene or a TCRγ variable region gene, expressed by the antigen-specific T cells, cloning the nucleic acid sequence of the T cell receptor, e.g., the first, second, third and/or fourth nucleic acid sequence respectively encoding the human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene, into an expression vector (e.g., a retroviral vector), e.g., optionally wherein the first, second, third and/or fourth nucleic acid sequence respectively encoding the human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene is respectively cloned in-frame with a human TCRα constant gene, human TCRβ constant gene, TCRδ constant gene or a TCRγ constant gene, introducing the vector into T cells derived from the subject such that the T cells express the antigen-specific T cell receptor, and infusing the T cells into the subject. In one embodiment, the T cell receptor nucleic acid sequence is further humanized prior to introduction into T cells derived from the subject, e.g., the sequence encoding the non-human constant region is modified to further resemble a human TCR constant region (e.g., the non-human constant region is replaced with a human constant region). In some embodiments, the disease or condition is cancer. In some embodiments, an antigen-specific T cell population is expanded prior to infusing into the subject. In some embodiments, the subject's immune cell population is immunodepleted prior to the infusion of antigen-specific T cells. In some embodiments, the antigen-specific TCR is a high avidity TCR, e.g., a high avidity TCR to a tumor associated antigen. In some embodiments, the T cell is a cytotoxic T cell. In other embodiments, the disease or condition is caused by a virus or a bacterium.
In another embodiment, a disease or condition is an autoimmune disease. TREG cells are a subpopulation of T cells that maintain tolerance to self-antigens and prevent pathological self-reactivity. Thus, also provided herein are methods of treating autoimmune disease that rely on generation of antigen-specific TREG cells in the non-human animal of the invention described herein.
Also provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject comprising introducing the cells affected by the disease or condition (e.g., cancer cells) from the subject into a non-human animal, allowing the animal to mount an immune response to the cells, isolating from the animal a population of T cells reactive to the cells, determining the nucleic acid sequence of a T cell receptor variable domain expressed by the T cells, cloning the T cell receptor variable domain encoding sequence into a vector (e.g., in-frame and operably linked to a human TCR constant gene), introducing the vector into T cells derived from the subject, and infusing the subject's T cells harboring the T cell receptor into the subject.
Also provided herein is the use of a non-human animal as described herein to make nucleic acid sequences encoding human TCR variable domains (e.g., TCR α and/or β variable domains). In one embodiment, a method is provided for making a nucleic acid sequence encoding a human TCR variable domain, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response to the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds the antigen of interest. In one embodiment, the method further comprises making a nucleic acid sequence encoding a human TCR variable domain that is operably linked to a non-human TCR constant region, comprising isolating a T cell from a non-human animal described herein and obtaining therefrom the nucleic acid sequence encoding the TCR variable domain linked to the non-human constant region TCR constant region, and cloning the nucleic acid sequence(s) encoding the TCR variable domain (e.g., a first, second, third or fourth nucleic acid sequence respectively encoding a human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene) in-frame with an appropriate human constant region (e.g., a human TCRα constant region gene, human TCRβ constant region gene, TCRδ constant region gene or a TCRγ variable region gene, respectively).
Thus, provided herein are TCR variable region nucleic acid sequences, such as rearranged TCR variable nucleic acid sequences, e.g., rearranged TCRα and/or TCRβ variable region nucleic acid sequences, generated in the non-human animals described herein, and encoded respectively by, e.g., a human rearranged Vα/Jα gene sequence and a rearranged human VβDβJβ gene sequence. Also, provided are TCR variable region amino acid sequences encoded by such rearranged TCR variable region nucleic acid sequences. Such rearranged TCR variable region nucleic acid sequences (TCRα and/or TCRβ variable region nucleic acid sequences) obtained in the non-human animals described herein may be cloned in operable linkage with human TCR constant region (TCRα and/or TCRβ constant region), and utilized for various uses described herein, e.g., as a human therapeutic, in a human.
Also provided herein is the use of a non-human animal as described herein to make a human therapeutic, comprising immunizing the non-human animal with an antigen of interest (e.g., a tumor associated antigen), allowing the non-human animal to mount an immune response, obtaining from the animal T cells reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized TCR protein or human TCR variable domain that binds the antigen of interest, and employing the nucleic acid sequence(s) encoding a humanized TCR protein or a human TCR variable domain in a human therapeutic.
Thus, also provided is a method for making a human therapeutic, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response, obtaining from the animal T cells reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized T cell receptor that binds the antigen of interest, and employing the humanized (or fully human) T cell receptor in a human therapeutic.
In one embodiment, the human therapeutic is a T cell (e.g., a human T cell, e.g., a T cell derived from a human subject) harboring a nucleic acid sequence of interest (e.g., transfected or transduced or otherwise introduced with the nucleic acid of interest) such that the T cell expresses the humanized TCR protein with affinity for an antigen of interest. In one aspect, a subject in whom the therapeutic is employed is in need of therapy for a particular disease or condition, and the antigen is associated with the disease or condition. In one aspect, the T cell is a cytotoxic T cell, the antigen is a tumor associated antigen, and the disease or condition is cancer. In one aspect, the T cell is derived from the subject.
In another embodiment, the human therapeutic is a T cell receptor. In one embodiment, the therapeutic receptor is a soluble T cell receptor. Much effort has been expanded to generate soluble T cell receptors or TCR variable regions for use therapeutic agents. Generation of soluble T cell receptors depends on obtaining rearranged TCR variable regions. One approach is to design single chain TCRs comprising TCRα and TCRβ, and, similarly to scFv immunoglobulin format, fuse them together via a linker (see, e.g., International Application No. WO 2011/044186). The resulting scTv, if analogous to scFv, would provide a thermally stable and soluble form of TCRα/β binding protein. Alternative approaches included designing a soluble TCR having TCRβ constant domains (see, e.g., Chung et al., (1994) Functional three-domain single-chain T-cell receptors, Proc. Natl. Acad. Sci. USA. 91:12654-58); as well as engineering a non-native disulfide bond into the interface between TCR constant domains (reviewed in Boulter and Jakobsen (2005) Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens, Clinical and Experimental Immunology 142:454-60; see also, U.S. Pat. No. 7,569,664). Other formats of soluble T cell receptors have been described. The non-human animals described herein may be used to determine a sequence of a T cell receptor that binds with high affinity to an antigen of interest, and subsequently design a soluble T cell receptor based on the sequence.
A soluble T cell receptor derived from the TCR receptor sequence expressed by the non-human animal can be used to block the function of a protein of interest, e.g., a viral, bacterial, or tumor associated protein. Alternatively, a soluble T cell receptor may be fused to a moiety that can kill an infected or cancer cell, e.g., a cytotoxic molecules (e.g., a chemotherapeutic), toxin, radionuclide, prodrug, antibody, etc. A soluble T cell receptor may also be fused to an immunomodulatory molecule, e.g., a cytokine, chemokine, etc. A soluble T cell receptor may also be fused to an immune inhibitory molecule, e.g., a molecule that inhibits a T cell from killing other cells harboring an antigen recognized by the T cell. Such soluble T cell receptors fused to immune inhibitory molecules can be used, e.g., in blocking autoimmunity. Various exemplary immune inhibitory molecules that may be fused to a soluble T cell receptor are reviewed in Ravetch and Lanier (2000) Immune Inhibitory Receptors, Science 290:84-89, incorporated herein by reference.
The present invention also provides methods for studying immunological response in the context of human TCR, including human TCR rearrangement, T cell development, T cell activation, immunological tolerance, etc.
Also provided are methods of testing vaccine candidates. In one embodiment, provided herein is a method of determining whether a vaccine will activate an immunological response (e.g., T cell proliferation, cytokine release, etc.), and lead to generation of effector, as well as memory T cells (e.g., central and effector memory T cells).
In one aspect, an in vitro preparation is provided that comprises a T cell that bears a chimeric CD8 protein on its surface and a second cell that binds the chimeric CD8. In one embodiment, the second cell is a cell expressing an MHC I polypeptide, e.g., a chimeric human/non-human MHC I protein. In one embodiment, the chimeric CD8 on the surface of the first cell interacts with chimeric MHC I on the surface of the second cell. In one embodiment, the chimeric CD8 protein retains interaction with endogenous cytosolic molecules, e.g., endogenous cytosolic signaling molecules (e.g., endogenous Lck, etc.).
In one aspect, an in vitro preparation is provided that comprises a T cell that bears a chimeric CD4 protein on its surface and a second cell that binds the chimeric CD4. In one embodiment, the second cell is a cell, e.g., an APC, expressing an MHC II polypeptide, e.g., a chimeric human/non-human MHC II protein. In one embodiment, the chimeric CD4 on the surface of the first cell interacts with chimeric MHC II on the surface of the second cell. In one embodiment, the chimeric CD4 protein retains interaction with endogenous cytosolic molecules, e.g., endogenous cytosolic signaling molecules (e.g., endogenous Lck, etc.).
Other uses of the genetically modified animals described herein, i.e., animals comprising a human or humanized T cell co-receptor (e.g., chimeric human/non-human CD4 or CD8), optionally further comprising a human or humanized MHC II or I protein, will be apparent from the present disclosure.
EXAMPLES
The following examples are provided so as to describe to those of ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.
Example 1
Generation of Humanized MHC Mice
The various steps involved in engineering a mouse comprising humanized MHC I and MHC II loci, with corresponding and additional endogenous MHC I and MHC II loci deletions (HLA-A2/H-2K, HLA-DR2/H-2E, H-2A-del, H-2D-del) are depicted in FIG. 3A. Detailed description of the steps appears below.
Example 1.1
Generation and Characterization of Humanized MHC I Mice
Generation of humanized MHC I mice has previously been described in U.S. Patent Publication No. 20130111617, incorporated herein by reference. Briefly, the mouse H-2K gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659). DNA from mouse BAC clone RP23-173k21 (Invitrogen) was modified by homologous recombination to replace the genomic DNA encoding the α1, α2 and α3 domains of the mouse H-2K gene with human genomic DNA encoding the α1, α2 and α3 subunits of the human HLA-A gene (FIG. 2A).
Specifically, the genomic sequence encoding the mouse the α1, α2 and α3 subunits of the H-2K gene is replaced with the human genomic DNA encoding the α1, α2 and α3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector comprising a hygromycin cassette flanked by loxP sites with a 5′ mouse homology arm containing sequence 5′ of the mouse H-2K locus including the 5′ untranslated region (UTR) and a 3′ mouse homology arm containing genomic sequence 3′ of the mouse H-2K α3 coding sequence.
The final construct for targeting the endogenous H-2K gene locus from 5′ to 3′ included (1) a 5′ homology arm containing ˜200 bp of mouse genomic sequence 5′ of the endogenous H-2K gene including the 5′UTR, (2) ˜1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the HLA-A*0201 leader/al intron, the HLA-A*0201 α1 exon, the HLA-A*0201 α1-α2 intron, the HLA-A*0201 α2 exon, ˜316 bp of the 5′ end of the α2-α3 intron, (3) a 5′ loxP site, (4) a hygromycin cassette, (5) a 3′ loxP site, (6) ˜580 bp of human genomic sequence including ˜304 bp of the 3′ end of the α2-α3 intron, the HLA-A*0201 α3 exon, and (7) a 3′ homology arm containing ˜200 bp of mouse genomic sequence including the intron between the mouse H-2K α3 and transmembrane coding sequences. The sequence of 149 nucleotides at the junction of the mouse/human sequences at the 5′ of the targeting vector is set forth in SEQ ID NO: 90, and the sequence of 159 nucleotides at the junction of the human/mouse sequences at the 3′ of the targeting vector is set forth in SEQ ID NO:91. Homologous recombination with this targeting vector created a modified mouse H-2K locus containing human genomic DNA encoding the α1, α2 and α3 domains of the HLA-A*0201 gene operably linked to the endogenous mouse H-2K transmembrane and cytoplasmic domain coding sequences which, upon translation, leads to the formation of a chimeric human/mouse MHC class I protein. The selection cassette present in the targeting construct may be later removed using various methods known in the art.
The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create modified ES cells for generating mice that express a chimeric MHC class I protein on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, neutrophils) (see, e.g., step 1 in the scheme depicted in FIG. 3A). ES cells containing an insertion of human HLA sequences were identified by a quantitative TAQMAN™ assay (Valenzuela et al. (2003), supra).
To generate mice expressing chimeric MHC I, targeted ES cells described herein are used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I gene are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human HLA-A*0201 gene sequences. Heterozygous mice generated by this method are bred to homozygosity. Expression of chimeric HLA-A2/H-2K is confirmed by flow cytometry using antibodies specific for HLA-A and H-2K.
Targeted ES cells described above comprising the chimeric HLA-A2/H-2K were used in further genetic engineering steps described in Examples 1.2-1.3 to generate mice comprising both humanized MHC I and MHC II loci and lacking endogenous MHC I and MHC II loci (See FIG. 3A).
Example 1.2
Generation of Mouse ES Cells Comprising MHC I and MHC II Loci Deletions
Deletion of endogenous MHC II loci is described in U.S. Patent Application Number No. 20130111616, incorporated herein by reference. Briefly, the targeting vector for introducing a deletion of the endogenous MHC class II H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea genes was made using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra). Bacterial Artificial Chromosome (BAC) RP23-458i22 (Invitrogen) DNA was modified to delete the endogenous MHC class II genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea.
Specifically, upstream and downstream homology arms were derived by PCR of mouse BAC DNA from locations 5′ of the H-2Ab1 gene and 3′ of the H-2Ea gene, respectively. These homology arms were used to make a cassette that deleted ˜79 kb of RP23-458i22 comprising genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea of the MHC class II locus by bacterial homologous recombination (BHR). This region was replaced with a neomycin cassette flanked by lox2372 sites. The final targeting vector from 5′ to 3′ included a 26 kb homology arm comprising mouse genomic sequence 5′ to the H-2Ab1 gene of the endogenous MHC class II locus, a 5′ lox2372 site, a neomycin cassette, a 3′ lox2372 site and a 63 kb homology arm comprising mouse genomic sequence 3′ to the H-2Ea gene of the endogenous MHC class II locus.
The BAC DNA targeting vector (described above) was used to electroporate mouse ES cells comprising humanized MHC I locus (from Example 1.1 above; see, e.g., step 2 in FIG. 3A) to create modified ES cells comprising a deletion of the endogenous MHC class II locus (both H-2A and H-2E were deleted). Positive ES cells containing a deleted endogenous MHC class II locus were identified by the quantitative PCR assay using TAQMAN™ probes (Lie and Petropoulos (1998) Curr. Opin. Biotechnology 9:43-48). The upstream region of the deleted locus was confirmed by PCR using primers 5111U F (CAGAACGCCAGGCTGTAAC; SEQ ID NO:1) and 5111U R (GGAGAGCAGGGTCAGTCAAC; SEQ ID NO:2) and probe 5111U P (CACCGCCACTCACAGCTCCTTACA; SEQ ID NO:3), whereas the downstream region of the deleted locus was confirmed using primers 5111 D F (GTGGGCACCATCTTCATCATTC; SEQ ID NO:4) and 5111 D R (CTTCCTTTCCAGGGTGTGACTC; SEQ ID NO:5) and probe 5111 D P (AGGCCTGCGATCAGGTGGCACCT; SEQ ID NO:6). The presence of the neomycin cassette from the targeting vector was confirmed using primers NEOF (GGTGGAGAGGCTATTCGGC; SEQ ID NO:7) and NEOR (GAACACGGCGGCATCAG;SEQ ID NO:8) and probe NEOP (TGGGCACAACAGACAATCGGCTG; SEQ ID NO:9). The nucleotide sequence across the upstream deletion point (SEQ ID NO:10) included the following, which indicates endogenous mouse sequence upstream of the deletion point (contained within the parentheses below) linked contiguously to cassette sequence present at the deletion point: (TTTGTAAACA AAGTCTACCC AGAGACAGAT GACAGACTTC AGCTCCAATG CTGATTGGTT CCTCACTTGG GACCAACCCT) ACCGGTATAA CTTCGTATAA GGTATCCTAT ACGAAGTTAT ATGCATGGCC TCCGCGCCGG. The nucleotide sequence across the downstream deletion point (SEQ ID NO:11) included the following, which indicates cassette sequence contiguous with endogenous mouse sequence downstream of the deletion point (contained within the parentheses below): CGACCTGCAG CCGGCGCGCC ATAACTTCGT ATAAGGTATC CTATACGAAG TTATCTCGAG (CACAGGCATT TGGGTGGGCA GGGATGGACG GTGACTGGGA CAATCGGGAT GGAAGAGCAT AGAATGGGAG TTAGGGAAGA).
Subsequently to generation of the ES cells comprising both the MHC I humanization and endogenous MHC II deletion described above, the loxed neomycin cassette was removed using CRE (see, e.g., step 3 in FIG. 3A). Specifically, a plasmid encoding Cre recombinase was electroporated into ES cells to remove the neomycin cassette. Neo cassette may also be removed using other methods known in the art.
To delete mouse H-2D locus, BHR was used to modify mouse BAC clone bMQ-218H21 (Sanger Institute), replacing 3756 bp of the H2-D gene (from the ATG start codon to 3 bp downstream of the TGA stop codon, exons 1-8 of mouse H-2D) with a 6,085 bp cassette containing from 5′ to 3′: a LacZ gene in frame with a 5′ loxp site, UbC promoter, Neomycin gene, and 3′ loxp site.
The BAC DNA targeting vector (described above) was used to electroporate mouse ES cells comprising humanized MHC I locus and a deletion of mouse MHC II, described above (see, e.g., step 4 in FIG. 3A). Positive ES cells containing a deleted endogenous H-2D locus were identified by the quantitative PCR assay, as described above. Table 2 contains primers and probes used for the quantitative PCR assay.
TABLE 2
TAQMAN ™ Loss of Allele Assay Primers and Probes for Detection of
Deleted H-2D Locus
Name
(location)
Forward Primer
Reverse Primer
Probe
5152 mTU
CGAGGAGCCCCG
AAGCGCACGAACTC
CTCTGTCGGCTAT
(upstream)
GTACA
CTTGTT
GTGG
(SEQ ID NO: 12)
(SEQ ID NO: 13)
(SEQ ID NO: 14)
5152 mTD
GGACTCCCAGAAT
GAGTCATGAACCATC
TGGTGGGTTGCTG
(downstream)
CTCCTGAGA
ACTGTGAAGA
GAA
(SEQ ID NO: 15)
(SEQ ID NO: 16)
(SEQ ID NO: 17)
Example 1.3
Introduction of Chimeric Human/Mouse MHC II Locus
To generate a vector comprising humanized HLA-DR2/H-2E, first, mouse H-2Ea gene was modified in accordance with the description in U.S. Pat. No. 8,847,005, issued Sep. 30, 2014, incorporated herein by reference, to generate a vector comprising sequence encoding a chimeric H-2Ea/HLA-DRA1*01 protein.
For mouse H-2Eb gene, synthesized human HLA-DR2 β chain (DRB1*1501) was used to generate a vector comprising DRβ1*02(1501) exons and introns, and swapped using bacterial homologous recombination into the vector comprising chimeric H-2Ea/HLA-DRA1*01 protein. H-2Eb1 gene was modified essentially as described in U.S. Patent Publication No. 20130185820, and U.S. Pat. No. 8,847,005, each incorporated herein by reference. A hygromycin selection cassette was used.
The resulting HLA-DR2/H-2E large targeting vector (LTVEC) is depicted in FIGS. 2B and 3B. The various nucleotide sequence junctions of the resulting LTVECs (e.g., mouse/human sequence junctions, human/mouse sequence junctions, or junctions of mouse or human sequence with selection cassettes) are summarized below in Table 3 and listed in the Sequence Listing; their locations are indicated in the schematic diagram of FIG. 3B. In Table 3 below, with the exception of sequences marked with asterisks (*, see Table legend) the mouse sequences are in regular font; the human sequences are in parentheses; the Lox sequences are italicized; and the restriction sites introduced during cloning steps and other vector-based sequences (e.g., multiple cloning sites, etc.) are bolded.
TABLE 3
Nucleotide Sequence Junctions of Chimeric HLA-DR2/H-2E Locus
SEQ ID
NO:
Nucleotide Sequence
18
CTGTTTCTTC CCTAACTCCC ATTCTATGCT CTTCCATCCC GA CCGCGG(CCCA
ATCTCTCTCC ACTACTTCCT GCCTACATGT ATGTAGGT)
19
(CAAGGTTTCC TCCTATGATG CTTGTGTGAA ACTCGG) GGCC GGCC
AGCATTTAAC AGTACAGGGA TGGGAGCACA GCTCAC
20*
(GAAAGCAGTC TTCCCAGCCT TCACACTCAG AGGTACAAAT) CCCCATTTTC
ATATTAGCGA TTTTAATTTA TTCTAGCCTC
21*
TCTTCCCTAA CTCCCATTCT ATGCTCTTCC ATCCCGA CCG CGG (CCCAATC
TCTCTCCACT ACTTCCTGCC TACATGTATG)
22
GAGTTCCTCCATCACTTCACTGGGTAGCACAGCTGTAACTGTCCAGCCTG
(TCCTGGGCTGCAGGTGGTGGGCGTTGCGGGTGGGGCCGGTTAAGGTTCCA)
23
(TCCCACATCCTATTTTAATTTGCTCCATGTTCTCATCTCCATCAGCACAG)
CTCGAG ATAACTTCGTATAATGTATGCTATACGAAGTTAT ATGCATGGCC
24
ATACGAAGTTAT GCTAGTAACTATAACGGTCCTAAGGTAGCGAGTGGCTT
ACAGGTAGGTGCGTGAAGCTTCTACAAGCACAGTTGCCCCCTGGGAAGCA
Sequences marked with asterisk are C57BL/6-BALB/c junction sequences where C57BL/6 sequences are in parentheses. During cloning of the chimeric H-2Ea gene, exon 1 and the remainder of intron 1 of the C57BL/6 allele of H-2Ea was replaced with the equivalent 2616 bp region from the BALB/c allele of H-2Ea. This was done because exon 1 of the C57BL/6 allele of H-2Ea contains a deletion which renders the gene nonfunctional, while exon 1 of BALB/c allele of H-2Ea is functional. For a more detailed description, see U.S. Pat. No. 8,847,005, incorporated herein by reference.
The targeted BAC DNA described above was used to electroporate mouse ES cells comprising humanized MHC I (HLA-A2), as well as MHC II and H-2D deletion to create modified ES cells for generating mice that express chimeric MHC I and MHC II genes and lack functional endogenous mouse H-2E, H-2A, H-2K, and H-2D loci (see, e.g., step 5 in FIG. 3A). ES cells containing an insertion of human HLA sequences were identified by a quantitative PCR (TAQMAN™) assay, using primers and probes in Table 4.
TABLE 4
TAQMAN ™ Primer and Probe Sequences for Detection of MHC I and MHC
II Loci Humanization
Name
(location)
Forward Primer
Reverse Primer
Probe
Hyg cassette
TGCGGCCGATCTT
TTGACCGATTCCTTG
ACGAGCGGGTTC
AGCC (SEQ ID
CGG (SEQ ID NO: 26)
GGCCCATTC (SEQ
NO: 25)
ID NO: 27)
7092 hTUP1
CCCCACAGCACGT
CGTCCCATTGAAGAA
TGGCAGCCTAAGA
(Exon 2 of
TTCCT (SEQ ID
ATGACACT (SEQ ID
GG (SEQ ID NO: 30)
DRB1*1501)
NO: 28)
NO: 29)
7092 hTUP2
CCCCACAGCACGT
ACCCGCTCCGTCCC
AGCCTAAGAGGG
(Exon 2 of
TTCCT (SEQ ID
ATT (SEQ ID NO: 32)
AGTGTC (SEQ ID
DRB1*1501)
NO: 31)
NO: 33)
7092 hTDP1
AGACCCTGGTGAT
CGCTTGGGTGCTCC
TCGAAGTGGAGA
(Exon 3 of
GCTGGAA (SEQ ID
ACTT (SEQ ID NO: 35)
GGTTTA (SEQ ID
DRB1*1501)
NO: 34)
NO: 36)
7092 hTDP2
TGGAATGGAGTGA
GCACGGTCCCCTTC
TGACTTCCTAAAT
(exon 3 of
GCAGCTTT (SEQ
TTAGTG (SEQ ID
TTCTC (SEQ ID
DRB1*1501)
ID NO: 37)
NO: 38)
NO: 39)
hDRAIU
CTGGCGGCTTGAA
CATGATTTCCAGGTT
CGATTTGCCAGCT
(exon 2 of
GAATTTGG (SEQ
GGCTTTGTC (SEQ ID
TTGAGGCTCAAGG
DRA)
ID NO: 40)
NO: 41)
(SEQ ID NO: 42)
1751jxn21
CCTCACTTGGGAC
TTGTCCCAGTCACCG
TGCATCTCGAGCA
(loss-of-
CAACCCTA (SEQ
TCCAT (SEQ ID
CAGGCATTTGG
allele
ID NO: 43)
NO: 44)
(SEQ ID NO: 45)
assay,
sequence
present in H-
2A and H-2E
delete only)
1A11 sequences except this one are used in the gain-of-allele assay.
The selection cassette may be removed by methods known by the skilled artisan. For example, ES cells bearing the chimeric human/mouse MHC class I locus may be transfected with a construct that expresses Cre in order to remove the “loxed” selection cassette introduced by the insertion of the targeting construct (see, e.g., step 6 in FIG. 3A). The selection cassette may optionally be removed by breeding to mice that express Cre recombinase. Optionally, the selection cassette is retained in the mice.
Targeted ES cells containing all of the modifications described herein (HLA-A2/H-2K, HLA-DR2/H-2E, H-2A-del, H-2D-del of FIG. 3A) were verified using a quantitative TAQMAN® assay described above using the primer/probe sets described herein for individual modifications. An additional primer/probe set was used to determine that during cassette-deletion step, no inverted clone was created due to lox sites present in opposing orientation.
Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007), supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I and MHC II genes were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human gene sequences. A schematic representation of the genotype of MHC loci in the resulting mice is depicted in FIG. 3C (** represents H-2L gene which is not present in all mouse strains). Expression of both chimeric human/mouse MHC I and MHC II proteins is confirmed using antibodies specific for human HLA-DR2 and HLA-A2. Heterozygous mice are bred to homozygosity.
Example 1.4
Generation of Humanized β2 Microglobulin Mice
Generation of β2 microglobulin mice was described in U.S. Patent Application Publication No. 20130111617, incorporated herein by reference. Briefly, mouse β2 microglobulin (β2m) gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).
Specifically, a targeting vector was generated by bacterial homologous recombination containing mouse β2m upstream and downstream homology arms from BAC clone 89C24 from the RPCI-23 library (Invitrogen). The mouse homology arms were engineered to flank a 2.8 kb human β2m DNA fragment extending from exon 2 to about 267 nucleotides downstream of non-coding exon 4 (FIG. 2C). A drug selection cassette (neomycin) flanked by recombinase recognition sites (e.g., loxP sites) was engineered into the targeting vector to allow for subsequent selection. The final targeting vector was linearized and electroporated into a F1H4 mouse ES cell line (Valenzuela et al., supra).
Targeted ES cell clones with drug cassette removed (by introduction of Cre recombinase) were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing the humanized β2m gene were identified by screening for loss of mouse allele and gain of human allele using a modification of allele assay (Valenzuela et al., supra). Heterozygous mice are bred to homozygosity. Expression of human β2 microglobulin was confirmed by flow cytometry using antibodies specific for human β2 microglobulin.
Example 2
Generation of Humanized T Cell Receptor Mice
Mice comprising a deletion of endogenous TCR (α or β) variable loci and replacement of endogenous V and J or V, D, and J segments are made using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, D. M., et al. (2003), supra), wherein human sequences derived from BAC libraries using bacterial homologous recombination are used to make large targeting vectors (LTVECs) comprising genomic fragments of human TCR variable loci flanked by targeting arms to target the LTVECs to endogenous mouse TCR variable loci in mouse ES cells. Detailed description of the humanization of the TCR alpha and beta loci is described in U.S. Pat. No. 9,113,616, incorporated herein by reference. LTVECs re linearized and electroporated into a mouse ES cell line according to Valenzuela et al. ES cells are selected for hygromycin or neomycin resistance, and screened for loss of mouse allele or gain of human allele.
Targeted ES cell clones are introduced into 8-cell stage (or earlier) mouse embryos by the VELOCIMOUSE® method (Poueymirou, W. T. et al. (2007, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing humanized TCR loci are identified by screening for loss of endogenous TCR variable allele and gain of human allele using a modification of allele assay (Valenzuela et al., supra). F0 pups are genotyped and bred to homozygosity. Mice homozygous for humanized TCRα and/or TCRβ variable loci are made as described herein.
Example 2.1
Humanization of TCR Alpha Locus
1.5 megabases of DNA at mouse TCRα locus corresponding to 110 V and 60 J mouse segments was replaced with 1 megabase of DNA corresponding to 54V and 61J segments of human TCRα using a progressive humanization strategy summarized in FIG. 4A and described in U.S. Pat. No. 9,113,616. Junctional nucleic acid sequences of various targeting vectors used for progressive humanization strategy of TCRα locus are summarized in Table 5, and included in the Sequence Listing.
TABLE 5
Junctional Nucleic Acid Sequences for Various TCRα Locus Targeting Vectors
MAID
SEQ ID
NO.
NO
Description
1626
46
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRα variable locus and the 5′ end of
loxP-Ub-Hyg-loxP cassette.
47
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Hyg-loxP cassette and the 5′ end of human TCRVα40-TCRVα41-
TCRJα1 insertion, including AsiSI site.
48
Junctional nucleic acid sequence between the 3′ end of human
TCRVα40-TCRVα41-TCRJα1 insertion and the 5′ end of the
mouse sequence downstream of the human TCRα variable locus,
including Notl site.
1767
49
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRα variable locus and the 5′ end of
loxP-Ub-Neo-loxP cassette.
50
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Neo-loxP cassette and the 5′ end of human TCRVα35-TCRVα39
insertion, including AsiSI site.
1979
51
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRα variable locus and the 5′ end of
frt-Pgk-Hyg-frt cassette.
52
Junctional nucleic acid sequence between the 3′ end of frt-Pgk-
Hyg-frt cassette and the 5′ end of human TCRVα22-TCRVα34
insertion, including AsiSI site.
1769
53
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRα variable locus and the 5′ end of
loxP-Ub-Neo-loxP cassette.
54
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Neo-loxP cassette and the 5′ end of human TCRVα13-2-
TCRVα21 insertion, including AsiSI site.
1770
55
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRα variable locus and the 5′ end of
loxP-Ub-Hyg-loxP cassette.
56
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Hyg-loxP cassette and the 5′ end of human TCRVα6-TCRVα8-5
insertion, including AsiSI site.
1771
57
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream and the TCRα variable locus to the 5′ end of
loxP-Ub-Neo-loxP cassette.
58
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Neo-loxP cassette and the 5′ end of human TCRVα1-1-TCRVα5
insertion, including AsiSI site.
Human TCRα variable region segments are numbered as in IMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.
First, DNA from mouse BAC clone RP23-6A14 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace TCRAJ1-TCRAJ28 region of the endogenous mouse TCRα locus with a Ub-hygromycin cassette followed by a loxP site. DNA from mouse BAC clone RP23-117i19 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace ˜15 kb region surrounding (and including) TCRAV1 of the endogenous mouse TCRα and δ locus with a PGK-neomycin cassette followed by a loxP site. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRα locus targeted with both of these targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase, thereby mediating the deletion of the region between the two loxP sites (i.e., the region consisting of the endogenous mouse TCRα locus from TCRAV1 to TCRAJ1) and leaving behind only a single loxP site, neomycin cassette and the mouse constant and enhancer regions. This strategy resulted in generation of a deleted mouse TCR a/5 locus (MAID 1540, FIG. 4A, second diagram).
The first human targeting vector for TCRα had 191,660 bp of human DNA from the CTD2216p1 and CTD2285m07 BAC clones (Invitrogen) that contained the first two consecutive human TCRα V gene segments (TRAV40 & 41) and 61 TCRα J (50 functional) gene segments. This BAC was modified by homologous recombination to contain a Not1 site 403 bp downstream (3′) of the TCRα J1 gene segment for ligation of a 3′ mouse homology arm and a 5′ AsiSI site for ligation of a 5′ mouse homology arm. Two different homology arms were used for ligation to this human fragment: the 3′ homology arm contained endogenous mouse TCRα sequences from the RP23-6A14 BAC clone and the 5′ homology arm contained endogenous TCRα sequence 5′ of mouse TCRα V from mouse BAC clone RP23-117i19. This mouse-human chimeric BAC was used as a targeting vector (MAID 1626) for making an initial insertion of human TCRα gene segments plus an upstream loxp-ub-hygromycin-loxp cassette at the mouse TCRα loci. The junctional nucleic acid sequences (SEQ ID NOs: 46-48) for the MAID 1626 targeting vector are described in Table 5.
Subsequently, a series of human targeting vectors were made that utilized the same mouse 5′ arm that contained endogenous TCRα sequence 5′ of mouse TCRα V from mouse BAC clone RP23-117i19 with alternating loxP-neomycin-loxP and loxP-hygromycin-loxP (or frt-hygromycin-frt for MAID 1979) selection cassettes. The specific constructs are described in U.S. Pat. No. 9,113,616, as well as depicted in FIG. 4A, with junctional sequences for each insertion included in Table 5 and the Sequence Listing. The final TCRα locus contained a 5′ loxp-ub-neomycin-loxP cassette plus a total of 54 human TCRα V (45 functional) and 61 human TCRα J gene segment operably linked to mouse TCRα constant genes and enhancers. The junctional nucleic acid sequences (SEQ ID NOs: 57 and 58) for the MAID 1771 targeting vector are described in Table 5.
In any of progressive humanization steps, the selection cassettes are removed by deletion with Cre or Flp recombinase. In addition, human TCRδ locus may be reintroduced into the TCR alpha sequence.
Example 2.2
Humanization of TCRβ Variable Locus
0.6 megabases of DNA at mouse TCRβ locus corresponding to 33 V, 2 D, and 14 J mouse segments were replaced with 0.6 megabases of DNA corresponding to 67 V, 2D, and 14 J segments of human TCRβ using a progressive humanization strategy summarized in FIG. 4B and described in detail in U.S. Pat. No. 9,113,616. Junctional nucleic acid sequences of various targeting vectors used for progressive humanization strategy of TCRβ locus are summarized in Table 6, and included in the Sequence Listing.
TABLE 6
Junctional Nucleic Acid Sequences for Various TCRβ Locus Targeting Vectors
MAID
SEQ ID
NO.
NO
Description
1625
59
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRβ variable locus (nearby the
upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Neo-
frt cassette.
60
Junctional nucleic acid sequence between the 3′ end of frt-Ub-
Neo-frt cassette and the 5′ end of human TCRVβ18-TCRVβ29-1
insertion.
61
Junctional nucleic acid sequence between the 3′ end of human
TCRVβ18-TCRVβ29-1 insertion and the 5′ end of the mouse
sequence downstream of the mouse TCRVβ segments (nearby
downstream mouse trypsinogen genes).
1715
62
Junctional nucleic acid sequence between 3′ of the downstream
mouse trypsinogen genes and the 5′ end of human TCRDβ1-
TCRJβ1-1-TCRJβ1-6 insertion, including lceul site.
63
Junctional nucleic acid sequence between the 3′ end of human
TCRDβ1-TCRJβ1-1-TCRJβ1-6 insertion and the 5′ end of loxP-
Ub-Hyg-loxP cassette.
64
Junctional nucleic acid sequence between the 3′ end of loxP-Ub-
Hyg-loxP cassette and the 5′ end of mouse sequence nearby the
mouse Cβ1 gene.
65
Junctional nucleic acid sequence between the 3′ end of the
mouse sequence nearby the mouse Cβ1 gene and the 5′ end of
human TCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion, including Notl
site.
66
Junctional nucleic acid sequence between the 3′ end of human
TCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion and the 5′ end of the
mouse sequence downstream of the TCRβ variable locus (nearby
the Cβ2 mouse sequence).
1791
67
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRβ variable locus (nearby the
upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Hyg-
frt cassette.
68
Junctional nucleic acid sequence between the 3′ end of frt-Ub-
Hyg-frt cassette and the 5′ end of human TCRVβ6-5-TCRVβ17
insertion.
1792
69
Junctional nucleic acid sequence between the 3′ end of mouse
sequence upstream of the TCRβ variable locus (nearby the
upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Neo-
frt cassette.
70
Junctional nucleic acid sequence between the 3′ end of frt-Ub-
Hyg-frt cassette and the 5′ end of human TCRVβ1-TCRVβ12-2
insertion.
6192
71
Junctional nucleic acid sequence between the 3′ end of mouse
sequence nearby the mouse Cβ2 gene and the 5′ end of the
human TCRBV30 exon 2 sequence.
72
Junctional nucleic acid sequence between the 3′ end human
TCRBV30 exon 1 sequence and the 5′ end of mouse sequence
downstream of TCRβ locus.
Human TCRβ variable region segments are numbered as in IMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.
Specifically, DNA from mouse BAC clone RP23-153p19 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace 17 kb region (including TCRBV30) just upstream of the 3′ trypsinogen (TRY) gene cluster in the endogenous mouse TCRβ locus with a PGK-neo cassette followed by a loxP site. DNA from mouse BAC clone RP23-461h15 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace 8355 bp region (including TCRBV2 and TCRBV3) downstream of 5′ trypsinogen (TRY) gene cluster in the endogenous mouse TCRβ locus with a Ub-hygromycin cassette followed by a loxP site. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase, mediating the deletion of the region between the 5′ and 3′ loxP sites (consisting of the endogenous mouse TCRβ locus from TCRBV2 to TCRBV30) and leaving behind only a single loxP site, hygromycin cassette and the mouse TCRBDs, TCRBJs, constant, and enhancer sequences. One mouse TCRVβ was left upstream of the 5′ cluster of trypsinogen genes, and one mouse TCRVβ was left downstream of the mouse Eβ, as noted in FIG. 4B.
The first human targeting vector for TCRβ had 125,781 bp of human DNA from the CTD2559j2 BAC clone (Invitrogen) that contained the first 14 consecutive human TCRβV gene segments (TRBV18-TRBV29-1); the junctional nucleic acid sequences (SEQ ID NOs: 59-61) for the MAID 1625 targeting vector are described in Table 6.
In order to replace mouse TCRβ D and J segments with human TCRβ D and J segments, DNA from mouse BAC clone RP23-302p18 (Invitrogen) and from human BAC clone RP11-701D14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1715) into the ES cells that contained the TCRβV mini-locus described above (i.e., MAID 1625). This modification replaced ˜18540 bp region (from 100 bp downstream of the polyA of the 3′ trypsinogen genes to 100 bp downstream from the J segments in the D2 cluster which included mouse TCRBD1-J1, mouse constant 1, and mouse TCRBD2-J2) in the endogenous mouse TCRβ locus with ˜25425 bp of sequence containing human TCRBD1-J1, loxP Ub-hygromycin-loxP cassette, mouse constant 1, human TCRBD2-J2. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase thereby mediating the deletion the hygromycin cassette leaving behind only a single loxP site downstream from human J segments in D1J cluster. The junctional nucleic acid sequences (SEQ ID NOs: 62-66) for the MAID 1715 targeting vector are described in Table 6.
Subsequently, a series of human targeting vectors were made that utilized the same mouse 5′ arm that contained endogenous TCRβ sequence surrounding the upstream mouse trypsinogen genes from mouse BAC clone RP23-461h15 with alternating selection cassette. The specific constructs are described in U.S. Pat. No. 9,113,616, as well as depicted in FIG. 4B, with junctional sequences for each insertion included in Table 6 and the Sequence Listing.
Finally, a human TCRβ mini-locus containing a total 66 human TCRβV (47 functional) and the human TCRβ D and J segments (MAID 1792) operably linked to mouse TCRβ constant genes and enhancers was generated. The junctional nucleic acid sequences (SEQ ID NOs: 69 and 70) for the MAID 1792 targeting vector are described in Table 6.
Mouse TCRBV31 is located ˜9.4 kb 3′ of TCRBC2 (second TCRB constant region sequence) and is in the opposite orientation to the other TCRBV segments. The equivalent human V segment is TCRBV30, which is located in a similar position in the human TCRB locus. To humanize TCRBV31, the mouse BAC clone containing mouse TCRBV31, was modified by bacterial homologous recombination to make LTVEC MAID 6192. The entire coding region, beginning at the start codon in exon 1, the intron, the 3′ UTR, and the recombination signal sequences (RSS) of TCRBV31 were replaced with the homologous human TCRBV30 sequences. FIG. 4B depicts the selection cassette located in the intron between exon 1 and exon 2 of the hTCRBV30 gene.
The junctional nucleic acid sequences (SEQ ID NOs: 71 and 72) for the MAID 6192 targeting vector are described in Table 6. MAID 6192 DNA is electroporated into MAID1792 ES cells, and cells are screened for loss of mouse TCRB31 allele and gain of human TCRB30 allele.
Similar engineering strategy is used to optionally delete the remaining 5′ mouse TCRβ V segment.
In any of the above steps, the selection cassettes are removed by deletion with Cre or Flp recombinase.
Mice homozygous for humanized TCRα variable locus are bred with mice homozygous for humanized TCRβ variable locus to form progeny comprising humanized TCRα and TCRβ variable loci. Progeny are bred to homozygosity with respect to humanized TCRα and humanized TCRβ loci.
Mice comprising humanized TCRα and TCRβ variable loci are confirmed to undergo normal T cell development and comprise T cell receptors that express variable domains derived from a variety of variable gene segments.
Example 3
Humanization of T Cell Co-Receptor Loci
Humanization of CD4 and CD8 loci (both CD8alpha and CD8 beta loci) is described in detail in U.S. Patent Application Publication No. 20140245466, incorporated herein in its entirety by reference.
Example 3.1
Humanization of CD4 Locus
Specifically, mouse CD4 locus was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003), supra). To generate the targeting vector, a series of bacterial homologous recombinations (BHRs) using Bacterial Artificial Chromosome (BAC) DNA, as well as other engineering steps, were carried out as described in detail in U.S. Patent Application Publication No. 20140245466.
The human CD4 Targeting Vector was linearized with Notl and electroporated into F1H4 mouse ES cells. Targeted ES cells bearing a humanized CD4 locus were identified by genotyping using a modification of allele assay (Valenzuela et al.) that detected the presence of the neomycin cassette and the human CD4 gene, as well as one copy of the mouse CD4 gene.
The final humanized CD4 locus derived from successful incorporation of humanized CD4 targeting vector into ES cells is depicted in FIG. 5A. The sequence across the human intron 3—lox-neo cassette junction (5′ end of the cassette) is set forth in SEQ ID NO:75, and the sequence across lox-neo cassette—human intron 3 junction (3′ end of the cassette) is set forth in SEQ ID NO:76; both sequences are also listed in Table 7. The complete nucleic acid sequence of the humanized CD4 piece, including the pgk-neo cassette depicted in FIG. 5A is set forth in SEQ ID NO:77. The pgk-neo cassette is spans residues 307-2176 of SEQ ID NO:77, the two lox sites are located at residues 267-300 and 2182-2215, while the human sequence spans residues 1-234 and 2222-18263. The amino acid sequence of complete humanized CD4 protein is set forth in SEQ ID NO:78, with human sequence spanning amino acids 27-319 (set forth in SEQ ID NO:79).
TABLE 7
Junction Sequences of the Chimeric CD4 Targeting Vector
SEQ ID
Junction
Sequence
NO
5′ mouse/
AGGGGAAACCCGCAAAGGATGGGACATAGGGAGACAGCTGT
73
human
TAACATCTGAAACATGACCTTCTTTTCTGTGCAGCACAACTCC
junction
TAGCTGTCACTCAAGGG(AAGAAAGTGGTGCTGGGCAAAAAA
GGGGATACAGTGGAACTGACCTGTACAGCTTCCCAGAAGAA
GAGCATACAATTCCACTGGAAAAACTCCAACCAGAT)
3′ human/
(CTGGTCACCTGGATGAAGTGAGGGAGGGCCCTCTGGGTTTG
74
mouse
GGGCTGGTTTTGAACTGAGACATCCATGAGCCAGCCTGGGG
junction
CTGGCTTCACTGAAGATC)ATCTATGTCGGGTGCGGAGAAAG
AGGTAATGAAATGGCACATGCTATGTACAAACTCTATTGCTG
AGCAGCACCCAGTCCTGAGCTGGCTCTGAATTGAGGGTGAA
ATTCACACATTCTCCCCCAACATCTATAATCTGG
Human/5′
(TATGGAGTGAAAGCCTTTGGTGTCTGAGATCTGGTCTTAGT
75
lox site
TAAACTCTGGGATC)GGCGCGCCGAATTCCTGCAGCCCGGG
CTCGAGATAACTTCGTATAATGTATGCTATACGAAGTTATATG
CATCCGGGTAGGGGAGGCGCTTTTCCC
3′ lox site/
AGTATTGTTTTGCCAAGTTCTAATTCCATCAGACCTCGACCTG
76
human
CAGCCCTAGATAACTTCGTATAATGTATGCTATACGAAGTTAT
CCTAGG(CCAGAGGGCTTGGGTTGACAGAAACTCAGTGGCAT
TCTTATCCAGAGTTTCTCTACACC)
Human sequences are in parenthesis and sequence containing restriction enzyme site (PI-Sce I) is bolded. Selection cassette sequences are italicized.
Floxed neomycin resistance cassette is removed by electroporation of plasmid expressing Cre recombinase into ES cells containing humanized CD4 locus.
Targeted ES cells bearing a humanized CD4 locus without resistance marker are identified by genotyping that detected absence of the neomycin cassette, the presence of one copy of the human CD4 gene and one copy of the mouse CD4 gene.
Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric CD4 gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human CD4 gene sequences. Expression of humanized CD4 proteins on the surface of T cells was detected using anti-human CD4 antibodies. Mice heterozygous for humanized CD4 protein described herein were bred to homozygosity.
Example 3.2
Humanization of CD8 Loci
CD8α and CD8β genes are colocalized in the genome, e.g., on mouse chromosome 6, they are located about 37 kb away from each other. Due to close linkage, sequential targeting, by first introducing one gene, e.g., CD8β, followed by introduction of the second gene, e.g., CD8α, is performed. Specific detailed steps of humanization are described in U.S. Patent Application Publication No. 20140245466, incorporated herein by reference.
Briefly, mouse CD8β locus was humanized in a single step by construction of a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology. DNA from BAC RP23-431M6 was modified by BHR to generate a large targeting vector (LTVEC), MAID 1737, to contain a replacement of mouse exons 2-3 encoding the CD8 ecto domain (from the 5′ junction in intron 1 to the 3′ junction in intron 3), with homologous human sequences (FIG. 5B). A loxp-Ub-Hyg cassette was inserted at the 3′ junction in intron 3. The nucleotide sequence at various junctions of the resulting vector are listed in Table 8 and set forth in Sequence Listing. The complete amino acid sequence of humanized CD8β protein is set forth in SEQ ID NO:83; with human sequences spanning amino acids 15-165 (set forth in SEQ ID NO:84).
TABLE 8
Junction Sequences of the Chimeric CD813 Targeting Vector
SEQ ID
Junction
Sequence
NO
Mouse/human in
TGTTTGCCTGTGACATGAACTCATTGTGACACAAA
80
intron 1
CCACTGTGCTAGGGGGGATCCACTAGTAACGGC
CGCCAGTGTGCTGGAATTCGCCC(TCGCAAGGG
CCAGGCATATAAGTACACAATAAACAAATGGCAG
CTCTCTCC)
Human/5′ of lox
(CCCCTCCTTCCTTCCCCAGGCACTTTCCAAGTGTC
81
site in intron 3
AACTCTAGAGCCTAT)CGCGGCCGCACCGGTATA
ACTTCGTATAATGTATGCTATACGAAGTTAT
3′ of lox site/
ATAACTTCGTATAATGTATGCTATACGAAGTTATGTCG
82
mouse in intron 3
ACGTAGCCTATTTCTCTAGATCCAAAATGATGACA
ACAAAAGGTACCTTGTG
Human sequences are in parenthesis, lox sites are italicized, and restriction enzyme sites, multiple cloning sites, and vector-derived sequences are bolded.
Targeting vector was electroporated into F1H4 mouse ES cells. Targeted ES cells bearing a humanized CD8β locus were identified by genotyping using a modification of allele assay (Valenzuela et al.) that detected the presence of the human CD8β gene.
Mouse CD8α locus was also humanized in a single step by construction of a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology. DNA from BAC RP23-431M6 was modified by BHR to generate a large targeting vector (LTVEC), MAID 1738, to contain a replacement of mouse exons 1-2 encoding the CD8α ecto domain (from the 5′ junction at Ala codon 27 in mouse exon 1 to the 3′ junction in mouse intron 2), with the homologous human sequences (from the 5′ junction in human exon 2 to the 3′ junction in intron 3 (FIG. 5A)). This retains the mouse leader sequence at the beginning of exon 1. A lox2372-Ub-Neo cassette was inserted at the 3′ junction of human/mouse sequences. The nucleotide sequences at various junctions of the resulting vector are listed in Table 9 and set forth in Sequence Listing. The complete amino acids sequence of humanized CD8α polypeptide is set forth in SEQ ID NO:88, with human sequence spanning amino acids 28-179 (set forth in SEQ ID NO:89).
TABLE 9
Junction Sequences of the Chimeric CD8a Targeting Vector
SEQ
ID
Junction
Sequence
NO
Mouse/human
TGAACCTGCTGCTGCTGGGTGAGTCGATTATCCTGGGGAGT
85
at exon 1
GGAGAAGCT(AGGCCGAGCCAGTTCCGGGTGTCGCCGCTGG
(mouse) and
ATCGGACCTGGAACCTGGG)
exon 2
(human)
Human/5′ of
(ATGCCAGGGACAGCCCTGATACTGTAGGTAGAGTCAAGG
86
lox 2372 site
GCTGTCCAAGT)ACCGGTATAACTTCGTATAAGGTATCCTAT
ACGAAGTTAT
3′ of lox 2372
ATAACTTCGTATAAGGTATCCTATACGAAGTTATCTCGACCTG
87
site/mouse
ATCTTGGAGGGAGACCTGGACCGGGAGACGTGCTGGGGGC
AGGGTT
Human sequences are in parenthesis, lox sites are italicized, and restriction enzyme sites, multiple cloning sites, and vector-derived sequences are bolded.
Humanized CD8α targeting vector described above was electroporated into mouse ES cells that contained a humanized CD8b locus to create modified ES cells that comprise humanized CD8b and CD8α loci (FIG. 5B). Targeted ES cells bearing a humanized CD8α and CD8b loci were identified by genotyping using a modification of allele assay (Valenzuela et al.).
Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing a chimeric CD8b gene and a chimeric CD8α gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human CD8b and CD8α gene sequences.
The selection cassettes in CD8α and CD8β loci may be removed by methods known by the skilled artisan. Mice heterozygous for humanized CD8α and CD8β loci as described herein are bred to homozygosity. Expression of humanized CD8α and CD8β on the surface of T cells is detected using anti-human CD8 antibodies.
Example 4
Generation of Mice Comprising Humanized Cellular Immune System Components
In order to generate mice comprising humanized cellular immune system components, mice homozygous for humanization of various components, e.g., MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M may be bred together in any combination to create mice that have different components of the T cell immune response humanized. For example, a mouse comprising a humanized MHC I may be bred with a mouse comprising a humanized β2M to generate a mouse expressing humanized MHC I/β2M. Mice homozygous for humanization of various components, e.g., MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M are bred together using methods known in the art to obtain a mouse comprising all nine humanizations (“TM I/II B C4/8” mice). Mice are bred to homozygosity using methods known in the art. Alternatively, targeting vectors comprising each humanized gene can be introduced via sequential targeting into the same ES cell to obtain an ES cell comprising all nine humanizations, and the resultant ES cell is introduced into 8-cell stage mouse embryo by the VELOCIMOUSE® method, described in Examples 1-3 above.
Example 5
Characterization of Mice Comprising Humanized Cellular Immune System Components
Mice homozygous for humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β and for humanized β2M were characterized. Specifically, spleen and thymic, from mice were harvested and single cell suspensions were obtained. Suspensions were centrifuged at 1200 rpm for 5 min at 4° C. to pellet cells, and cells from each tissue were lysed with 4 mL of ACK lysing buffer (GIBCO) to lyse red blood cells. Cells were filtered through cell strainer, centrifuged to pellet, resuspended in media and counted.
Cell surface expression of CD19, CD3, CD4 and CD8α depicted in FIGS. 6A-C and FIGS. 9A-C was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD3 (17A2, BD), anti-mouse CD19 (1D3, BD), anti-mouse F4/80 (BM8, Biolegend), anti-mouse CD8α (53-6.7, BD), anti-mouse CD4 (RM4-5, eBioscience), anti-human CD8α (SK1, BD), and anti-human CD4 (RPA-T4, BD). Cell surface expression of mouse H2Db, human HLA molecules (HLA-A2, B2m, and HLA-DR) and mouse MHC IAIE molecules in FIGS. 7A-F and 10A-F was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD19 (6D5, Biolegend), anti-mouse F4/80 (BM8, Biolegend), anti-mouse H2Db (KH95, Biolegend), anti-human HLA-A2 (BB7.2, BD), anti-human HLA-DR (G46-6, BD), anti-human B2-mibroglobulin (2M2, Biolegend) and anti-mouse IAIE (M5/114.15.2, eBioscience). Cell surface expression of mouse and human CD4 and CD8 in FIG. 7G and FIG. 10G was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD3 (17A2, Biolegend), anti-mouse CD4 (GK1.5, eBiosciences), anti-mouse CD8α (53-6.7, BD 2), anti-mouse CD8β (H35-17.2, eBioscience), anti-human CD4 (OKT4, eBioscience), anti-human CD8α (RPA-T8, BD 6), anti-human CD8β (2ST8.5H7, BD). Cell surface expression of FoxP3 and CD25 shown in FIG. 8 or FIG. 11 was analyzed by FACS anti-FoxP3 (FJK-16s, eBioscience) and anti-CD25 (PC61, Biolegend) Cell surface expression of CD44 and CD62L shown in FIGS. 9D-9E was analysed using anti-CD44 (IM7, BD) and anti-CD62L (MEL-14, Biolegend).
All flow cytometry was performed using BD Fortessa. Data was analyzed using FlowJo.
Expression in thymus is depicted in FIGS. 6A-C, 7A-G and 8. The absolute numbers of thymocytes and CD3+ cells, and the overall development of thymic T cells, were comparable in control mice and humanized TM I/II B C4/8 mice (data not shown). FIG. 6A shows that the proportion of B cells and T cells in the thymi of mice having a humanized cellular immune system (TM I/II B C4/8) is similar to the proportion found in control mice. The frequency and number of F4/80 cells in the thymi of TM I/II B C4/8 mice was compared to control mice (FIG. 6B and data not shown). Also, humanized CD4 and CD8 are expressed on thymic cells of a mouse humanized for all nine cellular immunity genes (TM I/II B C4/8), similar to the expression of mouse CD4 and CD8 in non-humanized control mice (FIG. 6C). Humanized β2M is expressed on the surface of B cells and macrophages in humanized TM I/II B C4/8 mice, while its expression is absent from the B cells and macrophages of control mice (FIGS. 7A and 7B). Similarly, humanized MHC I and II are present on the surface of both B cells and macrophages of humanized TM I/II B C4/8 mice (FIGS. 7C and 7D) whereas mouse MHC class I and II molecules were undetectable (FIGS. 7E and 7F). Humanized CD4, CD8 α and CD8β are expressed on the surface of CD3+ thymic cells obtained from humanized TM I/II B C4/8 mice while absent from CD3+ thymic cells in the control mice (FIG. 7G). Humanized TM I/II B CD4/8 express regulatory T cells (Treg) (FIG. 8), NK cells (CD335+CD3−) and monocytes (CD11b+) (data not shown).
Expression in the spleen is depicted in FIGS. 9A-D, and 10A-10G. Spleens of mice humanized for cellular immune system components (TM I/II B CD4/8) comprised comparable absolute numbers of CD3+ cells, and nearly normal proportion of B and T cells (FIG. 9A and data not shown). The frequency and number of F4/80 cells in the spleens of TM I/II B C4/8 mice were compared to control mice (FIG. 9B and data not shown). Mice humanized for cellular immune system components (TM I/II B CD4/8) expressed humanized CD4 and CD8α on CD3+ splenic cells (FIG. 9C). Humanized TM I/II B CD4/8 mice comprised memory effector (CD44+CD62L−) CD4+ and CD8+ T cells and central memory (CD44+ CD62L+) CD8+ T cells (FIGS. 9D and 9E).
As depicted in FIGS. 10A and 10B, humanized β2M is expressed on the surface of B cells and macrophages in the spleen of humanized TM I/II B C4/B mice, while its expression, and the expression of mouse MHC molecules, are absent from the B cells and macrophages in the spleen of control mice. Similarly, humanized MHC I and II are present on the surface of both B cells and macrophages in the spleen of humanized TM I/II B C4/B mice (FIGS. 10C and 10D) whereas mouse MHC class I and II molecules were undetectable (FIGS. 10E and 10F). Humanized CD4, CD8 α and CD8β are expressed on the surface of CD3+ splenic cells obtained from humanized TM I/II B C4/8 mice while absent from CD3+ splenic cells in the control mice (FIG. 10G). TM I/II B C4/8 mice have near normal expression of splenic regulatory T cells compared to control mice (FIG. 11), and express splenic NK cells (CD335+CD3+) and monocytes (CD11b+).
Example 6
Evaluation of Presentation to and Activation of T Cells with Human Peptide
To determine whether the mice comprising humanized cellular immune system components exhibited humanized T cell immune responses, the ability of splenocytes from mice humanized for cellular immune system components (TM I/II B CD4/8) to present and respond to MAGE-A3, a peptide presented specifically by human HLA-A2, was tested.
MAGE-A3, a peptide presented specifically by human HLA-A2, is synthesized (Celtek Biosciences), diluted in PBS, and mixed in equal volume with Complete Freund's Adjuvant (CFA; Chondrex, Inc.) such that 200 μg of the MAGE-A3 is contained in the 200 μl emulsion. 50 μl of emulsion is injected into 4 spots on each animal. Two spots are each in a hind flank and 2 spots each are near each shoulder of mice homozygous for humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B CD4/8) or control mice which express endogenous MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M.
Spleen suspensions from immunized mice are obtained and dissociated. Red blood cells are lysed in ACK lysis buffer (Life Technologies), and splenocytes are suspended in RPMI complete media. 2×105 of isolated splenocytes in the absence or in the presence of 10 μg/mL or 1 μg/mL of diluted MAGE-A3 peptide are tested per well of PVDF plates (Millipore) coated with 5 μg/mL of the mouse IFN-γ capture antibody (BD Biosciences) in an ELISPOT assay. After a 16-20 hour incubation with peptide, the plates are washed and incubated with biotinylated detection antibody (BD Biosciences), washed, treated with Streptavidin-HRP (MabTech), washed and developed with TMB substrate (Mabtech), and counted by AID Elispot reader.
While only one mouse per genotype is shown, several mice of each genotype were tested, and all samples were run in triplicate with standard deviation shown by error bars. As shown in FIG. 12, only samples from mice homozygous for each of humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B CD4/8) responded by secreting IFN-γ after treatment with HLA-A2-specific peptide MAGE-A3, indicating that T cells from these mice were activated after presentation of MAGE-A3 by humanized HLA-A2.
Example 7
Evaluation of T Cell Function Using LCMV Infection Model
To determine whether the mice comprising humanized cellular immune system components exhibited normal response to infection, the ability of humanized mice to clear lymphocytic choriomeningitis virus (LCMV) was tested. LCMV is a mouse tropic virus, where the fate of infection depends on the viral strain. Exposure to Armstrong strain results in an acute infection, where mice can quickly mount a T cell response against the virus and clear the infection in about a week. On the other hand, Clone 13 virus cannot be cleared, and T cells become “exhausted” (expressing markers associated with T cell exhaustion, e.g., PD1, Lag3, Tim3) and chronic infection is established. It has been shown that infection of CD8 depleted or MHC class I deficient mice with Armstrong strain results in maintenance of high viral titers (J. Virol. 68:8056-63 (1994)). Thus, since viral infection depends on T cell activity, LCMV is an ideal model to test for T cell function.
To determine if mice comprising humanized cellular immune system components, e.g., MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M, exhibit normal T cell function, both control and humanized (TM I/II B C4/8) mice were infected with 2×105 ffu of Armstrong virus strain i.p. on Day 0. On Days 3, 6, 9, and 12, organs were harvested and viral titers were measured. As shown in FIG. 13A, both control and humanized mice were able to clear Armstrong infection.
Both control and humanized mice were also infected with 4.5×105 ffu of Clone 13 virus i.v. on Day 0, and on Day 21 organs were harvested and viral titers measured. As depicted in FIG. 13B, both mouse strains were able to establish chronic LCMV infection. The ability of humanized mice to express PD1, Lag3, and Tim3, markers of T cell exhaustion, was also measured. Blood was taken from uninfected mice and infected humanized mice 3 weeks post-infection and stained using flow cytometry with PE-Cy7 conjugated anti-PD1 antibody (BIOLEGEND), PerCpCy5.5 conjugated Lag3 antibody (BIOLEGEND), and PE conjugated Tim3 antibody (R&D Systems). Data in FIG. 13C is a quantification of cells staining positive for the indicated receptors. Both humanized (TM I/II B C4/8) mice and control B6 mice expressed all three markers of T cell exhaustion 3 weeks after infection with chronic LCMV Clone 13 strain.
To evaluate memory T cell responses in mice humanized for cellular immune system components, 5 control and 4 humanized mice were infected with 2×105 ffu of Armstrong strain, and on Day 17 super-infected with 4.5×105 ffu Clone 13 strain (2 of each humanized and control mice were mock-infected as an additional control). On Day 31 post initial infection, organs were harvested and viral titers were analyzed. As depicted in FIG. 14, 5/5 control mice and 3/4 humanized mice that have encountered an acute LCMV infection were subsequently protected from chronic LCMV infection, demonstrating intact memory T cell responses in these animals.
To analyze the nature of the cellular responses, control and humanized mice were infected on Day 0 with 2×105 ffu of Armstrong virus strain. On Day 10 (FIGS. 15A-B) or at the indicated time points post infection (FIGS. 15C-D) the specificity of the cellular response was analyzed using three HLA-A2 restricted peptides known to activate human CD8+ T cells (GPC10-18, N69-77 or Z49-58), see Botten et al. (2007) J. ViroL 81:2307-17, or gp33, an immunodominant LCMV peptide recognized by mice on a H-2Db background. Specifically, CD8+ T cells were isolated from harvested spleens and pulsed with the peptides. CD8+ cells producing interferon-γ (IFNγ) were measured by ELISpot (FIGS. 15A-B) or by staining for intracellular IFNγ (FIGS. 15C-D).
CD8+ T cells isolated from control animals are specifically activated by the gp33 peptide (FIG. 15A), while CD8+ T cells isolated from humanized animals are activated by the HLA-A2 restricted peptides (FIG. 15B). The time course of CD8+ T cell activation, as monitored by their ability to express IFNγ when stimulated with the peptides, shows in both control and humanized mice CD8+ T cells expand during the first two weeks post infection and are undetectable after the virus is cleared (FIGS. 15C-D). Although the response to gp33 peptide appeared stronger in control animals, it should be noted that gp33 is a known immunodominant LCMV epitope while the immunodominant HLA-A2 restricted LCMV epitope has not been identified. In conclusion, animals comprising a humanized, or substantially humanized T cell immune system are capable of processing LCMV expressed protein, presenting them on humanized MHC molecules and activating T cells via a humanized T cell receptor.
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.
Entire contents of all non-patent documents, patent applications and patents cited throughout this application are incorporated by reference herein in their entirety.
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15564723
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Mar 12th, 2019 12:00AM
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Jun 11th, 2015 12:00AM
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https://www.uspto.gov?id=US10227401-20190312
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Production cell line enhancers
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The present invention relates to discovery of the ectopic expression of EDEM2 in a production cell to improve the yield of a useful multi-subunit protein. Thus, the present invention provides for production cell lines, such as the canonical mammalian biopharmaceutical production cell—the CHO cell, containing recombinant polynucleotides encoding EDEM2. Also disclosed is a production cell containing both an EDEM2-encoding polynucleotide as well an XBP1-encoding polynucleotide. Improved titers of antibodies produced by these cell lines are disclosed, as well as the improved cell densities attained by these cells in culture.
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10227401
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1. A recombinant host cell comprising an exogenous recombinant polynucleotide that encodes (i) an endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2) selected from (a) an amino acid sequence of SEQ ID NO: 8 or (b) an amino acid sequence that is at least 92% identical to SEQ ID NO: 1, and (ii) an antibody or antigen-binding fragment thereof.
2. The cell of claim 1, wherein the antibody consists of a heavy chain and a light chain.
3. The cell of claim 2, wherein the heavy chain of the antibody comprises an amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 44 and the light chain of the antibody comprises an amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 46.
4. The cell of claim 1, further comprising a polynucleotide that encodes an unfolded protein response transcription factor that operates upstream of EDEM2.
5. The cell of claim 4, wherein the transcription factor is a spliced form of X-box binding protein 1 (XBP-1).
6. The cell of claim 5, wherein the XBP-1 comprises an amino acid sequence of SEQ ID NO: 13.
7. The cell of claim 5, wherein the XBP-1 comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 9.
8. The cell of claim 2, wherein the cell is a mammalian cell.
9. The cell of claim 8, wherein the cell is a CHO cell.
10. A cell line comprising the cell of claim 9.
11. The cell line of claim 10, which produces the antibody or antigen-binding fragment thereof at a titer of at least 3 g/L.
12. The cell line of claim 10, which produces the antibody or antigen-binding fragment thereof at a titer of at least 5 g/L.
13. The cell line of claim 10, which produces the antibody or antigen-binding fragment thereof at a titer of at least 8 g/L.
14. The cell line of claim 10, wherein the integrated cell density is at least 30% greater than the integrated cell density of a cell line that does not comprise the recombinant polynucleotide that encodes an EDEM2.
15. The cell line of claim 10, wherein the integrated cell density is at least 50% greater than the integrated cell density of a cell line that does not comprise the recombinant polynucleotide that encodes an EDEM2.
16. The cell line of claim 10, wherein the integrated cell density is at least 60% greater than the integrated cell density of a cell line that does not comprise the recombinant polynucleotide that encodes an EDEM2.
17. The cell line of claim 10, wherein the integrated cell density is at least 90% greater than the integrated cell density of a cell line that does not comprise the recombinant polynucleotide that encodes an EDEM2.
18. The cell of claim 1, wherein the antibody is selected from the group consisting of an anti-GDF8 antibody, and anti-AGN2 antibody, and an anti-ANGPTL4 antibody.
19. The cell of claim 2, wherein a heavy chain of the antibody comprises an amino acid sequence of SEQ ID NO: 19 and a light chain of the antibody comprises an amino acid sequence of SEQ ID NO: 21.
20. The cell of claim 2, wherein a heavy chain of the antibody comprises an amino acid sequence of SEQ ID NO: 27 and a light chain of the antibody comprises an amino acid sequence of SEQ ID NO: 29.
21. The cell of claim 2, wherein a heavy chain of the antibody comprises an amino acid sequence of SEQ ID NO: 35 and a light chain of the antibody comprises an amino acid sequence of SEQ ID NO: 37.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/904,587, filed on May 29, 2013, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/652,549 filed 29 May 2012, which application is herein specifically incorporated by reference in its entirety.
SEQUENCE LISTING
This application incorporates by reference the sequence listing submitted in computer readable form as file 8150A-2_ST25.txt created on Aug. 6, 2013 (206,310 bytes).
FIELD
The invention relates to a cell or cells expressing a recombinant stress-response lectin for the improved production of a multi-subunit protein. Specifically, the invention provides a mammalian cell and cell-line derived therefrom containing a gene encoding EDEM2, and which yields antibody at a high titer.
BACKGROUND
The manufacture of therapeutically active proteins requires proper folding and processing prior to secretion. Proper folding is particularly relevant for proteins, such as antibodies, which consist of multiple subunits that must be properly assembled before secretion. Eukaryotic cells have adapted a system that ensures the proper folding of proteins and the removal of misfolded proteins from the secretory pathway. This system is called the unfolded protein response (UPR) pathway, and it is triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER).
An early event of the UPR is the activation of the transcription factor Xbp1, which in turn activates the transcription of endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2), a member of the endoplasmic reticulum associated degradation (ERAD) pathway. EDEM2 facilitates the removal of misfolded proteins. The ERAD pathway comprises five steps: (1) chaperone-mediated recognition of malformed proteins, (2) targeting of malformed proteins to the retrotranslocation machinery or E3-ligases, which involves EDEM2, (3) intitiation of retrotranslocation; (4) ubiquitylation and further retrotranslocation; and (5) proteosome targeting and degradation.
Antibodies are multi-subunit proteins comprising two heavy chains and two light chains, which must be properly folded and associated to form a functional heterotetramer. Any improvement in the efficient and accurate processing of the heavy and light chains to improve the yield or titer of functional antibody heterotetramers is desired.
SUMMARY
Applicants made the surprising discovery that the ectopic expression of EDEM2 in a protein-manufacturing cell line increases the average output of protein per cell, increases the titer of protein secreted into the media, and increases the integrated cell density of production cell lines.
Thus, in one aspect, the invention provides a cell containing (a) a recombinant polynucleotide that encodes a stress-induced mannose-binding lectin and (b) a polynucleotide that encodes a multi-subunit protein. In some embodiments, the stress-induced mannose-binding lectin is an EDEM2 protein, non-limiting examples of which are provided in Table 1, and the multi-subunit protein is an antibody. In other embodiments, the cell also contains a polynucleotide that encodes the active spliced form of XBP1, non-limiting examples of which are provided in Table 2. In one embodiment, the cell is a mammalian cell, such as a CHO cell used in the manufacture of biopharmaceuticals.
In another aspect, the invention provides a cell line derived from the cell described in the previous aspect. By “derived from”, what is meant is a population of cells clonally descended from an individual cell and having some select qualities, such as the ability to produce active protein at a given titer, or the ability to proliferate to a particular density. In some embodiments, the cell line, which is derived from a cell harboring the recombinant polynucleotide encoding a stress-induced mannose-binding lectin and a polynucleotide encoding a multi-subunit protein, is capable of producing the multi-subunit protein at a titer of at least 3 grams per liter of media (g/L), at least 5 g/L, or at least 8 g/L. In some embodiments, the cell line can attain an integrated cell density (ICD) that is at least 30% greater, at least 50% greater, at least 60% greater, or at least 90% greater than the integrated cell density attainable by a cell line derived from what is essentially the same cell but without the recombinant polynucleotide encoding the stress-induced mannose-binding lectin.
In another aspect, the invention provides an isolated or recombinant polynucleotide comprising a nucleic acid sequence encoding an EDEM2 protein, which is operably linked (cis) to a constitutive and ubiquitously expressed mammalian promoter, such as the ubiquitin C promoter. In some embodiments, the EDEM2 protein has the amino acid of SEQ ID NO: 8, or an amino acid sequence that is at least 92% identical to any one of SEQ ID NO: 1-7. In some embodiments, the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 16. In one particular embodiment, the polynucleotide consists of a nucleic acid sequence of SEQ ID NO: 14; and in another particular embodiment, SEQ ID NO: 15.
In another aspect, the invention provides an isolated or recombinant polynucleotide comprising a nucleic acid sequence encoding an XBP1 protein, which is operably linked to (in cis) a constitutive and ubiquitously expressed mammalian promoter, such as the ubiquitin C promoter. In some embodiments, the XBP1 protein has the amino acid of SEQ ID NO: 13, or an amino acid sequence that is at least 86% identical to any one of SEQ ID NO: 9-12. In some embodiments, the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 18. In one particular embodiment, the polynucleotide consists of a nucleic acid sequence of SEQ ID NO: 17.
In another aspect, the invention provides a cell that contains an EDEM2-encoding polynucleotide, as described in the prior aspect, and a polynucleotide that encodes a multi-subunit protein, such as an antibody. In some embodiments, the cell also contains an XBP1-encoding polynucleotide, as described in the preceding aspect. In one embodiment, the multi-subunit protein is an antibody, and the heavy chain of the antibody comprises an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, and the light chain of the antibody comprises an amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 46. In this and several embodiments, each polypeptide subunit of the multi-subunit protein is encoded by a separate polynucleotide. Thus, for example, a polynucleotide encoding an antibody may include a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain, hence two subunits. In some embodiments, the cell is a chinese hamster ovary (CHO) cell.
In one embodiment, the encoded multi-subunit protein is an anti-GDF8 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 20 and a light chain variable region amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-GDF8 antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 19 and a light chain having an amino acid sequence of SEQ ID NO: 21. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-GDF8 antibody comprises a nucleic acid sequence of SEQ ID NO: 23; and the polynucleotide that encodes the light chain of the anti-GDF8 antibody comprises a nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-GDF8 antibody consists of a nucleic acid sequence of SEQ ID NO: 24; and the polynucleotide that encodes the light chain of the anti-GDF8 antibody consists of a nucleic acid sequence of SEQ ID NO: 25.
In another embodiment, the encoded multi-subunit protein is an anti-ANG2 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 28 and a light chain variable region amino acid sequence of SEQ ID NO: 30. In one embodiment, the anti-ANG2 antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 27 and a light chain having an amino acid sequence of SEQ ID NO: 29. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-ANG2 antibody comprises a nucleic acid sequence of SEQ ID NO: 31; and the polynucleotide that encodes the light chain of the anti-ANG2 antibody comprises a nucleic acid sequence of SEQ ID NO: 33. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-ANG2 antibody consists of a nucleic acid sequence of SEQ ID NO: 32; and the polynucleotide that encodes the light chain of the anti-ANG2 antibody consists of a nucleic acid sequence of SEQ ID NO: 34.
In another embodiment, the encoded multi-subunit protein is an anti-ANGPTL4 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 36 and a light chain variable region amino acid sequence of SEQ ID NO: 38. In one embodiment, the anti-ANGPTL4 antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 35 and a light chain having an amino acid sequence of SEQ ID NO: 37. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-ANGPTL4 antibody comprises a nucleic acid sequence of SEQ ID NO: 39; and the polynucleotide that encodes the light chain of the anti-ANGPTL4 antibody comprises a nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the polynucleotide that encodes the heavy chain of the anti-ANGPTL4 antibody consists of a nucleic acid sequence of SEQ ID NO: 40; and the polynucleotide that encodes the light chain of the anti-ANGPTL4 antibody consists of a nucleic acid sequence of SEQ ID NO: 42.
In another aspect, the invention provides a method of manufacturing a multi-subunit protein, by culturing a cell of the previous aspect in a medium, wherein the multi-subunit protein is synthesized in the cell and subsequently secreted into the medium. In some embodiments, the multi-subunit protein is an antibody, such as for example anti-GDF8, anti-ANG2, anti-ANGPTL4, or an antibody having a heavy chain sequence of SEQ ID NO: 43 and 44, and a light chain sequence of SEQ ID NO: 45 and 46. In some embodiments, the multi-subunit protein attains a titer of at least 3 g/L, at least 5 g/L, at least 6 g/L, or at least 8 g/L. In some embodiments, the cell proliferates in the medium and establishes an integrated cell density of about ≥5×107 cell-day/mL, about ≥1×108 cell-day/mL, or about ≥1.5×108 cell-day/mL.
In another aspect, the invention provides a multi-subunit protein, which is manufactured according to the method described in the preceding aspect. In one embodiment, the manufactured protein is an antibody. In some embodiments, the antibody consists of a heavy chain, which comprises an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, and a light chain, which comprises an amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 46. In one specific embodiment, the manufactured multi-subunit protein is an anti-GDF8 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 20 and a light chain variable region amino acid sequence of SEQ ID NO: 22. In another specific embodiment, the manufactured multi-subunit protein is an anti-ANG2 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 28 and a light chain variable region amino acid sequence of SEQ ID NO: 30. In yet another specific embodiment, the manufactured multi-subunit protein is an anti-ANGPTL4 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 36 and a light chain variable region amino acid sequence of SEQ ID NO: 38.
DESCRIPTION
Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term “about”, when used in reference to a particular recited numerical value or range of values, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
As used herein, the term “recombinant polynucleotide”, which is used interchangeably with “isolated polynucleotide”, means a nucleic acid polymer such as a ribonucleic acid or a deoxyribonucleic acid, either single stranded or double stranded, originating by genetic engineering manipulations. A recombinant polynucleotide may be a circular plasmid or a linear construct existing in vitro or within a cell as an episome. A recombinant polynucleotide may be a construct that is integrated within a larger polynucleotide molecule or supermolecular structure, such as a linear or circular chromosome. The larger polynucleotide molecule or supermolecular structure may be within a cell or within the nucleus of a cell. Thus, a recombinant polynucleotide may be integrated within a chromosome of a cell.
As used herein, the term “stress-induced mannose-binding lectin” refers to a mannose-binding protein, which means a protein that binds or is capable of binding mannose, derivatives of mannose, such as mannose-6-phosphate, or a glycoprotein that expresses mannose or a mannose derivative in its glycocalyx; and whose activity is upregulated during stress. Cellular stress includes inter alia starvation, DNA damage, hypoxia, poisoning, shear stress and other mechanical stresses, tumor stress, and the accumulation of misfolded proteins in the endoplasmic reticulum. Exemplary stress-induced mannose-binding lectins include the EDEM proteins EDEM1, EDEM2 and EDEM3, Yos 9, OS9, and XTP3-B (see Vembar and Brodsky, Nat. Rev. Mol. Cell. Biol. 9(12): 944-957, 2008, and references cited therein).
As used herein, the term “EDEM2” means any ortholog, homolog, or conservatively substituted variant of endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein. EDEM2 proteins are generally known in the art to be involved in endoplasmic reticulum-associated degradation (ERAD), being up-regulated by Xbp-1 and facilitating the extraction of misfolded glycoproteins from the calnexin cycle for removal. (See Mast et al., Glycobiology 15(4): 421-436, 2004; Olivari and Molinari, FEBS Lett. 581: 3658-3664, 2007; Olivari et al., J. Biol. Chem. 280(4): 2424-2428, 2005; and Vembar and Brodsky 2008, which are herein incorporated by reference.) Exemplary EDEM2 sequences are depicted in Table 1, which is cross-referenced to the Sequence Listing.
TABLE 1
Animal
SEQ ID NO:
% id human
% id mouse
% id hamster
Mouse
1
93
100
96
Rat
2
94
98
96
Hamster
3
93
96
100
Human
4
100
93
93
Chimpanzee
5
99
94
93
Orangutan
6
97
92
92
Zebra fish
7
69
70
69
Consensus
8
100
100
100
As used herein, the term “Xbp1”, also known as XBP1 or X-box binding protein 1, means any ortholog, homolog, or conservatively substituted variant of Xbp1. Xbp1 is a transcription factor and functional element of the UPR. ER stress activates both (1) the transcription factor ATF6, which in turn upregulates the transcription of Xbp1 mRNA, and (2) the ER membrane protein IRE1, which mediates the splicing of the precursor Xbp1 mRNA to produce active Xbp1. As mentioned above, activated Xbp1 in turn upregulates the activity of EDEM2. (See Yoshida et al., Cell Structure and Function 31(2): 117-125, 2006; and Olivari, 2005.) Exemplary Xbp1 amino acid sequences are depicted in Table 2, which is cross-referenced to the Sequence Listing.
TABLE 2
Animal
SEQ ID NO
% id human
% id mouse
% id hamster
Mouse
9
86
100
92
Hamster
10
86
92
100
Human
11
100
86
86
Zebra fish
12
47
47
48
Consensus
13
100
100
100
As used herein, the term “antibody” is generally intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM); however, immunoglobulin molecules consisting of only heavy chains (i.e., lacking light chains) are also encompassed within the definition of the term “antibody”. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDRs), 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. An “isolated antibody” or “purified antibody” may be substantially free of other cellular material or chemicals.
The term “specifically binds”, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant of at least about 1×10−6 M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds human GDF8 (for example) may, however, have cross-reactivity to other antigens, such as GDF8 molecules from other species (orthologs).
Various antibodies are used as examples of multi-subunit proteins secreted by cells harboring the polynucleotide encoding a stress-induced mannose-binding lectin. Those examples include anti-GDF8, anti-ANG2, and anti-ANGPTL4 antibodies. These and similar antibodies are described in US Pat. Apps. No. 20110293630, 20110027286, and 20110159015 respectively, which are incorporated herein by reference.
As used herein, the term “cell” refers to a prokaryotic or eukaryotic cell capable of replicating DNA, transcribing RNA, translating polypeptides, and secreting proteins. Cells include animal cells used in the commercial production of biological products, such as insect cells (e.g., Schneider cells, Sf9 cells, Sf21 cells, Tn-368 cells, BTI-TN-5B1-4 cells; see Jarvis, Methods Enzymol. 463: 191-222, 2009; and Potter et al., Int. Rev. Immunol. 10(2-3): 103-112, 1993) and mammalian cells (e.g., CHO or CHO-K1 cells, COS or COS-7 cells, HEK293 cells, PC12 cells, HeLa cells, Hybridoma cells; Trill et al., Curr. Opin. Biotechnol. 6(5): 553-560, 1995; Kipriyanov and Little, Mo. Biotechnol. 12(2): 173-201, 1999). In one embodiment, the cell is a CHO-K1 cell containing the described UPR pathway polynucleotides. For a description of CHO-K1 cells, see also Kao et al., Proc. Nat'l. Acad. Sci. USA 60: 1275-1281, 1968.
As used herein, the term “promoter” means a genetic sequence generally in cis and located upstream of a protein coding sequence, and which facilitates the transcription of the protein coding sequence. Promoters can be regulated (developmental, tissue specific, or inducible (chemical, temperature)) or constitutively active. In certain embodiments, the polynucleotides that encode proteins are operably linked to a constitutive promoter. By “operably linked”, what is meant is that the protein-encoding polynucleotide is located three-prime (downstream) and cis of the promoter, and under control of the promoter. In certain embodiments, the promoter is a constitutive mammalian promoter, such as the ubiquitin C promoter (see Schorpp et al., Nucl. Acids Res. 24(9): 1787-1788, 1996); Byun et al., Biochem. Biophys. Res. Comm. 332(2): 518-523, 2005) or the CMV-IE promoter (see Addison et al., J. Gen. Virol. 78(7): 1653-1661, 1997; Hunninghake et al., J. Virol. 63(7): 3026-3033, 1989), or the hCMV-IE promoter (human cytomegalovirus immediate early gene promoter) (see Stinski & Roehr, J. Virol. 55(2): 431-441, 1985; Hunninghake et al., J. Virol. 63(7): 3026-3033, 1989).
As used herein, the phrase “integrated cell density”, or “ICD” means the density of cells in a culture medium taken as an integral over a period of time, expressed as cell-days per mL. In some embodiments, the ICD is measured around the twelfth day of cells in culture.
As used herein, the term “culture” means both (1) the composition comprising cells, medium, and secreted multi-subunit protein, and (2) the act of incubating the cells in medium, regardless of whether the cells are actively dividing or not. Cells can be cultured in a vessel as small as a 25 mL flask or smaller, and as large as a commercial bioreactor of 10,000 liters or larger. “Medium” refers to the culture medium, which comprises inter alia nutrients, lipids, amino acids, nucleic acids, buffers and trace elements to allow the growth, proliferation, or maintenance of cells, and the production of the multi-subunit protein by the cells. Cell culture media include serum-free and hydrolysate-free defined media as well as media supplemented with sera (e.g., fetal bovine serum (FBS)) or protein hydrolysates. Non-limiting examples of media, which can be commercially acquired, include RPMI medium 1640, Dulbecco's Modified Eagle Medium (DMEM), DMEM/F12 mixture, F10 nutrient mixture, Ham's F12 nutrient mixture, and minimum essential media (MEM).
As used herein, the phrase “conservatively substituted variant”, as applied to polypeptides, means a polypeptide having an amino acid sequence with one of more conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Embodiments—The Cell
In one aspect, the invention provides a cell useful in the production of a protein having therapeutic or research utility. In some embodiments, the protein consists of multiple subunits, which must be properly folded and assembled to produce sufficient quantities of active protein. Antibodies are an example of multi-subunit proteins having therapeutic or research utility. In some embodiments, the cell harbors a recombinant genetic construct (i.e., a polynucleotide) that encodes one or more of the individual subunits of the multi-subunit protein. In other embodiments, the genetic construct encoding the individual polypeptide subunits is naturally occurring, such as for example the nucleic acid sequences encoding the subunits of an antibody in a B cell.
To facilitate the proper assembly and secretion of the multi-subunit protein, the cell contains a recombinant polynucleotide that encodes a stress-induced mannose-binding lectin, which in some embodiments is a component of the ERAD. In some embodiments, the stress-induced mannose-binding lectin is an endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2). It is envisioned that any encoded EDEM2 or conservatively-substituted variant can be successfully employed in the instant invention. Table 1 lists some examples of vertebrate EDEM2 proteins. A multiple pairwise comparison of those protein sequences, which was performed using the Clustal W program of Thompson et al., Nucl. Acids Rev. 22(22): 4673-80, 1994 (see also Yuan et al., Bioinformatics 15(10): 862-3, 1999), revealed that each of the disclosed EDEM2 polynucleotide sequences is at least 69% identical to each other EDEM2 sequence. A Clustal W comparison of the disclosed mammalian EDEM2 sequences revealed that each sequence is at least 92% identical to the other. Thus, in some embodiments, the cell contains a polynucleotide that encodes an EDEM2 polypeptide having a sequence that is at least 92% to any one of a mammalian EDEM2. A consensus EDEM2 amino acid sequence was built by aligning a mouse, rat, hamster, chimpanzee, and human EDEM2 polypeptide amino acid sequences. That consensus sequence is depicted as SEQ ID NO: 8. Thus, in some embodiments, the cell contains a polynucleotide that encodes an EDEM2 polypeptide having an amino acid sequence of SEQ ID NO: 8.
In various embodiments, the cell contains a recombinant polynucleotide that encodes an EDEM2 polypeptide having an amino acid sequence that is at least 92% identical to the mouse EDEM2 (mEDEM2) amino acid sequence; and in a particular embodiment, the polypeptide is mEDEM2 or a conservatively substituted variant thereof.
In some embodiments, the multi-subunit protein is an antibody, and the cell contains a polynucleotide encoding any one or more of a polypeptide comprising an amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46. SEQ ID NO: 43 and 44 each represent consensus sequences of the roughly N-terminal and C-terminal portions, respectively, of particular antibody heavy chains. Thus, the polynucleotide encoding a protein subunit in one embodiment encodes a polypeptide comprising both SEQ ID NO: 43 and SEQ ID NO: 44. SEQ ID NO: 45 and 46 each represent consensus sequences of the roughly N-terminal and C-terminal portions, respectively, of particular antibody light chains. Thus, the polynucleotide encoding a protein subunit in one embodiment encodes a polypeptide comprising both SEQ ID NO: 45 and SEQ ID NO: 46. In some embodiments, in addition to the recombinant polynucleotide encoding the EDEM2 protein, the cell contains at least two polynucleotides, each of which encodes a particular subunit of the multi-subunit protein. For example, and as exemplified below, the cell contains a polynucleotide encoding an antibody heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, and another polynucleotide encoding an antibody light chain comprising an amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 46.
In some embodiments, the cell, in addition to containing the stress-response polynucleotide and one or more polynucleotides encoding a polypeptide subunit, as described above, also contains a polynucleotide that encodes an unfolded protein response transcription factor that operates upstream of EDEM2. The upstream transcription factor is in some cases the spliced form of an XBP1. It is envisioned that any encoded XBP1 can be successfully employed in the instant invention. Table 2 lists some examples of sequences of vertebrate XBP1 spliced-form polypeptides. A multiple pairwise comparison of those polypeptide sequences, which was performed using Clustal W (Thompson 1994; Yuan 1999), revealed that each of the disclosed spliced XBP1 polynucleotide sequences is at least 48% identical to each other XBP1 sequence. A Clustal W comparison of the disclosed mammalian XBP1 sequences revealed that each sequence is at least 86% identical to the other. Thus, in some embodiments, the cell contains a polynucleotide that encodes a spliced-form of an XBP1 polypeptide having a sequence that is at least 86% to any one of a mammalian spliced XBP1. A consensus XBP1 amino acid sequence was built by aligning a mouse, hamster, and human XBP1 amino acid sequences. That consensus sequence is depicted as SEQ ID NO: 13. Thus, in some embodiments, the cell contains a polynucleotide that encodes an XBP1 polypeptide having an amino acid sequence of SEQ ID NO: 13.
In various embodiments, the cell contains a polynucleotide that encodes an XBP1 polypeptide having an amino acid sequence that is at least 86% identical to the mouse XBP1 (mXBP1) amino acid sequence (SEQ ID NO: 9); and in a particular embodiment, the polypeptide is mXBP1, or a conservatively substituted variant thereof.
The invention envisions that any cell may be used to harbor the lectin-encoding polypeptide for the production of a properly folded and active multi-subunit protein. Such cells include the well-known protein production cells such as the bacterium Escherichia coli and similar prokaryotic cells, the yeasts Pichia pastoris and other Pichia and non-pichia yeasts, plant cell explants, such as those of Nicotiana, insect cells, such as Schneider 2 cells, Sf9 and Sf21, and the Trichoplusia ni-derived High Five cells, and the mammalian cells typically used in bioproduction, such as CHO, CHO-K1, COS, HeLa, HEK293, Jurkat, and PC12 cells. In some embodiments, the cell is a CHO-K1 or a modified CHO-K1 cell such as that which is taught in U.S. Pat. Nos. 7,435,553, 7,514,545, and 7,771,997, as well as U.S. Published Patent Application No. US 2010-0304436 A1, each of which is incorporated herein by reference in its entirety.
In some particular embodiments, the invention provides ex vivo a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequences of SEQ ID NO: 43 and 44, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequences of SEQ ID NO: 45 and 46.
In one particular embodiment, the invention provides ex vivo a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO:18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 23, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 25.
In another particular embodiment, the invention provides ex vivo a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 31, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 33.
In yet another particular embodiment, the invention provides ex vivo a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 39, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 41.
The Cell Line
In another aspect, the invention provides a cell line, which comprises a plurality of cells descended by clonal expansion from a cell described above. At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100% of the constituent cells of the cell line contain a recombinant polynucleotide that encodes a stress-induced mannose-binding lectin, which in some embodiments is a component of the ERAD. In some embodiments, the stress-induced mannose-binding lectin is an endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2). It is envisioned that any encoded EDEM2 or conservatively-substituted variant thereof can be successfully employed in the instant invention. Table 1, as discussed in the previous section, lists some examples of vertebrate EDEM2 proteins. In some embodiments, the constituent cell contains a polynucleotide that encodes an EDEM2 polypeptide having a sequence that is at least 92% identical to any mammalian EDEM2. In some embodiments, the constituent cell contains a polynucleotide that encodes an EDEM2 polypeptide having the mammalian consensus amino acid sequence of SEQ ID NO: 8. In some embodiments, the constituent cell contains a recombinant polynucleotide of SEQ ID NO: 1 or a conservatively substituted variant thereof.
In some embodiments, the multi-subunit protein that is produced by the cell line is an antibody, and the constituent cell of the cell line contains a polynucleotide encoding any one or more of a polypeptide comprising an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44 (which represent consensus sequences of the N-terminal and C-terminal portions, respectively, of particular antibody heavy chains), and SEQ ID NO: 45 and SEQ ID NO: 46 (which represent consensus sequences of the N-terminal and C-terminal portions, respectively, of particular antibody light chains). In some embodiments, in addition to the recombinant polynucleotide encoding the EDEM2 protein, the constituent cell of the cell line contains at least two polynucleotides, each of which encodes a particular subunit of the multi-subunit protein. For example, the constituent cell contains a polynucleotide encoding an antibody heavy chain comprising an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, and another polynucleotide encoding an antibody light chain comprising an amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 46.
In some embodiments, the constituent cell, in addition to containing the stress-response polynucleotide and one or more polynucleotides encoding a polypeptide subunit, as described above, also contains a polynucleotide that encodes an unfolded protein response transcription factor, which operates upstream of EDEM2, such as a spliced form of an XBP1. It is envisioned that any encoded XBP1 can be successfully employed in the instant invention. Table 2, as discussed in the preceding section, lists some examples of sequences of vertebrate XBP1 spliced-form polypeptides. Clustal W analysis of those sequences revealed that each of the disclosed spliced XBP1 polynucleotide sequences is at least 48% identical to each other XBP1 sequence; and a comparison of the mammalian XBP1 sequences revealed that each sequence is at least 86% identical to the other. Thus, in some embodiments, the constituent cell of the cell line contains a polynucleotide that encodes a spliced-form of an XBP1 polypeptide having a sequence that is at least 86% to any one of a mammalian spliced XBP1. In some embodiments, the constituent cell contains a polynucleotide that encodes an XBP1 polypeptide having a consensus amino acid sequence of SEQ ID NO: 13.
In various embodiments, the cell contains a polynucleotide that encodes an XBP1 polypeptide having an amino acid sequence that is at least 86% identical to the mouse XBP1 (mXBP1) amino acid sequence (SEQ ID NO: 9); and in a particular embodiment, the polypeptide is mXBP1 of SEQ ID NO: 9, or a conservatively substituted variant thereof.
The invention envisions that the cell line comprises constituent cells whose parent is selected from a list of well-known protein production cells such as, e.g., the bacterium Escherichia coli and similar prokaryotic cells, the yeasts Pichia pastoris and other Pichia and non-pichia yeasts, plant cell explants, such as those of Nicotiana, insect cells, such as Schneider 2 cells, Sf9 and Sf21, and the Trichoplusia ni-derived High Five cells, and the mammalian cells typically used in bioproduction, such as CHO, CHO-K1, COS, HeLa, HEK293, Jurkat, and PC12 cells. In some embodiments, the cell is a CHO-K1 or a modified CHO-K1 cell, such as that which is taught in U.S. Pat. Nos. 7,435,553, 7,514,545, and 7,771,997, as well as U.S. Published Patent Application No. US 2010-0304436 A1.
In some embodiments, the cell line, which is cultured in media, is capable of producing the multi-subunit protein and secreting the properly assembled multi-subunit protein into the media to a titer that is at least 3 g/L, at least 5 g/L, or at least 8 g/L.
Furthermore, the constituent cells of the cell line are capable proliferating in culture to such an extent as to attain an integrated cell density that is about 30% greater than the integrated cell density of a cell line that does not contain the recombinant polynucleotide encoding the stress-induced mannose-binding lectin. In some cases, the cell line is able to attain an integrated cell density that is at least about 50% greater, at least 60% greater, or at least 90% greater than the integrated cell density of a cell line that does not contain the recombinant polynucleotide that encodes a stress-induced mannose-binding lectin. In some embodiments, the integrated cell density of the cell line is assessed after about 12 days in culture.
In some particular embodiments, the invention provides a cell-line comprising clonally-derived constituent cells, wherein the constituent cell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequences of SEQ ID NO: 43 and 44, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequences of SEQ ID NO: 45 and 46.
In one particular embodiment, the invention provides a cell-line comprising clonally-derived constituent cells, wherein the constituent cell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 23, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 25.
In another particular embodiment, the invention provides a cell-line comprising clonally-derived constituent cells, wherein the constituent cell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 31, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 33.
In yet another particular embodiment, the invention provides a cell-line comprising clonally-derived constituent cells, wherein the constituent cell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) an antibody heavy chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 39, and (4) an antibody light chain-encoding polynucleotide comprising a nucleotide sequence of SEQ ID NO: 41.
The EDEM2 Polynucleotide
In another aspect, the invention provides a polynucleotide that encodes an EDEM2 protein. The EDEM2-encoding polynucleotide is recombinant and can be manufactured, stored, used or expressed in vitro, as in a test tube, or an in vitro translation system, or in vivo, such as in a cell, which can be ex vivo, as in a cell culture, or in vivo, as in an organism. In some embodiments, the EDEM2-encoding polynucleotide is within a gene, meaning that it is under the control of and down stream of a promoter, and up stream of a polyadenylation site. The EDEM2-encoding polynucleotide or gene can be within a plasmid or other circular or linear vector. The EDEM2-encoding polynucleotide or gene can be within a circular or linear DNA construct, which can be within a cell as an episome or integrated into the cellular genome.
As described above, the EDEM2-encoding polynucleotide encodes any ortholog, homolog or conservatively substituted EDEM2 polypeptide of Table 1, or an EDEM2 polypeptide having an amino acid sequence that is at least 92% identical to any one of SEQ ID NO: 1-5 and 8, including the mammalian consensus sequence of SEQ ID NO: 8.
In some cases, the recombinant or isolated EDEM2-encoding polynucleotide is operably linked to a mammalian promoter. The promoter can be any promoter, but in some cases it is a mammalian promoter, such as for example a ubiquitin C promoter.
In a particular embodiment, the EDEM2-encoding polynucleotide essentially consists of, from 5′ to 3′, a promoter, such as a ubiquitin C promoter, followed by an optional intron, such as a beta globin intron, followed by an EDEM2 coding sequence, followed by a polyadenylation sequence, such as an SV40pA sequence. A specific example, which is also a particular embodiment, of such an EDEM2-encoding polynucleotide is described by SEQ ID NO: 16. Conserved variants of that sequence are also envisioned to be embodiments of the invention.
In some cases, the recombinant EDEM2-encoding polynucleotide is part of a plasmid, which can be linear, circular, episomal, integrated, a static DNA construct, or a vector for delivering the EDEM2 gene or expressing the EDEM2 protein. In one particular embodiment, the plasmid contains (1) an EDEM2 gene, which is under the control of a ubiquitin C promoter and terminates with an SV40 polyadenylation signal, and (2) a selectable marker, such as a polynucleotide encoding a polypeptide that confers resistance to zeocin or a polynucleotide encoding a polypeptide that confers resistance to neomycin, under the control of a promoter, such as an SV40 promoter, and terminated with a polyadenylation sequence, such as a PGK pA sequence. In one particular embodiment, the plasmid comprises, in a circular format running in a 5′ to 3′ direction, a ubiquitin C promotor, a beta globin intron, an EDEM2 coding sequence, an SV40 pA sequence, an SV40 promoter, a neomycin-resistance coding sequence, and a PGK pA sequence. A specific example of this embodiment is exemplified by a plasmid having the sequence of SEQ ID NO: 14. In another particular embodiment, the plasmid comprises, in a circular format running in a 5′ to 3′ direction, a ubiquitin C promotor, a beta globin intron, an EDEM2 coding sequence, an SV40 pA sequence, an SV40 promoter, a zeocin-resistance coding sequence, and a PGK pA sequence. A specific example of this embodiment is exemplified by a plasmid having the sequence of SEQ ID NO: 15.
The XBP1 Polynucleotide
In another aspect, the invention provides a polynucleotide that encodes an XBP1 protein. The XBP1-encoding polynucleotide is recombinant and can be manufactured, stored, used or expressed in vitro, as in a test tube, or an in vitro translation system, or in vivo, such as in a cell, which can be ex vivo, as in a cell culture, or in vivo, as in an organism. In some embodiments, the XBP1-encoding polynucleotide is within a gene, meaning that it is under the control of and down stream of a promoter, and up stream of a polyadenylation site. The XBP1-encoding polynucleotide can be within a plasmid or other circular or linear vector. The XBP1-encoding polynucleotide or gene can be within a circular or linear DNA construct, which can be within a cell as an episome, or integrated into the cellular genome.
As described above, the XBP1-encoding polynucleotide encodes any ortholog, homolog or conservatively substituted XBP1 polypeptide of Table 2, or an XBP1 polypeptide having an amino acid sequence that is at least 86% identical to any one of SEQ ID NO: 9, 10, and 11, including the mammalian consensus sequence of SEQ ID NO: 13.
In some cases, the recombinant or isolated XBP1-encoding polynucleotide is operably linked to a mammalian promoter. The promoter can be any promoter, but in some cases it is a mammalian promoter, such as for example a ubiquitin C promoter.
In a particular embodiment, the XBP1-encoding polynucleotide essentially consists of, from 5′ to 3′, a promoter, such as a ubiquitin C promoter, followed by an optional intron, such as a beta globin intron, followed by an XBP1 coding sequence, followed by a polyadenylation sequence, such as an SV40 pA sequence. SEQ ID NO: 18 describes an example of an XBP1-encoding polynucleotide. Conserved variants of that exemplary sequence are also envisioned to be embodiments of the invention.
In some cases, the recombinant XBP1-encoding polynucleotide is part of a plasmid, which can be linear, circular, episomal, integrated, a static DNA construct, or a vector for delivering the XBP1 gene or expressing the spliced and active XBP1 protein. In one particular embodiment, the plasmid contains (1) an XBP1 gene, which is under the control of a ubiquitin C promoter and terminates with an SV40 polyadenylation signal, and (2) a selectable marker, such as a polynucleotide encoding a polypeptide that confers resistance to zeocin or a polynucleotide encoding a polypeptide that confers resistance to neomycin, under the control of a promoter, such as an SV40 promoter, and terminated with a polyadenylation sequence, such as a PGK pA sequence. In one particular embodiment, the plasmid comprises, in a circular format running in a 5′ to 3′ direction, a ubiquitin C promotor, a beta globin intron, an XBP1 coding sequence, an SV40 pA sequence, an SV40 promoter, a zeocin-resistance coding sequence, and a PGK pA sequence. A specific example of this embodiment is exemplified by a circular plasmid having the sequence of SEQ ID NO: 17.
The Antibody Heavy and Light Chain-Encoding Polynucleotides
In another aspect, the invention provides a polynucleotide that encodes an antibody heavy chain polypeptide (HC). The HC-encoding polynucleotide is recombinant and can be manufactured, stored, used or expressed in vitro, as in a test tube, or an in vitro translation system, or in vivo, such as in a cell, which can be ex vivo, as in a cell culture, or in vivo, as in an organism. In some embodiments, the HC-encoding polynucleotide is within a gene, meaning that it is under the control of and down stream from a promoter, and up stream of a polyadenylation site. The HC-encoding polynucleotide may be within a plasmid or other circular or linear vector. The HC-encoding polynucleotide or gene may be within a circular or linear DNA construct, which may be within a cell as an episome or integrated into the cellular genome.
In some cases, the recombinant or isolated HC-encoding polynucleotide is operably linked to a mammalian promoter. The promoter can be any promoter, but in some cases it is a mammalian promoter, such as for example a ubiquitin C promoter or an hCMV-IE promoter.
In a particular embodiment, the HC-encoding polynucleotide is an HC gene, which essentially comprises, from 5′ to 3′, a promoter, for example an hCMV-IE promoter, followed by an optional intron, such as a beta globin intron, followed by a heavy chain coding sequence, such as for example a sequence encoding an amino acid sequence of SEQ ID NO: 43 and 44, SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 35, followed by a polyadenylation sequence, for example an SV40 pA sequence. A specific example of an HC gene is described by SEQ ID NO: 23, SEQ ID NO: 31, or SEQ ID NO: 39. Conserved variants of any one of these sequences are also envisioned to be embodiments of the invention.
In some cases, the recombinant HC-encoding polynucleotide is part of a plasmid, which can be linear, circular, episomal, integrated, a static DNA construct, or a vector for delivering the heavy chain gene or expressing the heavy chain subunit. In one particular embodiment, the plasmid contains (1) an HC gene, which is under the control of an hCMV-IE promoter and terminates with an SV40 polyadenylation signal, and (2) a selectable marker, such as a polynucleotide encoding a polypeptide that confers resistance to hygromycin, under the control of a promoter, such as an SV40 promoter, and terminated with a polyadenylation sequence, such as a PGK pA sequence. In one particular embodiment, the plasmid comprises, in a circular format running in a 5′ to 3′ direction, an hCMV-IE promoter, a beta globin intron, an antibody heavy chain coding sequence (which encodes a HC having an amino acid of SEQ ID NO: 43 and 44, SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 35), an SV40 pA sequence, an SV40 promoter, a hygromycin-resistance coding sequence, and a PGK pA sequence. A specific example and particular embodiment of such a plasmid containing an HC gene is described by SEQ ID NO: 24, SEQ ID NO: 32, or SEQ ID NO: 40. Conserved variants of any one of these sequences are also envisioned to be embodiments of the invention.
In another aspect, the invention provides a polynucleotide that encodes an antibody light chain polypeptide (LC). The LC-encoding polynucleotide is recombinant and can be manufactured, stored, used or expressed in vitro, as in a test tube, or an in vitro translation system, or in vivo, such as in a cell, which can be ex vivo, as in a cell culture, or in vivo, as in an organism. In some embodiments, the LC-encoding polynucleotide is within a gene, meaning that it is under the control of and down stream from a promoter, and up stream of a polyadenylation site. The LC-encoding polynucleotide or gene may be within a plasmid or other circular or linear vector. The LC-encoding polynucleotide or gene may be within a circular or linear DNA construct, which may be within a cell as an episome or integrated into the cellular genome.
In some cases, the recombinant or isolated LC-encoding polynucleotide is operably linked to a mammalian promoter. The promoter can be any promoter, but in some cases it is a mammalian promoter, such as, e.g., a ubiquitin C promoter or an hCMV-IE promoter.
In a particular embodiment, the LC-encoding polynucleotide is an LC gene, which essentially comprises, from 5′ to 3′, a promoter, for example an hCMV-IE promoter, followed by an optional intron, such as a beta globin intron, followed by a light chain coding sequence, such as for example a sequence encoding an amino acid sequence of SEQ ID NO: 45 and 46, SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 37, followed by a polyadenylation sequence, such as an SV40 pA sequence. A specific example and particular embodiment of such an LC gene is described by SEQ ID NO: 25, SEQ ID NO: 33, or SEQ ID NO: 41. Conserved variants of any one of these sequences are also envisioned to be embodiments of the invention.
In some cases, the recombinant LC-encoding polynucleotide is part of a plasmid, which may be linear, circular, episomal, integrated, a static DNA construct, or a vector for delivering the light chain gene or expressing the light chain subunit. In one particular embodiment, the plasmid contains (1) an LC gene, which is under the control of an hCMV-IE promoter and terminates with an SV40 polyadenylation signal, and (2) a selectable marker, such as a polynucleotide encoding a polypeptide that confers resistance to hygromycin, under the control of a promoter, such as an SV40 promoter, and terminated with a polyadenylation sequence, such as a PGK pA sequence. In one particular embodiment, the plasmid comprises, in a circular format running in a 5′ to 3′ direction, an hCMV-IE promoter, a beta globin intron, an antibody light chain coding sequence (which encodes a LC having an amino acid of SEQ ID NO: 45 and 46, SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 37), an SV40 pA sequence, an SV40 promoter, a hygromycin-resistance coding sequence, and a PGK pA sequence. A specific example and particular embodiment of such a plasmid containing an LC gene is described by SEQ ID NO: 26, SEQ ID NO: 34, or SEQ ID NO: 42. Conserved variants of any one of these sequences are also envisioned to be embodiments of the invention.
Methods of Manufacturing Multi-Subunit Proteins
In another aspect, the invention provides a method for manufacturing a multi-subunit protein by culturing a cell, or a constituent cell of a cell line, which is capable of producing and secreting relatively large amounts of a properly assembled multi-subunit protein, in a medium, wherein the multi-subunit component is secreted into the medium at a relatively high titer. The cell utilized in this manufacturing process is a cell described in the foregoing aspects, which contains an ERAD lectin-encoding polynucleotide described herein.
Methods of culturing cells, and in particular mammalian cells, for the purpose of producing useful recombinant proteins is well-known in the art (e.g., see De Jesus & Wurm, Eur. J. Pharm. Biopharm. 78:184-188, 2011, and references cited therein). Briefly, cells containing the described polynucleotides are cultured in media, which may contain sera or hydrolysates, or may be chemically defined and optimized for protein production. The cultures may be fed-batch cultures or continuous cultures, as in a chemostat. The cells may be cultured in lab bench size flasks (˜25 mL), production scale-up bioreactors (1-5 L), or industrial scale bioreactors (5,000-25,000 L). Production runs may last for several weeks to a month, during which time the multi-subunit protein is secreted into the media.
The subject cell has an enhanced ability to produce and secrete properly assembled multi-subunit proteins. In some embodiments, the multi-subunit protein, for example an antibody, is secreted into the media at a rate of at least 94 ρg/cell/day, at least 37 ρg/cell/day, or at least 39 ρg/cell/day. In some embodiments, the multi-subunit protein attains a titer of at least at least 3 g/L, at least 5 g/L, at least 6 g/L, or at least 8 g/L after about twelve days of culture.
Furthermore, the subject cell has an enhanced ability to proliferate and attain a relatively high cell density, further optimizing productivity. In some embodiments, the cell or cell-line seed train attains an integrated cell density in culture of at least 5×107 cell-day/mL, at least 1×108 cell-day/mL or at least 1.5×108 cell-day/mL.
Optionally, the secreted multi-subunit protein is subsequently purified from the medium into which it was secreted. Protein purification methods are well-known in the art (see e.g., Kelley, mAbs 1(5):443-452). In some embodiments, the protein is harvested by centrifugation to remove the cells from the liquid media supernatant, followed by various chromatography steps and a filtration step to remove inter alia viruses and other contaminants or adulterants. In some embodiments, the chromatography steps include an ion exchange step, such as cation-exchange or anion-exchange. Various affinity chromatographic media may also be employed, such as protein A chromatography for the purification of antibodies.
Optionally, the manufacturing method may include the antecedent steps of creating the cell. Thus, in some embodiments, the method of manufacturing the multi-subunit protein comprises the step of transfecting the cell with the vector that encodes the stress-induced mannose-binding lectin, as described above, followed by selecting stable integrants thereof. Non-limiting examples of vectors include those genetic constructs that contain a polynucleotide that encodes an EDEM2 having an amino acid sequence of any one of SEQ ID NO: 1-8, an amino acid sequence that is at least 92% identical to any one of SEQ ID NO: 1-8, or any one of a conservatively substituted variant of SEQ ID NO: 1-8. Useful vectors also include, for example, a plasmid harboring the gene of SEQ ID NO: 16, the plasmid of SEQ ID NO: 15, and the plasmid of SEQ ID NO: 14. One should keep in mind that the plasmid sequences (e.g., SEQ ID NO: 14, 15, 17, 24, 26, 32, 34, 40, and 42) are circular sequences described in a linear manner in the sequence listing. Thus, in those cases, the 3-prime-most nucleotide of the written sequence may be considered to be immediately 5-prime of the 5-prime-most nucleotide of the sequence as written. In the example of the plasmid of SEQ ID NO: 14, transformants are selected through resistance to neomycin; for SEQ ID NO: 15, by selection through ZEOCIN resistance.
Detailed methods for the construction of polynucleotides and vectors comprising same, are described in U.S. Pat. Nos. 7,435,553 and 7,771,997, which are incorporated herein by reference, and in, e.g., Zwarthoff et al., J. Gen. Virol. 66(4):685-91, 1985; Mory et al., DNA. 5(3):181-93, 1986; and Pichler et al., Biotechnol. Bioeng. 108(2):386-94, 2011.
The starting cell, into which the vector that encodes the stress-induced mannose-binding lectin is placed, may already contain the constructs or genetic elements encoding or regulating the expression of the subunits of the multi-subunit protein, or XBP1 for those embodiments utilizing XBP1. Alternatively, the vector that encodes the stress-induced mannose-binding lectin may be put inside the cell first, and followed by the other constructs.
Multi-Subunit Proteins Manufactured by the Process
In another aspect, the invention provides a multi-subunit protein that is made according to the process disclosed herein. Given the inclusion of one or more elements that facilitate the proper folding, assembly, and post-translational modification of a multi-subunit protein, such as an antibody, one of ordinary skill in the art would reasonably expect such a protein to have distinct structural and functional qualities. For example, an antibody manufactured by the disclosed process is reasonably believed to have a particular glycosylation pattern and a quantitatively greater proportion of non-aggregated heterotetramers.
EXAMPLES
The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by mole, molecular weight is average molecular weight, percent concentration (%) means the mass of the solute in grams divided by the volume of the solution in milliliters times 100% (e.g., 10% substance X means 0.1 gram of substance X per milliliter of solution), temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.
Example 1: Cell Lines
CHO-K1 derived host cell line was transfected with two plasmids encoding heavy and light chain of a human antibody. Both plasmids contain the hph gene conferring resistance to hygromycin B (Asselbergs and Pronk, 1992, Mol. Biol. Rep., 17(1):61-70). Cells were transfected using LIPOFECTAMIN reagent (Invitrogen cat.#18324020). Briefly, one day before transfection 3.5 million cells were plated on a 10 cm plate in complete F12 (Invitrogen cat.#11765) containing 10% fetal bovine serum (FBS) (Invitrogen cat.#10100). On the day of transfection the cells were washed once and medium was replaced with OPTIMEM from (Invitrogen cat.#31985). DNA/Lipofectamin complexes were prepared in OPTIMEM medium and then added to the cells. The medium was changed again to the complete F12 with 10% FBS 6 hours later. The stable integration of the plasmids was selected using hygromycin B selection agent at 400 μg/ml. Clonal antibody expressing cell lines were isolated using the FASTR technology (described in the U.S. Pat. No. 6,919,183, which is herein incorporated by reference).
The antibody expressing lines were then re-transfected with the EDEM2 encoding plasmid. EDEM2 plasmids contained either neomycin phosphotransferase (plasmid construct designated “p3”) or sh ble (plasmid “p7”) genes to confer resistance to either G418 or zeocin respectively. The same transfection method was used. Depending on the selectable marker, cells were selected with either G418 or zeocin at 400 μg/ml or 250 μg/ml, respectively. The clonal cell lines were then isolated using FASTR technology.
TABLE 3
Cell Lines
Name
Enhancers
Constructs
Protein
C1
EDEM2 + XBP1
HC/LC = p1/p2
αAng2
C2
XBP1
EDEM2 = p3
XBP1 = p4
C3
EDEM2 + XBP1
HC/LC = p5/p6
αGDF8
C4
XBP1
EDEM2 = p7
C5
EDEM2
XBP1 = p4
C6
EDEM2 + XBP1
HC/LC = p8/p9
αAngPtl4
C7
XBP1
EDEM2 = p3
XBP1 = p4
Example 2
The antibody production was evaluated in a scaled-down 12-day fed batch process using shaker flasks. In this method the cells were seeded in a shaker flask at the density of 0.8 million cells per mL in the production medium (defined media with high amino acid). The culture was maintained for about 12 days, and was supplemented with three feeds as well as glucose. The viable cell density, and antibody titer were monitored throughout the batch.
To determine the effect of mEDEM2 on enhanced protein production, the production of proteins by CHO cell lines containing mEDEM2 and mXBP1 were compared to production by control cells that contained mXBP1, but not mEDEM2. Protein titers were higher in those cell lines expressing mEDEM2 versus those cell lines that did not express mEDEM2.
TABLE 4
TITERS
Cell
Production rate
Titre g/L
Line
Enhancers
(ρg/cell/day)
(% increase)
C1
EDEM2 + XBP1
39
8.1 (93)
C2
XBP1
39
4.2
C3
EDEM2 + XBP1
37
5.9 (55)
C8
XBP1
32
3.8
C6
EDEM2 + XBP1
94
5.3 (152)
C7
XBP1
52
2.1
C5
EDEM2
29
3.1 (343)
C9
—
9
0.7
Example 3: Integrated Cell Days
Integrated Cell Days (“ICD”) is a phrase used to describe the growth of the culture throughout the fed batch process. In the course of the 12-day production assay, we monitored viable cell density on days 0, 3, 5, 7, 10, and 12. This data was then plotted against time. ICD is the integral of viable cell density, calculated as the area under the cell density curve. EDEM2 transfected lines have higher ICD in a 12-day fed batch process (see Table 5).
TABLE 5
INTEGRATED CELL DENSITIES
ICD 106 cell-day/mL
Cell Line
Enhancers
(% increase)
C1
EDEM2 + XBP1
205 (93)
C2
XBP1
106
C3
EDEM2 + XBP1
157 (34)
C4
XBP1
117
C6
EDEM2 + XBP1
56 (51)
C7
XBP1
37
C5
EDEM2
116 (59)
C9
—
73
Example 4: Anti-GDF8 Antibody Production
The effect of ectopic expression of EDEM2, XBP1, or both on the production of an anti-GDF8 antibody having a heavy chain sequence of SEQ ID NO: 19 and a light chain sequence of SEQ ID NO: 21 was examined. Individual cell-lines were examined for titer and integrated cell density and placed into “bins”, or ranges of values. Ectopic expression of EDEM2 significantly increased the number of cell lines that express antibody in the 5-6 g/L titer range. The combination of XBP1 and EDEM2 showed more than an additive effect toward the increase in high titer cell lines. The expression of EDEM2 in the antibody secreting cells also significantly increased the number of cell lines that attain a high ICD (see Table 6).
TABLE 6
con-
Titre Bins (g/L)
ICD Bins (106 cell-day/mL)
struct
<1
1-3
3-5
5-6
30-50
50-100
100-200
E + X
0%
33.3%
44.4%
22.2%
11.1%
50%
38.9%
X
0%
37.5%
54%
8.3%
14.3%
85.7%
0%
E
0%
33%
60%
7%
0%
27%
73%
—
82%
18%
0%
0%
13%
67%
21%
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14737090
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Sep 22nd, 2020 12:00AM
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Jun 21st, 2018 12:00AM
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https://www.uspto.gov?id=US10779520-20200922
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Genetically modified major histocompatibility complex animals
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The invention provides genetically modified non-human animals that express chimeric human/non-human MHC I polypeptide and/or human or humanized β2 microglobulin polypeptide, as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified animals and methods of making the same. Methods of using the genetically modified animals to study various aspects of human immune system are provided.
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10779520
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1. A mouse comprising:
(i) two, three, four, five, or six nucleotide sequences, each encoding a different chimeric human/mouse MHC I polypeptide,
wherein each nucleotide sequence comprises from 5′ to 3′: a nucleic acid sequence encoding α1, α2, and α3 domains of a classical human MHC I polypeptide operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of an endogenous classical MHC I polypeptide,
wherein the nucleic acid sequence encoding α1, α2, and α3 domains of a classical human MHC I polypeptide replaces the sequence encoding α1, α2, and α3 domains of an endogenous classical MHC I polypeptide at an endogenous mouse MHC I locus, and
wherein each nucleotide sequence is operably linked to an endogenous mouse MHC I regulatory element and an MHC class I leader encoding sequence; and
(ii) at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide,
(a) wherein the nucleotide sequence encoding a human or humanized β2microglobulin polypeptide comprises from 5′ to 3′: exon 2, exon 3 and exon 4 of a human β2 microglobulin gene, wherein exon 2, exon 3 and exon 4 of a human β2 microglobulin gene replace, respectively, exon 2, exon 3 and exon 4 of an endogenous mouse β2 microglobulin gene, and
(b) wherein the nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide is operably linked to an endogenous mouse β2 microglobulin regulatory element, and
wherein the mouse expresses, on the surface of nucleated cells, the two, three, four, five, or six different chimeric human/mouse MHC I polypeptides and the human or humanized β2 microglobulin polypeptide.
2. The mouse of claim 1, wherein the classical human MHC I polypeptide is selected from the group consisting of HLA-A and HLA-B.
3. The mouse of claim 1, wherein the mouse does not express any classical endogenous MHC I polypeptides on a cell surface.
4. The mouse of claim 1, wherein the mouse does not express a functional endogenous mouse β2 microglobulin from its endogenous mouse locus.
5. The mouse of claim 1, wherein the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene, wherein the nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene replaces a nucleotide sequence set forth in exon 2 to exon 4 of the endogenous mouse β2 microglobulin gene.
6. The mouse of claim 1, wherein the mouse comprises two or three different nucleotides sequences encoding two or three different chimeric human/mouse MHC I polypeptides on at least one of the two chromosomes 17 of the mouse.
7. The mouse of claim 6, wherein each chimeric human/mouse MHC I polypeptide comprises (a) α1, α2, and α3 domains of a classical human MHC I polypeptide selected from the group consisting of HLA-A and HLA-B and (b) transmembrane and cytoplasmic domains of an endogenous classical MHC I polypeptide selected from the group consisting of H-2D and H-2K.
8. The mouse of claim 7, wherein the mouse does not express any classical endogenous MHC I polypeptides on a cell surface.
9. The mouse of claim 7, wherein the mouse comprises two different nucleotide sequences encoding different chimeric human/mouse MHC I polypeptides.
10. The mouse of claim 9, wherein the two nucleotide sequences respectively encode chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides, and
wherein the mouse expresses the chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides.
11. The mouse of claim 10, wherein the nucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide is located at an endogenous H-2K locus, and the nucleotide sequence encoding the chimeric HLA-B27/H-2D polypeptide is located at an endogenous H-2D locus.
12. A method for generating the mouse according to claim 1, comprising
replacing in a mouse embryonic stem (ES) cell
(i) at a first endogenous classical MHC I locus, a first nucleotide sequence encoding α1, α2, and α3 domains of a first endogenous classical MHC I polypeptide with a first nucleotide sequence encoding α1, α2, and α3 domains of a first classical human MHC I polypeptide to form a first nucleic acid sequence encoding a first chimeric human/mouse MHC I polypeptide comprising α1, α2, and α3 domains of the first classical human MHC I polypeptide operably linked to transmembrane and cytoplasmic domains of the first endogenous classical MHC I polypeptide, and
(ii) at a second endogenous classical MHC I locus, a second nucleotide sequence encoding α1, α2, and α3 domains of a second endogenous classical MHC I polypeptide with a second nucleotide sequence encoding α1, α2, and α3 domains of a second classical human MHC I polypeptide to form a second nucleic sequence encoding a second chimeric human/mouse MHC I polypeptide comprising α1, α2, and α3 domains of the second classical human MHC I polypeptide operably linked to transmembrane and cytoplasmic domains of the second endogenous classical MHC I polypeptide,
wherein the ES cell further comprises at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide
(a) wherein the nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide comprises from 5′ to 3′: exon 2, exon 3 and exon 4 of a human β2 microglobulin gene,
wherein exon 2, exon 3 and exon 4 of a human β2 microglobulin gene replace, respectively, exon 2, exon 3 and exon 4 of an endogenous mouse β2 microglobulin gene, and
(b) wherein the nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide is operably linked to an endogenous mouse β2 microglobulin regulatory element, and
generating a mouse from the ES cell,
wherein the first and second nucleic acid sequences are operably linked to an endogenous mouse regulatory element and an MHC class I leader encoding sequence at the first and second endogenous classical mouse MHC I loci, respectively,
wherein the mouse expresses at least two different chimeric human/mouse MHC I polypeptides and the human or humanized β2 microglobulin polypeptide.
13. The method of claim 12, wherein each chimeric human/mouse MHC I polypeptide comprises (a) α1, α2, and α3 domains of a classical human MHC I polypeptide selected from the group consisting of HLA-A and HLA-B and (b) transmembrane and cytoplasmic domains of an endogenous classical MHC I polypeptide selected from the group consisting of H-2D and H-2K.
14. The method of claim 13, wherein the first and second nucleic acid sequences respectively encode chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides, and
wherein the mouse expresses the chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides.
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14
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 16/415,544, filed, Jan. 25, 2017, now allowed, which is a divisional of U.S. patent application Ser. No. 13/793,812, filed Mar. 11, 2013, now U.S. Pat. No. 9,591,835, which is a continuation-in-part of U.S. patent application Ser. No. 13/661,159, filed Oct. 26, 2012, now U.S. Pat. No. 9,615,550 which claims benefit of priority to U.S. Provisional Patent Application Nos. 61/552,582 and 61/552,587, both filed Oct. 28, 2011, and U.S. Provisional Patent Application No. 61/700,908, filed Sep. 14, 2012, all of which are hereby incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
Present invention relates to a genetically modified non-human animal, e.g., a rodent (e.g., a mouse or a rat), that expresses a human or humanized Major Histocompatibility Complex (MHC) class I molecule. The invention also relates to a genetically modified non-human animal, e.g., a mouse or a rat, that expresses a human or humanized MHC I protein (e.g., MHC I α chain) and/or a human or humanized β2 microglobulin; as well as embryos, tissues, and cells expressing the same. The invention further provides methods for making a genetically modified non-human animal that expresses human or humanized MHC class I protein (e.g., MHC I α chain) and/or β2 microglobulin. Also provided are methods for identifying and evaluating peptides in the context of a humanized cellular immune system in vitro or in a genetically modified non-human animal, and methods of modifying an MHC I and/or a β2 microglobulin locus of a non-human animal, e.g., a mouse or a rat, to express a human or humanized MHC I and/or β2 microglobulin.
BACKGROUND OF THE INVENTION
In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptor or TCR). These foreign antigens are presented on the surface of cells as peptide fragments by specialized proteins, generically referred to as major histocompatibility complex (MHC) molecules. MHC molecules are encoded by multiple loci that are found as a linked cluster of genes that spans about 4 Mb. In mice, the MHC genes are found on chromosome 17, and for historical reasons are referred to as the histocompatibility 2 (H-2) genes. In humans, the genes are found on chromosome 6 and are called human leukocyte antigen (HLA) genes. The loci in mice and humans are polygenic; they include three highly polymorphic classes of MHC genes (class I, II and III) that exhibit similar organization in human and murine genomes (see FIG. 2 and FIG. 3, respectively).
MHC loci exhibit the highest polymorphism in the genome; some genes are represented by >300 alleles (e.g., human HLA-DRβ and human HLA-B). All class I and II MHC genes can present peptide fragments, but each gene expresses a protein with different binding characteristics, reflecting polymorphisms and allelic variants. Any given individual has a unique range of peptide fragments that can be presented on the cell surface to B and T cells in the course of an immune response.
Both humans and mice have class I MHC genes (see FIG. 2 and FIG. 3). In humans, the classical class I genes are termed HLA-A, HLA-B and HLA-C, whereas in mice they are H-2K, H-2D and H-2L. Class I molecules consist of two chains: a polymorphic α-chain (sometimes referred to as heavy chain) and a smaller chain called β2-microglobulin (also known as light chain), which is generally not polymorphic (FIG. 1). These two chains form a non-covalent heterodimer on the cell surface. The α-chain contains three domains (α1, α2 and α3). Exon 1 of the α-chain gene encodes the leader sequence, exons 2 and 3 encode the α1 and α2 domains, exon 4 encodes the α3 domain, exon 5 encodes the transmembrane domain, and exons 6 and 7 encode the cytoplasmic tail. The α-chain forms a peptide-binding cleft involving the α1 and α2 domains (which resemble Ig-like domains) followed by the α3 domain, which is similar to β2-microglobulin.
β2 microglobulin is a non-glycosylated 12 kDa protein; one of its functions is to stabilize the MHC class I α-chain. Unlike the α-chain, the β2 microglobulin does not span the membrane. The human β2 microglobulin locus is on chromosome 15, while the mouse locus is on chromosome 2. (32 microglobulin gene consists of 4 exons and 3 introns. Circulating forms of β2 microglobulin are present in the serum, urine, and other body fluids; thus, the non-covalently MHC I-associated β2 microglobulin can be exchanged with circulating β2 microglobulin under physiological conditions.
Class I MHC molecules are expressed on all nucleated cells, including tumor cells. They are expressed specifically on T and B lymphocytes, macrophages, dendritic cells and neutrophils, among other cells, and function to display peptide fragments (typically 8-10 amino acids in length) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs are specialized to kill any cell that bears an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell displays peptides derived from cellular proteins not normally present (e.g., of viral, tumor, or other non-self origin), such peptides are recognized by CTLs, which become activated and kill the cell displaying the peptide.
Typically, presentation of normal (i.e., self) proteins in the context of MHC I molecules does not elicit CTL activation due to the tolerance mechanisms. However, in some diseases (e.g., cancer, autoimmune diseases) peptides derived from self-proteins become a target of the cellular component of the immune system, which results in destruction of cells presenting such peptides. Although there has been advancement in recognizing some self-derived antigens that elicit cellular immune response (e.g., antigens associated with various cancers), in order to improve identification of peptides recognized by human CTLs through MHC class I molecules there remains a need for both in vivo and in vitro systems that mimic aspects of the human cellular immune system. Systems that mimic the human cellular immune system can be used in identifying disease-associated antigens in order to develop human therapeutics, e.g., vaccines and other biologics. Systems for assessing antigen recognition in the context of the human immune system can assist in identifying therapeutically useful CTL populations (e.g., useful for studying and combatting human disease). Such systems can also assist in enhancing the activity of human CTL populations to more effectively combat infections and foreign antigen-bearing entities. Thus, there is a need for biological systems (e.g., genetically engineered animals) that can generate an immune system that displays components that mimic the function of human immune system.
SUMMARY OF THE INVENTION
A biological system for generating or identifying peptides that associate with human MHC class I proteins and chimeras thereof, and bind to CD8+ T cells, is provided. Non-human animals comprising non-human cells that express human or humanized molecules that function in the cellular immune response are provided. Humanized rodent loci that encode human or humanized MHC I and β32 microglobulin proteins are also provided. Humanized rodent cells that express human or humanized MHC and β2 microglobulin molecules are also provided. In vivo and in vitro systems are provided that comprise humanized rodent cells, wherein the rodent cells express one or more human or humanized immune system molecules.
Provided herein is a non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprising in its genome a nucleotide sequence encoding a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide. Specifically, provided herein is a non-human animal comprising at an endogenous MHC I locus a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide, and wherein the animal expresses the chimeric human/non-human MHC I polypeptide. In one aspect, the animal does not express an extracellular domain (e.g., a functional extracellular domain) of an endogenous non-human MHC I polypeptide from an endogenous non-human MHC I locus. In one aspect of the invention, the non-human animal (e.g., a rodent, e.g., a mouse or a rat) comprises two copies of the MHC I locus comprising a nucleotide sequence encoding chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide. In another aspect of the invention, the animal comprises one copy of the MHC I locus comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide. Thus, the animal may be homozygous or heterozygous for the MHC I locus comprising a nucleotide sequence encoding chimeric human/non-human MHC I polypeptide. In various embodiments, the nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide is comprised in the germline of the non-human animal (e.g., rodent, e.g., rat or mouse). In one embodiment, the chimeric MHC I locus is comprised in the germline of a non-human animal.
In one aspect, the nucleotide sequence encoding the chimeric human/non-human MHC I is operably linked to endogenous non-human regulatory elements, e.g., promoter, enhancer, silencer, etc. In one embodiment, a human portion of the chimeric polypeptide comprises a human leader sequence. In another embodiment, the chimeric polypeptide comprises a non-human leader sequence, e.g., endogenous non-human leader sequence. In an additional embodiment, the human portion of the chimeric polypeptide comprises α1, α2, and α3 domains of the human MHC I polypeptide. The human MHC I polypeptide may be selected from a group consisting of HLA-A, HLA-B, and HLA-C. In one embodiment, the human MHC I polypeptide is an HLA-a polypeptide, e.g., HLA-A2 polypeptide, e.g., an HLA-A2.1 polypeptide. In another embodiment, the human MHC I polypeptide is an HLA-B polypeptide, e.g., HLA-B27 polypeptide.
In one aspect, the genetically engineered non-human animal is a rodent. In one embodiment, the rodent is a mouse. Thus, in one embodiment, the endogenous non-human locus is a mouse locus, e.g., a mouse H-2K, H-2D or H-2L locus. In one embodiment, the non-human portion of the chimeric human/non-human MHC I polypeptide comprises transmembrane and cytoplasmic domains of the endogenous non-human MHC I polypeptide. Thus, in an embodiment wherein the non-human animal is a mouse, the endogenous non-human MHC I locus may be an H-2K locus (e.g., H-2Kb locus) and the endogenous non-human MHC I polypeptide may be an H-2K polypeptide; therefore, the chimeric human/non-human MHC I polypeptide may comprise transmembrane and cytoplasmic domains of H-2K polypeptide. In another embodiment wherein the non-human animal is a mouse, the endogenous non-human MHC I locus may be an H-2D locus and the endogenous non-human MHC I polypeptide may be an H-2D polypeptide; therefore, the chimeric human/non-human MHC I polypeptide may comprise transmembrane and cytoplasmic domains of H-2D polypeptide. Similarly, in another embodiment, the endogenous non-human MHC I locus may be a mouse H-2L locus and the endogenous non-human MHC I polypeptide may be an H-2L polypeptide; therefore, the chimeric human/non-human MHC I polypeptide may comprise transmembrane and cytoplasmic domains of H-2L polypeptide.
Also provided herein is a mouse comprising at an endogenous H-2K locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., HLA-A2) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K polypeptide, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide. In some embodiments, the mouse does not express an extracellular domain (e.g., does not express a functional extracellular domain) of the mouse H-2K polypeptide from an endogenous H-2K locus. In one aspect, the nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide is operably linked to endogenous mouse regulatory elements. The human portion of the chimeric polypeptide may comprise a human leader sequence. It may also comprise α1, α2, and α3 domains of the human MHC I polypeptide. The human MHC I polypeptide may be HLA-A polypeptide, e.g., HLA-A2.1 polypeptide. In one aspect, the mouse H-2K locus is an H-2Kb locus.
Also provided herein is a mouse comprising at an endogenous H-2D locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-B (e.g., HLA-B27) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2D (e.g., H-2D1) polypeptide, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide. In some embodiments, the mouse does not express an extracellular domain (e.g., a functional extracellular domain) of the mouse H-2D polypeptide from an endogenous H-2D locus. In one aspect, the nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide is operably linked to endogenous mouse regulatory elements. The human portion of the chimeric polypeptide may comprise a human leader sequence. The chimeric polypeptide may also comprise a mouse leader sequence. The human portion of the chimeric polypeptide may also comprise α1, α2, and α3 domains of the human MHC I polypeptide. The human MHC I polypeptide may be HLA-B polypeptide, e.g., HLA-B27 polypeptide. In one aspect, the mouse H-2D locus is an H-2D1 locus.
In an additional embodiment, provided herein is a mouse that comprises at an endogenous mouse MHC I locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I selected from the group consisting of HLA-A2, HLA-A3, HLA-B27, and HLA-B7. In another embodiment, provided herein is a mouse that comprises at an endogenous MHC I locus one or more, e.g., one, two, three, four, five, or six, nucleotide sequence(s) encoding a chimeric human/mouse MHC I polypeptide(s), wherein a human portion of the chimeric polypeptide(s) comprises an extracellular domain of a human MHC I polypeptide, wherein a mouse portion of a chimeric human/mouse MHC I polypeptide(s) comprises transmembrane and cytoplasmic domain of a mouse MHC I polypeptide, and wherein the mouse expresses one or more, e.g., one, two, three, four, five, or six, chimeric human/mouse MHC I polypeptide(s). Thus, the mouse MHC I polypeptide may be selected from H-2D, H-2K, and H-2L. In one embodiment, the chimeric human/mouse MHC I polypeptide may be selected from HLA-A2/H-2K, HLA-A3/H-2K, HLA-B27/H-2D, HLA-B7/H-2D, and a combination thereof. In one embodiment, one, two, or three chimeric human/mouse MHC I polypeptide(s) may be encoded on each sister chromosome 17; thus, a mouse MHC I locus may comprise one or more, e.g., one, two, three, four, five, or six, nucleotide sequences encoding a chimeric human/mouse MHC I polypeptide(s). In one embodiment, the mouse comprises two nucleotide sequences encoding the chimeric human/mouse MHC I polypeptide(s), wherein the two nucleotide sequences encode HLA-A2/H-2K and HLA-B27/H-2D polypeptides, and wherein the mouse expresses the chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides. In one embodiment, the mouse provided herein does not express any functional endogenous mouse MHC I polypeptides from the endogenous mouse MHC I locus. Thus, in one aspect, the mouse expresses only humanized, e.g., chimeric human/mouse MHC I polypeptides, and the remaining MHC I loci that do not comprise chimeric human/mouse MHC I sequences (e.g., MHC I loci that do not comprise replacement of endogenous mouse MHC I nucleotide sequences with those encoding chimeric human/mouse polypeptides) are removed by deletion. In another embodiment, the mouse retains a nucleotide sequence encoding a functional endogenous mouse MHC I polypeptide (e.g., H-2L polypeptide).
Another aspect of the invention relates to a non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprising in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. Thus, provided herein is a non-human animal comprising at an endogenous non-human β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the animal expresses the human or humanized β2 microglobulin polypeptide. In one aspect, the animal does not express a functional endogenous non-human β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In one aspect, the animal comprises two copies of the β2 microglobulin locus encoding the human or humanized β2 microglobulin polypeptide; in another embodiment, the animal comprises one copy of the β2 microglobulin locus encoding the human or humanized β2 microglobulin polypeptide. Thus, the animal may be homozygous or heterozygous for the β2 microglobulin locus encoding the human or humanized β2 microglobulin polypeptide. In various embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide is comprised in the germline of the non-human animal (e.g., rodent, e.g., rat or mouse). In one embodiment, a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.
In some embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide is operably linked to endogenous non-human β2 microglobulin regulatory elements. In one aspect, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. In another aspect, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In a further aspect, the nucleotide sequence also comprises a nucleotide sequence set forth in exon 1 of a non-human β2 microglobulin gene. In some embodiments, the non-human animal is a rodent (e.g., mouse or a rat); thus, the non-human β2 microglobulin locus is a rodent (e.g., a mouse or a rat) β2 microglobulin locus.
Also provided is a mouse comprising at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the mouse expresses the human or humanized β2 microglobulin polypeptide. In some embodiments, the mouse does not express a functional endogenous mouse β2 microglobulin from an endogenous β2 microglobulin locus. The nucleotide sequence may be linked to endogenous mouse regulatory elements. In one aspect, the nucleotide sequence comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may further comprise a nucleotide sequence of exon 1 of a mouse β2 microglobulin gene. In one embodiment, a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.
The invention further provides a non-human animal (e.g., a rodent, e.g., a mouse or a rat) comprising in its genome a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one embodiment, the invention provides a non-human animal comprising in its genome a first nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide; and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous non-human MHC I locus, and the second nucleotide sequence is located at an endogenous non-human β2 microglobulin locus, and wherein the animal expresses the chimeric human/non-human MHC I polypeptide and the human or humanized β2 microglobulin polypeptide. In one aspect, the animal is a mouse. Thus, the endogenous MHC I locus may be selected from a group consisting of H-2K, H-2D, and H-2L locus. In one embodiment, the endogenous mouse locus is an H-2K locus (e.g., H-2Kb locus). In another embodiment, the endogenous mouse locus is an H-2D locus. In one embodiment, the human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C polypeptide. In one aspect, the human MHC I polypeptide is HLA-A, e.g., HLA-A2 (e.g., HLA-A2.1). In another embodiment, the human MHC I polypeptide is HLA-B, e.g., HLA-B27. In various embodiments, the first and the second nucleotide sequences are comprised in the germline of the non-human animal (e.g., rodent, e.g., mouse or rat).
Therefore, in one embodiment, the invention provides a mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A (e.g., HLA-A2) and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K; and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous H-2K locus and the second nucleotide sequence is located at an endogenous mouse β2 microglobulin locus, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide and the human or humanized β2 microglobulin polypeptide. In another embodiment, the invention provides a mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-B (e.g., HLA-B27) and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2D; and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the first nucleotide sequence is located at an endogenous H-2D locus and the second nucleotide sequence is located at an endogenous mouse β2 microglobulin locus, and wherein the mouse expresses the chimeric human/mouse MHC I polypeptide and the human or humanized β2 microglobulin polypeptide. In one embodiment, the non-human animal (e.g., the mouse) comprising both the chimeric MHC I polypeptide and human or humanized β2 microglobulin polypeptide does not express an extracellular domain (e.g., a functional extracellular domain) of an endogenous non-human MHC I polypeptide (e.g., the mouse H-2K or H-2D polypeptide) and/or a functional endogenous non-human (e.g., the mouse) β2 microglobulin polypeptides from their respective endogenous loci. In one aspect, the animal (e.g., the mouse) comprises two copies of each of the first and the second nucleotide sequence. In another aspect, the animal (e.g., the mouse) comprises one copy of the first and one copy of the second nucleotide sequences. Thus, the animal may be homozygous or heterozygous for both the first and the second nucleotide sequences.
In one aspect, the first nucleotide sequence is operably linked to endogenous non-human (e.g., mouse) MHC I regulatory elements, and the second nucleotide sequence is operably linked to endogenous non-human (e.g., mouse) β2 microglobulin elements. The human portion of the chimeric polypeptide may comprise α1, α2 and α3 domains of the human MHC I polypeptide. The second nucleotide sequence may comprise a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In one aspect, the mouse comprising both the chimeric MHC I polypeptide and human or humanized β2 microglobulin polypeptide may be such that the expression of human or humanized β2 microglobulin increases the expression of the chimeric human/mouse MHC I polypeptide as compared to the expression of the chimeric human/mouse MHC I polypeptide in the absence of expression of human or humanized β2 microglobulin polypeptide.
Additionally, provided herein is a mouse that comprises in its genome, e.g., at its endogenous MHC I locus, one or more, e.g., one, two, three, four, five, or six, nucleotide sequence(s) encoding a chimeric human/mouse MHC I polypeptide(s), and further comprising at its endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one embodiment, the human portion of the chimeric MHC I polypeptide(s) comprises an extracellular domain of a human MHC I polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse MHC I polypeptide. Thus, the mouse may express one or more, e.g., one, two, three, four, five or six, chimeric human/mouse MHC I polypeptide(s) and a human or a humanized β2 microglobulin polypeptide.
Also provided are methods of making genetically engineered non-human animals (e.g., rodents, e.g., mice or rats) described herein. Thus, in one embodiment, provided is a method of modifying an MHC I locus of a non-human animal, e.g., a rodent (e.g., a mouse or a rat) to express a chimeric human/non-human, e.g., human/rodent (e.g., human/mouse or human/rat) MHC I polypeptide, wherein the method comprises replacing at the endogenous MHC I locus a nucleotide sequence encoding an extracellular domain of a non-human, e.g., rodent MHC I polypeptide with a nucleotide sequence encoding an extracellular domain of a human MHC I polypeptide. In another embodiment, provided is a method of modifying a β2 microglobulin locus of a non-human animal, e.g., a rodent (e.g., a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide, wherein the method comprises replacing at the endogenous non-human, e.g., rodent (e.g., mouse or rat) β2 microglobulin locus a nucleotide sequence encoding a non-human, e.g., rodent (e.g., a mouse or a rat) β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In such methods, the replacement may be made in a single ES cell, and the single ES cell may be introduced into a non-human animal, e.g., rodent (e.g., a mouse or a rat) to make an embryo. The resultant non-human animal, e.g., rodent (e.g., a mouse or a rat) can be bred to generate a double humanized animal.
Thus, the invention also provides a method of making double humanized animals, e.g., rodents (e.g., mice or rats). In one embodiment, provided is a method of making a genetically modified mouse comprising (a) modifying an MHC I locus of a first mouse to express a chimeric human/mouse MHC I polypeptide comprising replacing at the endogenous mouse MHC I locus a nucleotide sequence encoding an extracellular domain of a mouse MHC I polypeptide with a nucleotide sequence encoding an extracellular domain of a human MHC I polypeptide, (b) modifying a β2 microglobulin locus of a second mouse to express a human or humanized β2 microglobulin polypeptide comprising replacing at the endogenous mouse β2 microglobulin locus a nucleotide sequence encoding a mouse β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; and (c) breeding the first and the second mouse to generate a genetically modified mouse comprising in its genome a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide, wherein the genetically modified mouse expresses the chimeric human/mouse MHC I polypeptide and the human or humanized β2 microglobulin polypeptide. In some embodiments, the MHC I locus is selected from H-2K, H-2D, and H-2L; in some embodiments, the human MHC I polypeptide is selected from HLA-A, HLA-B, and HLA-C. In one embodiment, the MHC I locus is an H-2K locus, the human MHC I polypeptide is HLA-A (e.g., HLA-A2), and the mouse expresses a chimeric HLA-A/H-2K polypeptide (e.g., HLA-A2/H-2K polypeptide). In one aspect, the chimeric HLA-A2/H-2K polypeptide comprises an extracellular domain of the HLA-A2 polypeptide and cytoplasmic and transmembrane domains of H-2K polypeptide. In another embodiment, the MHC I locus is an H-2D locus, the human MHC I polypeptide is HLA-B (e.g., HLA-B27), and the mouse expresses a chimeric HLA-B2/H-2D polypeptide (e.g., HLA-B27/H-2D polypeptide). In one aspect, the chimeric HLA-B27/H-2D polypeptide comprises an extracellular domain of the HLA-B27 polypeptide and cytoplasmic and transmembrane domains of H-2D polypeptide. In one aspect, the second nucleotide sequence comprises nucleotide sequences set forth in exons 2, 3, and 4 (e.g., exon 2 to exon 4) of a human in microglobulin gene, and a nucleotide sequence set forth in exon 1 of a mouse β2 microglobulin gene.
Also provided herein is a non-human chimeric MHC I locus encoding a chimeric human/non-human MHC I polypeptide, comprising a first nucleotide sequence encoding a human MHC I extracellular domain operably linked to a second nucleotide sequence encoding a non-human MHC I transmembrane and cytoplasmic domains. In one aspect, the chimeric MHC I locus is at an endogenous MHC I position in a genome of a non-human animal. In one aspect, the chimeric MHC I locus expresses a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse or human/rat) MHC I polypeptide. In one embodiment, the human MHC I is selected from HLA-A, HLA-B, and HLA-C (e.g., HLA-B27 or HLA-A2). In one embodiment, the non-human MHC I is a mouse MHC I selected from H-2D, H-2K, and H-2L (e.g., H-2D or H-2K). In one embodiment, the chimeric MHC I locus expresses one or more chimeric human/non-human MHC I polypeptide(s).
Also provided herein is a genetically modified β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the locus comprises a first nucleotide sequence comprising the sequence set forth in exons 2, 3, and 4 (e.g., exons 2-4) of a human β2 microglobulin gene, and a second nucleotide sequence comprising the sequence set forth in exon 1 of a non-human (e.g., rodent, e.g., rat or mouse) β2 microglobulin gene. In one embodiment, the second nucleotide sequence is operably linked to the first nucleotide sequence. In one aspect, the genetically modified β2 microglobulin locus is at an endogenous β2 microglobulin position in a genome of a non-human animal. In one aspect, the genetically modified β2 microglobulin locus expresses a human or humanized β2 microglobulin polypeptide.
In one aspect, the non-human chimeric MHC I locus is obtainable by any methods described herein for generating genetically modified non-human animals (e.g., rodents, e.g., mice or rats). In one aspect, the genetically modified β2 microglobulin locus is obtainable by any methods described herein for generating genetically modified non-human animals (e.g., rodents, e.g., mice or rats).
Also provided herein are cells, e.g., isolated antigen-presenting cells, derived from the non-human animals (e.g., rodents, e.g., mice or rats) described herein. Tissues and embryos derived from the non-human animals described herein are also provided.
In yet another embodiment, the invention provides methods for identification of antigens or antigen epitopes that elicit immune response, methods for evaluating a vaccine candidate, methods for identification of high affinity T cells to human pathogens or cancer antigens.
Any of the embodiments and aspects described herein can be used in conjunction with one another, unless otherwise indicated or apparent from the context. Other embodiments will become apparent to those skilled in the art from a review of the ensuing detailed description. The following detailed description includes exemplary representations of various embodiments of the invention, which are not restrictive of the invention as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate embodiments and not to limit the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing of the four domains of a class I MHC molecule: α-chain containing the α1, α2 and α3 domains and the non-covalently associated fourth domain, β2-microglobulin (β2m). The gray circle represents a peptide bound in the peptide-binding cleft.
FIG. 2 is a schematic representation (not to scale) of the relative genomic structure of the human HLA, showing class I, II and III genes.
FIG. 3 is a schematic representation (not to scale) of the relative genomic structure of the mouse MHC, showing class I, II and III genes.
FIG. 4 illustrates a viral vector construct containing a cDNA encoding a chimeric HLA-A/H-2K polypeptide with an IRES-GFP reporter (A); and histograms comparing expression of human HLA-A2 in MG87 cells transduced with HLA-A2 (dashed line), HLA-A2/H-2K (dotted line), or no transduction (solid line) either alone (left) or co-transduced with humanized β2 microglobulin (right) (B). Data from horizontal gates presented graphically in (B) is illustrated as percent of cells expressing the construct in the table in (C).
FIG. 5 is a schematic diagram (not to scale) of the targeting strategy used for making a chimeric H-2K locus that expresses an extracellular region of a human HLA-A2 protein. Mouse sequences are represented in black and human sequences are represented in white. L=leader, UTR=untranslated region, TM=transmembrane domain, CYT=cytoplasmic domain, HYG=hygromycin.
FIG. 6A demonstrates expression (% total cells) of HLA-A2 (left) and H-2K (right) in cells isolated from either a wild-type (WT) mouse or a heterozygous mouse carrying the chimeric HLA-A2/H-2K locus (HLA-A/H-2K HET).
FIG. 6B is a dot plot of in vivo expression of the chimeric HLA-A2/H-2K protein in a heterozygous mouse harboring a chimeric HLA-A2/H-2K locus.
FIG. 7 shows a targeting strategy (not to scale) for humanization of a β2 microglobulin gene at a mouse β2 microglobulin locus. Mouse sequences are in black and human sequences are in white. NEO=neomycin.
FIG. 8 shows a representative dot plot of HLA class I and human β2 microglobulin expression on cells isolated from the blood of wild-type (WT) mice, mice heterozygous for chimeric HLA-A2/H-2K, and mice heterozygous for chimeric HLA-A2/H-2K and heterozygous for humanized β2 microglobulin (double heterozygous; class I/β2m HET).
FIG. 9 shows a representative histogram of human HLA class I expression (X axis) on cells isolated from the blood of wild-type (WT), chimeric HLA-A2/H-2K heterozygous (class I HET), and chimeric HLA-A2/H2K/humanized β2 microglobulin double heterozygous (class I/β2m HET) mice.
FIG. 10 shows the results of IFNγ Elispot assays for human T cells exposed to antigen-presenting cells (APCs) from wild-type mice (WT APCs) or mice heterozygous for both chimeric HLA-A2/H-2K and humanized β2 microglobulin (double HET APCs) in the presence of flu (left) or EBV (right) peptides. Statistical analysis was performed using one way ANOVA with a Tukey's Multiple Comparison Post Test.
FIG. 11 is a schematic diagram (not to scale) of the targeting strategy for making a chimeric HLA-B27/H-2D1 locus that expresses an extracellular domain (in the particular embodiment depicted, human α1, α2, and α3 domains) of a human HLA-B27 gene, and endogenous mouse H-2D leader, transmembrane, and cytoplasmic domains. Locations of the probes used for genotyping (see Table 2) are also included. hEX1=human exon1; mEX1=mouse exon 1; h2, h3, h4=human exons 2, 3, and 4; m5, m6, m7, m8=mouse exons 5, 6, 7, and 8; mTM=mouse transmembrane domain in m5. Where not otherwise indicated, mouse sequences are represented in black and human sequences are represented in white.
FIG. 12, top two graphs, shows representative histograms of human HLA class I expression on cells isolated from the blood of wild-type (WT; right top figure) or chimeric HLA-B27/H-2D heterozygous/humanized β2 microglobulin heterozygous (B27/hB2M Het/Het; left top figure) mice; bottom two graphs depict representative histograms of human β2 microglobulin expression on cells isolated from the blood of WT or B27/hB2M Het/Het mice. MFI=mean fluorescent intensity; sec alone=secondary antibody staining only.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The present invention provides genetically modified non-human animals (e.g., mice, rats, rabbits, etc.) that express human or humanized MHC I and/or β2 microglobulin polypeptides; embryos, cells, and tissues comprising the same; methods of making the same; as well as methods of using the same. Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used.
The term “conservative,” when used to describe a conservative amino acid substitution, includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of MHC I to present a peptide of interest. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
Thus, also encompassed by the invention is a genetically modified non-human animal whose genome comprises a nucleotide sequence encoding a human or humanized MHC I polypeptide and/or β2 microglobulin polypeptide, wherein the polypeptide(s) comprises conservative amino acid substitutions of the amino acid sequence(s) described herein.
One skilled in the art would understand that in addition to the nucleic acid residues encoding a human or humanized MHC I polypeptide and/or β2 microglobulin described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptide(s) of the invention. Therefore, in addition to a genetically modified non-human animal that comprises in its genome a nucleotide sequence encoding MHC I and/or β2 microglobulin polypeptide(s) with conservative amino acid substitutions, a non-human animal whose genome comprises a nucleotide sequence(s) that differs from that described herein due to the degeneracy of the genetic code is also provided.
The term “identity” when used in connection with sequence includes identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular sequences. In various embodiments, identity is determined by comparing the sequence of a mature protein from its N-terminal to its C-terminal. In various embodiments when comparing a chimeric human/non-human sequence to a human sequence, the human portion of the chimeric human/non-human sequence (but not the non-human portion) is used in making a comparison for the purpose of ascertaining a level of identity between a human sequence and a human portion of a chimeric human/non-human sequence (e.g., comparing a human ectodomain of a chimeric human/mouse protein to a human ectodomain of a human protein).
The terms “homology” or “homologous” in reference to sequences, e.g., nucleotide or amino acid sequences, means two sequences which, upon optimal alignment and comparison, are identical in, e.g., at least about 75% of nucleotides or amino acids, e.g., at least about 80% of nucleotides or amino acids, e.g., at least about 90-95% nucleotides or amino acids, e.g., greater than 97% nucleotides or amino acids. One skilled in the art would understand that, for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.
The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In addition, various portions of the chimeric or humanized protein of the invention may be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless stated otherwise, various domains of the chimeric or humanized proteins of the invention are operably linked to each other.
The term “MHC I complex” or the like, as used herein, includes the complex between the MHC I α chain polypeptide and the β2-microglobulin polypeptide. The term “MHC I polypeptide” or the like, as used herein, includes the MHC I α chain polypeptide alone. Typically, the terms “human MHC” and “HLA” can be used interchangeably.
The term “replacement” in reference to gene replacement refers to placing exogenous genetic material at an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the Examples below, nucleic acid sequences of endogenous loci encoding portions of mouse MHC I and β2 microglobulin polypeptides were replaced by nucleotide sequences encoding portions of human MHC I and β2 microglobulin polypeptides, respectively.
“Functional” as used herein, e.g., in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non-human MHC I and/or β2 microglobulin locus) results in a locus that fails to express a functional endogenous polypeptide. Likewise, the term “functional” as used herein in reference to functional extracellular domain of a protein, refers to an extracellular domain that retains its functionality, e.g., in the case of MHC I, ability to bind an antigen, ability to bind a T cell co-receptor, etc. In some embodiments of the invention, a replacement at the endogenous MHC locus results in a locus that fails to express an extracellular domain (e.g., a functional extracellular domain) of an endogenous MHC while expressing an extracellular domain (e.g., a functional extracellular domain) of a human MHC.
Several aspects described herein below for the genetically modified MHC I non-human animals, e.g., animal type; animal strains; cell types; screening, detection and other methods; methods of use; etc., will be applicable to the genetically engineered β2 microglobulin and MHC I/β2 microglobulin animals.
Genetically Modified MHC I Animals
In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome a nucleotide sequence encoding a human or humanized MHC I polypeptide; thus, the animals express a human or humanized MHC I polypeptide.
MHC genes are categorized into three classes: class I, class II, and class III, all of which are encoded either on human chromosome 6 or mouse chromosome 17. A schematic of the relative organization of the human and mouse MHC classes is presented in FIGS. 2 and 3, respectively. The MHC genes are among the most polymorphic genes of the mouse and human genomes. MHC polymorphisms are presumed to be important in providing evolutionary advantage; changes in sequence can result in differences in peptide binding that allow for better presentation of pathogens to cytotoxic T cells.
MHC class I protein comprises an extracellular domain (which comprises three domains: α1, α2, and α3), a transmembrane domain, and a cytoplasmic tail. The α1 and α2 domains form the peptide-binding cleft, while the α3 interacts with β2-microglobulin.
In addition to its interaction with β2-microglobulin, the α3 domain interacts with the TCR co-receptor CD8, facilitating antigen-specific activation. Although binding of MHC class I to CD8 is about 100-fold weaker than binding of TCR to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366. Interestingly, increasing MHC class I binding to CD8 abrogated antigen specificity in CTL activation. Id.
CD8 binding to MHC class I molecules is species-specific; the mouse homolog of CD8, Lyt-2, was shown to bind H-2Dd molecules at the α3 domain, but it did not bind HLA-A molecules. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341. Differential binding was presumably due to CDR-like determinants (CDR1- and CDR2-like) on CD8 that was not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174:371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8αα and HLA-A2, Nature 387:630-634. It has been reported that CD8 binds HLA-A2 in a conserved region of the α3 domain (at position 223-229). A single substitution (V245A) in HLA-A reduced binding of CD8 to HLA-A, with a concomitant large reduction in T cell-mediated lysis. Salter et al. (1989), Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphism in the α3 domain of HLA-A molecules also affected binding to CD8. Id. In mice, amino acid substitution at residue 227 in H-2Dd affected the binding of mouse Lyt-2 to H-2Dd, and cells transfected with a mutant H-2Dd were not lysed by CD8+ T cells. Potter et al. (1989) Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75.
Therefore, due to species specificity of interaction between the MHC class I α3 domain and CD8, an MHC I complex comprising a replacement of an H-2K α3 domain with a human HLA-A2 α3 domain was nonfunctional in a mouse (i.e., in vivo) in the absence of a human CD8. In animals transgenic for HLA-A2, substitution of human α3 domain for the mouse α3 domain resulted in restoration of T cell response. Irwin et al. (1989) Species-restricted interactions between CD8 and the α3 domain of class I influence the magnitude of the xenogeneic response, J. Exp. Med. 170:1091-1101; Vitiello et al. (1991), supra.
The transmembrane domain and cytoplasmic tail of mouse MHC class I proteins also have important functions. One function of MHC I transmembrane domain is to facilitate modulation by HLA-A2 of homotypic cell adhesion (to enhance or inhibit adhesion), presumably as the result of cross-linking (or ligation) of surface MHC molecules. Wagner et al. (1994) Ligation of MHC Class I and Class II Molecules Can Lead to Heterologous Desensitization of Signal Transduction Pathways That Regulate Homotypic Adhesion in Human Lymphocytes, J. Immunol. 152:5275-5287. Cell adhesion can be affected by mAbs that bind at diverse epitopes of the HLA-A2 molecule, suggesting that there are multiple sites on HLA-A2 implicated in modulating homotypic cell adhesion; depending on the epitope bound, the affect can be to enhance or to inhibit HLA-A2-dependent adhesion. Id.
The cytoplasmic tail, encoded by exons 6 and 7 of the MHC I gene, is reportedly necessary for proper expression on the cell surface and for LIR1-mediated inhibition of NK cell cytotoxicity. Gruda et al. (2007) Intracellular Cysteine Residues in the Tail of MHC Class I Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1, J. Immunol. 179:3655-3661. A cytoplasmic tail is required for multimerizaton of at least some MHC I molecules through formation of disulfide bonds on its cysteine residues, and thus may play a role in clustering and in recognition by NK cells. Lynch et al. (2009) Novel MHC Class I Structures on Exosomes, J. Immunol. 183:1884-1891.
The cytoplasmic domain of HLA-A2 contains a constitutively phosphorylated serine residue and a phosphorylatable tyrosine, although—in Jurkat cells—mutant HLA-A2 molecules lacking a cytoplasmic domain appear normal with respect to expression, cytoskeletal association, aggregation, and endocytic internalization. Gur et al. (1997) Structural Analysis of Class I MHC Molecules: The Cytoplasmic Domain Is Not Required for. Cytoskeletal Association, Aggregation, and Internalization, Mol. Immunol. 34(2):125-132. Truncated HLA-A2 molecules lacking the cytoplasmic domain are apparently normally expressed and associate with β2 microglobulin. Id.
However, several studies have demonstrated that the cytoplasmic tail is critical in intracellular trafficking, dendritic cell (DC)-mediated antigen presentation, and CTL priming. A tyrosine residue encoded by exon 6 was shown to be required for MHC I trafficking through endosomal compartments, presentation of exogenous antigens, and CTL priming; while deletion of exon 7 caused enhancement of anti-viral CTL responses. Lizee et al. (2003) Control of Dendritic Cross-Presentation by the Major Histocompatibility Complex Class I Cytoplasmic Domain, Nature Immunol. 4:1065-73; Basha et al. (2008) MHC Class I Endosomal and Lysosomal Trafficking Coincides with Exogenous Antigen Loading in Dendritic Cells, PLoS ONE 3: e3247; and Rodriguez-Cruz et al. (2011) Natural Splice Variant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity, PLoS ONE 6:e22939.
In various embodiments, the invention provides a genetically modified non-human animal (e.g., mouse, rat, rabbit, etc.) that comprises in its genome a nucleotide sequence encoding a human or humanized MHC class I polypeptide. The non-human animal may comprise in its genome a nucleotide sequence that encodes an MHC I polypeptide that is partially human and partially non-human, e.g., a non-human animal that expresses a chimeric human/non-human MHC I polypeptide. In one aspect, the non-human animal only expresses the human or humanized MHC I polypeptide, e.g., chimeric human/non-human MHC I polypeptide, and does not express an endogenous non-human MHC I protein (e.g., a functional endogenous non-human MHC I protein) from an endogenous MHC I locus.
In one embodiment, provided herein is a non-human animal, e.g., a rodent, e.g., a rat or a mouse, comprising in its genome, e.g., at an endogenous non-human MHC I locus, a nucleotide sequence encoding a human MHC I polypeptide. In another embodiment, provided herein is a non-human animal, e.g., a rodent, e.g., a rat or a mouse, comprising in its genome, e.g., at an endogenous MHC I locus, a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide.
In one embodiment, the chimeric human/non-human MHC I polypeptide comprises in its human portion a peptide binding domain of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular domain of a human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises an extracellular domain of an α chain of a human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises α1 and α2 domains of a human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises α1, α2, and α3 domains of a human MHC I. In one embodiment, the human portion of the chimeric polypeptide (e.g., the extracellular domain of the chimeric polypeptide) comprises a human leader sequence. In another embodiment, the chimeric polypeptide (e.g., the extracellular domain of the chimeric polypeptide) comprises a non-human leader sequence (e.g., endogenous non-human leader sequence).
The human or humanized MHC I polypeptide may be derived from a functional human HLA molecule encoded by any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. A list of commonly used HLA antigens is described in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), incorporated herein by reference. Shankarkumar et al. also present a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75:291-455, both incorporated herein by reference. Thus, the human or humanized MHC I polypeptide may be derived from any functional human HLA class I molecules described therein.
In one specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., and HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is commonly used amongst the North American population. Although the present Examples describe this particular HLA sequence, any suitable HLA-A sequence is encompassed herein, e.g., polymorphic variants of HLA-A2 exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequence described herein due to the degeneracy of genetic code, etc.
In one aspect, a non-human animal that expresses a human HLA-A2 sequence is provided, wherein the human HLA-A2 sequence comprises one or more conservative or non-conservative modifications.
In one aspect, a non-human animal that expresses a human HLA-A2 sequence is provided, wherein the human HLA-A2 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human HLA-A2 sequence. In a specific embodiment, the human HLA-A2 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human HLA-A2 sequence described in the Examples. In one embodiment, the human HLA-A2 sequence comprises one or more conservative substitutions. In one embodiment, the human HLA-A2 sequence comprises one or more non-conservative substitutions.
In another specific aspect, the human or humanized MHC I polypeptide is derived from human MHC I selected from HLA-B and HLA-C. In one aspect, the human or humanized MHC I is derived from HLA-B, e.g., HLA-B27. In other embodiments, the human or humanized MHC I is derived from HLA-A3, HLA-B7, HLA-B27, HLA-Cw6, etc.
Thus, in one aspect, the human or humanized MHC I is derived from HLA-B. In a specific embodiment, the HLA-B polypeptide is an HLA-B27. In one embodiment, the HLA-B27 polypeptide is a polypeptide encoded by HLA-B27 allele subtypes B*2701-2759. The HLA-B27 usage is commonly correlated with ankylosing spondylitis in human population. Although the present Examples describe this particular HLA sequence, any suitable HLA-B27 sequence is encompassed herein, e.g., polymorphic variants of HLA-B27 exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequence described herein due to the degeneracy of genetic code, etc.
In one aspect, a non-human animal that expresses a human HLA-B27 sequence is provided, wherein the human HLA-B27 sequence comprises one or more conservative or non-conservative modifications.
In one aspect, a non-human animal that expresses a human HLA-B27 sequence is provided, wherein the human HLA-27 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human HLA-B27 sequence. In a specific embodiment, the human HLA-B27 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human HLA-B27 sequence described in the Examples. In one embodiment, the human HLA-B27 sequence comprises one or more conservative substitutions. In one embodiment, the human HLA-B27 sequence comprises one or more non-conservative substitutions.
In one aspect, the non-human portion of the chimeric human/non-human MHC I polypeptide comprises transmembrane and/or cytoplasmic domains of the non-human MHC I polypeptide. In one embodiment, the non-human animal is a mouse, and the non-human MHC I polypeptide is selected from H-2K, H-2D, and H-2L. In one embodiment, the non-human MHC I polypeptide is H-2K, e.g., H-2Kb. In another embodiment, the non-human MHC I polypeptide is H-2D, e.g., H-2D1. Although specific H-2K and H-2D sequences are described in the Examples, any suitable H-2K or H-2D sequences, e.g., polymorphic variants, conservative/non-conservative amino acid substitutions, etc., are encompassed herein.
The non-human animal described herein may comprise in its genome a nucleotide sequence encoding a human or humanized MHC I polypeptide, e.g., chimeric human/non-human MHC I polypeptide, wherein the nucleotide sequence encoding such polypeptide is located at an endogenous non-human MHC I locus (e.g., H-2K or H-2D locus). In one aspect, this results in a replacement of an endogenous MHC I gene or a portion thereof with a nucleotide sequence encoding a human or humanized MHC I polypeptide, e.g., resulting in a chimeric gene encoding a chimeric human/non-human MHC I polypeptide described herein. In one embodiment, the replacement comprises a replacement of an endogenous nucleotide sequence encoding a non-human MHC I peptide binding domain or a non-human MHC I extracellular domain with a human nucleotide sequence (e.g., HLA-A2 or HLA-B27 nucleotide sequence) encoding the same. In this embodiment, the replacement does not comprise a replacement of an MHC I sequence encoding transmembrane and/or cytoplasmic domains of a non-human MHC I polypeptide (e.g., H-2K or H-2D polypeptide). Thus, the non-human animal contains chimeric human/non-human nucleotide sequence at an endogenous non-human MHC I locus, and expresses chimeric human/non-human MHC polypeptide from the endogenous non-human MHC I locus.
A chimeric human/non-human polypeptide may be such that it comprises a human or a non-human leader (signal) sequence. In one embodiment, the chimeric polypeptide comprises a leader sequence of a human MHC I protein, e.g., HLA-A2 protein (for instance, HLA-A2.1 leader sequence). Thus, the nucleotide sequence encoding the chimeric MHC I polypeptide may be operably linked to a nucleotide sequence encoding a human MHC I leader sequence.
In another embodiment, the chimeric polypeptide comprises a non-human leader sequence of an MHC I protein, e.g., a non-human leader sequence of an endogenous MHC I protein. In one embodiment, the chimeric polypeptide comprises a leader sequence of a non-human MHC I protein, e.g., mouse H-2D protein (for instance, mouse H-2D1 leader sequence). Thus, the nucleotide sequence encoding the chimeric MHC I polypeptide may be operably linked to a nucleotide sequence encoding a non-human MHC I leader sequence.
A chimeric human/non-human MHC I polypeptide may comprise in its human portion a complete or substantially complete extracellular domain of a human MHC I polypeptide. Thus, the human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more of the amino acids encoding an extracellular domain of a human MHC I polypeptide (e.g., HLA-A2 polypeptide, HLA-B27 polypeptide). In one example, substantially complete extracellular domain of the human MHC I polypeptide lacks a human MHC I leader sequence. In another example, the chimeric human/non-human MHC I polypeptide comprises a human MHC I leader sequence.
Moreover, the chimeric MHC I polypeptide may be expressed under the control of endogenous non-human regulatory elements, e.g., rodent MHC I regulatory animals. Such arrangement will facilitate proper expression of the chimeric MHC I polypeptide in the non-human animal, e.g., during immune response in the non-human animal.
The genetically modified non-human animal may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.
In a specific embodiment, the non-human animal is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and another aforementioned strain.
In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
Thus, in one embodiment, the invention relates to a genetically modified mouse that comprises in its genome a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises a peptide binding domain or an extracellular domain (e.g., a functional peptide binding or extracellular domain) of a human MHC I (e.g., human HLA-A or HLA-B, e.g., human HLA-A2 or HLA-B27 (e.g., human HLA-A2.1)). In some embodiments, the mouse does not express a peptide binding or an extracellular domain of an endogenous mouse polypeptide from its endogenous mouse locus. The peptide binding domain of the human MHC I may comprise α1 and α2 domains. Alternatively, the peptide binding domain of the human MHC I may comprise α1, α2, and α3 domains. In one aspect, the extracellular domain of the human MHC I comprises an extracellular domain of a human MHC I α chain. In one embodiment, the endogenous mouse locus is an H-2K or an H-2D locus (e.g., H-2Kb or H-2D1), and the mouse portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a mouse H-2K or H-2D polypeptide (e.g., H-2Kb or H-2D1), respectively.
Thus, in one embodiment, a genetically modified mouse is provided, wherein the mouse comprises at an endogenous H-2K (e.g., H-2Kb) locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. In one aspect, the mouse does not express an extracellular domain, e.g., a functional extracellular domain, of the mouse H-2K (e.g., H-2Kb) polypeptide from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-A2/H-2K (e.g., a chimeric HLA-A2.1/H-2Kb) polypeptide from an endogenous H-2K (e.g., H-2Kb) locus. In various embodiments, expression of the chimeric gene is under control of endogenous mouse MHC class I regulatory elements. In some aspects, the mouse comprises two copies of the chimeric MHC I locus containing a nucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide; while in other aspects, the mouse comprises one copy of the chimeric MHC I locus containing a nucleotide sequence encoding a chimeric HLA-A2/H-2K polypeptide. Thus, the mouse may be homozygous or heterozygous for the nucleotide sequence encoding the chimeric HLA-A2/H-2K polypeptide.
In some embodiments described herein, a mouse is provided that comprises a chimeric MHC I locus located at an endogenous mouse H-2K locus. The chimeric locus comprises a nucleotide sequence that encodes an extracellular domain of a human HLA-A2 protein, e.g., α1, α2, and α3 domains of a human HLA-A2 gene. The chimeric locus lacks a nucleotide sequence that encodes an extracellular domain of a mouse H-2K protein (e.g., α1, α2, and α3 domains of the mouse H-2K). In one aspect, the chimeric locus lacks a nucleotide sequence that encodes a leader peptide, α1, α2, and α3 domains of a mouse H-2K; and comprises a leader peptide, α1, α2, and α3 domains of a human HLA-A2, and transmembrane and cytoplasmic domains of a mouse H-2K. The various domains of the chimeric locus are operably linked to each other such that the chimeric locus expresses a functional chimeric human/mouse MHC I protein.
In another embodiment, a genetically modified mouse is provided, wherein the mouse comprises at an endogenous H-2D (e.g., H-2D1) locus a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-B (e.g., HLA-B27) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2D (e.g., H-2D1) polypeptide. In one aspect, the mouse does not express an extracellular domain, e.g., a functional extracellular domain, of the mouse H-2D polypeptide from an endogenous MHC I locus. In one embodiment, the mouse expresses a chimeric HLA-B27/H-2D polypeptide from an endogenous H-2D locus. In various embodiments, expression of the chimeric gene is under control of endogenous mouse MHC class I regulatory elements. In some aspects, the mouse comprises two copies of the chimeric MHC I locus containing a nucleotide sequence encoding a chimeric HLA-B27/H-2D polypeptide; while in other aspects, the mouse comprises one copy of the chimeric MHC I locus containing a nucleotide sequence encoding a chimeric HLA-B27/H-2D polypeptide. Thus, the mouse may be homozygous or heterozygous for the nucleotide sequence encoding the chimeric HLA-B27/H-2D polypeptide.
In some embodiments described herein, a mouse is provided that comprises a chimeric MHC I locus located at an endogenous mouse H-2D locus. The chimeric locus comprises a nucleotide sequence that encodes an extracellular domain of a human HLA-B27 protein, e.g., α1, α2, and α3 domains of a human HLA-B27 gene. The chimeric locus lacks a nucleotide sequence that encodes an extracellular domain of a mouse H-2D protein (e.g., α1, α2, and α3 domains of the mouse H-2D). In one aspect, the chimeric locus lacks a nucleotide sequence that encodes α1, α2, and α3 domains of a mouse H-2D; and comprises α1, α2, and α3 domains of a human HLA-B27, and a leader sequence and transmembrane and cytoplasmic domains of a mouse H-2D. The various domains of the chimeric locus are operably linked to each other such that the chimeric locus expresses a functional chimeric human/mouse MHC I protein.
In various embodiments, a non-human animal, e.g., a rodent (e.g., a mouse or a rat), that expresses a functional chimeric MHC I protein from a chimeric MHC I locus as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expresses the chimeric MHC I protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the cell displays a peptide fragment (an antigen fragment) bound to an extracellular portion (e.g., human HLA-A2 or HLA-B27 extracellular portion) of the chimeric MHC I protein. In an embodiment, the extracellular portion of such chimeric protein interacts with other proteins on the surface of said cell, e.g., β2-microglobulin.
In various embodiments, a cell displaying the chimeric MHC I protein, e.g., HLA-A2/H-2K protein or HL-B27/H-2D protein, is a nucleated cell. In various aspects, the cell is an antigen-presenting cell (APC). Although most cells in the body can present an antigen in the context of MHC I, some nonlimiting examples of antigen presenting cells include macrophages, dendritic cells, and B cells. Other antigen presenting cells, including professional and nonprofessional APCs, are known in the art, and are encompassed herein. In some embodiments, the cell displaying the chimeric MHC I protein is a tumor cell, and a peptide fragment presented by the chimeric protein is derived from a tumor. In other embodiments, the peptide fragment presented by the chimeric MHC I protein is derived from a pathogen, e.g., a bacterium or a virus.
The chimeric MHC I protein described herein may interact with other proteins on the surface of the same cell or a second cell. In some embodiments, the chimeric MHC I protein interacts with endogenous non-human proteins on the surface of said cell. The chimeric MHC I protein may also interact with human or humanized proteins on the surface of the same cell or a second cell.
On the same cell, HLA class I molecules may functionally interact with both non-human (e.g., rodent, e.g., mouse or rat) and human β2-microglobulin. Thus, in one embodiment, the chimeric MHC I protein, e.g., HLA-A2/H-2K or HLA-B27/H-2D protein, interacts with a mouse β2-microglobulin. Although interaction between some human HLA class I molecules and mouse β2-microglobulin is possible, it nevertheless may be greatly reduced in comparison to interaction between human HLA class I and human β2-microglobulin. Thus, in the absence of human β2-microglobulin, expression of human MHC I on the cell surface may be reduced. Perarnau et al. (1988) Human β2-microglobulin Specifically Enhances Cell-Surface Expression of HLA Class I Molecules in Transfected Murine Cells, J. Immunol. 141:1383-89. Other HLA molecules, e.g., HLA-B27, do not interact with mouse β2-microglobulin; see, e.g., Tishon et al. (2000) Transgenic Mice Expressing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection, Virology 275:286-293, which reports that HLA-B27 function in transgenic mice requires both human β2-microglobulin and human CD8. Therefore, in another embodiment, the chimeric MHC I protein interacts with a human or humanized β2-microglobulin. In some such embodiments, as described herein below, the non-human animal, e.g., a rodent (e.g., a mouse or a rat), comprises in its genome a human or humanized β2-microglobulin gene, and the animal expresses a functional human or humanized β2-microglobulin polypeptide; therefore, the chimeric MHC I protein interacts with a human or humanized β2-microglobulin polypeptide.
In various aspects, the chimeric protein (e.g., HLA-A2/H-2K or HLA-B27/H-2D protein) also interacts with proteins on the surface of a second cell (through its extracellular portion). The second cell may be a cell of a non-human, e.g., a mouse, or a human origin. The second cell may be derived from the same non-human animal or the same non-human animal specie as the cell expressing the chimeric MHC I polypeptide. Nonlimiting examples of proteins with which the extracellular portion of a chimeric protein (e.g., HLA-A2/H-2K or HLA-B27/H-2D) may interact include T cell receptor (TCR) and its co-receptor CD8. Thus, a second cell may be a T cell. In addition, the extracellular portion of the chimeric MHC I protein may bind a protein on the surface of Natural Killer (NK) cells, e.g., killer immunoglobulin receptors (KIRs) on the surface of NK cells.
A T cell or NK cell may bind a complex formed between the chimeric MHC I polypeptide and its displayed peptide fragment. Such binding may result in T cell activation or inhibition of NK-mediated cell killing, respectively. One hypothesis is that NK cells have evolved to kill either infected or tumor cells that have evaded T cell mediated cytotoxicity by downregulating their MHC I complex. However, when MHC I complex is expressed on cell surface, NK cell receptors recognize it, and NK-mediated cell killing is inhibited. Thus, in some aspects, when an NK cell binds a complex formed between the chimeric MHC I polypeptide (e.g., HLA-A2/H-2K or HLA-B27/H-2D polypeptide) and a displayed peptide fragment on the surface of infected or tumor cell, the NK-mediated cell killing is inhibited.
In one example, the chimeric MHC I polypeptide described herein, e.g., a chimeric HLA-A2/H-2K or HLA-B27/H-2D polypeptide, interacts with CD8 protein on the surface of a second cell. In one embodiment, the chimeric MHC I polypeptide, e.g., HLA-A2/H-2K or HLA-B27/H-2D polypeptide, interacts with endogenous rodent (e.g., mouse or rat) CD8 protein on the surface of a second cell. In one embodiment, the second cell is a T cell. In another embodiment, the second cell is engineered to express CD8. In certain aspects, the chimeric MHC I polypeptide, e.g., HLA-A2/H-2K or HLA-B27/H-2D polypeptide, interacts with a human CD8 on the surface of the second cell (e.g., a human cell or a rodent cell). In some such embodiments, the non-human animal, e.g., a mouse or a rat, comprises a human CD8 transgene, and the mouse or the rat expresses a functional human CD8 protein.
The chimeric MHC I polypeptide described herein may also interact with a non-human (e.g., a mouse or a rat) TCR, a human TCR, or a humanized TCR on a second cell. The chimeric MHC I polypeptide may interact with an endogenous TCR (e.g., mouse or rat TCR) on the surface of a second cell. The chimeric MHC I polypeptide may also interact with a human or humanized TCR expressed on the surface of a second cell, wherein the cell is derived from the same animal or animal specie (e.g., mouse or rat) as the cell expressing the chimeric MHC I polypeptide. The chimeric MHC I polypeptide may interact with a human TCR expressed on the surface of a human cell.
In addition to genetically engineered non-human animals, a non-human embryo (e.g., a rodent embryo, e.g., mouse or a rat embryo) is also provided, wherein the embryo comprises a donor ES cell that is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein. In one aspect, the embryo comprises an ES donor cell that comprises the chimeric MHC I gene, and host embryo cells.
Also provided is a tissue, wherein the tissue is derived from a non-human animal (e.g., a mouse or a rat) as described herein, and expresses the chimeric MHC I polypeptide (e.g., HLA-A2/H-2K polypeptide or HLA-B27/H-2D polypeptide).
In addition, a non-human cell isolated from a non-human animal as described herein is provided. In one embodiment, the cell is an ES cell. In one embodiment, the cell is an antigen-presenting cell, e.g., dendritic cell, macrophage, B cell. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is a lymphocyte.
Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.
In one aspect, a non-human induced pluripotent cell comprising gene encoding a chimeric MHC I polypeptide (e.g., HLA-A2/H-2K or HLA-B27/H-2D polypeptide) as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.
In one aspect, a hybridoma or quadroma is provided, derived from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.
Also provided is a method for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or a rat) described herein. The method for making a genetically engineered non-human animal results in the animal whose genome comprises a nucleotide sequence encoding a chimeric MHC I polypeptide. In one embodiment, the method results in a genetically engineered mouse, whose genome comprises at an endogenous MHC I locus, e.g., H-2K or H-2D locus, a nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric MHC I polypeptide comprises an extracellular domain of a human HLA-A2 or human HLA-B27 and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K or a mouse H-2D, respectively. In some embodiments, the method utilizes a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. In one embodiment, the ES cells are a mix of 129 and C57BL/6 mouse strains; in another embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.
Thus, a nucleotide construct used for generating genetically engineered non-human animals described herein is also provided. In one aspect, the nucleotide construct comprises: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human HLA-A or HLA-B gene sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human HLA-A or HLA-B gene. In one embodiment, the non-human homology arms are homologous to a non-human MHC class I locus (e.g., a mouse H-2K or H-2D locus).
In one embodiment, the genomic fragment comprises a human HLA-A (e.g., HLA-A2) leader, an α1 domain, an α2 domain and an α3 domain coding sequence. In one embodiment, the human DNA fragment comprises, from 5′ to 3′: an HLA-A leader sequence, an HLA-A leader/α1 intron, an HLA-A α1 exon, an HLA-A α1-α2 intron, an HLA-A α2 exon, an HLA-A α2-α3 intron, and an HLA-A α3 exon.
In another embodiment, the genomic fragment comprises a human HLA-B (e.g., HLA-B27) α1 domain, α2 domain and α3 domain coding sequence. Thus, the nucleotide sequence for generating genetically engineered animals may also comprise a non-human, e.g., a mouse, e.g., a mouse H-2D, leader sequence. In one embodiment, the human DNA fragment comprises, from 5′ to 3′: a human HLA-B27 α1 exon, an HLA-B27 α1-α2 intron, an HLA-B27 α2 exon, an HLA-B27 α2-α3 intron, and an HLA-B27 α3 exon.
A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Commonly, a selection cassette enables positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may be flanked by recombination sites, which allow deletion of the selection cassette upon treatment with recombinase enzymes. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, but others are known in the art.
In one embodiment, the selection cassette is located at the 5′ end the human DNA fragment. In another embodiment, the selection cassette is located at the 3′ end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located within the α2-α3 intron. In yet another embodiment, the selection cassette may be located within the non-human sequence that is a part of the sequence being inserted into the genome of the non-human animal.
In one embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence at 5′ and 3′ locations of an endogenous non-human (e.g., murine) MHC class I gene locus, respectively (e.g., 5′ of the first leader sequence and 3′ of the α3 exon of the non-human MHC I gene). In another embodiment, the 5′ non-human homology arm may comprise genomic sequence upstream of the α1 exon of the non-human MHC I gene, e.g., genomic sequence comprising the non-human leader sequence exon; in this embodiment, the chimeric human/non-human MHC I protein retains the non-human leader sequence. In one embodiment, the endogenous MHC class I locus is selected from mouse H-2K, H-2D and H-2L.
In a specific embodiment, the endogenous MHC class I locus is mouse H-2K. Thus, in one aspect, a nucleotide construct is provided, comprising, from 5′ to 3′; a 5′ homology arm containing mouse genomic sequence 5′ of the endogenous mouse H-2K locus, a first human DNA fragment comprising a first genomic sequence of an HLA-A gene, a 5′ recombination sequence site (e.g., loxP), a selection cassette, a 3′ recombination sequence site (e.g., loxP), a second human DNA fragment comprising a second genomic sequence of an HLA-A gene and a 3′ homology arm containing mouse genomic sequence 3′ of an endogenous H-2K α3 exon. In one embodiment, the nucleotide construct comprises, from 5′ to 3′: a 5′ homology arm containing mouse genomic sequence 5′ of the endogenous mouse H-2K locus, a human genomic sequence including an HLA-A leader, an HLA-A leader/a1 intron sequence, an HLA-A al exon, an HLA-A α1-α2 intron, an HLA-A α2 exon, a first 5′ portion of an α2-α3 intron, a selection cassette flanked by recombination sites, a second 3′ portion of an α2-α3 intron, an HLA-A α3 exon, and a 3′ homology arm containing non-mouse genomic sequence 3′ of the endogenous mouse H-2K α3 exon. In one embodiment, a 5′ homology arm sequence is set forth in SEQ ID NO:1, and a 3′ homology arm sequence is set forth in SEQ ID NO:2.
In another specific embodiment, the endogenous MHC class I locus is a mouse H-2D locus. Thus, in one aspect, a nucleotide construct is provided, comprising, from 5′ to 3′: a 5′ homology arm containing mouse genomic sequence 5′ of the endogenous mouse H-2D1 gene, a human DNA fragment comprising a genomic sequence of an HLA-B27 gene and a 3′ homology arm containing mouse genomic sequence 3′ of an endogenous H-D gene. In one embodiment, the nucleotide construct comprises, from 5′ to 3′: a 5′ homology arm containing mouse genomic sequence 5′ of the endogenous mouse H-2D gene including the leader sequence exon and the H-2D1 leader-α1 intron, an HLA-B27 α1 exon, an HLA-B27 α1-α2 intron, an HLA-B27 α2 exon, an HLA-B27 α2-α3 intron with a 5′ loxP site insertion, an HLA-B27 α3 exon, a mouse H-2D α3-transmembrane domain intron, a mouse transmembrane and cytoplasmic domain genomic sequence and a polyA tail, a 5′ FRT site, a hygromycin cassette, a 3′ FRT site, a 3′ loxP site and a 3′ homology arm containing genomic sequence downstream of the mouse H-2D gene. In one embodiment, a 5′ homology arm sequence spans mouse genomic sequence 49.8 kb upstream of endogenous mouse H-2D gene and includes the H-2D leader sequence, and a 3′ homology arm spans mouse genomic sequence 155.6 kb downstream of endogenous mouse H-2D gene.
Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) bearing the genetic modification of interest can be identified by screening for loss of mouse allele and/or gain of human allele using a modification of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.
The disclosure also provides a method of modifying an MHC I locus of a non-human animal to express a chimeric human/non-human MHC I polypeptide described herein. In one embodiment, the invention provides a method of modifying an MHC I locus of a mouse to express a chimeric human/mouse MHC I polypeptide wherein the method comprises replacing at an endogenous MHC I locus a nucleotide sequence encoding a peptide binding domain of a mouse MHC polypeptide with a nucleotide sequence encoding a peptide binding domain of a human MHC I polypeptide. In some aspects, a nucleotide sequence of an extracellular domain of a mouse MHC I is replaced by a nucleotide sequence of an extracellular domain of a human MHC I. The mouse may fail to express the peptide binding or the extracellular domain of the mouse MHC I from an endogenous MHC I locus. In one embodiment, a nucleotide sequence of an extracellular domain of a mouse H-2K is replaced by a nucleotide sequence of an extracellular domain of a human HLA-A2, such that the modified mouse MHC I locus expresses a chimeric HLA-A2/H-2K polypeptide. In one embodiment, a nucleotide sequence of an extracellular domain of a mouse H-2D is replaced by a nucleotide sequence of an extracellular domain of a human HLA-B27, such that the modified mouse MHC I locus expresses a chimeric HLA-B27/H-2D polypeptide.
In one aspect, a method for making a chimeric human HLA class I/non-human MHC class I molecule is provided, comprising expressing in a single cell a chimeric HLA-A/H-2K or HLA-B/H-2D protein from a nucleotide construct, wherein the nucleotide construct comprises a cDNA sequence that encodes an α1, α2, and α3 domain of an HLA-A or HLA-B protein, respectively, and a transmembrane and cytoplasmic domain of a non-human H-2K or H-2D protein, e.g., mouse H-2K or H-2D protein, respectively. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
In one aspect, a cell that expresses a chimeric human HLA Class I/non-human MHC I protein (e.g., HLA-A/H-2K or HLA-B/H-2D protein) is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric MHC class I gene, wherein the chimeric MHC class I gene comprises a sequence of a human HLA-A or HLA-B gene fused in operable linkage with a sequence of a non-human H-2K or H-2D gene, e.g., mouse H-2K or H-2D gene, respectively. In one embodiment, the sequence of the human HLA-A or HLA-B gene comprises the exons that encode α1, α2 and α3 domains of an HLA-A or HLA-B protein. In one embodiment, the sequence of the non-human H-2K or H-2D gene comprises the exons that encode transmembrane and cytoplasmic domains of an H-2K or H-2D protein, respectively. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).
A chimeric MHC class I molecule made by a non-human animal as described herein is also provided, wherein the chimeric MHC class I molecule comprises α1, α2, and α3 domains from a human HLA-A or HLA-B protein and transmembrane and cytoplasmic domains from a non-human, e.g., mouse, H-2K or H-2D protein. The chimeric HLA-A/H-2K polypeptide described herein maybe detected by anti-HLA-A antibodies. Thus, a cell displaying chimeric human/non-human HLA-A/H-2K polypeptide may be detected and/or selected using anti-HLA-A antibody. In some instances, the chimeric HLA-A2/H-2K polypeptide described herein maybe detected by an anti-HLA-A2 antibody. In another embodiment, the chimeric HLA-B/H-2D polypeptide described herein may be detected by anti-HLA-B antibodies; for example, the chimeric HLA-B27/H-2D polypeptide may be detected by anti-HLA-B27 antibodies.
Although the following Examples describe a genetically engineered animal whose genome comprises a replacement of a nucleotide sequence encoding an extracellular domain of mouse H-2K or H-2D polypeptide with the sequence encoding an extracellular domain of a human HLA-A2 at the endogenous mouse H-2K locus or human HLA-B27 at the endogenous H-2D locus, respectively, one skilled in the art would understand that a similar strategy may be used to replace other mouse MHC I loci (e.g., H-2L) with their corresponding human HLA loci (e.g., HLA-C). Thus, a non-human animal comprising in its genome a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide wherein a human portion of the polypeptide is derived from another HLA class I protein is also provided.
The replacement at multiple MHC I loci is also provided. Thus, also provided herein is a non-human animal, e.g., a rodent, e.g., a mouse, that comprises at an endogenous MHC I locus one or more, e.g., one, two, three, four, five, or six, nucleotide sequence(s) encoding a human or humanized MHC I polypeptide(s), e.g., a chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) MHC I polypeptide(s). In one instance, each of the two mouse sister chromosomes 17 contains an MHC I locus that comprises H-2K, H-2L, and H-2D genes; thus, in one aspect, each sister chromosome may encode up to three chimeric human/mouse polypeptides at their endogenous genomic positions. Therefore, in one embodiment, a genetically modified non-human animal, e.g., a mouse, may comprise up to six different nucleotide sequences encoding up to six human or humanized MHC I polypeptide(s), e.g., up to six chimeric human/non-human, e.g., human/mouse, MHC I polypeptides at their endogenous MHC loci. In another instance, each of the two mouse sister chromosomes 17 contains an MHC I locus that comprises H-2K and H-2D genes; thus, in one aspect, each sister chromosome may encode up to two chimeric human/mouse polypeptides at their endogenous genomic positions; and the genetically modified non-human animal, e.g., a mouse, may comprise up to four different nucleotide sequences encoding up to four human or humanized MHC I polypeptide(s).
In one embodiment, provided herein is a mouse comprising at an endogenous MHC I locus one or more, e.g., one, two, three, four, five, or six, nucleotide sequence(s) encoding a human or humanized MHC I polypeptide(s); e.g., chimeric human/mouse MHC I polypeptide(s), wherein a human portion of the chimeric polypeptide(s) comprises an extracellular domain of a human MHC I polypeptide and wherein a mouse portion of the chimeric polypeptide comprises a transmembrane and cytoplasmic domain of a mouse MHC I polypeptide, and wherein the mouse expresses one or more, e.g., one, two, three, four, five, or six, chimeric human/mouse MHC I polypeptide(s). In one embodiment, the mouse MHC I is selected from H-2D, H-2K, and H-2L. In one embodiment, the human MHC is selected from HLA-A, HLA-B, and HLA-C.
Thus, provided herein is a mouse that comprises at an endogenous MHC I locus two nucleotide sequences encoding a chimeric human/mouse MHC I polypeptides, wherein the two nucleotide sequences encode chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides, and wherein the mouse expresses the chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides. In one embodiment, the nucleotide sequences encoding HLA-A2/H-2K and HLA-B27/H-2D are located at endogenous H-2K and H-2D loci, respectively. In one aspect, the mouse does not express any functional endogenous mouse MHC I polypeptides. In another aspect, the mouse retains a nucleotide sequence encoding an endogenous mouse MHC I polypeptide, e.g., expresses a functional mouse MHC polypeptide (e.g., H-2L polypeptide).
Furthermore, provided herein is a method for generating a non-human animal, e.g., a mouse, comprising replacements at multiple endogenous MHC loci, e.g., a non-human, e.g., a mouse, comprising one or more, e.g., one, two, three, four, five, or six, nucleotide sequence(s) encoding a chimeric human/non-human, e.g., human/mouse, MHC I polypeptide(s). Due to close linkage of the various MHC I loci on mouse chromosome 17, in some embodiments, the methods comprise successive replacements at the locus. In one embodiment, the method comprises replacing a nucleotide sequence encoding a first mouse MHC I polypeptide with a nucleotide sequence encoding a first chimeric human/mouse MHC I polypeptide in an ES cell, generating a mouse expressing the first chimeric MHC I polypeptide, generating an ES cell from said mouse, replacing in said ES cell a nucleotide sequence encoding a second mouse MHC I polypeptide with a nucleotide sequence encoding a second chimeric human/mouse MHC I polypeptide, and generating a mouse expressing two chimeric human/mouse MHC I polypeptides. A mouse comprising a nucleotide sequence encoding a third chimeric human/mouse MHC I polypeptide can be generated in a similar fashion, by replacing a nucleotide sequence encoding a third mouse MHC I polypeptide with a nucleotide sequence encoding a third chimeric human/mouse MHC I polypeptide, performed in an ES cell comprising two chimeric MHC I genes. Alternatively, the method may comprise replacing a nucleotide sequence encoding a first mouse MHC I polypeptide with a nucleotide sequence encoding a first chimeric human/mouse MHC I polypeptide in an ES cell, followed by replacing in same ES cell a nucleotide sequence encoding a second mouse MHC I polypeptide with a nucleotide sequence encoding a second chimeric human/mouse MHC I polypeptide, and generating a mouse expressing two chimeric human/mouse MHC I polypeptides; a mouse comprising three chimeric human/mouse MHC I polypeptides can be generated in the same ES cell. The mouse comprising one, two, or three chimeric MHC I polypeptides generated by successive replacement may be heterozygous or homozygous for the chimeric MHC I sequences. A mouse comprising four, five, and six chimeric MHC I polypeptides may be generated by breeding two animals each comprising nucleotide sequences encoding chimeric MHC I polypeptides, resulting in an animal that, in one embodiment, is heterozygous for each of the chimeric MHC I sequences. One skilled in the art would understand that a mouse comprising two, three, or four chimeric MHC I polypeptides may also be generated by breeding rather than successive replacement; this animal may be heterozygous for all of the chimeric MHC I sequences (e.g., a mouse comprising a different chimeric gene on each of its sister chromosomes will be heterozygous for the two MHC genes, etc.).
Genetically Modified β2 Microglobulin Animals
The invention generally provides genetically modified non-human animals that comprise in their genome a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide; thus, the animals express a human or humanized β2 microglobulin polypeptide.
β2 microglobulin or the light chain of the MHC class I complex (also abbreviated “β2M”) is a small (12 kDa) non-glycosylated protein, that functions primarily to stabilize the MHC I α chain. The human β2 microglobulin gene encodes a protein of 119 amino acids, with 20 N-terminal amino acids encoding a leader sequence. The mature protein comprises 99 amino acids. The gene contains 4 exons, with the first exon containing the 5′ untranslated region, the entire leader sequence and the first two amino acids of the mature polypeptide; the second exon encoding the majority of the mature protein; the third exon encoding the last four amino acids of the mature protein and a stop codon; and the fourth exon containing the 3′ non-translated region. Gussow et al. (1987) The β2-Microglobulin Gene. Primary Structure and Definition of the Transcriptional Unit, J. Immunol. 139:3131-38. β2 microglobulin is non-covalently associated with MHC I. Unbound β2 microglobulin is found in body fluids, such as plasma, and is carried to the kidney for excretion. Kidney dysfunction causes accumulation of β2 microglobulin, which can be pathogenic (e.g., Dialysis Related Amyloidosis); the accumulated protein forms filamentous fibrils resembling amyloid plaques in joints and connective tissues.
In addition to Dialysis Related Amyloidosis, β2 microglobulin has been implicated in a number of other disorders. Elevated levels of β2 microglobulin were detected in lymphocytic malignancies, e.g., non-Hodgkin's lymphoma and multiple myeloma. See, e.g.; Shi et al. (2009) β2 Microglobulin: Emerging as a Promising Cancer Therapeutic Target, Drug Discovery Today 14:25-30. Some other malignancies with elevated levels of β2 microglobulin include breast cancer, prostate cancer, lung cancer, renal cancer, gastrointestinal and nasopharyngeal cancers. Overexpression of β2 microglobulin has been suggested to have tumor growth promoting effects. Id. It has also been recently shown that β2 microglobulin drives epithelial to mesenchymal transition, promoting cancer bone and soft tissue metastasis in breast, prostate, lung and renal cancers. Josson et al. (2011) β2 microglobulin Induces Epitelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells. Cancer Res. 71(7): 1-11. β2 microglobulin interacts with a non-classical MHC I member, hemochromatosis (HFE) protein, and with the transferrin receptor, and modulates iron homeostasis. Id. Involvement of β2 microglobulin in other hallmarks of malignancy (self-renewal, angiogenesis enhancement, resistance to treatment) is widely documented in the art.
Mice deficient in β2 microglobulin have been reported. See, Koller et al. (1990) Normal development of mice deficient in β2m, MHC class I proteins, and CD8+ T cells, Science 248: 1227-1230. As reported in Koller et al., these mice appeared healthy, however, MHC class I expression was not detected. Further, most T cell populations appeared normal in some tissues, while a marked decrease of CD8+ T cells was observed in others. This purported lack of MHC I expression disagrees with previous results obtained by Allen et al. ((1986) β2 microglobulin Is Not Required for Cell Surface Expression of the Murine Class I Histocompatibility Antigen H-2Db or of a Truncated H-2Db, Proc. Natl. Acad. Sci. USA 83:7447-7451). Allen et al. reported that β2 microglobulin was not absolutely required for cell surface expression of all MHC I complexes, because cells lacking β2 microglobulin were able to express H-2Db. However, the function of H-2Db in these cells was presumably compromised, and conformation of H-2Db was different from the native protein, which explains the inability of Koller and colleagues to detect this protein using antibodies against native H-2Db. However, cells lacking β2 microglobulin can reportedly present endogenous antigen to CD8+ T cells (including exogenous CD8+ T cells from normal mice), and β2 microglobulin is reportedly not required in order to develop high levels of H-2d MHC class I-restricted CD8+ CTLs in response to antigen challenge in mice, although it is required in order to sustain an effective immune response. Quinn et al. (1997) Virus-Specific, CD8+ Major Histocompatibility Complex Class I-Restricted Cytotoxic T Lymphocytes in Lymphocytic Choriomeningitis Virus-Infected β2-Microglobulin-Deficient Mice, J. Virol. 71:8392-8396. It is of note that the ability to generate high levels of such T cells in the absence of β2 microglobulin is reportedly limited to an H-2d MHC class I-restricted response. β2 microglobulin deficient mice have been reported to have a host of dramatic characteristics, such as, for example, an increased susceptibility to some parasitic diseases, an increased susceptibility to hepatitis infections, a deficiency in iron metabolism, and an impaired breeding phenotype. Cooper et al. (2007) An impaired breeding phenotype in mice with a genetic deletion of Beta-2 microglobulin and diminished MHC class I expression: Role in reproductive fitness, Biol. Reprod. 77:274-279.
Mice that express human β2 microglobulin as well as human HLA class I molecules (i.e., HLA-B7) on a randomly inserted transgene have been reported. Chamberlain et al. (1988) Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (β2-microglobulin) chain genes in transgenic mice, Proc. Natl. Acad. Sci. USA 85:7690-7694. The expression of human HLA class I was consistent with that of endogenous class I with a marked decrease in the liver. Id. The expression of human β2 microglobulin was also consistent with the endogenous β2 microglobulin, while expression of the human HLA class I molecule was increased 10- to 17-fold in double transgenic mice. Id. However, the authors did not attempt a replacement of a mouse endogenous β2 microglobulin locus with a human β2 microglobulin locus.
Therefore, disclosed herein is a genetically engineered non-human animal (e.g., a rodent, e.g., a mouse or a rat) whose genome comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the animal does not express an endogenous non-human β2 microglobulin from an endogenous non-human β2 microglobulin locus. In some embodiments, the nucleotide sequence encodes a β2 microglobulin polypeptide that is partially human and partially non-human, e.g., it contains some amino acids that correspond to human and some amino acids that correspond to non-human β2 microglobulin. In one aspect, the non-human animal does not express an endogenous non-human β2 microglobulin polypeptide from an endogenous non-human locus, and only expresses the human or humanized β2 microglobulin polypeptide. In one example, the non-human animal does not express a complete endogenous non-human β2 microglobulin polypeptide but only expresses a portion of a non-human endogenous β2 microglobulin polypeptide from an endogenous β2 microglobulin locus. Thus, in various embodiments, the animal does not express a functional non-human β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In a specific aspect, the nucleotide sequence encoding the human or humanized β2 microglobulin is located at an endogenous non-human β2 microglobulin locus. In one aspect, the animal comprises two copies of β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In another aspect, the animal comprises one copy of β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. Thus, the animal may be homozygous or heterozygous for β2 microglobulin locus comprising a nucleotide sequence that encodes a human or humanized β2 microglobulin polypeptide. The nucleotide sequence of the human or humanized β2 microglobulin may be derived from a collection of β2 microglobulin sequences that are naturally found in human populations. In various embodiments, the genetically engineered non-human animal of the invention comprises in its germline a nucleotide sequence encoding a human or humanized β2 microglobulin. In one embodiment, a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide comprises a nucleotide sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence. In one embodiment, the polypeptide is capable of binding to an MHC I protein.
The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin protein (i.e., amino acid residues corresponding to the mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin protein, for example, amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin protein. The nucleic and amino acid sequences of human β2 microglobulin are described in Gussow et al., supra, incorporated herein by reference.
Thus, the human or humanized β2 microglobulin polypeptide may comprise amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.
In some embodiments, the nucleotide sequence encoding a human or humanized β2 microglobulin comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to allow for normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises about 2.8 kb of a human β2 microglobulin gene.
Thus, the human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin, e.g., nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In a specific embodiment, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8 kb of a human β2 microglobulin gene. As exon 4 of the β2 microglobulin gene contains the 5′ untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.
It would be understood by those of ordinary skill in the art that although specific nucleic acid and amino acid sequences to generate genetically engineered animals are described in the present examples, sequences of one or more conservative or non-conservative amino acid substitutions, or sequences differing from those described herein due to the degeneracy of the genetic code, are also provided.
Therefore, a non-human animal that expresses a human β2 microglobulin sequence is provided, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human β2 microglobulin sequence. In a specific embodiment, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence described in the Examples. In one embodiment, the human β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence comprises one or more non-conservative substitutions.
In addition, provided are non-human animals wherein the nucleotide sequence encoding a human or humanized β2 microglobulin protein also comprises a nucleotide sequence set forth in exon 1 of a non-human β2 microglobulin gene. Thus, in a specific embodiment, the non-human animal comprises in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin wherein the nucleotide sequence comprises exon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of a non-human β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2 and 3 of a human β2 microglobulin gene).
Similarly to a non-human animal comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, the non-human animal comprising a nucleotide sequence encoding a human or humanized β2 microglobulin may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). In some embodiments of the invention, the non-human animal is a mammal. In a specific embodiment, the non-human animal is a murine, e.g., a rodent (e.g., a mouse or a rat). In one embodiment, the animal is a mouse.
Thus, in some aspects, a genetically engineered mouse is provided, wherein the mouse comprises a nucleotide sequence encoding a human or a humanized β2 microglobulin polypeptide as described herein. A genetically engineered mouse is provided, wherein the mouse comprises at its endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide (e.g., a human or substantially human β2 microglobulin polypeptide). In some embodiments, the mouse does not express an endogenous β2 microglobulin polypeptide (e.g., a functional endogenous β2 microglobulin polypeptide) from an endogenous β2 microglobulin locus. In some embodiments, the genetically engineered mouse comprises a nucleotide sequence comprising exon 1 of a mouse β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene. In some embodiments, the mouse expresses the human or humanized β2 microglobulin polypeptide.
In one aspect, a modified non-human β2 microglobulin locus is provided that comprises a heterologous β2 microglobulin sequence. In one embodiment, the heterologous β2 microglobulin sequence is a human or a humanized sequence.
In one embodiment, the modified locus is a rodent locus. In a specific embodiment, the rodent locus is selected from a mouse or rat locus. In one embodiment, the non-human locus is modified with at least one human β2 microglobulin coding sequence.
In one embodiment, the heterologous β2 microglobulin sequence is operably linked to endogenous regulatory elements, e.g., endogenous promoter and/or expression control sequence. In a specific embodiment, the heterologous β2 microglobulin sequence is a human sequence and the human sequence is operably linked to an endogenous promoter and/or expression control sequence.
In one aspect, a modified non-human β2 microglobulin locus is provided that comprises a human sequence operably linked to an endogenous promoter and/or expression control sequence.
In various aspects, the human or humanized β2 microglobulin expressed by a genetically modified non-human animal, or cells, embryos, or tissues derived from a non-human animal, preserves all the functional aspects of the endogenous and/or human β2 microglobulin. For example, it is preferred that the human or humanized β2 microglobulin binds the α chain of MHC I polypeptide (e.g., endogenous non-human or human MHC I polypeptide). The human or humanized β2 microglobulin polypeptide may bind, recruit or otherwise associate with any other molecules, e.g., receptor, anchor or signaling molecules that associate with endogenous non-human and/or human β2 microglobulin (e.g., HFE, etc.).
In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), also provided is a tissue or cell, wherein the tissue or cell is derived from a non-human animal as described herein, and comprises a heterologous β2 microglobulin gene or β2 microglobulin sequence, i.e., nucleotide and/or amino acid sequence. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or human or humanized β2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express MHC I complex, e.g., an antigen presenting cell. The human or humanized β2 microglobulin polypeptide expressed by said cell may interact with endogenous non-human MHC I (e.g., rodent MHC I), to form a functional MHC I complex. The resultant MHC I complex may be capable of interacting with a T cell, e.g., a cytotoxic T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a T cell.
Also provided are non-human cells that comprise human or humanized β2 microglobulin gene or sequence, and an additional human or humanized sequence, e.g., chimeric MHC I polypeptide presently disclosed. In such an instance, the human or humanized β2 microglobulin polypeptide may interact with, e.g., a chimeric human/non-human MHC I polypeptide, and a functional MHC I complex may be formed. In some aspects, such complex is capable of interacting with a TCR on a T cell, e.g., a human or a non-human T cell. Thus, also provided in an in vitro complex of a cell from a non-human animal as described herein and a human or a non-human T cell.
Another aspect of the disclosure is a rodent embryo (e.g., a mouse or a rat embryo) comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence as described herein. In one embodiment, the embryo comprises an ES donor cell that comprises the heterologous β2 microglobulin gene or β2 microglobulin sequence, and host embryo cells. The heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or β2 microglobulin sequence.
This invention also encompasses a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein (e.g., wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide). The non-human cell may comprise a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.
In one aspect, a non-human induced pluripotent cell comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized gene or sequence.
Also provided is a hybridoma or quadroma, derived from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.
The disclosure also provides methods for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or a rat) described herein. The methods result in an animal whose genome comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the methods result in a genetically engineered mouse, whose genome comprises at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some instances, the mouse does not express a functional mouse β2 microglobulin from an endogenous mouse β2 microglobulin locus. In some aspects, the methods utilize a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. In one embodiment, the ES cells are mix of 129 and C57BL/6 mouse strains; in another embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.
Also provided is a nucleotide construct used for generating genetically engineered non-human animals. The nucleotide construct may comprise: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human β2 microglobulin sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human β2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to a non-human β2 microglobulin locus. The genomic fragment may comprise exons 2, 3, and 4 of the human β2 microglobulin gene. In one instance, the genomic fragment comprises, from 5′ to 3′: exon 2, intron, exon 3, intron, and exon 4, all of human β2 microglobulin sequence. The selection cassette may be located anywhere in the construct outside the β2 microglobulin coding region, e.g., it may be located 3′ of exon 4 of the human β2 microglobulin. The 5′ and 3′ non-human homology arms may comprise genomic sequence 5′ and 3′ of endogenous non-human β2 microglobulin gene, respectively. In another embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence 5′ of exon 2 and 3′ of exon 4 of endogenous non-human gene, respectively.
Another aspect of the invention relates to a method of modifying a β2 microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide described herein. One method of modifying a β2 microglobulin locus of a mouse to express a human or humanized β2 microglobulin polypeptide comprises replacing at an endogenous β2 microglobulin locus a nucleotide sequence encoding a mouse β2 microglobulin with a nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide. In one embodiment of such method, the mouse does not express a functional β2 microglobulin polypeptide from an endogenous mouse β2 microglobulin locus. In some specific embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequence set forth in exons 2 to 4 of the human β2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequences set forth in exons 2, 3, and 4 of the human β2 microglobulin gene.
Genetically Modified MHC I/β2 Microglobulin Animals
In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome nucleotide sequences encoding both human or humanized MHC I and β2 microglobulin polypeptides; thus, the animals express both human or humanized MHC I and β2 microglobulin polypeptides.
Functional differences arise in the use of mixed human/non-human system components. HLA class I binds β2 microglobulin tighter than mouse class I. Bernabeu (1984) β2-microgobulin from serum associates with MHC class I antigens on the surface of cultured cells, Nature 308:642-645. Attempts to abrogate functional differences are reflected in the construction of particular humanized MHC mice. H-2 class I and class 2 knockout mice (in a mouse β2 microglobulin KO background) that express a randomly integrated human HLA-A2.1/HLA-DR1 chimeric transgene having an α1 and α2 of human HLA-A2.1, and α3 of mouse H-2Db, attached at its N-terminal via a linker to the C-terminus of human β2-microglobulin have been developed. See, e.g., Pajot et al. (2004) A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DRI-transgenic H-2 class I-/class I/-knockout mice, Eur. J. Immunol. 34:3060-3069. These mice reportedly generate antigen-specific antibody and CTL responses against hepatitis B virus, whereas mice merely transgenic for HLA-A2.1 or H-2 class I/class II knockout mice do not. The deficiency of mice that are merely transgenic for the genes presumably stems from the ability of such mice to employ endogenous class I and/or class II genes to circumvent any transgene, an option not available to MHC knockout mice. However, the mice may express at least H-2Db, presumably due to breedings into the mouse β2 microglobulin knockout mouse background (see, Pajot et al., supra; which apparently comprised an intact endogenous class I and class II locus).
Cell surface expression of the chimeric fusion with human β2 microglobulin is reportedly lower than endogenous MHC expression, but survivability/rate of NK killing is not reported, nor is the rate of NK self-killing. Pajot et al., supra. Some improvement in CD8+T cell numbers was observed over MHC class I-deficient β2-microglobulin knockout mice (2-3% of total splenocytes, vs. 0.6-1% in the β2 KO mice). However, T cell variable region usage exhibited altered profiles for BV 5.1, BV 5.2, and BV 11 gene segments. Both CD8+ and CD4+ T cell responses were reportedly restricted to the appropriate hepatitis B antigen used to immunize the mice, although at least two mice killed cells bearing either of the antigens, where the mice were immunized with only one antigen, which might be due to a lack of NK cell inhibition or lack of NK cell selectivity.
As mentioned above, mice transgenic for both human MHC I and human β2 microglobulin comprise a nucleotide sequence encoding a chimeric MHC I/β2 microglobulin protein, wherein the MHC I and β2 microglobulin portions are contained within a single polypeptide chain, resulting in MHC I α chain and β2 microglobulin being covalently linked to each other and thereby tethered at the cell surface. A mouse which comprises in its genome two independent nucleotide sequences, one encoding a human or humanized MHC I polypeptide and the other encoding a human or humanized β2 microglobulin polypeptide is provided. The mouse provided herein would express an MHC I complex that more closely resembles an MHC I complex present in nature, wherein MHC I α chain and β2 microglobulin are provided on two separate polypeptide chains with β2 microglobulin non-covalently associating with the MHC I α chain.
Thus, the present disclosure provides a non-human animal comprising in its genome: a first nucleotide sequence encoding a human or humanized MHC I polypeptide, and a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In one aspect, provided is a non-human animal comprising in its genome: (a) a first nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein the human portion of the chimeric polypeptide comprises a peptide binding domain or an extracellular domain of a human MHC I (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2 or HLA-B27), and (b) a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.
The first nucleotide sequence may be located at an endogenous non-human MHC I locus such that the animal comprises in its genome a replacement at the MHC I locus of all or a portion of endogenous MHC I gene (e.g., a portion encoding a peptide binding domain or an extracellular domain) with the corresponding human MHC I sequence. Thus, the animal may comprise at an endogenous MHC I locus a nucleotide sequence encoding an extracellular domain of a human MHC I (e.g., HLA-A, HLA-B, or HLA-C; e.g., HLA-A2 or HLA-B27) and transmembrane and cytoplasmic domains of endogenous non-human MHC I (e.g., H-2K, H-2D, H-2L, e.g., H-2K or H-2D). In one aspect, the animal is a mouse, and the first nucleotide sequence comprises a nucleotide sequence encoding an extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) and transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb). In another aspect, the animal is a mouse and the first nucleotide sequence comprises a nucleotide sequence encoding an extracellular domain of a human HLA-B27 and transmembrane and cytoplasmic domains of a mouse H-2D (e.g., H-2D1).
The second nucleotide sequence may be located at an endogenous non-human β2 microglobulin locus such that the animal comprises in its genome a replacement at the β2 microglobulin locus of all or a portion of endogenous β2 microglobulin gene with the corresponding human β2 microglobulin sequence. The second nucleotide sequence may comprise a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the second nucleotide sequence may comprise nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In this embodiment, nucleotide sequences are operably linked to each other. The second nucleotide sequence may further comprise the sequence of exon 1 of a non-human β2 microglobulin gene.
In one aspect, the animal does not express a functional MHC I from an endogenous non-human MHC I locus (e.g., does not express either a functional peptide binding domain or a functional extracellular domain of the endogenous MHC I); in one aspect, the animal does not express a functional β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In some aspects, the animal is homozygous for both an MHC I locus comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin. In other aspects, the animal is heterozygous for both an MHC I locus comprising a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and a β2 microglobulin locus comprising a nucleotide sequence encoding a human or humanized β2 microglobulin.
Preferably, the first and the second nucleotide sequences are operably linked to endogenous expression control elements (e.g., promoters, enhancers, silencers, etc.).
Various other embodiments of the first and second nucleotide sequences (and the polypeptides they encode) encompassed herein may be readily understood from the embodiments described throughout the specification, e.g., those described in the sections related to genetically engineered MHC I animals and genetically engineered β2 microglobulin animals.
In one aspect, the disclosure provides a mouse comprising in its genome (a) a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide (specifically, either HLA-A2/H-2K or HLA-B27/H-2D polypeptide), wherein the human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 or HLA-B27 and the mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K or mouse H-2D, respectively, and (b) a second nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide (e.g., wherein the nucleotide sequence comprises a nucleotide sequence set forth in exon 2 to exon 4 of the human β2 microglobulin gene or nucleotide sequences set forth in exon 2, 3, and 4 of the human β2 microglobulin gene), wherein the first nucleotide sequence is located at an endogenous H-2K or H-2D locus, and the second sequence is located at an endogenous β2 microglobulin locus. In one embodiment, the mouse does not express functional H-2K or H-2D and mouse β2 microglobulin polypeptides from their respective endogenous loci. In one embodiment, the mouse expresses both the chimeric human/mouse MHC I polypeptide and the human or humanized β2 microglobulin polypeptide.
As shown in the following Examples, animals genetically engineered to co-express both the human or humanized MHC I and β2 microglobulin displayed increased expression of chimeric MHC class I on cell surface in comparison to animals humanized for MHC I alone. In some embodiments, co-expression of human or humanized MHC I and β2 microglobulin increases cell surface expression of human or humanized MHC I by more than about 10%, e.g., more than about 20%, e.g., about 50% or more, e.g., about 70%, over the expression of human or humanized MHC I in the absence of human or humanized β2 microglobulin.
The disclosure also provides a method of making genetically engineered non-human animals (e.g., rodents, e.g., rats or mice) whose genome comprises a first and a second nucleotide sequence as described herein. The method generally comprises generating a first genetically engineered non-human animal whose genome comprises a first nucleotide sequence described herein (i.e., a human or humanized MHC I sequence), generating a second genetically engineered non-human animal whose genome comprises a second nucleotide sequence described herein (i.e., a human or humanized β2 microglobulin sequence), and breeding the first and the second animal to obtain progeny whose genome contains both nucleotide sequences. In one embodiment, the first and the second animal are heterozygous for the first and the second nucleotide sequence, respectively. In one embodiment, the first and the second animal are homozygous for the first and the second nucleotide sequence, respectively. In one embodiment, the first and second animals are generated through replacement of endogenous non-human loci with the first and the second nucleotide sequences, respectively. In one aspect, the first and the second animals are generated through utilization of constructs generated via VELOCIGENE® technology, and introducing targeted ES cell clones bearing such constructs into an embryo (e.g., a rodent embryo, e.g., a mouse or a rat embryo) via the VELOCIMOUSE® method.
In yet another aspect, the invention provides a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that comprises in its genome nucleotide sequence(s) encoding one or more (e.g., one, two, three, four, five, six) chimeric human/non-human (e.g., chimeric human/mouse) MHC I polypeptide(s) and a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. Various aspects of the chimeric human/non-human MHC I polypeptide(s) and the human or humanized β2 microglobulin are disclosed throughout the specification and would be clear from the disclosure to those skilled in the art. Methods for generating non-human animals comprising nucleotide sequence(s) encoding one or more (e.g., one, two, three, four, five, six) chimeric human/non-human (e.g., chimeric human/mouse) MHC I polypeptide(s) and a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide are also provided herein.
Use of Genetically Modified Animals
In various embodiments, the genetically modified non-human animals described herein make APCs with human or humanized MHC I and/or β2 microglobulin on the cell surface and, as a result, present peptides derived from cytosolic proteins as epitopes for CTLs in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in vaccine development; for identification of high affinity T cells to human pathogens or cancer antigens (i.e., T cells that bind to antigen in the context of human MHC I complex with high avidity), e.g., for use in adaptive T cell therapy; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC expression.
The MHC I complex binds peptides and presents them on cell surface. Once presented on the surface in the context of such a complex, the peptides are recognizable by T cells. For example, when the peptide is derived from a pathogen or other antigen of interest (e.g., a tumor antigen), T cell recognition can result in T cell activation, macrophage killing of cells bearing the presented peptide sequence, and B cell activation of antibodies that bind the presented sequence.
T cells interact with cells expressing MHC I complex through the peptide-bound MHC class I ectodomain and the T cell's CD8 ectodomain. CD8+ T cells that encounter APC's that have suitable antigens bound to the MHC class I molecule will become cytotoxic T cells. Thus, antigens that in the context of MHC class I bind with high avidity to a T cell receptor are potentially important in the development of treatments for human pathologies. However, presentation of antigens in the context of mouse MHC I is only somewhat relevant to human disease, since human and mouse MHC complexes recognize antigens differently, e.g., a mouse MHC I may not recognize the same antigens or may present different epitopes than a human MHC I. Thus, the most relevant data for human pathologies is obtained through studying the presentation of antigen epitopes by human MHC I.
Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the capacity of an antigen to initiate an immune response in a human, and for generating a diversity of antigens and identifying a specific antigen that may be used in human vaccine development.
In one aspect, a method for determining antigenicity in a human of a peptide sequence is provided, comprising exposing a genetically modified non-human animal as described herein to a molecule comprising the peptide sequence, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell that binds a sequence of the peptide presented by a chimeric human/non-human MHC I, or a humanized MHC I complex (comprising a chimeric human/non-human MHC I and a human or humanized β2 microglobulin) as described herein.
In one aspect, a method for determining whether a peptide will provoke a cellular immune response in a human is provided, comprising exposing a genetically modified non-human animal as described herein to the peptide, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell that binds a sequence of the peptide by a chimeric human/non-human MHC class I molecule as described herein. In one embodiment, the non-human animal following exposure comprises an MHC class I-restricted CD8+ cytotoxic T lymphocyte (CTL) that binds the peptide. In one embodiment, the CTL kills a cell bearing the peptide.
In one aspect, a method for identifying a human CTL epitope is provided, comprising exposing a non-human animal as described herein to an antigen comprising a putative CTL epitope, allowing the non-human animal to mount an immune response, isolating from the non-human animal an MHC class I-restricted CD8+ CTL that binds the epitope, and identifying the epitope bound by the MHC class I-restricted CD8+ CTL.
In one aspect, a method is provided for identifying an HLA class I-restricted peptide whose presentation by a human cell and binding by a human lymphocyte (e.g., human T cell) will result in cytotoxicity of the peptide-bearing cell, comprising exposing a non-human animal (or MHC class I-expressing cell thereof) as described herein to a molecule comprising a peptide of interest, isolating a cell of the non-human animal that expresses a chimeric human/non-human class I molecule that binds the peptide of interest, exposing the cell to a human lymphocyte that is capable of conducting HLA class I-restricted cytotoxicity, and measuring peptide-induced cytotoxicity.
In one aspect, a method is provided for identifying an antigen that generates a cytotoxic T cell response in a human, comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying the antigen bound by the HLA class I-restricted molecule.
In one embodiment, the antigen comprises a bacterial or viral surface or envelope protein. In one embodiment, the antigen comprises an antigen on the surface of a human tumor cell. In one embodiment, the antigen comprises a putative vaccine for use in a human. In one embodiment, the antigen comprises a human epitope that generates antibodies in a human. In another embodiment, the antigen comprises a human epitope that generates high affinity CTLs that target the epitope/MHC I complex.
In one aspect, a method is provided for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA class I-restricted immune response, e.g., HLA-A-restricted immune response (e.g., HLA-A2-restricted response) or HLA-B-restricted response (e.g., HLA-B27-restricted response), comprising exposing a mouse as described herein to the putative antigen and measuring an antigen-specific HLA class I-restricted, e.g., HLA-A- or HLA-B-restricted (e.g., HLA-A2-restricted or HLA-B27-restricted) immune response in the mouse.
In one embodiment, the putative antigen is selected from a biopharmaceutical or fragment thereof, a non-self protein, a surface antigen of a non-self cell, a surface antigen of a tumor cell, a surface antigen of a bacterial or yeast or fungal cell, a surface antigen or envelope protein of a virus.
In addition, the genetically engineered non-human animals described herein may be useful for identification of T cell receptors, e.g., high-avidity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. The method may comprise: exposing the non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, isolating from the non-human animal a T cell comprising a T cell receptor that binds the antigen presented by a human or humanized MHC I, and determining the sequence of said T cell receptor.
In one aspect, a method for identifying a T cell receptor variable domain having high affinity for a human tumor antigen is provided, comprising exposing a mouse comprising humanized MHC I α1, α2, and α3 domains (e.g., HLA-A2 or HLA-B27 α1, α2, and α3 domains) to a human tumor antigen; allowing the mouse to generate an immune response; and, isolating from the mouse a nucleic acid sequence encoding a T cell receptor variable domain, wherein the T cell receptor variable domain binds the human tumor antigen with a KD of no higher than about 1 nanomolar.
In one embodiment, the mouse further comprises a replacement at the endogenous mouse T cell receptor variable region gene locus with a plurality of unrearranged human T cell receptor variable region gene segments, wherein the unrearranged human T cell receptor variable region gene segments recombine to encode a chimeric human-mouse T cell receptor gene comprising a human variable region and a mouse constant region. In one embodiment, the mouse comprises a human CD8 transgene, and the mouse expresses a functional human CD8 protein.
T cell receptors having high avidity to tumor antigens are useful in cell-based therapeutics. T cell populations with high avidity to human tumor antigens have been prepared by exposing human T cells to HLA-A2 that has been mutated to minimize CD8 binding to the α3 subunit, in order to seelect only those T cells with extremely high avidity to the tumor antigen (i.e., T cell clones that recognize the antigen in spite of the inability of CD8 to bind α3). See, Pittet et al. (2003) α3 Domain Mutants of Peptide/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells, J. Immunol. 171:1844-1849. The non-human animals, and cells of the non-human animals, are useful for identifying peptides that will form a complex with human HLA class I that will bind with high avidity to a T cell receptor, or activate a lymphocyte bearing a T cell receptor.
Antigen/HLA class I binding to a T cell, or activation of a T cell, can be measured by any suitable method known in the art. Peptide-specific APC-T cell binding and activation are measurable. For example, T cell engagement of antigen-presenting cells that express HLA-A2 reportedly causes PIP2 to accumulate at the immunosynapse, whereas cross-linking MHC class I molecules does not. See, Fooksman et al. (2009) Cutting Edge: Phosphatidylinositol 4,5-Bisphosphate Concentration at the APC Side of the Immunological Synapse Is Required for Effector T Cell Function, J. Immunol. 182:5179-5182.
Functional consequences of the interaction of a lymphocyte bearing a TCR, and a class I-expressing APC, are also measurable and include cell killing by the lymphocyte. For example, contact points on the α2 subunit of HLA-A2 by CD8+ CTLs reportedly generate a signal for Fas-independent killing. HLA-A2-expressing Jurkat cells apoptose when contacted (by antibodies) at epitopes on the HLA-A2 molecule known (from crystallographic studies) to contact CD8, without any apparent reliance on the cytoplasmic domain. See, Pettersen et al. (1998) The TCR-Binding Region of the HLA Class I α2 Domain Signals Rapid Fas-Independent Cell Death: A Direct Pathway for T Cell-Mediated Killing of Target Cells? J. Immunol. 160:4343-4352. It has been postulated that the rapid killing induced by HLA-A2 α2 contact with a CD8 of a CD8+ CTL may primarily be due to this Fas-independent HLA-A2-mediated pathway (id.), as distinguished from TCR-independent α3 domain-mediated killing—which by itself can induce apoptosis (see, Woodle et al. (1997) Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway, J. Immunol. 158:2156-2164).
The consequence of interaction between a T cell and an APC displaying a peptide in the context of MHC I can also be measured by a T cell proliferation assay. Alternatively, it can be determined by measuring cytokine release commonly associated with activation of immune response. In one embodiment, IFNγ ELISPOT can be used to monitor and quantify CD8+ T cell activation.
As described herein, CD8+ T cell activation can be hampered in the genetically modified non-human animals described herein due to species-specific binding of CD8 to MHC I. For embodiments where a species-specific CD8 interaction is desired, a cell of a genetically modified animal as described herein (e.g., a rodent, e.g., a mouse or a rat) is exposed (e.g., in vitro) to a human cell, e.g., a human CD8-bearing cell, e.g., a human T cell. In one embodiment, an MHC class I-expressing cell of a mouse as described herein is exposed in vitro to a T cell that comprises a human CD8 and a T cell receptor. In a specific embodiment, the T cell is a human T cell. In one embodiment, the MHC class I-expressing cell of the mouse comprises a peptide bound to a chimeric human/mouse MHC I or a humanized MHC I complex (which includes human β2 microglobulin), the T cell is a human T cell, and the ability of the T cell to bind the peptide-displaying mouse cell is determined. In one embodiment, activation of the human T cell by the peptide-displaying mouse cell is determined. In one embodiment, an in vitro method for measuring activation of a human T cell by the peptide-displaying cell is provided, comprising exposing a mouse or a mouse cell as described herein to an antigen of interest, exposing a cell from said mouse or said mouse cell (presumably bearing a peptide derived from the antigen in complex with human or humanized MHC I) to a human T cell, and measuring activation of the human T cell. In one embodiment, the method is used to identify a T cell epitope of a human pathogen or a human neoplasm. In one embodiment, the method is used to identify an epitope for a vaccine.
In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic, comprising exposing a genetically modified animal as described herein to a putative human therapeutic (or e.g., exposing a human or humanized MHC I-expressing cell of such an animal to a peptide sequence of the putative therapeutic), exposing a cell of the genetically modified animal that displays a human or humanized MHC I/peptide complex to a T cell comprising a human T cell receptor and a CD8 capable of binding the cell of the genetically modified animal, and measuring activation of the human T cell that is induced by the peptide-displaying cell of the genetically modified animal.
In various embodiments, a complex formed between a human or humanized MHC class I-expressing cell from an animal as described herein is made with a T cell that comprises a human CD8 sequence, e.g., a human T cell, or a T cell of a non-human animal that comprises a transgene that encodes human CD8. Mice transgenic for human CD8 are known in the art. Tishon et al. (2000) Trangenic Mice Expressing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection, Virology 275(2):286-293; also, LaFace et al. (1995) Human CD8 Transgene Regulation of HLA Recognition by Murine T Cells, J. Exp. Med. 182:1315-1325.
In addition to the ability to identify antigens and antigen epitopes from human pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. For example, Takaki et al. ((2006) HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes, J. Immunol. 176:3257-65) describe the utility of NOD mice bearing HLA/β2 microglobulin monochain in identifying type 1 diabetes autoantigens. Also, the genetically modified animals of the invention can be used to study various aspects of human autoimmune disease. As some polymorphic alleles of human MHC I are known to be associated with development of certain diseases, e.g., autoimmune diseases (e.g., Graves' disease, myasthenia gravis, psoriasis, etc.; see Bakker et al. (2006) A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106:18680-85, both incorporated herein by reference), a genetically modified animal of the invention comprising a humanized MHC I locus including such an allele may be useful as an autoimmune disease model. In one embodiment, the disease allele is HLA-B27, and the disease is ankylosing spondylitis or reactive arthritis; thus, in one embodiment, the animal used for the study of these diseases comprises a human or humanized HLA-B27. Other human disease alleles are known, e.g., HLA class I alleles associated with HIV, Ebola infection, etc., and these alleles or combination of alleles may be useful in for disease model creation in a genetically modified animal described herein.
In addition, the genetically modified animals of the invention and the human or humanized HLA molecules expressed by the same can be used to test antibodies that block antigen presentation by human HLA molecules associated with human disease progression. Thus, provided herein is a method of determining whether an antibody is capable of blocking presentation of an antigen by an HLA molecule linked to a human disease, e.g., a human disease described above, comprising exposing a cell expressing a human or humanized HLA described herein to a test antibody and determining whether the test antibody is capable of blocking the presentation of the antigen by a human or humanized HLA to immune cells (e.g., to T cells), e.g., by measuring its ability to block human or humanized HLA-restricted immune response. In one embodiment, the method is conducted in an animal expressing the human or humanized HLA, e.g., an animal expressing disease-associated human or humanized HLA that serves as a disease model for the disease.
Other aspects of cellular immunity that involve MHC I complexes are known in the art; therefore, genetically engineered non-human animals described herein can be used to study these aspects of immune biology. For instance, binding of TCR to MHC class I is modulated in vivo by additional factors. Leukocyte immunoglobulin-like receptor subfamily B member (LILRB1, or LIR-1) is expressed on MHC Class I-restricted CTLs and down-regulates T cell stimulation by binding a specific determinant on the α3 subunit of MHC class I molecules on APCs. Structural studies show that the binding site for LIR-1 and CD8 overlap, suggesting that inhibitory LIR-1 competes with stimulatory CD8 for binding with MHC class I molecules. Willcox et al. (2003) Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor, Nature Immunology 4(9):913-919; also, Shirioshi et al. (2003) Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G, Proc. Natl. Acad. Sci. USA 100(15):8856-8861. LIR-1 transduces inhibitory signals through its (intracellular) immunoreceptor tyrosine-based inhibitory motif (ITIM). In NK cells, studies have shown that KIRs (inhibitory killer cell Ig-like receptors) lacking ITIMs (normally incapable of inhibition) can inhibit in the presence of LIR-1 (presumably through the LIR-1 ITIM) bound to the α3 domain of an MHC class I molecule (see, Kirwin et al. (2005) Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR-1) J. Immunol. 175:5006-5015), suggesting cooperation between LIR-1 bound to MHC class I and KIRs and thus a role for HLA α3 domain binding in modulating NK cell inhibition.
As described above, MHC molecules interact with cells that do not express a TCR. Among these cells are NK cells. NK cells are cytotoxic lymphocytes (distinguished from CTLs, or cytotoxic T lymphocytes) that play a central role in the cellular immune response, and in particular innate immunity. NK cells are the first line of defense against invading microorganisms, viruses, and other non-self (e.g., tumor) entities. NK cells are activated or inhibited through surface receptors, and they express CD8 but do not express TCRs. NK cells can interact with cells that express MHC class I, but interaction is through the CD8-binding α3 domain rather than the TCR-binding, peptide-bearing α1 and α2 domains. A primary function of NK cells is to destroy cells that lack sufficient MHC class I surface protein.
Cross-linking MHC class I molecules on the surface of human natural killer (NK) cells results in intracellular tyrosine phosphorylation, migration of the MHC class I molecule from the immunosynapse, and down-regulation of tumor cell killing. Rubio et al. (2004) Cross-linking of MHC class I molecules on human NK cells inhibits NK cell function, segregates MHC I from the NK cell synapse, and induces intracellular phosphotyrosines, J. Leukocyte Biol. 76:116-124.
Another function of MHC class I in NK cells is apparently to prevent self-killing. NK cells bear both activating receptor 2B4 and the 2B4 ligand CD48; MHC class I appears to bind 2B4 and prevent its activation by CD48. Betser-Cohen (2010) The Association of MHC Class I Proteins with the 284 Receptor Inhibits Self-Killing of Human NK Cells, J. Immunol. 184:2761-2768.
Thus, the genetically engineered non-human animals described herein can be used to study these non-TCR or non-CTL mediated processes and to design approaches for their modulation.
EXAMPLES
The invention will be further illustrated by the following nonlimiting examples. These Examples are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to, limit its scope in any way. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.
Example 1
Construction and Characterization of Genetically Modified HLA-A2 Mice
Example 1.1
Expression of HLA-A2/H-2K in MG87
A viral construct containing a chimeric HLA-A2/H-2K gene sequence (FIG. 4A) was made using standard molecular cloning techniques known to a skilled artisan in order to analyze chimeric human/mouse MHC I expression in transfected cells.
Briefly, a chimeric human HLA-A/mouse H-2K viral construct was made using the exon sequences encoding the α1, α2 and α3 domains of the α chain and cloning them in frame with the mouse coding sequences for the transmembrane and cytoplasmic domains from the H-2K gene (FIG. 4A, pMIG-HLA-A2/H2K). As illustrated in FIG. 4, the construct contained an IRES-GFP reporter sequence, which allowed for determining if the construct was able to express in cells upon transfection.
Viruses containing the chimeric construct described above were made and propagated in human embryonic kidney 293 (293T) cells. 293T cells were plated on 10 cm dishes and allowed to grow to 95% confluency. A DNA transfection mixture was prepared with 25 μg of pMIG-HLA-A2/H2K, pMIG-human HLA-A2, or pMIG-humanized β2 microglobulin, and 5 μg of pMDG (envelope plasmid), 15 μg of pCL-Eco (packaging construct without packaging signal Ψ), 1 mL of Opti-MEM (Invitrogen). Added to this 1 mL DNA mixture was 80 μL of Lipofectamine-2000 (Invitrogen) in 1 mL of Opti-MEM, which was previously mixed together and allowed to incubate at room temperature for 5 minutes. The Lipofectamine/DNA mixture was allowed to incubate for an additional 20 minutes at room temperature, and then was added to 10 cm dishes, and the plates were incubated at 37° C. Media from the cells was collected after 24 hours and a fresh 10 mL of R10 (RPMI 1640+10% FBS) media was added to the cells. This media exchange was repeated twice. After a total of four days, the collected media was pooled, centrifuged and passed through a sterile filter to remove cellular debris.
The propagated viruses made above were used to transduce MG87 (mouse fibroblast) cells. MG87 cells from a single T-75 flask were washed once with PBS. 3 mL of 0.25% Trypsin+EDTA was added to the cells and allowed to incubate at room temperature for three minutes. 7 mL of D10 (high glucose DMEM; 10% Fetal Bovine Serum) was added to the cells/trypsin mixture and transferred to a 15 mL tube to centrifuge at 1300 rpm for five minutes. After centrifuging the cells, the media was aspirated and the cells resuspended in 5 mL D10. Cells were counted and ˜3.0×105 cells were placed per well in a 6-well plate. pMIG-human HLA-A2 or pMIG-HLA-A2/H-2K either alone or with pMIG-humanized β2 microglobulin virus were added to the wells, with non-transduced cells as a control. Cells were incubated at 37° C. with 5% CO2 for 2 days. Cells were prepared for FACS analysis (using anti-HLA-A2 antibody, clone BB7.2) for HLA-A2 expression with or without β2 microglobulin.
The graphs (FIG. 4B), as well as the table summarizing the data obtained from the graphs (FIG. 4C) demonstrate that co-transduction with humanized β2 microglobulin increases the expression of human HLA-A2 or chimeric human/non-human HLA-A2/H-2K, as demonstrated by the shift of curves to the right.
Example 1.2
Engineering a Chimeric HLA-A2/H-2K Locus
The mouse H-2K gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659). DNA from mouse BAC clone RP23-173k21 (Invitrogen) was modified by homologous recombination to replace the genomic DNA encoding the α1, α2 and α3 domains of the mouse H-2K gene with human genomic DNA encoding the α1, α2 and α3 subunits of the human HLA-A gene (FIG. 5).
Briefly, the genomic sequence encoding the mouse the α1, α2 and α3 subunits of the H-2K gene is replaced with the human genomic DNA encoding the α1, α2 and α3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector comprising a hygromycin cassette flanked by IoxP sites with a 5′ mouse homology arm containing sequence 5′ of the mouse H-2K locus including the 5′ untranslated region (UTR; 5′ homology arm is set forth in SEQ ID NO: 1) and a 3′ mouse homology arm containing genomic sequence 3′ of the mouse H-2K α3 coding sequence β3′ homology arm is set forth in SEQ ID NO: 2).
The final construct for targeting the endogenous H-2K gene locus from 5′ to 3′ included (1) a 5′ homology arm containing ˜200 bp of mouse genomic sequence 5′ of the endogenous H-2K gene including the 5′UTR, (2) ˜1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the HLA-A*0201 leader/α1 intron, the HLA-A*0201 α1 exon, the HLA-A*0201 α1-α2 intron, the HLA-A*0201 α2 exon, ˜316 bp of the 5′ end of the α2-α3 intron, (3) a 5′ /oxP site, (4) a hygromycin cassette, (5) a 3′/oxP site, (6) ˜580 bp of human genomic sequence including ˜304 bp of the 3′ end of the α2-α3 intron, the HLA-A*0201 α3 exon, and (7) a 3′ homology arm containing ˜200 bp of mouse genomic sequence including the intron between the mouse H-2K α3 and transmembrane coding sequences (see FIG. 5 for schematic representation of the H-2K targeting vector). The sequence of 149 nucleotides at the junction of the mouse/human sequences at the 5′ of the targeting vector is set forth in SEQ ID NO: 3, and the sequence of 159 nucleotides at the junction of the human/mouse sequences at the 3′ of the targeting vector is set forth in SEQ ID NO:4. Homologous recombination with this targeting vector created a modified mouse H-2K locus containing human genomic DNA encoding the α1, α2 and α3 domains of the HLA-A*0201 gene operably linked to the endogenous mouse H-2K transmembrane and cytoplasmic domain coding sequences which, upon translation, leads to the formation of a chimeric human/mouse MHC class I protein.
The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create modified ES cells for generating mice that express a chimeric MHC class I protein on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, neutrophils). ES cells containing an insertion of human HLA sequences were identified by a quantitative TAQMAN™ assay. Specific primer sets and probes were designed for detecting insertion of human HLA sequences and associated selection cassettes (gain of allele, GOA) and loss of endogenous mouse sequences (loss of allele, LOA). Table 1 identifies the names and locations detected for each of the probes used in the quantitative PCR assays.
TABLE 1
Probes Used For Confirming Chimeric HLA-A2/H-2K Gene
Probe
Assay
Region Detected by Probe
Sequence
SEQ ID NO
HYG
GOA
Hygromycin cassette
ACGAGCGGGT TCGGCCCATT C
5
1665H1
GOA
Human HLA-A2
AGTCCTTCAG CCTCCACTCA
6
α2-α3 intron
GGTCAGG
1665H2
GOA
Human HLA-A2
TACCACCAGT ACGCCTACGA
7
α2 exon
CGGCA
5112H2
GOA
Human HLA-A2
CACTCTCTGGTACAGGAT
8
α2-α3 intron
The selection cassette may be removed by methods known by the skilled artisan. For example, ES cells bearing the chimeric human/mouse MHC class I locus may be transfected with a construct that expresses Cre in order to remove the “loxed” hygromycin cassette introduced by the insertion of the targeting construct containing human HLA-A*0201 gene sequences (See FIG. 5). The hygromycin cassette may optionally be removed by breeding to mice that express Cre recombinase. Optionally, the hygromycin cassette is retained in the mice.
Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) FO generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human HLA-A*0201 gene sequences.
Example 1.3
In Vivo Expression of Chimeric HLA-A/H-2K in Genetically Modified Mice
A heterozygous mouse carrying a genetically modified H-2K locus as described in Example 1.2 was analyzed for expression of the chimeric HLA-A/H-2K protein in the cells of the animal.
Blood was obtained separately from a wild-type and a HLA-A/H-2K chimeric heterozygote (A2/H2K) mouse. Cells were stained for human HLA-A2 with a phycoerythrin-conjugated (PE) anti-HLA-A antibody, and exposed to an allophycocyanin-conjugated anti-H-2Kb antibody for one hour at 4° C. Cells were analyzed for expression by flow cytometry using antibodies specific for HLA-A and H-2Kb. FIG. 6A shows the expression of H-2Kb and HLA-A2 in the wild-type and chimeric heterozygote, with chimeric heterozygote expressing both proteins. FIG. 6B shows expression of both the H-2Kb and the chimeric HLA-A2/H2K in the heterozygous mouse.
Example 2
Construction and Characterization of Genetically Modified β2 Microglobulin Mice
Example 2.1
Engineering a Humanized β2 Microglobulin Locus
The mouse β2 microglobulin (β2m) gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).
Briefly, a targeting vector was generated by bacterial homologous recombination containing mouse β2m upstream and downstream homology arms from BAC clone 89C24 from the RPCI-23 library (Invitrogen). The mouse homology arms were engineered to flank a 2.8 kb human β2m DNA fragment extending from exon 2 to about 267 nucleotides downstream of non-coding exon 4 (FIG. 7). A drug selection cassette (neomycin) flanked by recombinase recognition sites (e.g., IoxP sites) was engineered into the targeting vector to allow for subsequent selection. The final targeting vector was linearized and electroporated into a F1H4 mouse ES cell line (Valenzuela et al., supra).
Targeted ES cell clones with drug cassette removed (by introduction of Cre recombinase) were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing the humanized β2m gene were identified by screening for loss of mouse allele and gain of human allele using a modification of allele assay (Valenzuela et al., supra).
Example 2.2
Characterization of Humanized β2 Microglobulin Mice
Mice heterozygous for a humanized β2 microglobulin (β2m) gene were evaluated for expression using flow cytometry (FIGS. 8 and 9).
Briefly, blood was isolated from groups (n=4 per group) of wild type, humanized β2m, humanized MHC (i.e., human HLA) class I, and double humanized β2m and MHC class I mice using techniques known in art. The blood from each of the mice in each group was treated with ACK lysis buffer (Lonza, Walkersville, Md., USA) to eliminate red blood cells. Remaining cells were stained using fluorochrome conjugated anti-CD3 (17A2), anti-CD19 (1D3), anti-CD11 b (M1/70), anti-human HLA class I, and anti-human β2 microglobulin (2M2) antibodies. Flow cytometry was performed using BD-FACSCANTO™ (BD Biosciences).
Expression of human HLA class I was detected on cells from single humanized and double humanized animals, while expression of β2 microglobulin was only detected on cells from double humanized mice (FIG. 8). Co-expression of human β2m and human HLA class I resulted in an increase of detectable amount of human HLA class I on the cell surface compared to human HLA class I expression in the absence of human β2m (FIG. 9; mean fluorescent intensity of 2370 versus 1387).
Example 3
Immune Response to Flu an Epstein-Barr Virus (EBV) Peptides Presented by APCs from Genetically Modified Mice Expressing HLA-A2/H-2K and Humanized β2 Microglobulin
PBMCs from several human donors were screened for both HLA-A2 expression and their ability to mount a response to flu and EBV peptides. A single donor was selected for subsequent experiments.
Human T cells are isolated from PBMCs of the selected donor using negative selection. Splenic non-T cells were isolated from a mouse heterozygous for a chimeric HLA-A2/H-2K and heterozygous for a humanized β2-microglobulin gene, and a wild-type mouse. About 50,000 splenic non-T cells from the mice were added to an Elispot plate coated with anti-human IFNγ antibody. Flu peptide (10 micromolar) or a pool of EBV peptides (5 micromolar each) was added. Poly IC was added at 25 micrograms/well, and the wells were incubated for three hours at 37° C. at 5% CO2. Human T cells (50,000) and anti-human CD28 were added to the splenic non T cells and the peptides, and the wells were incubated for 40 hours at 37° C. at 5% CO2, after which an IFNγ Elispot assay was performed.
As shown in FIG. 10, human T cells were able to mount a response to flu and EBV peptides when presented by mouse APCs that expressed the chimeric HLA-A2/H-2K and humanized β2 microglobulin on their surface.
Example 4
Construction and Characterization of Genetically Modified HLA-B27 Mice
Example 4.1
Engineering a Chimeric HLA-B27/H-2D1 Locus
The mouse H-2D1 (Histocompatibility 2, D region locus 1) gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003), supra). DNA from mouse BAC clone bMQ300c10 (Invitrogen) was modified by homologous recombination to replace the genomic DNA encoding the α1, α2 and α3 domains of the mouse H-2D1 gene with human genomic DNA encoding the α1, α2 and α3 subunits of the human HLA-B27 gene (FIG. 11).
Briefly, the genomic sequence encoding the mouse the α1, α2 and α3 subunits of the H-2D1 gene is replaced with the human genomic DNA encoding the α1, α2 and α3 of the human HLA-B27 (subtypes B*2701-2759) gene in a single targeting event using a targeting vector comprising a 5′ mouse homology arm containing sequence 5′ of the mouse H-2D1 locus including the leader sequence exon and a 3′ mouse homology arm containing genomic sequence 3′ of the mouse H-2D1 polyA sequence.
The final construct for targeting the endogenous H-2D1 gene locus from 5′ to 3′ included (1) a 5′ homology arm containing 49.8 kb of mouse genomic sequence 5′ of the endogenous H-2D1 gene including the leader sequence exon and intron (2) 1.67 kb of human genomic sequence including the HLA-B27 α1 exon, the HLA-B α1-α2 intron, the HLA-B α2 exon, the HLA-B α2-α3 intron with a 5′ /oxP site insertion, the HLA-B α3 exon, (3) 1.8 kb of the mouse H-2D1 α3 intron, the H-2D1 transmembrane and cytoplasmic coding exons and polyA sequence, (4) a 5′ FRT site, (5) a hygromycin cassette, (6) a 3′ FRT site, (7) a 3′ IoxP site and (8) a 3′ homology arm containing 155.6 kb of mouse genomic sequence downstream of the mouse H-2D1 gene (see FIG. 11 for schematic representation of the H-2D1 targeting vector). The sequence of 199 nucleotides at the junction of the mouse/human sequences at the 5′ of the targeting vector is set forth in SEQ ID NO: 9, the sequence of 134 nucleotides comprising loxP insertion with its surrounding human sequences within the targeting vector is set forth in SEQ ID NO:10, the sequence of 200 nucleotides at the junction of the human/mouse sequences at the 3′ of the targeting vector is set forth in SEQ ID NO:11, the sequence at the 5′ junction of the mouse sequence and the FRT-HYG-FRT selection cassette is set forth in SEQ ID NO:12, and the sequence at the 3′ junction of the FRT-HYG-FRT cassette and the mouse sequence is set forth in SEQ ID NO:13. Homologous recombination with this targeting vector created a modified mouse H-2D1 locus containing human genomic DNA encoding the α1, α2 and α3 domains of the HLA-B27 gene operably linked to the endogenous mouse H-2D1 transmembrane and cytoplasmic domain coding sequences which, upon translation, leads to the formation of a chimeric human/mouse MHC class I protein.
The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create modified ES cells for generating mice that express a chimeric MHC class I protein on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, neutrophils). ES cells containing an insertion of human HLA sequences were identified by a quantitative TAQMAN™ assay. Specific primer sets and probes were designed for detecting insertion of human HLA sequences and associated selection cassettes (gain of allele, GOA) and loss of endogenous mouse sequences (loss of allele, LOA). Table 2 identifies the names and locations detected for each of the probes used in the quantitative PCR assays; these probes are schematically depicted in FIG. 11.
TABLE 2
Probes Used For Confirming Chimeric HLA-B27/H-2D Gene
Probe
Assay
Region Detected by Probe
Sequence
SEQ ID NO
HYG
GOA
Hygromycin cassette
ACGAGCGGGT TCGGCCCATTC C
5
936hTU
GOA
Human HLA-B27 α1
TGCAAGGCCAAGGCACAGACT
14
exon
936hTD
GOA
Human HLA-B27 α2-α3
TGCAAAGCGCCTGAATTTTCTGACTC
15
intron
5152TUP
LOA
Mouse H-2D1 α1 exon
CTCTGTCGGCTATGTGG
16
5152TDP
Retention
Mouse H-2D1 cytoplasmic
TGGTGGGTTGCTGGAA
17
region
The HLA-B27/H2-D1 allele may be induced to be conditionally deleted by crossing to a Cre deletor mouse strain. For example, mice bearing the chimeric human/mouse MHC class I locus may be crossed to transgenic mice that express Cre recombinase in specific cell lineage in order to remove the “Loxed” human HLA-B27a3 and mouse H2-D1 transmembrane and cytoplasmic regions flanked by the 5′ and 3′ loxP sites in the targeting construct (See FIG. 11).
The selection cassette may be removed by methods known by a skilled artisan. For example, ES cells bearing the chimeric human/mouse MHC class I locus may be transfected with a construct that expresses FIpO in order to remove the “FRTed” hygromycin cassette introduced by the insertion of the targeting construct containing human HLA-B27 gene sequences (See FIG. 11). The hygromycin cassette may optionally be removed by breeding to mice that express FIpO recombinase. Optionally, the hygromycin cassette is retained in the mice.
Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007), supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human HLA-B27 gene sequences.
Example 4.2
Expression of Chimeric HLA-827/H-2D1 in Genetically Modified Mice
Blood from mice heterozygous for both humanized B2M and chimeric HLA-B27/H-2D1 and their wild type littermates was collected in microtainer tubes with EDTA (BD). Blood was stained with 1 mg/ml of primary antibody (W6/32-PE, pan HLA-Class I antibody, Abcam; or anti-human b2m antibody) for 25 min at 4° C. followed by washing with FACS buffer and incubation with 1:300 dilution of APC-labeled Anti-Human IgG secondary antibody (Jackson Immunoresearch) for 20 min at 4° C. Red blood cells were lysed with 1-step Fix/lyse solution (eBiosciences) and cells were fixed and resuspended in 1× BD Stabilizing Fixative. Cells were acquired on a FACS Canto machine and data was analyzed using FlowJo software.
As depicted in FIG. 12, chimeric HLA-B27/H-2D1 protein and humanized B2M were expressed on blood cells of genetically engineered animals (as evidenced by detection by antibodies raised against human HLA-B27 and human B-2M), while their expression was not detected in wild-type animals.
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.
Entire contents of all non-patent documents, patent applications and patents cited throughout this application are incorporated by reference herein in their entirety.
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16015159
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Sep 7th, 2021 12:00AM
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Mar 18th, 2016 12:00AM
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https://www.uspto.gov?id=US11111314-20210907
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Non-human animals that select for light chain variable regions that bind antigen
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Non-human animals, cells, methods and compositions for making and using the same are provided, wherein the non-human animals and cells comprise an immunoglobulin heavy chain locus that includes unrearranged human immunoglobulin light chain gene segments and an immunoglobulin light chain locus that includes a single rearranged human light chain variable region nucleotide sequence. The unrearranged human light chain gene segments may be operably linked to a heavy chain constant region nucleotide sequence and the rearranged human immunoglobulin light chain variable region nucleotide sequence may be operably linked to a light chain constant region nucleotide sequence. Also provided are methods for obtaining nucleic acid sequences that encode immunoglobulin light chain variable domains capable of binding an antigen in the absence of a cognate variable domain, and expressing such nucleic acid sequences in a host cell, e.g., to generate a multispecific antigen-binding protein.
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11111314
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1. A mouse comprising in its germline genome:
(i) at an endogenous immunoglobulin (Ig) heavy chain locus, an unrearranged human Ig light chain variable kappa (Vκ) gene segment and an unrearranged human Ig light chain joining kappa (Jκ) gene segment operably linked to an endogenous Ig heavy chain constant region nucleic acid sequence comprising at least one intact Ig heavy chain constant region gene encoding a functional CH1 domain, wherein the at least one intact Ig heavy chain constant region gene is an Igμ gene, Igδ gene, Igγ gene, Igα gene or an Igε gene, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment rearrange in a B cell to form a hybrid sequence comprising a rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy κ chain constant region nucleic acid sequence;
(ii) at an endogenous Ig light chain κ locus, a human universal Ig light chain variable region nucleotide sequence comprising a single human Ig Vκ gene segment rearranged with a single human Ig Jκ gene segment operably linked to an endogenous Ig light chain κ constant region nucleic acid sequence;
wherein the mouse expresses an antigen-binding protein that comprises a human Ig hybrid chain derived from the rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy chain constant region nucleic acid sequence and a cognate light chain derived from the human universal Ig light chain variable region nucleotide sequence at the endogenous Ig light chain locus, wherein the human Ig hybrid chain comprises a human Ig light chain variable κ (hVκ/CHxULC) domain fused to an endogenous heavy chain constant IgM, IgD, IgG, IgE or IgA region comprising a functional CH1 domain, and wherein the cognate light chain comprises a human Ig light variable κ domain chain fused to an endogenous light chain κ constant domain.
2. A B cell expressing the antigen-binding protein obtained from the mouse of claim 1.
3. A method of making the mouse of claim 1, comprising modifying a genome of a mouse embryonic stem (ES) cell to comprise
(i) at an endogenous Ig heavy chain locus, an unrearranged human Ig Vκ gene segment and an unrearranged human Ig Jκ gene segment operably linked to an endogenous Ig heavy chain constant region nucleic acid sequence comprising at least one intact Ig heavy chain constant region gene encoding a functional CH1 domain, wherein the intact Ig heavy chain constant region gene is an Igμ gene, an Igδ gene, an Igγ gene, an Igα gene, or an Igε gene,
wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment rearrange in a B cell to form a hybrid sequence comprising a rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy chain constant region nucleic acid sequence; and
(ii) at an endogenous Ig light chain κ locus, a human universal Ig light chain variable region nucleotide sequence comprising a single human Ig Vκ gene segment rearranged with a single human Ig Jκ gene segment operably linked to an endogenous Ig light chain κ constant region nucleic acid sequence.
4. The method of claim 3, wherein modifying the genome of the mouse ES cell comprises
(i) replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with the unrearranged human Ig Vκ gene segment and Jκ gene segment, and
(ii) replacing endogenous Ig Vκ gene segments and Jκ gene segments at the endogenous Ig light chain κ locus with the human universal Ig light chain variable region nucleotide sequence.
5. A method of obtaining an hVκ/CHxULC domain or a nucleic acid encoding the hVκ/CHxULC domain, the method comprising
isolating from the mouse of claim 1 a cell expressing the nucleic acid that encodes the hVκ/CHxULC domain,
and obtaining from the cell the hVκ/CHxULC domain or the nucleic acid encoding the hVκ/CHxULC domain.
6. The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments, respectively, and wherein the pluralities of unrearranged human Ig Vκ ene segments and unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
7. The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
8. The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
9. The mouse of claim 1, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
10. The mouse of claim 1,
wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.
11. The mouse of claim 10, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
12. The mouse of claim 8, wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.
13. The mouse of claim 12, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
14. The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments.
15. The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments.
16. The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments.
17. The method of claim 4, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
18. The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments, respectively, and wherein the pluralities of unrearranged human Ig Vκ gene segments and unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
19. The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
20. The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.
21. The method of claim 5, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
22. The method of claim 5,
wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.
23. The method of claim 22, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
24. The method of claim 20, wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.
25. The mouse of claim 24, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.
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25
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application filed under 35 U.S.C. § 371 of PCT Application No. PCT/US2016/023289, filed 18 Mar. 2016, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/135,419, filed 19 Mar. 2015, which applications are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
An official copy of the sequence listing is submitted concurrently with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of 2016-03-28-1150WO01-CORRECTED-SEQ-LIST_ST25.txt, a creation date of Mar. 28, 2016, and a size of about 9 kilobytes. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF INVENTION
Provided herein are immunoglobulin light chain variable (VL/CHxULC) domains that are derived from a immunoglobulin hybrid chain that is cognate to a universal light chain, and that may bind antigen independently (e.g., in absence of) a cognate variable domain of the universal light chain, genetically modified non-human animals and cells that express VL/CHxULC domains, nucleic acids that encode VL/CHxULC domains, antigen-binding proteins (e.g., multispecific antigen-binding proteins) comprising one or more VL/CHxULC domains, and in vitro methods of generating antigen-binding protein (e.g., multispecific antigen-binding proteins) comprising one or more VL/CHxULC domains.
BACKGROUND
A number of promising novel diagnostics and therapies are biologics, commonly based on a traditional antibody format. However, traditional antibody-based design may be limited as antigen binding typically requires an antibody molecule that includes four polypeptides: two identical immunoglobulin heavy chains and two identical immunoglobulin light chains. The present invention encompasses the recognition that there remains a need for improvement and diversification of immunoglobulin-based therapeutic design.
SUMMARY
The present invention provides improved technologies for the development, production, and/or use of antigen-binding proteins based on immunoglobulin format. The present invention encompasses the recognition that conventional antibody-based format imposes certain constraints on the technology. For example, the present invention recognizes that requiring an antigen-binding site to be comprised of heavy and light chain variable domains can restrict the available affinity and/or specificity that can be achieved with respect to some antigenic determinants.
The present invention provides technologies that solve these problems. Among other things, the present invention provides genetically engineered non-human animals that express a “universal” or “common” immunoglobulin light chain variable domain and are useful, for example, in the development and/or production of novel antigen-binding protein formats. Moreover, the present invention surprisingly demonstrates that use of such an animal expressing a universal immunoglobulin light chain can direct selection of partner immunoglobulin chains whose variable domain binding characteristics can dominate within an antigen-binding site, even when the partner (or cognate) immunoglobulin chain's variable domain is a light chain variable domain. Thus, contrary to expectations in the art, the present invention demonstrates that it is possible to develop immunoglobulin light chain variable regions that determine or control specificity and/or affinity of antigen-binding sites in which they participate, e.g., that bind antigen when associated with a universal light chain variable domain and/or in the absence of, i.e., independently of, a cognate universal light chain variable domain.
Thus, in some embodiments, the present invention provides antigen-binding proteins, including multispecific antigen-binding proteins, comprising one or more imunoglobulin light chain variable domains that bind antigen when associated with a universal light chain variable domain and/or independently of a cognate universal light chain variable domain. Also provided are technologies, e.g., non-human animals and in vitro recombinant methods, for the development, production, and or use of such immunoglobulin light chain variable domain sequences in which antigen specificity and affinity results solely or primarily from, and/or resides solely or primarily in, immunoglobulin light chain variable domain diversity.
Various aspects and embodiments described herein are based in part on the surprising discovery that genetically modified non-human animals that express binding proteins that contain immunoglobulin light chain variable domains that are operably linked to a heavy chain constant region and immunoglobulin light chain variable domains encoded by a rearranged light chain variable gene sequence (e.g., a rearranged light chain VLJL sequence) can solve various problems recognized herein and/or can provide surprising results. Non-human animals whose genome includes (i) a hybrid immunoglobulin chain locus containing unrearranged human light chain gene segments (e.g., VL and JL gene segments) operably linked to a heavy chain constant region sequence, e.g., at an endogenous heavy chain locus; and (ii) an immunoglobulin light chain locus containing a rearranged immunoglobulin light chain variable sequence (e.g., a single rearranged immunoglobulin light chain variable region sequence, such as for example a universal light chain variable region sequence) operably linked to a light chain constant gene can focus the mechanisms of antibody diversification on the unrearranged (i.e., diversifiable) immunoglobulin light chain variable gene segment(s) operably linked to the heavy chain constant region. Upon rearrangement, the unrearranged human light chain gene segments form a light chain variable region gene sequence that is operably linked to a heavy chain constant region gene sequence to form a sequence that encodes a immunoglobulin hybrid chain, i.e., an immunoglobulin polypeptide comprising a light chain variable domain fused with a heavy chain constant region. Non-human animals with the genomes described herein are able to generate antigen-binding proteins comprising dimeric immunoglobulin hybrid chains, each associated with cognate universal light chains in typical tetrameric antibody format, wherein the immunoglobulin hybrid chains comprise a light chain variable domain that is cognate with the light chain variable domain of the universal light chain, e.g., a VL/CHxULC variable domain.
As shown herein, a light chain variable VL/CHxULC domain that is derived from an immunoglobulin hybrid chain (e.g., is encoded by a VL/JL gene sequence that encodes a variable domain of an immunoglobulin hybrid chain) and that is cognate to a universal light chain variable domain is capable of binding an antigen of interest in the presence or absence of the cognate universal light chain variable domain. The immunoglobulin hybrid chain from which the VL/CHxULC domain is derived is preferably somatically hypermutated and is not a single domain antibody, e.g., preferably has a heavy chain constant region that has an isotype selected from the group consisting of IgD, IgG, IgE and IgA and comprises a functional CH1 domain. Such a variable VL/CHxULC domain is also able to bind antigen when associated with a second and noncognate variable domain specific for a different epitope, and regardless of whether the variable VL/CHxULC domain is operably fused to a a heavy chain constant region or a light chain constant region.
Accordingly, provided herein are antigen-binding proteins comprising at least a first binding component comprising an immunoglobulin light chain variable domain, e.g., a light chain variable VL/CHxULC domain, wherein the VL/CHxULC domain is (1) derived from a immunoglobulin hybrid chain encoded by a light chain sequence operably linked to one or more heavy chain constant region genes, e.g., Igμ, Igδ, Igγ, Igα and/or Igε, each of which comprises a nucleotide sequence that encodes a functional CH1 domain, and (2) cognate to a universal light chain encoded by a rearranged light chain sequence operably linked to a light chain constant region gene.
In some embodiments, the a light chain variable VL/CHxULC domain is a VκOHxULC domain, e.g., is derived from and/or encoded by, a κ light chain variable region nucleotide sequence, e.g., a human κ light chain variable region nucleotide sequence, e.g., a Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, or Vκ7-3 gene segment sequence, which may be rearranged with a (human) Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 gene segment, or somatically hypermutated variant thereof. In some embodiments, the light chain variable VL/CHxULC domain is a VλOHxULC domain, e.g., is derived from and/or encoded by, a λ light chain variable region nucleotide sequence, e.g., a human λ light chain variable region nucleotide sequence, e.g., a Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 or Vλ4-69 gene segment sequence, which may be rearranged with a (human) Jλ1, Jλ2, Jλ3 or Jλ7 gene segment sequence, or a somatically hypermutated variant thereof.
Notably, rearrangement in a hybrid immunoglobulin locus of the unrearranged immunoglobulin VL and JL gene segments may result in a rearranged immunoglobulin light chain variable VL/CHxULC domain encoding gene sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions. In one embodiment, the N additions and/or the somatic mutations observed in the rearranged immunoglobulin light chain gene encoding a VL/CHxULC domain are 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or at least 5-fold more than the number of N additions and/or somatic mutations observed in a rearranged light chain variable sequence (derived from the same VL gene segment and the same JL gene segment) that is rearranged at an endogenous light chain locus. The increased N additions in the VL/CHxULC encoding gene sequence may encode a light chain variable VL/CHxULC domain having more amino acids in the CDR3 compared to a light chain variable VL domain encoded by a VL/JL gene sequence recombined at an endogenous light chain locus. Accordingly, in some embodiments, an antigen-binding protein provided herein comprises an immunoglobulin light chain variable VL/CHxULC domain, wherein the variable VL/CHxULC domain comprises a CDR3 having a length of 9, 10, 11, 12 or more amino acids. In some embodiments, the VL/CHxULC domain comprises a CDR3 that is 9 amino acids in length. In some embodiments, the VL/CHxULC domain comprises a CDR3 that is 10 amino acids in length. In some embodiments, the VL/CHxULC domain comprises a CDR3 that is 11 amino acids in length. In some embodiments, the VL/CHxULC domain comprises a CDR3 that is 12 amino acids in length.
In preferred embodiments, an antigen-binding protein as described herein is not a single domain binding protein, e.g., is not a heavy chain only binding protein. Accordingly, in some embodiments, a first binding component as described comprises a light chain variable VL/CHxULC domain fused to a constant region, e.g., a heavy chain constant region comprising at least a functional CH1 domain or a light chain constant domain, wherein the variable VL/CHxULC domain is derived from a immunoglobulin hybrid chain that (1) comprises a functional CH1 domain and (2) is cognate to a universal light chain, e.g., wherein the immunoglobulin light chain variable VL/CHxULC domain is a VκOHxULC or VλOHxULC domain (respectively encoded by a rearranged (human) Vλ/Jκ or Vλ/Jλ sequence) that is or was operably linked to a heavy chain constant region gene sequence encoding at least a functional CH1 domain, and thus, may comprise a CDR3 that is 9, 10, 11, 12 or more amino acids in length.
Accordingly, in some embodiments, an antigen-binding protein as described herein comprises at least a first binding component comprising an immunoglobulin light chain variable VL/CHxULC domain fused to a heavy chain constant region comprising at least a functional CH1 domain (e.g., the immunoglobulin light chain VL/CHxULC domain is fused to a CH1 domain capable of forming a disulfide bond with a light chain constant region) and optionally further comprising a hinge region, a CH2 domain, a CH3 domain, a CH4 domain or a combination thereof. In some embodiments, the heavy chain constant region is a non-human heavy chain constant region comprising at least a functional CH1 domain. In some embodiments, the non-human heavy chain constant region is a rodent (e.g., rat or mouse) or chicken heavy chain constant region comprising at least a functional CH1 domain. In some embodiments, the heavy chain constant region is a human heavy chain constant region comprising at least a functional CH1 domain. In some embodiments, the heavy chain constant region (or CH1 domain) has an isotype selected from the group consisting of IgM, IgD, IgG, IgE and IgA. In some embodiments, the variable VL/CHxULC domain is fused to an IgG heavy chain constant region (or CH1 domain) having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the variable VL/CHxULC domain is fused to a human and mutated IgG1, IgG2, or IgG4 heavy chain constant region (or CH1 domain) comprising a CH3 domain, wherein the mutation is in the CH3 domain of the IgG1, IgG2 or IgG4 heavy chain constant region and reduces or eliminates binding of the CH3 domain to Protein A, e.g., wherein the mutation is selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT numbering system, or (a′) 435R, and (b′) 435R and 436F in the EU numbering system. In some embodiments, the human and mutated heavy chain constant region is a human and mutated IgG1 constant region and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system. In some embodiments, the human heavy chain constant region is a human IgG2 constant region and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one or two modifications selected from the group consisting of 44S, 52N, 82I in the IMGT exon numbering system, or 348S, 392N and 422I in the EU numbering system. In other embodiments, the human heavy chain constant region is a human IgG4 constant region and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one to seven modifications selected from the group consisting of 15R, 44S, 52N, 57M, 69K, 79Q and 82I in the IMGT exon numbering system or 355R, 384S, 392N, 397M, 409K, 419Q and 422I in the EU numbering system and/or the modification 105P in the IGMT exon numbering system or 445P in the EU numbering system.
Additionally, in some embodiments, an antigen-binding protein as described herein comprises at least a first binding component comprising an immunoglobulin light chain variable VL/CHxULC domain fused to a light chain constant domain. In some embodiments, the light chain constant domain is a non-human light chain constant domain. In some embodiments, the non-human light chain constant domain is a rodent (e.g., rat or mouse) or chicken light chain constant domain. In some embodiments, the light chain constant domain is a human light chain constant domain. In some embodiments, the light chain constant domain is a light chain κ constant domain. In some embodiments, the light chain constant domain is a light chain λ constant domain.
In some embodiments, an immunoglobulin light chain variable VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain as described herein binds an antigen of interest in the absence of a cognate universal light chain. Accordingly, a first binding component as described herein may consist essentially or consist of the immunoglobulin light chain variable VL/CHxULC domain or the immunoglobulin light chain variable VL/CHxULC domain fused to a constant region, e.g., a heavy chain constant region comprising at least a functional CH1 domain or a light chain constant region, wherein the variable VL/CHxULC domain is derived from a immunoglobulin hybrid chain that is cognate to a universal light chain, e.g., the immunoglobulin light chain variable VL/CHxULC domain is encoded by a rearranged (human) Vκ/Jκ or Vλ/Jλ sequence that is or was operably linked to a heavy chain constant region gene sequence, and thus, may comprise a CDR3 that is 9, 10, 11, 12 or more amino acids in length.
In other embodiments, the first binding component further comprises a cognate universal light chain variable domain in association with the immunoglobulin light chain VL/CHxULC variable domain, wherein the variable VL/CHxULC domain is derived from a immunoglobulin hybrid chain (e.g., the immunoglobulin light chain variable VL/CHxULC domain is encoded by a rearranged (human) Vκ/Jκ or Vλ/Jλ sequence that is or was operably linked to a heavy chain constant region gene sequence, and thus, may comprise a CDR3 that is 9, 10, 11, 12 or more amino acids in length), and wherein the immunoglobulin hybrid chain is cognate to a universal light chain encoded by a rearranged VL/JL gene sequence and wherein the universal light chain variable domain is encoded by the rearranged VL/JL gene sequence or a somatically hypermutated variant thereof.
In some embodiments, the universal light chain variable domain is encoded by or derived from a κ sequence, e.g., a human κ sequence, e.g., a Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, or Vκ7-3 gene segment sequence rearranged with a (human) Jκ1, Jκ2, Jκ3, Jκ4, or Jκ5 gene segment sequence, or a somatically hypermutated variant thereof. In some embodiments, the universal light chain variable domain is encoded by or derived from a nucleotide sequence comprising a human Vκ1-39 gene segment sequence rearranged with a human Jκ5 gene segment sequence, or a somatically hypermutated variant thereof. In some embodiments, the universal light chain variable domain is encoded by or derived from a nucleotide sequence comprising a human Vκ3-20 gene segment sequence rearranged with a human Jκ1 gene segment sequence, or a somatically hypermutated variant thereof. In some embodiments, the universal light chain variable domain is encoded by or derived from a lambda sequence, e.g., a human Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 or Vλ4-69 gene segment sequence rearranged with a (human) Jλ1, Jλ2, Jλ3 or Jλ7 gene segment sequence, or a somatically hypermutated variant thereof. In some embodiments the universal light chain variable domain is encoded by or derived from a nucleotide sequence comprising a Vλ2-14 gene segment sequence rearranged with a Jλ3 gene segment sequence, or a somatically hypermutated variant thereof. In some embodiments the universal light chain variable domain is encoded by or derived from a nucleotide sequence comprising a Vλ2-14 gene segment sequence rearranged with a Jλ7 gene segment sequence, or a somatically hypermutated variant thereof.
A first binding component as described herein may comprise the immunoglobulin light chain variable VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain and the cognate universal light chain variable domain associated, e.g., linked, by a disulfide bond or a peptide linker. In some embodiments, a first binding component as described herein comprises a VL/CHxULC variable domain linked to a cognate universal light chain variable domain via a peptide linker, e.g., in an scFv-type format. In some embodiments, a first binding component as described herein comprises (i) an immunoglobulin light chain variable VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain fused to a heavy chain constant region comprising at least a functional CH1 domain, e.g., the immunoglobulin light chain variable VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain fused to the functional CH1 domain and (ii) a universal light chain variable domain fused to a light chain constant domain, wherein the CH1 domain is linked to the light chain constant domain by a disulfide bond or a peptide linker. In one embodiment, the VL/CHxULC variable domain is fused to heavy chain constant region comprising at least a functional CH1 domain (and optionally further comprising a hinge region, a CH2 domain, a CH3 domain, a CH4 domain, or a combination thereof), the universal light chain variable domain is fused to a light chain constant domain, and the functional CH1 domain is linked to the light chain constant domain by a disulfide bond. In another embodiment, the first binding component is in an scFab format, e.g., the VL/CHxULC variable domain is fused to heavy chain constant region comprising at least a functional CH1 domain, the universal light chain variable domain is fused to a light chain constant domain, and the functional CH1 domain is linked to the light chain constant domain by a peptide linker.
In some embodiments, a first binding component comprises a human VL/CHxULC (e.g., a human VκOHxULC or a human VλOHxULC) domain, optionally fused with a human heavy chain comprising a CH1 domain or a human light chain constant domain. In one embodiment, an antigen-binding protein provided herein consists essentially or consists of only a first binding component as described herein, wherein the first binding component binds an antigen of interest.
Also provided are an antigen-binding proteins that, in addition to comprising a first binding component comprising a VL/CHxULC (e.g., VκOHxULC or VλOHxULC) variable domain as described herein, further comprise a second binding component that comprises a second immunoglobulin variable domain that is derived from a heavy or hybrid chain, wherein both the VL/CHxULC variable domain of the first component and the second variable domain of second binding component may be, and preferably are, cognate to a universal light chain variable domain derived from, e.g., encoded by, an identical rearranged light chain variable region gene sequence. As such, any differences in the universal light chain variable domains to which the VL/CHxULC and second variable domains are respectively cognate are the result of somatic hypermutation(s), e.g., may be determined to have arisen from somatic hypermutation or affinity maturation processes.
An antigen-binding protein as provided herein may comprise first and second binding components as described herein, wherein the first and second binding components comprise identical VL/CHxULC variable domains, and wherein the antigen-binding protein is monospecific, e.g., may specifically bind a single epitope of interest.
In some embodiments, the first and second binding components are not identical, e.g., bind different epitopes, which may be on the same antigen or may be on different antigens. Accordingly, an antigen-binding protein as described herein may be a multi-specific antigen-binding protein and comprise a (i) first binding component comprising a first variable domain, e.g., a VL/CHxULC domain (e.g., VκOHxULC or VλOHxULC), specific for a first epitope; and (ii) a second binding component comprising a second variable domain specific for a second epitope, wherein the second variable domain is either a second VL/CHxULC domain or a VHxULC domain (a heavy chain variable domain derived from a heavy chain encoded by VHDJH gene sequence operably linked to a heavy chain constant region gene, wherein the heavy chain variable domain is cognate to a universal light chain variable domain), wherein the first and second epitopes are not identical, and wherein the first and second variable domains are each cognate to universal light chain variable domains that are derived from the same single rearranged light chain variable region gene sequence, and thus, are identical or are somatically hypermutated variants, e.g., differ in amino acid sequence only through somatic hypermutation. In some embodiments, the second variable domain is a second VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain that binds a second epitope that is different than the first epitope, although the first and second variable VL/CHxULC domains are cognate to universal light chain variable domains that are derived from the same single rearranged light chain variable region gene sequence, and thus, are identical or somatically hypermutated variants. In some embodiments, the second variable domain is an immunoglobulin heavy chain variable (VHxULC) domain that binds a second epitope different than the first epitope, wherein the VL/CHxULC variable domain of the first binding component and the VHxULC variable domain of the second binding component are cognate to universal light chain variable domains that are derived from same single rearranged light chain variable region gene sequence, and thus, are identical or somatically hypermutated variants. A multispecific antigen-binding protein provided herein comprising first and second binding components that are not identical may specifically bind more than one epitope of interest.
In some embodiments, wherein the second binding component comprises a second variable domain that is a VHxULC domain, the VH domain is encoded by a heavy chain variable region nucleotide sequence, e.g., a human heavy chain variable region nucleotide sequence, e.g., any human VH, D, and JH gene segment sequence present in the human repertoire, e.g., any human heavy chain variable gene segments described in IMGT database, www.imgt.org, or somatically hypermutated variants thereof.
Additionally, the first binding component and the second binding component may be associated by one or more peptide linkers, one or more disulfide bonds and/or one or more leucine zippers such that a multiple specific antigen-binding protein provided herein is in a form selected from the group consisting of a Fab-like structure, an scFab-like structure, a diabody-like structure, an scFv-like structure, an scFv-Fc like structure, an scFv-zipper like structure, or a tetrameric structure that is similar to a typical antibody that includes the cognate universal light chain. Accordingly, in some embodiments, either or both the VL/CHxULC (e.g., VκOHxULC or VλOHxULC) and second variable domains may be or may not be fused to a constant region (e.g., a heavy chain constant region comprising a functional CH1 domain or a light chain constant domain) and/or may or may not be associated with a cognate universal light chain variable domain.
In some embodiments, the variable VL/CHxULC (e.g., VκOHxULC or VλOHxULC) domain of the first component is linked to the second variable (VL/CHxULC or VHxULC) domain of second binding component by a peptide linker such that the antigen-binding protein may have a diabody-like structure, an scFv-like structure, an scFv-Fc like structure, or an scFv-zipper like structure. In some embodiments, (1) at least one of (a) the VL/CHxULC variable domain of the first component or (b) the second variable domain of second binding component is fused to a (non-human or human) heavy chain constant region comprising at least a functional CH1 domain (and optionally further comprising a hinge region, a CH2 domain, a CH3 domain, a CH4 domain, or a combination thereof) and (2) the other of (a) the VLICHxULC variable domain of the first component or (b) the second variable domain of second binding component is fused to a (non-human or human) light chain constant (CL) domain, wherein the CH1 domain is linked to the CL domain by a disulfide bond such that the antigen-binding protein has a Fab-like structure, or wherein the CH1 domain is linked to the CL domain by a peptide linker such that the antigen-binding protein may have a scFab-like structure.
In some embodiments, both the first VL/CHxULC (VκOHxULC or VλOHxULC) and second (VL/CHxULC or VHxULC) variable domains are respectively fused to a first and second heavy chain constant regions, wherein each of the first and second heavy chain constant regions respectively comprises a first functional CH1 domain and a second functional CH1 domain (each heavy chain constant region optionally further comprising a hinge region, a CH2 domain, a CH3 domain, a CH4 domain, or a combination thereof), and wherein the first and second heavy chain constant regions are linked, e.g., by a disulfide bond or a peptide linker. In some embodiments, at least one (or both) of the heavy chain constant regions is a non-human heavy chain constant region, e.g., a rodent (e.g., rat or mouse) or chicken heavy chain constant region. In some embodiments, at least one (or both) of the heavy chain constant regions is a human heavy chain constant region. In some embodiments, at least one (or both) of the heavy chain constant regions has an isotype selected from the group consisting of IgM, IgD, IgG, IgE and IgA. In some embodiments, at least one (or both) of the first variable VL/CHxULC and the second variable domains is fused to an IgG heavy chain constant region having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first variable VL/CHxULC and the second variable domains are fused to heavy chain constant regions having an identical isotype and/or subclass, but optionally, wherein the heavy chain constant regions differ in their affinity to Protein A. In some embodiments, wherein both the first variable VL/CHxULC and the second variable domains are fused to a human IgG1, IgG2, or IgG4 heavy chain constant region, only one of the first variable VL/CHxULC and the second variable domain is fused to a human IgG1, IgG2, or IgG4 heavy chain constant region comprising a mutation in the CH3 domain that reduces or eliminates binding of the CH3 domain to Protein A, e.g., a mutation selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT numbering system, or (a′) 435R, and (b′) 435R and 436F in the EU numbering system. In some embodiments, the human heavy chain constant region is a human IgG1 constant region and further comprises one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system. In some embodiments, the human heavy chain constant region is a human IgG2 constant region and further comprises one or two modifications selected from the group consisting of 44S, 52N, 82I in the IMGT exon numbering system, or 348S, 392N and 422I in the EU numbering system. In other embodiments, the human heavy chain constant region is a human IgG4 constant region and further comprises one to seven modifications selected from the group consisting of 15R, 44S, 52N, 57M, 69K, 79Q and 82I in the IMGT exon numbering system or 355R, 384S, 392N, 397M, 409K, 419Q and 422I in the EU numbering system and/or the modification 105P in the IGMT exon numbering system or 445P in the EU numbering system.
In some embodiments, the first and second binding components may each respectively further comprise a first and second universal light chain variable domain respectively fused to a first and second light chain constant (CL) domain, wherein the first and second CL domains are respectively linked, e.g., by a disulfide bond, to the first and second CH1 domains of the first and second heavy chain constant regions, wherein the first and second universal light chain variable domains are derived from same single rearranged light chain variable region gene sequence, and thus, are identical or somatically hypermutated variants.
In some embodiments, an antigen-binding protein as described herein comprises a first binding component comprising a human VL/CHxULC (VκOHxULC or VλOHxULC) domain, optionally fused with a human heavy chain comprising at least a CH1 domain or a human light chain constant domain, a second binding component comprising a second human VL/CHxULC or a human VHxULC domain, optionally fused with a human heavy chain comprising a CH1 domain or a human light chain constant domain, and optionally, a human universal light chain comprising a human universal light chain variable domain fused with a human light chain constant domain.
Non-human animals include, e.g., mammals and, in particular embodiments, rodents (e.g., mice, rats, or hamsters). In some embodiments, non-human animals include birds, e.g., chickens. The present invention provides non-human animals engineered to contain (e.g., in their germline genome and/or in genomes of their B cells) nucleic acid sequences as described herein and/or to express antigen-binding proteins (e.g., immunoglobulin chains and/or antibodies) as described herein, are provided by the present invention.
In some embodiments, the present invention particularly encompasses the recognition that it is desirable to engineer non-human animals to provide improved in vivo systems for the generation of immunoglobulin light chain domains in which antigen specificity and affinity is dominated by (e.g., results solely or primarily from, and/or resides solely or primarily in), immunoglobulin light chain variable domain diversity. In some embodiments, the present invention encompasses the recognition that it is desirable to engineer non-human animals to permit improved in vivo affinity maturation and/or selection for immunoglobulin light chain variable domains that bind antigen independent from an immunoglobulin heavy chain variable domain. In some embodiments, the present invention encompasses the recognition that non-human animals whose genome comprises unrearranged human light chain variable region gene segments operably linked to a heavy chain constant region and a rearranged human light chain variable region nucleic acid sequence are desirable, for example for use in selection immunoglobulin light chain variable domains (VκOHxULC or VλOHxULC) having some or all of the aforementioned characteristics.
In some embodiments, the present invention provides a non-human animal capable of generating a VL/CHxULC variable domain, wherein the non-human animal comprises in its germline genome (a) a first hybrid immunoglobulin locus, e.g., at an endogenous non-human heavy chain locus, comprising unrearranged (human) immunoglobulin light chain (VL and JL) gene segments capable of rearranging to form a rearranged (human) VL/JL gene sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene, wherein the rearranged human VL/JL gene sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence encodes a hybrid immunoglobulin chain; and (b) a second light chain immunoglobulin locus, e.g., at an endogenous non-human light chain locus, comprising a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence, wherein the rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence encodes a universal light chain, and wherein the non-human animal is capable of producing or does produce a cell, e.g., a lymphocyte, e.g., a B cell that expresses an antigen-binding protein comprising the immunoglobulin hybrid chain and the universal light chain, and wherein an immunoglobulin light chain variable domain of the immunoglobulin hybrid chain is a VL/CHxULC domain. In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, a non-human animal of the present invention is homozygous for the rearranged human immunoglobulin light chain variable region nucleotide sequence. In some embodiments, a non-human animal of the present invention is heterozygous for the rearranged human immunoglobulin light chain variable region nucleotide sequence. In some embodiments, a non-human animal of the present invention is homozygous for the hybrid immunoglobulin locus. In some embodiments, a non-human animal of the present invention is heterozygous for the hybrid immunoglobulin locus. In some embodiments, the unrearranged (human) immunoglobulin light chain variable gene segments are operably linked to a non-human heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact IgE gene, and an intact Igα gene. In some embodiments, the non-human heavy chain constant region nucleic acid sequence is a mouse, rat, or chicken heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the non-human animal is a rodent, and the unrearranged human immunoglobulin light chain variable gene segments are operably linked to a human heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a non-human light chain constant region nucleic acid sequence. In some certain embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a human light chain constant region nucleic acid sequence.
In some certain embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a lambda sequence.
In some embodiments, the second immunoglobulin locus is a light chain kappa locus. In some embodiments, the second immunoglobulin locus is a light chain lambda locus.
In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are Vκ and Jκ gene segments. In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are Vλ and Jλ gene segments.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human κ light chain variable region nucleotide sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human λ light chain variable region nucleotide sequence.
In some embodiments, the first locus comprises one or more unrearranged human immunoglobulin VL gene segments selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the first locus comprises unrearranged human immunoglobulin JL gene segments that include Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the Vκ gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is a (human germline) Vκ gene segment selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segment is selected from the group consisting of Vκ1-39 and Vκ3-20. In some embodiments, the Vκ gene segment is selected from the group consisting of a human germline Vκ1-39 gene segment and a human germline Vκ3-20 gene segment.
In some embodiments, the JL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5, e.g., a human germline Jκ1 gene segment, a human germline Jκ2 gene segment, a human germlin e Jκ3 gene segment, a human germline Jκ4 gene segment, and a human germline Jκ5 gene segment.
In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vκ1-39 and Jκ5. In some certain embodiments, the Vκ1-39 is rearranged with the Jκ5. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is set forth as SEQ ID NO: 1. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vκ3-20 and Jκ1. In some certain embodiments, the Vκ3-20 is rearranged with Jκ1. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is set forth as SEQ ID NO:2.
In some embodiments the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, each of which encodes a functional CH1 domain.
In some embodiments, the first locus comprises one or more unrearranged human immunoglobulin VL gene segments selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some embodiments, the first locus comprises unrearranged human immunoglobulin JL gene segment that include Jλ1, Jλ2, Jλ3 and Jλ7.
In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is a (human germline) Vλ gene segment selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some certain embodiments the VL gene segment is Vλ2-14, e.g., a human germline Vλ2-14 gene segment.
In some embodiments, the JL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3 and Jλ7.
In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ1. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is Vλ2-14Jλ2. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is Vλ2-14Jλ3. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is Vλ2-14Jλ7. In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cλ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, substantially all endogenous functional variable heavy chain VH, D, and JH gene segments are deleted from an endogenous immunoglobulin heavy chain locus of the non-human animal or rendered non-functional. In some embodiments, substantially all endogenous functional light chain VL and JL gene segments are deleted from an endogenous immunoglobulin light chain locus of the non-human animal or rendered non-functional.
In some embodiments, the non-human animal comprises an integrated Adam6a gene, an Adam6b gene, or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
In some embodiments, the first immunoglobulin locus comprises a plurality of copies of the rearranged human immunoglobulin light chain variable nucleotide sequence.
In some embodiments, the present invention provides a method of making a non-human animal, the method generally comprising modifying a germline genome of the non-human animal to comprise (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) capable of rearranging to form a rearranged human VL/JL gene sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence. In some embodiments, the method comprises (a) modifying a genome of a non-human animal to delete or render non-functional all or substantially all (i) endogenous functional immunoglobulin heavy chain VH, D, and JH gene segments and (ii) endogenous functional light chain VL and JL gene segments; (b) placing unrearranged human immunoglobulin VL and JL gene segments in the genome so that the unrearranged light chain variable gene segments are operably linked to a heavy chain constant region nucleic acid sequence; and (c) placing a rearranged human immunoglobulin light chain variable region nucleotide sequence in the genome so that the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a light chain constant region nucleic acid sequence. In some embodiments, the method comprises (a) replacing all endogenous functional immunoglobulin heavy chain VH, D, and JH gene segments at an endogenous heavy chain locus with unrearranged human immunoglobulin VL and JL gene segments so that the unrearranged light chain variable gene segments are operably linked to an endogenous heavy chain constant region nucleic acid sequence, and (b) replacing all endogenous functional light chain VL and JL gene segments at an endogenous light chain locus with a rearranged human immunoglobulin light chain variable region nucleotide sequence so that the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to an endogenous light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are operably linked to a non-human immunoglobulin heavy chain constant region nucleic acid sequence. In some embodiments, the non-human immunoglobulin heavy chain constant region nucleic acid sequence is a mouse or rat immunoglobulin heavy chain constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and the unrearranged human immunoglobulin light chain variable VL and JL gene segments are operably linked to a human heavy chain constant region nucleic acid sequence.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a non-human light chain constant region nucleic acid sequence. In some embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a human light chain constant region nucleic acid sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a lambda sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is placed in a kappa light chain locus. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is placed in a lambda light chain locus.
In some certain embodiments, the unrearranged human immunoglobulin VL and JL gene segments are Vκ and Jκ gene segments. In some certain embodiments, the unrearranged human immunoglobulin VL and JL gene segments are Vλ and Jλ gene segments.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human κ light chain variable region nucleotide sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human λ light chain variable region nucleotide sequence.
In some embodiments, the unrearranged human immunoglobulin VL gene segments include one or more of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the unrearranged human immunoglobulin JL gene segments include Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises VL and JL gene segments. In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region is a (human germline) Vκ gene segment selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segment is selected from the group consisting of Vκ1-39 and Vκ3-20. In some embodiments, the Vκ gene segment is a human germline Vκ gene segment, e.g., a human germline Vκ1-39 gene segment or a human germline Vκ3-20 gene segment. In some embodiments, the JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5, e.g., the group consisting of a human germline Jκ1 gene segment, a human germline Jκ2 gene segment, a human germline Jκ3 gene segment, a human germline Jκ4 gene segment, and a human germline Jκ5 gene segment. In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ1-39 and Jκ5 (e.g., the Vκ1-39 is rearranged with the Jκ5). In some embodiments, the rearranged immunoglobulin light chain variable region nucleotide sequence comprises the sequence set forth as SEQ ID NO: 1. In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ3-20 and Jκ1 (e.g., the Vκ3-20 is rearranged with the Jκ1). In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises the sequence set forth as SEQ ID NO:2.
In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, the non-human animal comprises one or more unrearranged human immunoglobulin VL gene segments selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some embodiments, the non-human animal comprises unrearranged human immunoglobulin JL gene segments that include Jλ1, Jλ2, Jλ3 and Jλ7.
In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is a (human germline) Vλ gene segment selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some certain embodiments, the VL gene segment is Vλ2-14, e.g., a human germline Vλ2-14 gene segment. In some embodiments, the JL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3 and Jλ7. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is Vλ2-14Jλ1. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ2. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ3. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ7.
In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cλ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is placed at an endogenous immunoglobulin light chain locus in the genome. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is present in a germline genome of the non-human animal. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is placed at an ectopic locus in the genome. In some embodiments, the non-human animal comprises a plurality of copies of the rearranged human immunoglobulin light chain variable region nucleotide sequence.
In some embodiments, the non-human animal comprises an Adam6a gene, an Adam6b gene or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
In some embodiments, the nucleic acid sequence encoding the universal light chain comprises one or more histidine codons that are not encoded by a corresponding human germline light chain variable gene segment.
In some embodiments, the present invention provides methods of using a genetically modified non-human animal provided herein or made according to a method disclosed herein, wherein the methods generally comprise isolating from the non-human animal a cell, e.g., a lymphocyte, e.g., a B cell, that expresses a hybrid immunoglobulin chain that comprises a VL/CHxULC domain fused to a heavy chain constant region, wherein the hybrid immunoglobulin chain is cognate to a universal light chain and/or obtaining from cell a nucleic acid encoding the VL/CHxULC domain of the hybrid immunoglobulin chain. In some embodiments, a method for obtaining a nucleic acid sequence that encodes an immunoglobulin light chain variable VL/CHxULC domain comprises (a) optionally immunizing a non-human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the non-human animal comprises in its genome (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence, and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence such that the non-human animal mounts an immune response; and isolating from the immunized non-human animal a cell that expresses a nucleic acid sequence that encodes a light chain variable VL/CHxULC domain that can bind the antigen and/or the nucleic acid sequence that encodes a light chain variable VL/CHxULC domain that can bind the antigen.
In some embodiments, the nucleic acid sequence that encodes the light chain variable VL/CHxULC domain that can bind the antigen is derived from the unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to a heavy chain constant region nucleic acid sequence.
In some embodiments, the isolating step is carried out via fluorescence-activated cell sorting (FACS) or flow cytometry. In some embodiments, the isolating step comprises obtaining from the immunized non-human animal a cell and obtaining from said cell the nucleic acid sequence that encodes the light chain VL/CHxULC domain that can bind the antigen, and wherein the cell is a lymphocyte. In some certain embodiments, the lymphocyte comprises natural killer cells, T cells, or B cells.
In some embodiments, the method further comprises fusing the lymphocyte with a cancer cell to form a hybridoma. In some certain embodiments, the cancer cell is a myeloma cell.
In some embodiments, the isolated nucleic acid sequence is fused with a nucleic acid sequence encoding an immunoglobulin constant region nucleic acid sequence.
In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiment, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the unrearranged human immunoglobulin light chain variable VL and JL gene segments are operably linked to a non-human immunoglobulin heavy chain constant region nucleic acid sequence. In some embodiments, the non-human immunoglobulin heavy chain constant region nucleic acid sequence is a mouse or rat immunoglobulin heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene.
In some embodiments, the non-human animal is a rodent, and the unrearranged human immunoglobulin light chain variable VL and JL gene segments are operably linked to a human heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a non-human light chain constant region nucleic acid sequence. In some embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a human light chain constant region nucleic acid sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a lambda sequence.
In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vκ and Jκ gene segments. In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vλ and Jλ gene segments.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human κ light chain variable region nucleotide sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human λ light chain variable region nucleotide sequence.
In some embodiments, the unrearranged human immunoglobulin VL gene segments include one or more of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the unrearranged human immunoglobulin JL gene segments include one or more of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region is a (human germline) Vκ gene segment selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segment is selected from the group consisting of Vκ1-39 (e.g., a human germline Vκ1-39 gene segment) and Vκ3-20 (e.g., a human germline Vκ3-20 gene segment). In some embodiments, the JL gene segment is selected from the group consisting of Jκ1 (e.g., a human germline Jκ1 gene segment), Jκ2 (e.g., a human germline Jκ2 gene segment), Jκ3 (e.g., a human germline Jκ3 gene segment), Jκ4 (e.g., a human germline Jκ4 gene segment), and Jκ5 (e.g., a human germline Jκ5 gene segment). In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ1-39 and Jκ5 (e.g., the Vκ1-39 is rearranged with the Jκ5, e.g., a human germline Vκ1-38 gene segment is rearranged with a human germline Jκ5 gene segment). In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a sequence set forth as SEQ ID NO:1. In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ3-20 and Jκ1 (e.g., the Vκ3-20 is rearranged with the Jκ1, e.g., a human germline Vκ3-20 gene segment rearranged with a human germline Jκ1 gene segment). In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a sequence set forth as SEQ ID NO:2. In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, the unrearranged human immunoglobulin VL gene segments include one or more of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some embodiments, the non-human animal comprises unrearranged human immunoglobulin JL gene segments that include Jλ1, Jλ2, Jλ3 and Jλ7.
In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is a (human germline) Vλ gene segment selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In some certain embodiments the VL gene segment is Vλ2-14, e.g., a human germline Vλ2-14 gene segment. In some embodiments, the JL gene segment in the rearranged human immunoglobulin light chain variable region nucleotide sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3 and Jλ7. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ1. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ2. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ3. In some certain embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises Vλ2-14Jλ7. In some embodiments, the non-human animal is a rodent and the light chain constant region nucleic acid sequence is a rat or a mouse Cλ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is at an endogenous immunoglobulin light chain locus in the genome. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is present in a germline genome of the non-human animal. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is integrated into a transcriptionally active locus in the genome. In some embodiments, the non-human animal comprises a plurality of copies of the rearranged human immunoglobulin light chain variable region nucleotide sequence.
In some embodiments, the non-human animal comprises an integrated Adam6a gene, an Adam6b gene or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
In some embodiments, the nucleic acid sequence encoding the universal light chain comprises one or more histidine codons that are not encoded by a corresponding human germline light chain variable gene segment.
In some embodiments, the present invention provides a method for making an antigen-binding protein that comprises a VL/CHxULC (VκOHxULC or VλOHxULC) domain, the method generally comprising expressing in a host cell a first nucleic acid comprising a nucleic acid sequence that encodes a VL/CHxULC domain, optionally operably linked with a heavy chain constant region gene comprising a functional CH1 domain encoding sequence or a light chain constant region gene, wherein the VL/CHxULC domain is cognate to a universal light chain variable domain, and wherein the antigen-binding protein is not a single domain antigen binding protein. In some embodiments, the nucleic acid sequence that encodes the VL/CHxULC domain is isolated from non-human animal comprising in its genome (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence, wherein the nucleic acid sequence that encodes the VL/CHxULC domain is derived from the unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the method further comprises (a) optionally immunizing a non-human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the non-human animal comprises in its genome (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) capable of rearranging to form a rearranged VL/JL gene sequence operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence, such that the non-human animal mounts an immune response to the epitope or immunogenic portion thereof prior to (b) isolating from the non-human animal a nucleic acid sequence that encodes a light chain variable domain that specifically binds the epitope or immunogenic portion thereof and is derived from the rearranged VL/JL gene sequence, which is operably linked to an immunoglobulin heavy chain constant region nucleic acid. Additional embodiments include methods comprising (c) employing the isolated nucleic acid sequence in an expression construct optionally operably linked to a human immunoglobulin constant region nucleic acid sequence; prior to (d) expressing the nucleic acid sequence or expression construct comprising same in a production cell line, e.g., a host cell, to obtain an antigen-binding protein.
In some embodiments, the method for making an antigen-binding protein that comprises a VL/CHxULC domain comprises co-expressing in a host cell (i) a first nucleic acid comprising a nucleic sequence that encodes a first binding component comprising a first variable domain, e.g., a VL/CHxULC domain, specific for a first epitope, optionally operably linked with a first heavy chain constant region gene comprising a functional CH1 domain encoding sequence or a first light chain constant region gene, and (ii) a second nucleic acid encoding a second component comprising a second variable domain specific for a second epitope, wherein the second variable domain is either a second VL/CHxULC domain or a VHxULCdomain, wherein the first and second epitopes are not identical, and wherein the first and second variable domains are each cognate to universal light chain variable domains that are derived from the same single rearranged light chain variable region gene sequence, and thus, are identical or are somatically hypermutated variants, e.g., differ in amino acid sequence only through somatic hypermutation.
Thus, in some embodiments, the method comprises (a) immunizing a second non human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the second non-human animal comprises in its genome (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence; and (ii) either unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) capable of rearranging to form a rearranged VL/JL gene sequence (that encodes the second VL/CHxULC domain of the second binding component) or unrearranged human immunoglobulin heavy chain variable region gene segments (VH, D and JH) capable of rearranging to form a rearranged VH/D/JH gene sequence (that encodes the VHxULC domain of the second binding component) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence such that the non-human animal mounts an immune response to the epitope or immunogenic portion thereof prior to (b) isolating from the non-human animal a second nucleic acid sequence that encodes the second VL/CHxULC or VHxULC domain that specifically binds the second epitope or immunogenic portion thereof. Additional embodiments include methods comprising (c) employing the isolated second nucleic acid sequence in an expression construct, optionally operably linked to a human immunoglobulin constant region nucleic acid sequence; prior to (d) expressing of the first and second nucleic acid sequences or expression construct(s) comprising same in a production cell line, e.g., a host cell, to obtain an antigen-binding protein, wherein the antigen binding protein is not a single domain antigen binding protein.
In additional embodiments, the methods further comprise co-expressing in the production host cell the first nucleic acid encoding a first VL/CHxULC domain (or expression construct comprising same), optionally the second nucleic acid encoding a second VL/CHxULC domain or VHxULC domain (or expression construct comprising same) and a nucleotide sequence comprising a rearranged VL/JL gene sequence that encodes a human universal light chain variable domain, or somatically hypermutated variant thereof, that is cognate to the VL/CHxULC domain and the optional second VL/CHxULC domain or VHxULC domain. In some embodiments, the nucleotide sequence encodes the universal light chain variable domain fused to a human light chain constant domain.
The first nucleic acid sequence, and either or both the second nucleic acid sequence and the nucleotide sequence comprising a rearranged VL/JL gene sequence encoding the human universal light chain variable domain, may be employed in the same or different expression constructs, wherein the one or more expression constructs express the antigen binding protein, e.g., the first binding component, and either or both the second binding component and universal light chain, in a format selected from the group consisting of a Fab-like structure, an scFab-like structure, a diabody like structure, an scFv-like structure, an scFv-Fc like structure, an scFv-zipper like structure, and a tetrameric structure that is similar to a typical antibody and that includes the cognate universal light chain. Accordingly, in some embodiments, either or both the first and second nucleic acid sequences may respectively encode the first variable VL/CHxULC and second variable (VL/CHxULC or VHxULC) domain fused or not fused to a constant region, e.g., a (human) heavy chain constant region comprising a functional CH1 domain or a (human) light chain constant domain.
In some embodiments, either or both first and second nucleic acid sequences comprise a heavy chain constant region nucleic acid that encodes a human heavy chain constant region having an isotype selected from the group consisting of IgM, IgD, IgG, Igε and IgA, e.g., an IgG heavy chain constant region having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first and second nucleic acid sequence encode the first variable VL/CHxULC and the second variable (VL/CHxULC or VHxULC) domain fused to heavy chain constant regions having an identical isotype and/or subclass, but optionally, wherein the heavy chain constant regions differ in their affinity to Protein A. In some embodiments, wherein both the first variable VL/CHxULC and the second variable (VL/CHxULC or VHxULC) domains are fused to a human IgG1, IgG2, or IgG4 heavy chain constant region, only one of the first variable VL/CHxULC and the second variable (VL/CHxULC or VHxULC) domain is fused to a human IgG1, IgG2, or IgG4 heavy chain constant region comprising a mutation in the CH3 domain that reduces or eliminates binding of the CH3 domain to Protein A, e.g., a mutation selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT numbering system, or (a′) 435R, and (b′) 435R and 436F in the EU numbering system. In some embodiments, the human heavy chain constant region is a human IgG1 constant region and further comprises one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system. In some embodiments, the human heavy chain constant region is a human IgG2 constant region and further comprises one or two modifications selected from the group consisting of 44S, 52N, 82I in the IMGT exon numbering system, or 348S, 392N and 422I in the EU numbering system. In other embodiments, the human heavy chain constant region is a human IgG4 constant region and further comprises one to seven modifications selected from the group consisting of 15R, 44S, 52N, 57M, 69K, 79Q and 82I in the IMGT exon numbering system or 355R, 384S, 392N, 397M, 409K, 419Q and 422I in the EU numbering system and/or the modification 105P in the IGMT exon numbering system or 445P in the EU numbering system.
In some embodiments, at least one of the unrearranged human immunoglobulin light chain VL or JL gene segments encode one or more histidine residues that are not encoded by a corresponding human germline light chain variable gene segment.
In some embodiments, the first and/or second non-human animal from which the first and second nucleic acid sequences are derived is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the human immunoglobulin light chain variable VL and JL gene segments are operably linked to a non-human immunoglobulin heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the non-human immunoglobulin heavy chain constant region nucleic acid sequence is a mouse or rat immunoglobulin heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the non-human animal is a rodent, and the human immunoglobulin light chain variable VL and JL gene segments are operably linked to a human heavy chain constant region nucleic acid sequence, e.g., comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene.
In some embodiments, the heavy chain constant region nucleic acid sequence comprises a nucleotide sequence that encodes a CH1, a hinge, a CH2, a CH3, or a combination thereof. In some embodiments, heavy chain constant region nucleic acid sequence comprises a sequence that encodes a functional CH1 domain.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a non-human light chain constant region nucleic acid sequence. In some embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a human light chain constant region nucleic acid sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some certain embodiments, the light chain constant region nucleic acid sequence is a lambda sequence.
In some embodiments, the unrearranged human immunoglobulin light chain variable VL and JL gene segments are Vκ and Jκ gene segments. In some embodiments, the unrearranged human immunoglobulin light chain variable VL and JL gene segments are Vλ and Jλ gene segments.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human κ light chain variable region nucleotide sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a human λ light chain variable domain gene sequence.
In some embodiments, the unrearranged human immunoglobulin VL gene segments include one or more of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the unrearranged human immunoglobulin JL gene segments include Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises VL and JL gene segments. In some embodiments, the VL gene segment in the rearranged human immunoglobulin light chain variable region is a (human germline) Vκ gene segment selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segment is selected from the group consisting of Vκ1-39 (e.g., a human germline Vκ1-39 gene segment) and Vκ3-20 (e.g., a human germline Vκ3-20 gene segment). In some embodiments, the JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ1-39 and Jκ5 (e.g., the Vκ1-39 is rearranged with the Jκ5, e.g., a human germline Vκ1-39 gene segment is rearranged with a human germline Jκ5 gene segment). In some embodiments, the rearranged human immunoglobulin light chain variable region gene sequence comprises a sequence set forth as SEQ ID NO:1. In some certain embodiments, the rearranged human immunoglobulin light chain variable nucleotide sequence comprises Vκ3-20 and Jκ1 (e.g., the Vκ3-20 is rearranged with the Jκ1, e.g., a human germline Vκ3-20 gene segment is rearranged with a human germline Jκ1 gene segment). In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence comprises a sequence set forth as SEQ ID NO:2.
In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes at least a functional CH1 domain.
In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is at an endogenous immunoglobulin light chain locus in the genome. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is present in a germline genome of the non-human animal. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence is at a transcriptionally active locus in the genome.
In some embodiments, the non-human animal comprises a plurality of copies of the rearranged human immunoglobulin light chain variable region nucleotide sequence.
In some embodiments, the non-human animal comprises an Adam6a gene, an Adam6b gene or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
In some embodiments, the present invention provides a non-human animal whose genome comprises (a) a first immunoglobulin locus comprising unrearranged human immunoglobulin light chain variable VL and JL gene segments operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence; and (b) a second immunoglobulin locus comprising a rearranged non-human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the rearranged non-human immunoglobulin light chain variable region nucleotide sequence comprises rodent immunoglobulin Vκ and Jκ gene segments. In some certain embodiments, the rodent immunoglobulin Vκ and Jκ gene segments are mouse gene segments. In some certain embodiments, the rodent immunoglobulin Vκ and Jκ gene segments are rat gene segments.
In some embodiments, the present invention provides a method of making a non-human animal, the method comprising (a) modifying a genome of a non-human animal to delete or render non-functional all or substantially all (i) endogenous functional immunoglobulin heavy chain VH, D, and JH gene segments and (ii) endogenous functional light chain VL and JL gene segments; (b) placing unrearranged human immunoglobulin light chain variable VL and JL gene segments in the genome so that the unrearranged light chain variable gene segments are operably linked to a heavy chain constant region nucleic acid sequence; and (c) placing a rearranged non-human immunoglobulin light chain variable region nucleotide sequence in the genome so that the rearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a light chain constant region nucleic acid sequence.
In some embodiments, the present invention provides a method for obtaining a nucleic acid sequence that encodes an immunoglobulin light chain variable domain (VL) capable of binding an antigen independently from a heavy chain variable domain, the method comprising (a) immunizing a non-human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the non-human animal comprises in its genome (i) a rearranged non-human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence, and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence; (b) allowing the non-human animal to mount an immune response; and (c) obtaining from the immunized non-human animal a nucleic acid sequence that encodes the light chain variable domain (VL domain) that can bind the antigen.
In some embodiments, the present invention provides a method for making an antigen-binding protein that comprises an immunoglobulin light chain variable domain that can bind an antigen independently from a heavy chain variable domain, the method comprising (a) immunizing a non-human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the non-human animal comprises in its genome (i) a rearranged non-human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence; (b) allowing the non-human animal to mount an immune response to the first epitope or immunogenic portion thereof; (c) obtaining from the non-human animal a nucleic acid sequence that encodes the light chain variable domain that specifically binds the epitope or immunogenic portion thereof; (d) employing the nucleic acid sequence of (c) in an expression construct, fused to a human immunoglobulin constant region nucleic acid sequence; and (e) expressing the nucleic acid sequence of (c) in a production cell line to form an antigen-binding protein whose light chain is encoded by the nucleic acid of (c) and that binds the epitope or immunogenic portion thereof independently from a heavy chain.
In some embodiments, the present invention provides a non-human animal that comprises in its germline genome (a) a hybrid immunoglobulin chain locus comprising unrearranged human immunoglobulin light chain variable VL and JL gene segments operably linked to a heavy chain constant region nucleic acid sequence; and (b) an immunoglobulin light chain locus comprising two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments operably linked to an immunoglobulin light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the non-human animal is homozygous for the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments operably linked to an immunoglobulin light chain constant region nucleic acid sequence.
In some embodiments, the unrearranged human immunoglobulin light chain variable gene segments are operably linked to a non-human heavy chain constant region nucleic acid sequence. In some certain embodiments, the non-human heavy chain constant region nucleic acid sequence is a mouse or a rat heavy chain constant region nucleic acid sequence. In some embodiments, the non-human animal is a rodent, and the unrearranged human immunoglobulin light chain variable gene segments are operably linked to a human heavy chain constant region nucleic acid sequence. In some embodiments, the heavy chain constant region nucleic acid sequence comprises a nucleotide sequence that encodes a CH1, a hinge, a CH2, a CH3, or a combination thereof.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are operably linked to a non-human light chain constant region nucleic acid sequence. In some embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent and the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are operably linked to a human light chain constant region nucleic acid sequence. In some embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some embodiments, the light chain constant region nucleic acid sequence is a lambda sequence. In some embodiments, the immunoglobulin light chain locus is a kappa locus. In some embodiments, the immunoglobulin light chain locus is a lambda locus.
In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vκ and Jκ gene segments. In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vλ and Jλ gene segments.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise a human κ light chain variable region nucleotide sequence. In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise a human λ light chain variable region nucleotide sequence.
In some embodiments, the unrearranged human immunoglobulin light chain variable VL gene segment is selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the unrearranged human immunoglobulin light chain variable JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprises VL and JL gene segments.
In some embodiments, the VL gene segments of the two or more variable region gene segments are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segments are selected from the group consisting of Vκ1-39, Vκ3-20, and a combination thereof. In some embodiments, the JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise two or more but less than wild type number of human VL gene segments and one or more human JL gene segments. In some certain embodiments, the two or more but less than the wild type number of VL gene segments comprises Vκ1-39 and Vκ3-20 gene segments and one or more JL gene segments comprises Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combination thereof.
In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, and a combination thereof, each of which encodes a functional CH1 domain.
In some embodiments, substantially all endogenous variable heavy chain VH, D, and JH gene segments are deleted from the immunoglobulin heavy chain locus of the non-human animal or rendered non-functional.
In some embodiments, substantially all endogenous light chain VL and JL gene segments are deleted from the immunoglobulin light chain locus of the non-human animal or rendered non-functional.
In some embodiments, the non-human animal comprises an Adam6a gene, an Adam6b gene, or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
In some embodiments, the immunoglobulin light chain locus comprises a plurality of copies of the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments (VL and JL).
In some embodiments, the present invention provides a method of making a non-human animal, the method comprising (a) modifying a genome of a non-human animal to delete or render non-functional all or substantially all (i) endogenous immunoglobulin heavy chain VH, D, and JH gene segments and (ii) endogenous light chain VL and JL gene segments; (b) placing unrearranged human immunoglobulin light chain variable VL and JL gene segments in the genome such that the unrearranged light chain variable gene segments are operably linked to a heavy chain constant region nucleic acid sequence; and (c) placing two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments in the genome such that the human immunoglobulin light chain variable region gene segments are operably linked to a light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a mammal or a bird. In some certain embodiments, the bird is a chicken. In some certain embodiments, the mammal is a rodent. In some embodiments, the rodent is selected from the group consisting of a mouse, a rat, and a hamster.
In some embodiments, the unrearranged human immunoglobulin light chain variable VL and JL gene segments are operably linked to a non-human immunoglobulin heavy chain constant region nucleic acid sequence. In some certain embodiments, the non-human immunoglobulin heavy chain constant region nucleic acid sequence is a mouse or rat immunoglobulin heavy chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and the unrearranged human immunoglobulin light chain variable VL and JL gene segments are operably linked to a human heavy chain constant region nucleic acid sequence. In some embodiments, the heavy chain constant region nucleic acid sequence comprises a nucleotide sequence that encodes a CH1, a hinge, a CH2, a CH3, or a combination thereof. In some embodiments, the heavy chain constant region nucleic acid sequence comprises a nucleotide sequence that encodes a functional CH1 domain.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are operably linked to a non-human light chain constant region nucleic acid sequence. In some embodiments, the non-human light chain constant region nucleic acid sequence is a mouse or a rat light chain constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent and the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are operably linked to a human light chain constant region nucleic acid sequence. In some embodiments, the light chain constant region nucleic acid sequence is a kappa sequence. In some embodiments, the light chain constant region nucleic acid sequence is a lambda sequence.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are placed in a kappa light chain locus. In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are placed in a lambda light chain locus.
In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vκ and Jκ gene segments. In some embodiments, the unrearranged human immunoglobulin VL and JL gene segments are human Vλ and Jλ gene segments.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise a human κ light chain variable region nucleotide sequence. In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise a human λ light chain variable region nucleotide sequence.
In some embodiments, the unrearranged human immunoglobulin light chain variable VL gene segment is selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the unrearranged human immunoglobulin JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments comprise VL and JL gene segments.
In some embodiments, the VL gene segments of the two or more human immunoglobulin light chain variable region gene segments are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some certain embodiments, the VL gene segments are selected from the group consisting of Vκ1-39, Vκ3-20, and a combination thereof. In some embodiments, the JL gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof.
In some embodiments, the two or more but less than wild type number of human immunoglobulin light chain variable region gene segments comprises two or more but less than wild type number of human VL gene segments and one or more JL gene segments. In some certain embodiments, the two or more but less than the wild type number of human VL gene segments comprises Vκ1-29 and Vκ3-20 gene segments.
In some embodiments, the non-human animal is a rodent, and wherein the light chain constant region nucleic acid sequence is a rat or a mouse Cκ constant region nucleic acid sequence.
In some embodiments, the non-human animal is a rodent, and wherein the heavy chain constant region nucleic acid sequence is a rat or mouse constant region sequence selected from the group consisting of Igμ, Igδ, Igγ, Igε, Igα, each of which encodes a functional CH1 domain.
In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are placed at an endogenous immunoglobulin light chain locus in the genome. In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are present in a germline genome of the non-human animal. In some embodiments, the two or more but less than the wild type number of human immunoglobulin light chain variable region gene segments are present at an ectopic locus in the genome.
In some embodiments, the non-human animal comprises an Adam6a gene, an Adam6b gene or both. In some embodiments, a non-human animal comprises a functional ectopic mouse Adam6 gene.
Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The Drawings included herein, which is comprised of the following Figures, is for illustration purposes only not for limitation.
FIG. 1 illustrates a schematic (not to scale) of the mouse heavy chain locus (at top), and a schematic (not to scale) of the human κ light chain locus (at bottom). The mouse heavy chain locus is about 3 Mb in length and contains approximately 200 heavy chain variable (VH) gene segments, 13 heavy chain diversity (DH) gene segments and 4 heavy chain joining (JH) gene segments as well as enhancers (Enh) and heavy chain constant (CH) regions. The human κ light chain locus is duplicated into distal and proximal contigs of opposite polarity spanning about 440 kb and 600 kb, respectively. Between the two contigs is about 800 kb of DNA that is believed to be free of Vκ gene segments. The human κ light chain locus contains about 76 Vκ gene segments, 5 Jκ gene segments, an intronic enhancer (Enh) and a single constant region (Cκ).
FIG. 2 shows a targeting strategy for progressive insertion of 40 human Vκ and 5 human Jκ gene segments into a mouse heavy chain locus. Hygromycin (hyg) and Neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.). A modified mouse heavy chain locus, e.g., a hybrid immunoglobulin locus comprising human Vκ and Jκ gene segments operably linked to mouse CH regions, is shown at the bottom.
FIG. 3 shows an exemplary targeting strategy for progressive insertion of human Vλ and a human Jλ gene segment (or four human Jλ gene segments) into the mouse heavy chain locus. Hygromycin (hyg) and Neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.). A modified mouse heavy chain locus, e.g., a hybrid immunoglobulin locus, comprising human Vλ and Jλ gene segments (one or four) operably linked to mouse CH regions is shown at the bottom.
FIG. 4 illustrates an exemplary targeting strategy for replacing endogenous mouse immunoglobulin κ light chain variable region gene segments with a rearranged human VL/JL sequence.
FIG. 5 illustrates two exemplary targeting vectors for replacing endogenous mouse immunoglobulin light chain Vκ and Jκ gene segments with a rearranged human Vκ1-39Jκ5 sequence (MAID 1633; SEQ ID NO:1) or a rearranged human Vκ3-20Jκ1 sequence (MAID 1635; SEQ ID NO:2).
FIG. 6 shows a modified mouse heavy chain locus, e.g., a hybrid immunoglobulin locus comprising human Vκ and Jκ gene segments operably linked to mouse CH regions and a modified mouse κ light chain locus comprising a rearranged human VκJκ sequence. In one particular embodiment, a mouse having the modified heavy chain locus and modified κ light chain locus as shown (KOH×ULC) is created by breeding a “KOH” mouse and a “ULC” mouse.
FIG. 7 shows representative contour plots of bone marrow stained for B and T cells (top row; CD19+ and CD3+, respectively) and bone marrow gated on CD19+ and stained for ckit+ and CD43+ (bottom row) from a VELOCIMMUNE® mouse (VI3), a mouse homozygous for unrearranged human immunoglobulin light chain variable Vκ and Jκ gene segments at the heavy chain locus, homozygous for unrearranged human immunoglobulin Vκ and Jκ gene segments at the κ light chain locus and an integrated Adam6 gene (“KOH” mouse; 1994HO 1242HO), and a mouse homozygous for a rearranged light chain variable region nucleotide sequence at the κ light chain locus (either VK3-20JK1 or VK1-39JK5) and homozygous for unrearranged human immunoglobulin Vκ and Jκ gene segments at the heavy chain locus and an integrated Adam6 gene (1994HO 1635HO for VK3-20JK1; 1994HO 1633HO for VK1-39JK5; “KOH×ULC” mouse). Pro and Pre B cells are noted on the contour plots. Percentage of cells within each gated region is shown.
FIG. 8 shows the total number of cells (top left), the total number of B (CD19+) cells (top, right), the number of Pro B cells (CD19+CD43+ckit+), and the number of Pre B cells (CD19+CD43−ckit−) in bone marrow isolated from the femurs of KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), KOH mice (1994HO 1242HO) and VELOCIMMUNE® humanized mice (VI3).
FIG. 9 shows representative contour plots (top row) of bone marrow gated on singlets stained for immunoglobulin M (IgM) and B220 from KOH×ULC mice (1994HO 1633HO, 1994HO 1635HO), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3). Immature, mature and pro/pre B cells are noted on each of the contour plots. Percentage of cells within each gated region is shown; the bottom row shows the total number and immature B (left, IgM+B220int) cells and mature B (IgM+B220hi) in bone marrow isolated from the femurs of KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), KOH mice (1994HO 1242HO) and VELOCIMMUNE® humanized mice (VI3).
FIG. 10 shows representative contour plots (left column) of bone marrow gated on singlets stained for immunoglobulin M (IgM) and B220 from KOH×ULC mice (1994HO 1633HO, 1994HO 1635HO), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3). Immature, mature and pro/pre B cells are noted on each of the contour plots; the right two columns shows representative contour plots of bone marrow gated on immature B cells (left, IgM+B220int) and mature B cells (right, IgM+B220hi) stained for Igκ and Igλ expression isolated from the femurs of KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), a KOH mouse (1994HO 1242HO) and VELOCIMMUNE® humanized mouse (VI3). Percentage of cells within each gated region is shown.
FIG. 11 shows representative contour plots of splenocytes stained for B and T cells (top row; CD19+ and CD3+, respectively) and splenocytes gated on CD19+ and stained for Igκ+ and Igλ+ expression from KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3). Percentage of cells within each gated region is shown.
FIG. 12 shows the total number of B cells (CD19+), Igκ+B cells (CD19+Kappa+) and Igλ+B cells (CD19+Lambda+) in harvested spleens from KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), KOH mice (1994HO 1242HO) and VELOCIMMUNE® humanized mice (VI3).
FIG. 13 shows representative contour plots of splenocytes gated on CD19+ and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3). Percentage of cells within each gated region is shown. Mature (CD19+IgMloIgDhi) and transitional/immature (CD19+IgDintIgMhi) B cells are noted in each contour plot. Percentage of cells within each gated region is shown.
FIG. 14 shows the absolute number of splenocytes (top left), the total number of B cells (top right; CD19+), Transitional B cells (bottom left; CD19+IgDloIgMhi), and mature B cells (CD19+IgDhiIgMlo) in harvested spleens from KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3).
FIG. 15 shows representative contour plots of the peripheral B cell development KOH×ULC mice (1994HO 1633HO:Vκ1-39Jκ5; 1994HO 1635HO:Vκ3-20Jκ1), a KOH mouse (1994HO 1242HO) and a VELOCIMMUNE® humanized mouse (VI3). The first column (left) of contour plots show CD93+ and B220+ splenocytes gated on CD19+ indicating immature and mature B cells. The second column (middle) of contour plot shows IgM+ and CD23+ expression in immature B cells indicating T1 (IgD-IgM+CD21loCD23−), T2 (IgDhiIgMhiCD21midCD23+) and T3 B cell populations. The third column (right) of contour plots shows CD21+ (CD35+) and IgM+ expression of mature B cells indicating a smaller first population that give rise to marginal zone B cells and a second population that gives rise to follicular (FO) B cells. Percentage of cells within each gated region is shown.
FIG. 16 shows anti-Antigen 1 antibody titers in different KOH×ULC mice following immunization, resting phase and boost protocols. KOH×ULC mice mount a strong, high titer antigen-specific antibody response comparable to VI3 and ULC mice following a resting phase and additional boosts. Mice were immunized by the footpad route. The 2nd bleed is following six boosts; 3rd bleed is following four additional boosts. Mice were resting for 4.5 weeks after the 2nd bleed. Antigen 1 is a carrier protein. VI3 mice are disclosed in U.S. Pat. No. 8,642,835, herein incorporated by reference. 1633 is a ULC mouse with Vκ1-39. 1635 is a ULC mouse with Vκ3-20.
FIG. 17 shows a schematic of antigen specific binding protein Fabs constructed from KOH×ULC mice. Antigen positive B cells were sorted from two KOH×ULC mice following immunization protocol with immunogen Antigen 2. Positive KOH variable domains were cloned into Fab plasmids. KOH variable domains were cloned into heavy constants. Transient transfections were carried out to produce protein for antigen positive screening by ELISA. KOH-CH1 Fab was transfected with human Vκ3-20 germ line (GL) ULC. Fabs were assayed by ELISA and BIACORE™ for binding to Antigen 2, cell surface protein. A number of antigen specific binders were identified by ELISA and BIACORE™ assays. Fourteen (14) samples bound antigen 2 at neutral pH as determined by ELISA. Binding was confirmed by BIACORE™ for 13 of the 14 ELISA binders.
FIG. 18 shows a Table with BIACORE™ binding data for representative VL domains that retain binding to Antigen 2 when paired with a VHxULC domain from an antibody to an unrelated enzyme, anti-Antigen 3 antibody. The data shows that binding proteins from KOH×ULC mice have specificity solely in a single VL domain.
FIG. 19 shows schematic representation of different multispecific antigen-binding protein formats. (A) shows a schematic of a the generation of a multispecific antigen-binding protein comprising (1) a first heavy chain that has a human Vκ (hVκ/CHxULC) domain fused with a human heavy chain constant region, the hVκ/CHxULC being cognate to a first universal light chain variable domain and capable of binding a first antigen A (Ag A) and (2) a second heavy chain that has a human VH (hVHxULC) domain fused with a second human heavy chain constant region, the hVHxULC domain being cognate to a second universal light chain variable domain and capable of binding a second antigen B (Ag B), each of which heavy chains is paired with an identical universal light chain that comprises a third universal light chain variable domain fused with a human light chain constant region, wherein the third universal light chain is encoded by a rearranged VL/JL gene sequence from which the first and second universal light chains were derived. The hVκ/CHxULC domain of the final multispecific antigen binding protein is derived from antigen-binding protein raised against antigen A in a KOH×ULC mouse, which generates the hVκ/CHxULC domain fused to a mouse heavy chain constant region and paired with a universal light chain comprising a human universal light chain variable domain fused with a mouse light chain constant domain. The hVHxULC domain of the final multispecific antigen binding protein is derived from antigen-binding protein raised against antigen B in a ULC mouse, which generates the hVHxULC domain fused to a mouse heavy chain constant region and paired with a universal light chain comprising a human universal light chain variable domain fused with a mouse light chain constant domain. (B) shows a schematic of a the generation of a multispecific antigen-binding protein comprising (1) a first heavy chain that has a first human Vκ (hVκ/CHxULC) domain fused with a human heavy chain constant region, the first hVκ/CHxULC being cognate to a first universal light chain variable domain and capable of binding a first antigen A (Ag A) and (2) a second heavy chain that has a second human Vκ (hVκ/CHxULC) domain fused with a second human heavy chain constant region, the second hVκ/CHxULC domain being cognate to a second universal light chain variable domain and capable of binding a second antigen B (Ag B), each of which heavy chains is paired with an identical universal light chain that comprises a third universal light chain variable domain fused with a human light chain constant region, wherein the third universal light chain is encoded by a rearranged VL/JL gene sequence from which the first and second universal light chains were derived. The first hVκ/CHxULC domain of the final multispecific antigen binding protein is derived from antigen-binding protein raised against antigen A in a KOH×ULC mouse, which generates the first hVκ/CHxULC domain fused to a mouse heavy chain constant region and paired with a universal light chain comprising a human universal light chain variable domain fused with a mouse light chain constant domain. The second hVκ/CHxULC domain of the final multispecific antigen binding protein is derived from antigen-binding protein raised against antigen B in a KOH×ULC mouse (e.g., second KOH×ULC mouse), which generates the second hVκ/CHxULC domain fused to a mouse heavy chain constant region and paired with a universal light chain comprising a human universal light chain variable domain fused with a mouse light chain constant domain.
FIG. 20 shows a Table with BIACORE™ binding data for representative antigen-binding proteins having a structure depicted in FIG. 19A (B1-B3). Binding data for control antibodies (CKOH1-CKOH2, CVH, C1, and C) are also included. NT=not tested, NA=not applicable, NB=not bound.
DEFINITIONS
This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.
Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described. All publications mentioned are hereby incorporated by reference.
The term “antibody”, as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain and a heavy chain constant region (CH). The heavy chain constant region comprises several domains, e.g., CH1, a hinge region, CH2, CH3 and, optionally CH4. Each light chain comprises a light chain variable domain and a light chain constant region (CL). The heavy chain and light chain variable domains 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 heavy and light chain variable domain comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3). The term “high affinity” antibody refers to an antibody that has a KD with respect to its target epitope about of 10-9 M or lower (e.g., about 1×10−9 M, 1×10−10 M, 1×10−11 M, or about 1×10−12 M). In one embodiment, KD is measured by surface plasmon resonance, e.g., BIACORE™; in another embodiment, KD is measured by ELISA.
The term “biologically active” as used herein includes a characteristic of any agent that has activity in a biological system, in vitro or in vivo (e.g., in an organism). For instance, an agent that, when present in an organism, has a biological effect within that organism, is considered to be biologically active. In particular embodiments, where a protein or polypeptide is biologically active, a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a “biologically active” portion.
The phrase “antigen-binding protein” includes a mono-specific, a bi-specific or higher order antigen-binding protein that respectively and selectively binds one, two or more antigenic determinants. Bispecific antigen-binding proteins generally comprise two nonidentical binding components, with each binding component specifically binding a different epitope—either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule (e.g., different epitopes on the same immunogen). If a bispecific antigen-binding protein is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first binding component for the first epitope will generally be at least one to two or three or four or more orders of magnitude lower than the affinity of the first binding component for the second epitope, and vice versa. Epitopes specifically bound by a bispecific antigen-binding protein can be on the same or a different target (e.g., on the same or a different protein). Exemplary bispecific antigen-binding protein include those with a first binding component specific for a tumor antigen and a second binding component specific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI, FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.). Further, the second binding component can be substituted with a binding component having a different desired specificity. For example, a bispecific antigen-binding protein with a first binding component specific for a tumor antigen and a second binding component specific for a toxin can be paired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) to a tumor cell. Other exemplary bispecific antigen-binding protein include those with a first binding component specific for an activating receptor (e.g., B cell receptor, FcγRI, FcγRIIA, FcγRIIIA, FcγRI, FcεRI, T cell receptor, etc.) and a second binding component specific for an inhibitory receptor (e.g., FcγRIIB, CD5, CD22, CD72, CD300a, etc.). Such bispecific antigen-binding proteins can be constructed for therapeutic conditions associated with cell activation (e.g. allergy and asthma). Bispecific antigen-binding proteins can be made, for example, by combining binding components that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding binding components (e.g., light or heavy chain variable sequences) that recognize different epitopes of the same immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, the same or different light chains, or respectively a heavy chain constant region and a light chain constant region, and such sequences can be expressed in a cell as a multispecific antigen-binding protein in a format that is similar to a Fab structure, scFab structure, a diabody structure, an scFv structure, an scFv-Fc structure, an scFv-zipper structure, or a tetrameric structure similar to a typical antibody that includes the cognate universal light chain. An exemplary bispecific antigen-binding protein has two heavy chains each having three light chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer epitope-binding specificity but that can associate with each light chain, or that can associate with each light chain and that can bind one or more of the epitopes bound by the light chain epitope-binding regions, or that can associate with each light chain and enable binding of one or both of the light chains to one or both epitopes. Similarly, the term “trispecific antibody” includes an antigen-binding protein capable of selectively binding three or more epitopes.
The phrase “complementarity determining region,” or the term “CDR,” includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally (i.e., in a wild-type animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germ line sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. A CDR can be somatically mutated (e.g., vary from a sequence encoded in an animal's germ line), humanized, and/or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., for a CDR3), CDRs can be encoded by two or more sequences (e.g., germ line sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V-D-J recombination to form a heavy chain CDR3).
The term “comparable”, as used herein, includes two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another but that are sufficiently similar to permit comparison there between so that conclusions may reasonably be drawn based on differences or similarities observed. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.
The term “conservative” as used herein to describe a conservative amino acid substitution includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a receptor to bind to a ligand. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is one that that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45, hereby incorporated by reference. In some embodiments, a substitution is deemed to be “moderately conservative” if it has a nonnegative value in the PAM250 log-likelihood matrix.
In some embodiments, residue positions in an immunoglobulin light chain or heavy chain differ by one or more conservative amino acid substitutions. In some embodiments, residue positions in an immunoglobulin light chain or functional fragment thereof (e.g., a fragment that allows expression and secretion from, e.g., a B cell) are not identical to a light chain whose amino acid sequence is listed herein, but differs by one or more conservative amino acid substitutions.
The term “disruption” as used herein includes the result of an event that interrupts (e.g., via homologous recombination) a DNA. In some embodiments, a disruption may achieve or represent a deletion, insertion, inversion, modification, replacement, substitution, or any combination thereof, of a DNA sequence(s). In some embodiments, a disruption may achieve or represent introduction of a mutation, such as a missense, nonsense, or frame-shift mutation, or any combination thereof, in a coding sequence(s) in DNA. In some embodiments, a disruption may occur in a gene or gene locus endogenous to a cell. In some embodiments, insertions may include the insertion of entire genes or fragments of genes, e.g. exons, into an endogenous site in a cell or genome. In some embodiments, insertions may introduce sequences that are of an origin other than that of an endogenous sequence into which they are inserted. In some embodiments, a disruption may increase expression and/or activity of a gene or gene product (e.g., of a protein encoded by a gene). In some embodiments, a disruption may decrease expression and/or activity of a gene or gene product. In some embodiments, a disruption may alter sequence of a gene or gene product (e.g., an encoded protein). In some embodiments, a disruption may truncate or fragment a gene or gene product (e.g., an encoded protein). In some embodiments, a disruption may extend a gene or gene product; in some such embodiments, a disruption may achieve assembly of a fusion protein. In some embodiments, a disruption may affect level but not activity of a gene or gene product. In some embodiments, a disruption may affect activity but not level of a gene or gene product. In some embodiments, a disruption may have no significant effect on level of a gene or gene product. In some embodiments, a disruption may have no significant effect on activity of a gene or gene product. In some embodiments, a disruption may have no significant effect on either level or activity of a gene or gene product.
The phrase “endogenous locus” or “endogenous gene” as used herein includes a genetic locus found in a parent or reference organism prior to introduction of a disruption (e.g., deletion, insertion, inversion, modification, replacement, substitution, or a combination thereof as described herein). In some embodiments, an endogenous locus has a sequence found in nature. In some embodiments, an endogenous locus is wild type. In some embodiments, a reference organism that contains an endogenous locus as described herein is a wild-type organism. In some embodiments, a reference organism that contains an endogenous locus as described herein is an engineered organism. In some embodiments, a reference organism that contains an endogenous locus as described herein is a laboratory-bred organism (whether wild-type or engineered).
The phrase “endogenous promoter” includes a promoter that is naturally associated, e.g., in a wild-type organism, with an endogenous gene.
The phrase “epitope-binding protein” includes a protein having at least one CDR and that is capable of selectively recognizing an epitope, e.g., is capable of binding an epitope with a KD that is at about one micromolar or lower (e.g., a KD that is about 1×10−6M, 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, or about 1×10−12 M). Therapeutic epitope-binding proteins (e.g., therapeutic antibodies) frequently require a KD that is in the nanomolar or the picomolar range.
“Functional” as used herein, e.g., in reference to a functional polypeptide, includes a polypeptide that retains at least one biological activity normally associated with the native protein. In another instance, a functional immunoglobulin gene segment may include a variable gene segment that is capable of productive rearrangement to generate a rearranged immunoglobulin gene sequence.
The phrase “functional fragment” includes fragments of epitope-binding proteins that can be expressed, secreted, and specifically bind to an epitope with a KD in the micromolar, nanomolar, or picomolar range. Specific recognition includes having a KD that is at least in the micromolar range, the nanomolar range, or the picomolar range.
The term “germ line” in reference to an immunoglobulin nucleic acid sequence includes a nucleic acid sequence that can be passed to progeny.
The term “heterologous” as used herein includes an agent or entity from a different source. For example, when used in reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the relevant polypeptide, gene, or gene product 1) was engineered by the hand of man; 2) was introduced into the cell or organism (or a precursor thereof) through the hand of man (e.g., via genetic engineering); and/or 3) is not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type).
The term “host cell”, as used herein, includes a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to a particular subject cell, but also are used to refer to progeny of that 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 understood by those skilled in the art to be included within the scope of the term “host cell” as used herein. In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. In general, a host cell is any cell that is suitable for receiving and/or producing a heterologous nucleic acid or protein, regardless of the Kingdom of life to which the cell is designated. Exemplary cells that may be utilized as host cells in accordance with the present disclosure include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In some embodiments, a host cell is or comprises an isolated cell. In some embodiments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism.
The term “humanized” is used herein in accordance with its art-understood meaning and includes nucleic acids or proteins whose structures (i.e., nucleotide or amino acid sequences) include portions that correspond substantially or identically with versions of the relevant nucleic acids or proteins that are found in nature in non-human animals and that are distinguishable from corresponding versions that are found in nature in humans, and also include portions whose structures differ from those present in the non-human-animal versions and instead correspond more closely with comparable structures found in the human versions. In some embodiments, a “humanized” gene is one that encodes a polypeptide having substantially the amino acid sequence as that of a human polypeptide (e.g., a human protein or portion thereof—e.g., characteristic portion thereof). To give but one example, in the case of a membrane receptor, a “humanized” gene may encode a polypeptide with an extracellular portion whose amino acid sequence is identical or substantially identical to that of a human extracellular portion, and whose remaining sequence is identical or substantially identical to that of a non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene comprises at least a portion of a DNA sequence of a human gene. In some embodiment, a humanized gene comprises an entire DNA sequence found in a human gene. In some embodiments, a humanized protein has an amino acid sequence that comprises a portion that appears in a human protein. In some embodiments, a humanized protein has an amino acid sequence whose entire sequence is found in a human protein. In some embodiments (including, for example, some in which a humanized protein has an amino acid sequence whose entire sequence is found in a human protein), a humanized protein is expressed from an endogenous locus of a non-human animal, which endogenous locus corresponds to the homolog or ortholog of the relevant human gene encoding the protein.
The term “identity” as used herein in connection with a comparison of sequences, includes identity as determined by any of a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments, identities as described herein are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent identity between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Representative algorithms and computer programs useful in determining the percent identity between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), 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. The percent identity between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
The term “isolated”, as used herein, includes a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be “isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an “isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
The phrase “light chain” includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes human κ and λ light chains and a VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from amino terminus to carboxyl terminus, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. A light chain variable domain is encoded by a light chain variable region gene sequence, which generally comprises VL and JL segments, derived from a repertoire of V and J segments present in the germ line. Sequences, locations and nomenclature for V and J light chain segments for various organisms can be found in IMGT database, www.imgt.org. Light chains include those, e.g., that do not selectively bind either a first or a second epitope selectively bound by the epitope-binding protein in which they appear. Light chains also include those that bind and recognize, or assist the heavy chain or another light chain with binding and recognizing, one or more epitopes selectively bound by the epitope-binding protein in which they appear. Common or universal light chains include those derived from a human Vκ1-39Jκ gene or a human Vκ3-20Jκ gene, and include somatically mutated (e.g., affinity matured) versions of the same. Exemplary human VL segments include a human Vκ1-39 gene segment, a human Vκ3-20 gene segment, a human Vλ1-40 gene segment, a human Vλ1-44 gene segment, a human Vλ2-8 gene segment, a human Vλ2-14 gene segment, and human Vλ3-21 gene segment, and include somatically mutated (e.g., affinity matured) versions of the same. Light chains can be made that comprise a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken) and a constant region from the same or a different organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken).
“Neutral pH” includes pH between about 7.0 and about 8.0, e.g., pH between about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4, e.g., physiological pH. “Acidic pH” includes pH of 6.0 or lower, e.g., pH between about 5.0 and about 6.0, pH between about 5.75 and about 6.0, e.g., pH of endosomal or lysosomal compartments.
The phrase “non-human animal” as used herein includes a vertebrate organism that is not a human. In some embodiments, a non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal (e.g., a rodent, e.g., a mouse or a rat), or a bird (e.g., a chicken). In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent such as a rat or a mouse.
The phrase “operably linked”, as used herein, includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or permits such interaction and/or coordination. To give but one example, a control sequence (e.g., an expression control sequence) in a nucleic acid is said to be “operably linked” to a coding sequence when it is located relative to the coding sequence such that its presence or absence impacts expression and/or activity of the coding sequence. In many embodiments, “operable linkage” involves covalent linkage of relevant components or elements with one another. Those skilled in the art will readily appreciate, however, that in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, in some embodiments, nucleic acid control sequences that are operably linked with coding sequences that they control are contiguous with the gene of interest. Alternatively or additionally, in some embodiments, one or more such control sequences acts in trans or at a distance to control a coding sequence of interest. In some embodiments, the term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary and/or sufficient to effect the expression and processing of coding sequences to which they are ligated. In some embodiments, expression control sequences may be or comprise appropriate transcription initiation, termination, promoter and/or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and/or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or exclusively active in a particular host cell or organism, or type thereof. To give but one example, in prokaryotes, control sequences typically include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and/or transcription termination sequences. Those of ordinary skill in the art will appreciate from context that, in many embodiments, the term “control sequences” refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and/or fusion partner sequences).
The term “recombinant”, as used herein, includes polypeptides (e.g., B cell activating factor proteins as described herein) that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial human polypeptide library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370) or polypeptides prepared, expressed, created or isolated by any other means that involves splicing selected sequence elements to one another. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source. For example, in some embodiments, a recombinant polypeptide is comprised of sequences found in the genome of a source organism of interest (e.g., human, mouse, etc.). In some embodiments, a recombinant polypeptide has an amino acid sequence that resulted from mutagenesis (e.g., in vitro or in vivo, for example in a non-human animal), so that the amino acid sequences of the recombinant polypeptides are sequences that, while originating from and related to polypeptides sequences, may not naturally exist within the genome of a non-human animal in vivo.
The term “replacement” is used herein includes a process through which a “replaced” nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from that locus and a different, “replacement” nucleic acid is located in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequences are comparable to one another in that, for example, they are homologous to one another and/or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, a replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice receiver site, an intron, an exon, an untranslated region (UTR); in some embodiments, a replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, a replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, a replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, a replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, including where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse sequence). The nucleic acid sequence so placed may include one or more regulatory sequences that are part of source nucleic acid sequence used to obtain the sequence so placed (e.g., promoters, enhancers, 5′- or 3′-untranslated regions, etc.). For example, in various embodiments, the replacement is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence; the replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a protein that has a similar function as a protein encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a variable domain, and the DNA fragment encodes one or more human variable domains). In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, or is substantially similar or the same in structure and/or function, as the endogenous gene or fragment thereof that is replaced.
The term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, that is unable to associate with a light chain because the heavy chain constant region typically lacks a functional CH1 domain. Accordingly, the term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like encompasses a both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region lacking a functional CH1 domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprising a variable domain operably linked to a heavy chain constant region lacking a functional CH1 domain. In various aspects, a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional CH1 domain. Additionally, each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., VH, DH, JH), light chain gene segments (e.g., VL, JL), or a combination thereof, linked to a heavy chain constant region (CH) gene sequence comprising a deletion or inactivating mutation in a CH1 encoding sequence (and, optionally, a hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof. A single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a “VH-single domain antibody” or “VH-single domain antigen binding protein”, see, e.g., U.S. Pat. No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated in its entirety by reference. A single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a or “VL-single domain antigen binding protein,” see, e.g., U.S. Publication No. 20150289489, incorporated in its entirety by reference.
“Somatically mutated” includes reference to nucleic acid or amino acid sequences from affinity-matured B cells that are not identical to corresponding immunoglobulin variable region sequences in B cells that are not affinity-matured (i.e., sequences in the genome of germline cells). The phrase “somatically mutated” also includes reference to an immunoglobulin variable region nucleic acid or amino acid sequence from a B cell after exposure of the B cell to an epitope of interest, wherein the nucleic acid or amino acid sequence differs from the corresponding nucleic acid or amino acid sequence prior to exposure of the B cell to the epitope of interest. The phrase “somatically mutated” refers to sequences from binding proteins that have been generated in an animal, e.g., a mouse having human immunoglobulin variable region nucleic acid sequences, in response to an immunogen challenge, and that result from the selection processes inherently operative in such an animal.
The term “substantially” as used herein includes the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
The phrase “substantial homology” as used herein includes a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially homologous” if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant stretch includes contiguous residues along a complete sequence. In some embodiments, the relevant stretch includes discontinuous residues along a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.
The phrase “substantial identity” as used herein includes a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.
The phrase “targeting vector” or “targeting construct” as used herein includes a polynucleotide molecule that comprises a targeting region. A targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue or animal and provides for integration of the targeting construct into a position within the genome of the cell, tissue or animal via homologous recombination. Targeting regions that target using site-specific recombinase recognition sites (e.g., loxP or Frt sites) are also included. In some embodiments, a targeting construct of the present invention further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated through exogenous addition of proteins that aid in or facilitate recombination involving such sequences. In some embodiments, a targeting construct of the present invention further comprises a gene of interest in whole or in part, wherein the gene of interest is a heterologous gene that encodes a protein in whole or in part that has a similar function as a protein encoded by an endogenous sequence.
The term “unrearranged,” with reference to a nucleic acid sequence, includes nucleic acid sequences that exist, e.g., in a wild-type germ line of an animal cell.
The phrase “variable domain” includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The term “variant”, as used herein, includes an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocycle core) and/or one or more characteristic pendent moieties so that a variant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and/or in types of bonds present (single vs. double, E vs. Z, etc.) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a variant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide. In many embodiments, a polypeptide of interest is considered to be a “variant” of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be understood by those of ordinary skill in the art, a plurality of variants of a particular polypeptide of interest may commonly be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide.
The term “vector”, as used herein, includes a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiment, vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and/or prokaryotic cell. Vectors capable of directing the expression of operatively linked genes are referred to herein as “expression vectors.”
The term “wild-type”, as used herein, includes an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
The present invention provides, among other things, improved and/or engineered non-human animals having human genetic material encoding light chain variable domains (e.g., VL regions). In certain embodiments, such non-human animals are useful, for example, for the production and isolation of (human) VL domains that bind antigen independently. It is contemplated that such non-human animals provide a novel in vivo system for the generation and affinity maturation of human VL domains that exhibit unique antigen-binding characteristics. Therefore, the present invention is particularly useful for the development of unique antigen-binding proteins in non-human animals. In particular, the present invention encompasses the humanization of a rodent immunoglobulin loci resulting in expression of antigen-binding proteins that resemble naturally occurring immunoglobulins in structure yet differ in binding characteristics, and resulting in expression of said antigen-binding proteins on the membrane surface of cells of the non-human animal. Such antigen-binding proteins have the capacity to recognize foreign antigens that may elude natural immunoglobulins in the generation of unique binding surfaces provided by the antigen-binding proteins. In some embodiments, non-human animals of the present invention are capable of generating (human) VL/CHxULC domains that bind to antigen independent of a cognate variable domain (e.g., a heavy chain variable domain); in some embodiments, such non-human animals develop and/or have a B cell population that express binding proteins that resemble immunoglobulins in structure yet are devoid of any heavy chain variable sequences. In some embodiments, antigen-binding proteins expressed by such non-human animals are characterized in that the antigen-binding portion comprises exclusively of (human) VLxULC domains. In some embodiments, non-human animals of the present invention comprise an endogenous immunoglobulin heavy chain locus that contains genetic material from the non-human animal and a heterologous species (e.g., a human) and comprise an endogenous immunoglobulin light chain locus that contains genetic material from the non-human animal and a heterologous species (e.g., human). In some embodiments, non-human animals of the present invention comprise a hybrid immunoglobulin chain locus that includes unrearranged human VL and JL gene segments operably linked to a heavy chain constant region encoding sequence and an immunoglobulin light chain locus that includes a single rearranged human or non-human VLJL sequence. In some embodiments, the expression of the antigen-binding proteins is under the control of non-human immunoglobulin genetic material (e.g., a non-human immunoglobulin promoter and/or enhancer).
Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention.
Non-Human Animals Comprising a High Diversity Hybrid Chain Locus Containing Unrearranged Light Chain Variable Region Gene Segments and a Low Diversity Light Chain Locus Containing a Rearranged Light Chain Variable Region Sequence
Generation of light chain variable regions that have an ability to bind an antigen independently from a cognate chain variable region can be useful for making light chain variable domains (VLS) for use in antigen-binding molecules.
One approach to produce such light chain variable domains that can bind to an antigen independently from a cognate chain variable region is to apply a selective pressure on nucleotide sequences that encode a variable region or domain of a light chain (VL) to generate light chain CDR3s with more diverse antigenic binding repertoire. As disclosed herein, this can be achieved by generating a genetically modified non-human animal that contains, in its genome, an immunoglobulin hybrid chain locus that contains a high diversity of unrearranged light chain gene segments, see, e.g., U.S. Patent Publication No. 20120096572, incorporated herein by reference, and an immunoglobulin light chain locus that has a low diversity in that the locus contains a single rearranged human immunoglobulin light chain variable region nucleotide sequence. Alternatively, in some embodiments, non-human animals as described herein contain an immunoglobulin light chain locus that has a low diversity in that the locus contains two or more but less than the wild type number of unrearranged human VL gene segments (e.g., 2, 3 or 4). Since the light chain sequence (or the limited number of VL gene segments) at the immunoglobulin light chain locus is restricted to a common or universal (i.e., the same or a very similar) sequences in these animals, the unrearranged light chain variable region nucleotide sequences (i.e., genes) at the hybrid locus will be forced to make light chain CDR3s with more diverse and efficient antigenic binding properties, which can bind an antigenic determinant independently from the cognate variable regions. Furthermore, as disclosed herein, the precise replacement of germ line variable region gene segments (e.g., by homologous recombination-mediated gene targeting) allows for making animals (e.g., mice, rats, or chickens) that have partly human immunoglobulin loci. Because the partly human immunoglobulin loci rearrange, hypermutate, and somatically mutate (e.g., class switch) normally, the partly human immunoglobulin loci generate binding proteins in the animal that comprise human variable domains (i.e., human VL domains). These animals exhibit a humoral immune system that is substantially similar to wild type animals, and display normal cell populations and normal lymphoid organ structures-even where the animals lack a full repertoire of human variable region gene segments (at an immunoglobulin light chain locus). Immunizing these animals (e.g., mice, rats, or chickens) results in robust humoral responses that display a wide diversity of light chain variable gene segment usage. Nucleotide sequences that encode the variable regions can be identified and cloned, then fused (e.g., in an in vitro system) with any sequences of choice, e.g., any immunoglobulin isotype suitable for a particular use, resulting in an antibody or antigen-binding protein derived wholly from human sequences.
In addition, by utilizing animals (e.g., mice or rats or chickens) that have a restricted (limited) immunoglobulin light chain locus, e.g., a restricted immunoglobulin light chain locus comprising a rearranged light chain variable region nucleotide sequence (e.g., a universal light chain or “ULC,” US Patent Application Publication No. 2011-0195454 A1, US 2012-0021409A1, US 2012-0192300A1, US 2013-0045492A1, US 2013-0185821A1 and US 2013-0302836A1, incorporated by reference herein in their entireties) or a restricted (limited) immunoglobulin light chain variable region gene segment repertoire (e.g., a restricted immunoglobulin light chain variable segment repertoire comprising two or more but less than the wild type number of human VL gene segments; for example, a dual light chain, or “DLC”, U.S. Patent Application Publication No. US-2013-0198880-A1, incorporated by reference herein in its entirety) in combination with a high diversity hybrid immunoglobulin chain locus containing unrearranged light chain variable region gene segments described above, an immunoglobulin light chain variable (VL/CHxULC) domain that binds antigen in the absence of a heavy chain variable domain can be produced. Furthermore, by introducing histidine codons, e.g., via addition of one or more histidine codons or substitution of one or more non-histidine codons with histidine codons, into the rearranged light chain variable region nucleotide sequence (or into the limited VL gene segments) in the genome of the non-human animals described herein, light chain variable region amino acid sequences that can confer improved pH-dependent recyclability to the antigen-binding proteins can be generated.
In some embodiments, the genetically modified non-human animals as described herein provide a greater yield of binding proteins, while limiting diversity at the same time, thereby increasing the probability of successful production of light chain variable domains from the hybrid locus that bind antigen independent of a cognate variable domain. In some embodiments, the light chains may themselves exhibit antigen-binding properties. In some embodiments, the non-human animal may be induced to produce antigen-binding proteins exhibiting antigen specificity that resides in their light chains (e.g., by limiting a mouse or rat's immunoglobulin light chain repertoire and maximizing the immunoglobulin hybrid chain repertoire; e.g., by creating a hybrid immunoglobulin chain repertoire, e.g., by replacing the mouse or rat heavy chain variable region locus with a locus comprising a high diversity of unrearranged human VL and JL gene segments and replacing the mouse or rat light chain variable region locus a single rearranged human immunoglobulin light chain variable region nucleotide sequence). In some embodiments, antigen-binding proteins (e.g., antibodies) produced in such animals will be specific for a particular epitope (e.g., effector antigens, cytotoxic molecules, Fc receptors, toxins, activating or inhibitory receptors, T cell markers, immunoglobulin transporters, etc.) through their light chain binding.
In various aspects, a non-human animal is provided comprising in its germ line genome a hybrid immunoglobulin chain locus that comprises unrearranged (human) VL and JL gene segments operably linked to a heavy chain constant region encoding sequence and an immunoglobulin light chain locus that comprises a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (i.e., a rearranged light chain VJ sequence). In some embodiments, the unrearranged (human) VL and JL gene segments are operably linked to a human or non-human heavy chain constant region sequence comprising one or more heavy chain constant region genes, each of which encodes at least a functional CH1 domain, and the rearranged (human) immunoglobulin light chain variable region nucleotide sequence is operably linked to a human or a non-human light chain constant region sequence. In some embodiments, an immunoglobulin light chain variable domain encoded by the rearranged light chain variable region nucleotide sequence is not immunogenic to the non-human animal. In some embodiments, the non-human animal is modified to comprise a nucleotide sequence that encodes two copies, three copies, four copies or more of the rearranged light chain variable domain operably linked to a light chain constant domain. In some embodiments, the nucleotide sequence encodes a plurality of copies of the rearranged (human) immunoglobulin light chain variable region nucleotide sequence. For example, the nucleotide sequence can encode at least one, two, three, four, five copies of the rearranged human immunoglobulin light chain variable region nucleotide sequence. In some embodiments, the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the rearranged (human) immunoglobulin light chain variable region nucleotide sequence. In some embodiments, the immunoglobulin light chain locus comprises a plurality of copies of the rearranged (human) immunoglobulin light chain variable region nucleotide sequence operably linked to a light chain constant region gene sequence.
In various aspects, the immunoglobulin light chain locus of the non-human animals described herein comprises a single rearranged human immunoglobulin light chain variable region nucleotide sequence, e.g., a rearranged human VLJL sequence, operably linked to a non-human light chain constant region nucleotide sequence (e.g., a non-human light chain constant region nucleic acid sequence). Thus, genetically modified non-human animals are provided comprising in their genomes: (i) a hybrid immunoglobulin chain locus that comprises unrearranged human VL and JL gene segments operably linked to a human or non-human heavy chain constant region nucleic acid sequence; and (ii) an immunoglobulin light chain locus comprising a rearranged human light chain variable region nucleotide sequence operably linked to a light chain constant region nucleic acid sequence. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region. In some embodiments, the human VL and JL gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) are present as a plurality of gene segments (more than one human VL and more than one human JL gene segment) and capable of rearranging and encoding human VL domains in the context of heavy chain constant regions of an antibody, and the non-human animal does not comprise an endogenous VH and/or VL gene segment. In some embodiments, the non-human animal comprises six, 16, 30, 40 or more unrearranged human Vκ gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises five unrearranged human Jκ gene segments, e.g., Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises 12, 28, 40 or more unrearranged human Vλ gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises 1, 2, 3, 4 or more unrearranged human Jλ gene segments, e.g., Jλ1, Jλ2, Jλ3, Jλ7, etc., at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus).
In some embodiments, the immunoglobulin light chain locus of the non-human animals described herein comprises a rearranged human VκJκ nucleotide sequence. In some embodiments, the immunoglobulin light chain locus comprises a rearranged human VλJλ nucleotide sequence. In some embodiments, the rearranged human VκJκ nucleotide sequence or rearranged human VλJλ nucleotide sequence is present at an endogenous light chain locus, e.g., at an endogenous κ light chain locus. In some embodiments, the mouse comprises a functional λ light chain locus. In some embodiments, the mouse comprises a non-functional λ light chain locus. In some embodiments, the one or more human VL and one or more human JL gene segments at the immunoglobulin heavy chain locus are operably linked to a mouse or a rat heavy chain constant region sequence (e.g., in a hybrid immunoglobulin chain locus). In some embodiments, the rearranged human VκJκ nucleotide sequence is a rearranged human Vκ1-39Jκ nucleotide sequence, e.g., Vκ1-39Jκ5 sequence (e.g., as set forth in SEQ ID NO:1). In some embodiments, the rearranged human VκJκ nucleotide sequence is a rearranged human Vκ3-20Jκ nucleotide sequence, e.g., Vκ3-20Jκ1 sequence (e.g., as set forth in SEQ ID NO:2). In some embodiments, the rearranged human VλJλ nucleotide sequence is a rearranged human Vλ2-14Jλ1 nucleotide sequence. As persons of skill will recognize the use of other JL sequences may be employed in a rearranged light chain sequence.
In various aspects, the immunoglobulin light chain locus of the non-human animals described herein comprises a limited repertoire of immunoglobulin light chain variable gene segments, e.g., one or more but less than the wild type number of human VL gene segments; and one or more human JL gene segments, operably linked to a non-human light chain constant region nucleotide sequence. Thus, genetically modified non-human animals are provided comprising in their genomes: (i) an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) that comprises unrearranged human VL and JL gene segments operably linked to a human or non-human heavy chain constant region nucleic acid sequence (e.g., a non-human heavy chain constant region nucleic acid sequence encoding a CH1, hinge, CH2, CH3, CH4, or a combination thereof, e.g., a CH1, a hinge, an CH2, and a CH3); and (ii) an immunoglobulin light chain locus comprising two or more but less than the wild type number of human immunoglobulin VL and JL gene segments operably linked to a light chain constant region nucleic acid sequence. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region. In some embodiments, the human VL and JL gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) are present as a plurality of gene segments (more than one human VL and more than one human JL gene segment) and capable of rearranging and encoding human VL domains in the context of heavy chain constant regions of an antibody, and the non-human animal does not comprise an endogenous VH and/or VL gene segment. In some embodiments, the non-human animal comprises six, 16, 30, 40 or more unrearranged human Vκ gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises five unrearranged human Jκ gene segments, e.g., Jλ1, Jκ2, Jκ3, Jκ4, and Jκ5 gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises 12, 28, 40 or more unrearranged human Vλ gene segments at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises 1, 2, 3, 4 or more unrearranged human Jλ gene segments, e.g., Jλ1, Jλ2, Jλ3, Jλ7, etc., at an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus). In some embodiments, the non-human animal comprises two unrearranged human Vκ gene segments at an immunoglobulin light chain locus. In some embodiments, the non-human animal comprises two unrearranged human Vλ gene segments at an immunoglobulin light chain locus.
In some embodiments, genetically modified mice comprising in their genomes (i) an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) that comprises unrearranged human VL and JL gene segments operably linked to a human or non-human heavy chain constant region nucleic acid sequence, and (ii) an immunoglobulin light chain locus comprising rearranged human light chain variable region nucleic acid sequence operably linked to a light chain constant region nucleic acid sequence, demonstrate CD19+B cell numbers and mature B cell numbers that are substantially the same as the numbers observed in wild type mice or mice containing other modifications of their immunoglobulin loci (i.e., genetically modified control mice; e.g., VELOCIMMUNE® humanized mice, in which the humoral immune system of the mouse functions like that of a wild type mouse). In some embodiments, such mice also demonstrate a functional silencing of endogenous lambda light chains in splenic B cells. In some embodiments, the mice exhibit normal or nearly normal B cell development in the bone marrow and the spleen. In some embodiments, such mice exhibit a lack of detectable expression and/or usage (or functional silencing) of lambda light chains compared to genetically modified control mice.
In another aspect, a non-human animal is provided comprising (a) a genetically modified immunoglobulin heavy chain locus comprising: a first nucleotide sequence that encodes a light chain variable domain (e.g., where the first nucleotide sequence contains unrearranged human immunoglobulin light chain variable region gene segments), wherein the first nucleotide sequence is operably linked to a heavy chain constant region gene sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene (thus, resulting in, e.g., a hybrid immunoglobulin chain locus); and (b) genetically modified immunoglobulin light chain locus comprising a second nucleotide sequence that encodes a human light chain variable domain (e.g., where the second nucleotide sequence is a rearranged human immunoglobulin light chain variable region nucleotide sequence or where the second nucleotide sequence contains a limited number of human VL gene segments; e.g., two or more but less than the wild type number of human VL gene segments), wherein the second nucleotide sequence is operably linked to a light chain constant region gene sequence. For example, in some embodiments, a rearranged light chain from a pre-designed VJ region (i.e., a rearranged human immunoglobulin light chain variable region nucleotide sequence; i.e., a common or universal light chain sequence) or a limited number of human VL gene segments (e.g., two or more but less than the wild type number of human VL gene segments) can be operably linked to a light chain constant region gene sequence by targeting the rearranged light chain sequence into a mouse light chain locus, either κ or λ. Thus, as in other embodiments, this genetically engineered immunoglobulin light chain locus may be present in the germ line genome of the non-human animal. Genetically modified non-human animals comprising unrearranged human immunoglobulin light chain variable region nucleotide sequences in operable linkage with a heavy chain constant region gene sequences are described in U.S. Patent Application Publication No. 2012-0096572 A1, which is incorporated herein by reference. In some embodiments, the second nucleotide sequence that encodes the human light chain variable domain is operably linked to a κ light chain constant (i.e., Cκ) region gene sequence. In some embodiments, the second nucleotide sequence that encodes the human light chain variable domain is operably linked to a mouse or rat Cκ region gene sequence. In some embodiments, the second nucleotide sequence that encodes the light chain variable domain is operably linked to a human Cκ region gene sequence. In some embodiments, the second nucleotide sequence that encodes the human light chain variable domain is operably linked to a Cλ region gene sequence. In some embodiments, the second nucleotide sequence that encodes the human light chain variable domain is operably linked to a mouse or rat Cλ region gene sequence. In some embodiments, the second nucleotide sequence that encodes the human chain variable domain is operably linked to a human Cλ region gene sequence.
In some embodiments, the non-human animal is a mammal. Although embodiments employing a rearranged human light chain variable region (or a limited number of human VL gene segments) and unrearranged human light chain variable region gene segments in a mouse (i.e., a mouse with an immunoglobulin light locus comprising a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments) and an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising unrearranged human light chain variable region gene segments) are extensively discussed herein, other non-human animals that comprise a genetically modified immunoglobulin heavy and light chain loci as described herein are also provided. Such non-human animals include any of those which can be genetically modified to express the rearranged human immunoglobulin light chain variable region nucleotide sequence (or a human light chain variable domain from the limited number of human VL gene segments) as disclosed herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. For example, for those non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome to a suitable cell, e.g., an enucleated oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. Methods for modifying a non-human animal genome (e.g., a pig, cow, rodent, chicken, etc. genome) include, e.g., employing a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) to modify a genome to include an immunoglobulin light chain locus that contains a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) and an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) that contains unrearranged human light chain variable region gene segments.
In some embodiments, the non-human animal is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. In some embodiments, the rodent is selected from a mouse, a rat, and a hamster. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some embodiments, the genetically modified mouse is from a member of the family Muridae. In some embodiments, the animal is a rodent. In specific embodiments, the rodent is selected from a mouse and a rat. In some embodiments, the non-human animal is a mouse.
In some embodiments, the non-human animal is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6N, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain. In some embodiments, the 129 strain is selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al. (2000) Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In some embodiments, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL strain (e.g., a C57BL/6 strain). In another embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned C57BL/6 strains. In some embodiments, the 129 strain of the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a mix of a 129/SvEv- and a C57BL/6-derived strain. In a specific embodiment, the mouse is a mix of a 129/SvEv- and a C57BL/6-derived strain as described in Auerbach et al. 2000 BioTechniques 29:1024-1032. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In another embodiment, the mouse is a mix of a BALB strain (e.g., BALB/c strain) and another aforementioned strain.
In some embodiments, the non-human animal is a rat. In some embodiments, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, ACI, and Dark Agouti (DA). In some embodiments, the rat strain is a mix of two or more of a strain selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, ACI and Dark Agouti (DA).
In some embodiments, such a genetically modified mouse uses lambda gene sequences with a frequency that is half or less than half of the frequency that lambda gene sequences are used in wild type.
In various embodiments, as described herein, the rearranged light chain variable domain is derived from a human VL and JL gene sequence or segment. In other embodiments, the rearranged light chain variable domain is derived from a non-human VL and JL gene sequence or segment. In some embodiments, the rearranged light chain variable domain is derived from a human germ line VL segment and a human germ line JL segment. In some embodiments, the human VL segment corresponds to observed variants in the human population.
In various embodiments, as described herein, the human VL gene segment of the rearranged light chain variable region nucleotide sequence is a human Vκ gene segment. In some embodiments, the human Vκ gene segment is selected from the group consisting of Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6 Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40, and a polymorphic variant thereof. In some embodiments, the human Vκ segment is Vκ1-39 or polymorphic variant thereof. In some embodiments, the human Vκ gene segment is Vκ3-20.
In various embodiments, as described herein, the human VL gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire (e.g., a restricted immunoglobulin light chain variable segment repertoire comprising two or more but less than the wild type number of human VL gene segments) are human Vκ gene segments. In some embodiments, the human Vκ gene segments are selected from human Vκ gene segments described herein. In some certain embodiments, the human Vκ gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire include a human Vκ1-39 gene segment and a human Vκ3-20 gene segment. In various embodiments of the restricted (limited) immunoglobulin light chain variable gene segment non-human animal, the restricted light chain variable gene segments (e.g., a human Vκ1-39 gene segment and a human Vκ3-20 gene segment) are operably linked to one, two, three, four, or more human JL gene segments; such that the restricted immunoglobulin light chain variable gene segments recombine with one of the one or two or three or four or more human JL gene segments (i.e., Jκ gene segments) to form a rearranged VκJκ light chain variable gene.
In various embodiments, as described herein, the human VL gene segment of the rearranged light chain variable region nucleotide sequence is a human Vλ gene segment. In some embodiments, the human Vλ gene segment is selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61, Vλ4-69, and a polymorphic variant thereof. In some embodiments, the human Vλ segment is Vλ2-14.
In various embodiments, as described herein, the human VL gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire (e.g., a restricted immunoglobulin light chain variable segment repertoire comprising two or more but less than the wild type number of human VL gene segments) are human Vλ gene segments. In some embodiments, the human Vλ gene segments are selected from human Vλ gene segments described herein. In some certain embodiments, the human Vλ gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire include a human Vλ2-14 gene segment.
In various embodiments, as described herein, the human JL gene segment of the rearranged light chain variable region nucleotide sequence is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, Jλ1, Jλ2, Jλ3, Jλ7, and a polymorphic variant thereof.
In various embodiments, as described herein, the human JL gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire include human Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a polymorphic variant thereof. In various embodiments, as described herein, the human JL gene segments of the restricted (limited) immunoglobulin light chain variable region gene segment repertoire include human Jλ1, Jλ2, Jλ3, Jλ7, and a polymorphic variant thereof.
In some embodiments, the human or non-human animal light chain constant region sequence comprises a sequence selected from a Cκ and a Cλ region.
Various embodiments utilize or encompass features or sequence information derived from VELOCIMMUNE® humanized mice. VELOCIMMUNE® humanized mice contain a precise, large-scale replacement of germ line variable regions of mouse immunoglobulin heavy chain (IgH) and immunoglobulin light chain (e.g., κ light chain, Ig) with corresponding human immunoglobulin variable regions, at the endogenous loci (see, e.g., U.S. Pat. Nos. 6,596,541 and 8,502,018, the entire contents of which are incorporated herein by reference). In total, about six megabases of mouse loci are replaced with about 1.5 megabases of human genomic sequence. This precise replacement results in a mouse with hybrid immunoglobulin loci that make heavy and light chains that have a human variable regions and a mouse constant region. The precise replacement of mouse VH-D-JH and Vκ-Jκ segments leave flanking mouse sequences intact and functional at the hybrid immunoglobulin loci. The humoral immune system of the mouse functions like that of a wild type mouse. B cell development is unhindered in any significant respect and a rich diversity of human variable regions is generated in the mouse upon antigen challenge. Moreover, VELOCIMMUNE® humanized mice display an essentially normal, wild-type response to immunization that differs only in one significant respect from wild type mice—the variable regions generated in response to immunization are fully human. VELOCIMMUNE® humanized mice are possible because immunoglobulin gene segments for heavy and κ light chains rearrange similarly in humans and mice. Although the loci are not identical, they are similar enough that humanization of the heavy chain variable gene locus can be accomplished by replacing about three million base pairs of contiguous mouse sequence that contains all the VH, D, and JH gene segments with about one million bases of contiguous human genomic sequence covering basically the equivalent sequence from a human immunoglobulin locus.
In particular embodiments, a humanized mouse comprising an immunoglobulin heavy chain locus that contains unrearranged human light chain variable region gene segments (i.e., comprising an immunoglobulin heavy chain locus that comprises unrearranged human immunoglobulin VL and JL gene segments) is provided. A humanized mouse so modified comprises a replacement of mouse immunoglobulin heavy chain variable region gene segments with unrearranged human immunoglobulin light chain variable region gene segments (i.e., unrearranged VL and JL gene at an endogenous heavy chain locus), and a replacement of mouse immunoglobulin light chain variable gene segments with a rearranged human VLJL nucleotide sequence or a replacement of mouse immunoglobulin light chain variable gene segments with a restricted (limited) immunoglobulin light chain variable region gene segment repertoire (e.g., two or more but less than the wild type number of human VL gene segments).
In some embodiments, the mouse so modified comprises a replacement of mouse immunoglobulin heavy chain variable region gene segments with at least 40 unrearranged human Vκ gene segments and five unrearranged human Jκ gene segments. In some embodiments, the unrearranged human Vκ gene segments are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the human Vκ gene segments comprise Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, and Vκ1-6. In one embodiment, the Vκ gene segments comprise Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15 and Vκ1-16. In some embodiments, the human Vκ gene segments comprise Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, and Vκ2-30. In some embodiments, the human Vκ gene segments comprise Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, and Vκ2-40. In specific embodiments, the Vκ gene segments comprise contiguous human immunoglobulin κ gene segments spanning the human immunoglobulin κ light chain locus from Vκ4-1 through Vκ2-40, and the Jκ gene segments comprise contiguous gene segments spanning the human immunoglobulin κ light chain locus from Jκ1 through Jκ5. In some embodiments, the rearranged human light chain variable region nucleotide sequence (i.e., rearranged human VκJκ nucleotide sequence) is operably linked to a mouse light chain constant region sequence (e.g., a Cκ sequence). A humanized mouse comprising an immunoglobulin heavy chain locus encoding human light chain variable domains (i.e., comprising an immunoglobulin heavy chain locus that comprises unrearranged human immunoglobulin light chain variable region gene segments) can be used in any of the aspects, embodiments, methods, etc. described herein.
In some embodiments, the mouse so modified comprises a replacement of mouse immunoglobulin heavy chain variable region gene segments with at least 40 unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments; in some certain embodiments, at least 40 unrearranged human Vλ gene segments and four unrearranged human Jλ gene segments. In some embodiments, the unrearranged human Vκ gene segments are selected from the group consisting of Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61, Vλ4-69, and a polymorphic variant thereof. In some embodiments, the unrearranged human Vλ gene segments include Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11 and Vλ3-12. In some embodiments, the unrearranged human Vλ gene segments include V Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25 and Vλ3-27. In some embodiments, the unrearranged human Vλ gene segments include V Vλ1-36, Vλ5-37, Vλ5-39, Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52, Vλ6-57, Vλ4-60, Vλ8-61 and Vλ4-69. In specific embodiments, the Vλ gene segments comprise contiguous human immunoglobulin λ gene segments spanning the human immunoglobulin λ light chain locus from Vλ3-1 through Vλ3-12, and the Jλ gene segments include Jλ1. In specific embodiments, the Vλ gene segments comprise contiguous human immunoglobulin λ gene segments spanning the human immunoglobulin λ light chain locus from Vλ3-12 through Jλ1. In specific embodiments, the Vλ gene segments comprise contiguous human immunoglobulin λ gene segments spanning the human immunoglobulin λ light chain locus from Vλ3-1 through Vλ3-12, and the Jλ gene segments include Jλ1, Jλ2, Jλ3 and Jλ7. In specific embodiments, the Vλ gene segments comprise contiguous human immunoglobulin λ gene segments spanning the human immunoglobulin λ light chain locus from Vλ3-12 through Vλ3-27, and the Jλ gene segments include Jλ1 or Jλ1, Jλ2, Jλ3 and Jλ7. In specific embodiments, the Vλ gene segments comprise contiguous human immunoglobulin λ gene segments spanning the human immunoglobulin λ light chain locus from Vλ1-40 through Vλ5-52, and the Jλ gene segments include Jλ1 or Jλ1, Jλ2, Jλ3 and Jλ7. In some embodiments, the rearranged human light chain variable region nucleotide sequence is a rearranged human VλJλ nucleotide sequence and is operably linked to a mouse light chain constant region sequence (e.g., a Cλ sequence). A humanized mouse comprising an immunoglobulin heavy chain locus encoding human light chain variable domains (i.e., comprising an immunoglobulin heavy chain locus that comprises unrearranged human immunoglobulin light chain variable region gene segments) can be used in any of the aspects, embodiments, methods, etc. described herein.
In various embodiments, the unrearranged human immunoglobulin light chain variable region gene segments are operably linked to a human or mouse heavy chain constant region gene sequence (e.g., a heavy chain constant region gene sequence that encodes an immunoglobulin isotype selected from IgM, IgD, IgA, IgE, IgG, and combinations thereof, wherein each heavy chain constant region gene encodes a functional CH1 domain). For example, genetically modified non-human animals are provided comprising (a) an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) that contains a first nucleotide sequence which contains unrearranged human light chain variable region gene segments (i.e., where the first nucleotide sequence comprises at least 40 human Vκ gene segments and 5 human Jκ gene segments), wherein the first nucleotide sequence is operably linked to a human or non-human heavy chain constant region gene sequence; and (b) an immunoglobulin light chain locus that contains a second nucleotide sequence that encodes a light chain variable domain (i.e., where the second nucleotide sequence is a rearranged human immunoglobulin light chain variable region nucleotide sequence or where the second nucleotide sequence contains a limited number of human VL gene segments; e.g., two or more but less than the wild type number of human VL gene segments), wherein the second nucleotide sequence is operably linked to a human or non-human light chain constant region gene sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the human heavy chain constant region gene further encodes a hinge, a CH2, a CH3, and combinations thereof. In some embodiments, a mouse heavy chain constant region gene further encoedes a hinge, a CH2, a CH3, and combinations thereof. In some embodiments, further replacement of certain non-human animal constant region gene sequences with human gene sequences (e.g., replacement of mouse CH1 sequence with human CH1 sequence, and replacement of mouse CL sequence with human CL sequence) results in genetically modified non-human animals with chimeric (and hybrid) immunoglobulin loci that make antibodies that have human variable regions and partly human constant regions, suitable for, e.g., making fully human antibody fragments, e.g., fully human Fab's. In some embodiments, the unrearranged human light chain variable region gene segments are operably linked to a rat heavy chain constant region gene sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the rat heavy chain constant region gene further encodes a CH2, a CH3, and combinations thereof. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments) is operably linked with a human Cκ region sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments) is operably linked with a mouse or rat Cκ region sequence. In various embodiments, the genetically modified immunoglobulin light chain locus of the non-human animal comprises two copies, three copies, four copies or more of the rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to a light chain constant region gene sequence. In particular embodiments, the immunoglobulin light chain locus comprises a plurality of copies of the rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to a light chain constant region gene sequence.
In various embodiments, a (human) IgG1, IgG2, or IgG4 heavy chain constant region gene (e.g., cloned in an expression vector, at an endogenous locus) etc., comprises one or more modification(s) in a CH3 encoding sequence of the gene, wherein the modification reduces or eliminates affinity of the CH3 domain encoded by the modified encoding sequence to Protein A (see, e.g., U.S. Pat. No. 8,586,713, incorporated herein in its entirety by reference). Such modification includes, but is not limited to a mutation selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT numbering system, or (a′) 435R, and (b′) 435R and 436F in the EU numbering system. In some embodiments, the (human and) mutated heavy chain constant region is a (human and) mutated IgG1 constant region and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system. In some embodiments, the heavy chain constant gene is a (human) IgG2 constant gene and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one or two modifications selected from the group consisting of 44S, 52N, 82I in the IMGT exon numbering system, or 348S, 392N and 422I in the EU numbering system. In other embodiments, the (human) heavy chain constant gene is a (human) IgG4 constant gene and, in addition to the (a) 95R or (b) 95R and 96F mutation (in the IMGT numbering system), further comprises one to seven modifications selected from the group consisting of 15R, 44S, 52N, 57M, 69K, 79Q and 82I in the IMGT exon numbering system or 355R, 384S, 392N, 397M, 409K, 419Q and 422I in the EU numbering system and/or the modification 105P in the IGMT exon numbering system or 445P in the EU numbering system.
In various embodiments, the heavy chain constant region nucleotide sequence comprises a modification in a CH2 or a CH3, wherein the modification increases the affinity of the heavy chain constant region amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a modification at position 250 by EU numbering (263 by Kabat numbering) (e.g., E or Q); 250 by EU numbering (263 by Kabat numbering) and 428 by EU numbering (459 by Kabat numbering) (e.g., L or F); 252 by EU numbering (265 by Kabat numbering) (e.g., L/Y/F/W or T), 254 by EU numbering (267 by Kabat numbering) (e.g., S or T), and 256 by EU numbering (269 by Kabat numbering) (e.g., S/R/Q/E/D or T); or a modification at position 428 by EU numbering (459 by Kabat numbering) and/or 433 by EU numbering (464 by Kabat numbering) (e.g., L/R/S/P/Q or K) and/or 434 by EU numbering (465 by Kabat numbering) (e.g., H/F or Y); or a modification at position 250 by EU numbering (263 by Kabat numbering) and/or 428 by EU numbering (459 by Kabat numbering); or a modification at position 307 by EU numbering (326 by Kabat numbering) or 308 by EU numbering (327 by Kabat numbering) (e.g., 308F, V308F), and 434 by EU numbering (465 by Kabat numbering). In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification by EU numbering (a 459, e.g., M459L, and 465S (e.g., N465S) modification by Kabat numbering); a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification by EU numbering (a 459L, 272I (e.g., V272I), and 327F (e.g., V327F) modification by Kabat numbering; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification by EU numbering (a 464K (e.g., H464K) and a 465 (e.g., 465Y) modification by Kabat numbering; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification by EU numbering (a 265, 267, 269 (e.g., 265Y, 267T, and 269E) modification by Kabat numbering; a 250Q and 428L modification (e.g., T250Q and M428L) by EU numbering (a 263Q and 459L modification, e.g., T263Q and M459L, by Kabat numbering); and a 307 and/or 308 modification (e.g., 307F or 308P) by EU numbering (326 and/or 327 modification, e.g., 326F or 308P, by Kabat numbering), wherein the modification increases the affinity of the heavy chain constant region amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH2 amino acid sequence comprising at least one modification between amino acid residues at positions 252 and 257 by EU numbering (i.e., at least one modification between amino acid positions 265 and 270 by Kabat numbering), wherein the modification increases the affinity of the human CH2 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH2 amino acid sequence comprising at least one modification between amino acid residues at positions 307 and 311 (i.e., at least one modification between amino acid positions 326 and 330 by Kabat numbering), wherein the modification increases the affinity of the CH2 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH3 amino acid sequence, wherein the CH3 amino acid sequence comprises at least one modification between amino acid residues at positions 433 and 436 by EU numbering (i.e., at least one modification between amino acid residues at positions 464 and 467 by Kabat numbering), wherein the modification increases the affinity of the CH3 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M428L by EU numbering (459 by Kabat numbering), N434S by EU numbering (465 by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M428L by EU numbering (M459L by Kabat numbering), V259I by EU numbering (V272I by Kabat numbering), V308F by EU numbering (V327 by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising an N434A mutation by EU numbering (an N465A mutation by Kabat numbering). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M252Y by EU numbering (M265Y by Kabat numbering), S254T by EU numbering (S267T by Kabat numbering), T256E by EU numbering (T269E by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of T250Q by EU numbering (T263Q by Kabat numbering), M428L by EU numbering (M459L by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of H433K by EU numbering (H464K by Kabat numbering), N434Y by EU numbering (N465Y by Kabat numbering), and a combination thereof.
In various embodiments, a non-human animal as described herein is immunized with an antigen of interest, and a B cell expressing an antigen-binding protein that specifically binds the antigen of interest is identified, and a nucleic acid sequence of the B cell which encodes a light chain variable domain in a polypeptide comprising a heavy chain constant region is identified and determined. The nucleic acid sequence of the light chain variable domain is expressed, in a suitable cell and employing a suitable expression vector, with a heavy chain constant nucleic acid sequence comprising one, two, three, or more modifications. In some embodiments, the light chain variable region is human, and the heavy chain sequence is human. In some embodiments, the heavy chain constant region nucleotide sequence comprises a modification in a CH2 or a CH3, wherein the modification increases the affinity of the heavy chain constant region amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a modification at position 250 by EU numbering (263 by Kabat numbering) (e.g., E or Q); 250 by EU numbering (263 by Kabat numbering) and 428 by EU numbering (459 by Kabat numbering) (e.g., L or F); 252 by EU numbering (265 by Kabat numbering) (e.g., L/Y/F/W or T), 254 by EU numbering (267 by Kabat numbering) (e.g., S or T), and 256 by EU numbering (269 by Kabat numbering) (e.g., S/R/Q/E/D or T); or a modification at position 428 by EU numbering (459 by Kabat numbering) and/or 433 by EU numbering (464 by Kabat numbering) (e.g., L/R/S/P/Q or K) and/or 434 by EU numbering (465 by Kabat numbering) (e.g., H/F or Y); or a modification at position 250 by EU numbering (263 by Kabat numbering) and/or 428 by EU numbering (459 by Kabat numbering); or a modification at position 307 by EU numbering (326 by Kabat numbering) or 308 by EU numbering (327 by Kabat numbering) (e.g., 308F, V308F), and 434 by EU numbering (465 by Kabat numbering). In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification by EU numbering (a 459, e.g., M459L, and 465S (e.g., N465S) modification by Kabat numbering); a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification by EU numbering (a 459L, 272I (e.g., V272I), and 327F (e.g., V327F) modification by Kabat numbering; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification by EU numbering (a 464K (e.g., H464K) and a 465 (e.g., 465Y) modification by Kabat numbering; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification by EU numbering (a 265, 267, 269 (e.g., 265Y, 267T, and 269E) modification by Kabat numbering; a 250Q and 428L modification (e.g., T250Q and M428L) by EU numbering (a 263Q and 459L modification, e.g., T263Q and M459L, by Kabat numbering); and a 307 and/or 308 modification (e.g., 307F or 308P) by EU numbering (326 and/or 327 modification, e.g., 326F or 308P, by Kabat numbering), wherein the modification increases the affinity of the heavy chain constant region amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH2 amino acid sequence comprising at least one modification between amino acid residues at positions 252 and 257 by EU numbering (i.e., at least one modification between amino acid positions 265 and 270 by Kabat numbering), wherein the modification increases the affinity of the human CH2 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH2 amino acid sequence comprising at least one modification between amino acid residues at positions 307 and 311 (i.e., at least one modification between amino acid positions 326 and 330 by Kabat numbering), wherein the modification increases the affinity of the CH2 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human CH3 amino acid sequence, wherein the CH3 amino acid sequence comprises at least one modification between amino acid residues at positions 433 and 436 by EU numbering (i.e., at least one modification between amino acid residues at positions 464 and 467 by Kabat numbering), wherein the modification increases the affinity of the CH3 amino acid sequence to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M428L by EU numbering (459 by Kabat numbering), N434S by EU numbering (465 by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M428L by EU numbering (M459L by Kabat numbering), V259I by EU numbering (V272I by Kabat numbering), V308F by EU numbering (V327 by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising an N434A mutation by EU numbering (an N465A mutation by Kabat numbering). In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of M252Y by EU numbering (M265Y by Kabat numbering), S254T by EU numbering (S267T by Kabat numbering), T256E by EU numbering (T269E by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of T250Q by EU numbering (T263Q by Kabat numbering), M428L by EU numbering (M459L by Kabat numbering), and a combination thereof. In some embodiments, the heavy chain constant region nucleotide sequence encodes a human heavy chain constant region amino acid sequence comprising a mutation selected from the group consisting of H433K by EU numbering (H464K by Kabat numbering), N434Y by EU numbering (N465Y by Kabat numbering), and a combination thereof.
In various embodiments, Fc domains are modified (in the non-human animal; or, in an expression system that expresses together in a single polypeptide a light chain variable domain derived from a heavy chain of a non-human animal as described herein and a heavy chain constant sequence (e.g., a human sequence)) to have altered Fc receptor binding, which in turn affects effector function. In some embodiments, an engineered heavy chain constant region (CH), which includes the Fc domain, is chimeric. As such, a chimeric CH region combines CH domains derived from more than one immunoglobulin isotype. For example, a chimeric CH region comprises part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. In some embodiments, a chimeric CH region contains a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering; amino acid residues from positions 226 to 240 according to Kabat numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering; amino acid positions from positions 241 to 249 according to Kabat numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. In some embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.
In some embodiments, the Fc domain may be engineered to activate all, some, or none of the normal Fc effector functions, without affecting the Fc-containing protein's (e.g. antibody's) desired pharmacokinetic properties. For examples of proteins comprising chimeric CH regions and having altered effector functions, see International Patent Application No. PCT/US2014/14175, filed Jan. 31, 2014, which is herein incorporated in its entirety.
In various aspects, the genome of the non-human animals is modified (i) to delete or render nonfunctional (e.g., via insertion of a nucleotide sequence (e.g., an exogenous nucleotide sequence)) in the immunoglobulin locus or via non-functional rearrangement or inversion of all, or substantially all, endogenous functional immunoglobulin VH, D, JH gene segments; and (ii) to comprise unrearranged human immunoglobulin light chain variable region gene segments, wherein the gene segments are present at an endogenous locus (i.e., where the gene segments are located in a wild type non-human animal). In some embodiments, the unrearranged human immunoglobulin light chain variable region gene segments are integrated in the genome (e.g., at a locus different from the endogenous immunoglobulin heavy chain locus in its genome, or within its endogenous locus, e.g., within an immunoglobulin variable locus, wherein the endogenous locus is placed or moved to a different location in the genome). In some embodiments, e.g., about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of all endogenous functional heavy chain V, D, or J gene segments are deleted or rendered non-functional. In some embodiments, e.g., at least 95%, 96%, 97%, 98%, or 99% of endogenous functional heavy chain V, D, or J gene segments are deleted or rendered non-functional. In some embodiments, the unrearranged human immunoglobulin light chain variable region gene segments are operably linked to a human or non-human heavy chain constant region gene sequence.
In some embodiments, the genetically modified non-human animal comprises a modification that deletes or renders non-functional endogenous functional VH, D, and JH heavy chain variable gene segments and endogenous functional light chain variable VL and JL gene segments; and comprises (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) and (ii) a nucleotide sequence encoding unrearranged human immunoglobulin light chain V gene segments (VL) and unrearranged human immunoglobulin light chain J gene segments (JL) at an endogenous immunoglobulin locus (e.g., an endogenous immunoglobulin heavy chain locus comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene) or integrated elsewhere in the genome (e.g., at a locus different from the endogenous immunoglobulin locus in its genome, or within its endogenous locus, e.g., within an immunoglobulin variable region locus, wherein the endogenous locus is placed or moved to a different location in the genome). In some embodiments, the genetically modified non-human animal comprises a modification that deletes or renders non-functional endogenous VH, D, and JH heavy chain variable gene segments and endogenous light chain variable VL and JL gene segments; and comprises (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) and (ii) one or more unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) at an endogenous location (e.g., an endogenous immunoglobulin heavy chain locus) or integrated elsewhere in the genome (e.g., at a locus different from the endogenous immunoglobulin chain locus in its genome, or within its endogenous locus, e.g., within an immunoglobulin variable region locus, wherein the endogenous locus is placed or moved to a different location in the genome). In some embodiments, e.g., about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of all endogenous functional heavy chain V, D, or J gene segments are deleted or rendered non-functional. In some embodiments, e.g., at least 95%, 96%, 97%, 98%, or 99% of endogenous functional heavy chain V, D, or J gene segments are deleted or rendered non-functional. In some embodiments, the unrearranged human immunoglobulin light chain variable region gene segments are operably linked to a human or non-human heavy chain constant region gene sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) is operably linked to a human or non-human light chain constant region gene sequence, either kappa or lambda.
Various embodiments encompass light chain variable domains derived from immunoglobulin hybrid chains encoded by a hybrid immunoglobulin locus. Nucleic acid sequences encoding light chain variable domains may be used in making the genetically modified non-humans described herein, may be expressed by such animals, and/or may encode amino acids present in antibodies produced by (or derived from sequences diversified by) such animals. In some embodiments, the light chain variable domain is a human Vκ domain. In some embodiments, the light chain variable domain is a mouse Vκ domain. In some embodiments, the light chain variable domain is a rat Vκ domain. In some embodiments, the light chain variable domain is a human Vλ domain. In some embodiments, the light chain variable domain is a mouse Vλ domain. In some embodiments, the light chain variable domain is a rat Vλ domain.
In various embodiments, the light chain variable domains produced by the genetically modified non-human animals described herein are encoded by one or more mouse or human immunoglobulin κ light chain variable gene segments. In some embodiments, the one or more mouse immunoglobulin κ light chain variable gene segments comprise about three megabases of the mouse immunoglobulin κ light chain locus. In some embodiments, the one or more mouse immunoglobulin κ light chain variable gene segments comprises at least 137 Vκ gene segments, at least five Jκ gene segments or a combination thereof of the mouse immunoglobulin κ light chain locus. In some embodiments, the one or more human immunoglobulin κ light chain variable gene segments comprises about one-half megabase of a human immunoglobulin κ light chain locus. In specific embodiments, the one or more human immunoglobulin κ light chain variable gene segments comprise the proximal repeat (with respect to the immunoglobulin κ constant region) of a human immunoglobulin κ light chain locus. In some embodiments, the one or more human immunoglobulin κ light chain variable gene segments comprises at least 40 Vκ gene segments, at least five Jκ gene segments or a combination thereof of a human immunoglobulin κ light chain locus.
In particular embodiments, the genetically modified non-human animals further comprise a nucleotide sequence encoding an unrearranged human immunoglobulin light chain (VL) gene segment and an unrearranged human immunoglobulin light chain (JL) gene segment. In some embodiments, the nucleotide sequence encoding the unrearranged light chain V gene segment and the unrearranged light chain J gene segment is operably linked to an immunoglobulin heavy chain constant region gene sequence. In some embodiments, the unrearranged human immunoglobulin light chain V (VL) gene segment and the unrearranged human immunoglobulin J (JL) gene segment are operably linked, at an endogenous rodent locus, to a rodent immunoglobulin heavy chain constant region gene; e.g., an IgM or IgG heavy chain constant region gene, each of which encode a functional CH1 domain.
In various embodiments, the unrearranged human variable region gene segments (e.g., human Vκ gene segments) are capable of rearranging and encoding human variable domains of an antibody. In some embodiments, the non-human animal does not comprise an endogenous VL gene segment. In some embodiments, the human Vκ gene segments expressed by the non-human animals are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. In some embodiments, the genetically modified non-human animals described herein express all functional human Vκ genes. In some embodiments, the human Vκ gene segments comprise Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, and Vκ1-6. In some embodiments, the Vκ gene segments comprise Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15 and Vκ1-16. In some embodiments, the human Vκ gene segments comprise Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, and Vκ2-30. In some embodiments, the human Vκ gene segments comprise Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, and Vκ2-40. In various embodiments, the non-human animal comprises five human Jκ gene segments, e.g., Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 gene segments. In specific embodiments, the Vκ gene segments comprise contiguous human immunoglobulin κ gene segments spanning the human immunoglobulin κ light chain locus from Vκ4-1 through Vκ2-40, and the Jκ1 gene segments comprise contiguous gene segments spanning the human immunoglobulin κ light chain locus from Jκ1 through Jκ5. In some embodiments, the immunogloboulin light chain locus of the non-human animal comprise two human VL gene segments, Vκ1-39 and Vκ3-20. In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) human VL gene segments and two or more human JL gene segments are present at an endogenous heavy chain locus. In some embodiments, the genetically modified non-human animal is a mouse that comprises a functional λ light chain locus. In other embodiments, the mouse comprises a non-functional λ light chain locus.
In some embodiments, a genetically modified non-human animal (e.g., mouse or rat) as described herein expresses a rearranged human immunoglobulin light chain variable region nucleotide sequence (i.e., produces an antigen-binding protein comprising a rearranged light chain variable domain) and one or more, two or more, three or more, four or more, five or more, etc. light chain variable domains encoded by Vκ genes selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3.
In various embodiments, the rearranged human light chain variable region nucleotide sequence encodes one or more histidine codons that are not encoded by a corresponding human germ line light chain variable gene segment. In some embodiments, the light chain variable domain as described herein exhibits a decrease in dissociative half-life (t1/2) at an acidic pH as compared to neutral pH of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold. In some embodiments, the decrease in t1/2 at an acidic pH as compared to a neutral pH is about 30 fold or more. In some embodiments, the rearranged human light chain variable region nucleotide sequence (or at least one of the limited number of human VL gene segments) comprises a substitution of at least one non-histidine codon encoded by the corresponding human germ line VL gene segment with a histidine codon. In some embodiments, the substitution is of one, two, three, or four codons (e.g., three or four codons). In some embodiments, the substitution is in the CDR3 codon(s). In some embodiments, the human VL gene segments is a human Vκ1-39 or human Vκ3-20 gene segment, and the human Vκ1-39 or human Vκ3-20 gene segment comprises a substitution of at least one non-histidine codon encoded by a corresponding human germ line VL gene segment with the histidine codon. In some embodiments, the human Vκ1-39 or human Vκ3-20 gene segment comprises a substitution of three or four histidine codons. In some embodiments, the three or four substitutions are in the CDR3 region. In some embodiments, the substitution is of three non-histidine codons of the human Vκ1-39 gene segment, wherein the substitution is designed to express histidines at positions 106, 108, and 111. In some embodiments, the substitution is of four non-histidine codons of the human Vκ1-39 gene segment, and the substitution is designed to express histidines at positions 105, 106, 108, and 111 (see, e.g., U.S. Patent Application Publication No. 2013-0247234 A1 and WO 2013/138680, incorporated by reference herein). In some embodiments, the substitution is of three non-histidine codons of the human Vκ3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, and 109. In yet additional embodiments, the substitution is of four non-histidine codons of the human Vκ3-20 gene segment, and the substitution is designed to express histidines at positions 105, 106, 107, and 109. In some embodiments, the immunoglobulin light chain locus comprises a rearranged human light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments), wherein the nucleotide sequence (or at least one of the limited number of human VL gene segments) comprises at least one histidine codon that is not encoded by the corresponding human germ line VL gene segment. In various embodiments, the non-human animal comprising the genetically modified immunoglobulin loci as described herein, upon stimulation by an antigen of interest, expresses an antigen-binding protein comprising an amino acid sequence derived from human VL gene segments, wherein the antigen-binding protein retains at least one histidine residue at an amino acid position encoded by the at least one histidine codon introduced into the rearranged human light chain variable region nucleotide sequence (or the at least one of the limited number of human VL gene segments). In some embodiments, the animal expresses a population of antigen-binding proteins in response to an antigen, wherein all antigen-binding proteins in the population comprise (a) immunoglobulin light chain variable domains derived from a rearrangement of the human VL gene segments and the JL gene segments, and (b) immunoglobulin light chains comprising human light chain variable domains encoded by the rearranged human immunoglobulin light chain variable region nucleotide sequence (or encoded by one of the limited number of human VL gene segments), wherein rearranged human immunoglobulin light chain variable region nucleotide sequence (or at least one of the limited number of human VL gene segments) encodes one or more histidine codons that are not encoded by the corresponding human germ line VL gene segment.
Various embodiments encompass light chain constant region sequences. In some embodiments, for example, a first nucleotide sequence that encodes a human light chain variable domain (i.e., where the first nucleotide sequence contains unrearranged human immunoglobulin light chain variable region gene segments) is operably linked to a heavy chain constant region gene sequence, and a second nucleotide sequence that encodes a human light chain variable domain (i.e., where the second nucleotide sequence is a rearranged human immunoglobulin light chain variable nucleotide sequence or where the second sequence includes a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) is operably linked to a light chain constant region gene sequence. In various embodiments, the light chain constant region sequence operably linked to the rearranged human immunoglobulin light chain variable region nucleotide sequence (or limited number of human VL gene segments) is a human κ light chain constant region sequence. In some embodiments, the light chain constant region sequence operably linked to the rearranged light chain variable region nucleotide sequence (limited number of human VL gene segments) is a mouse κ light chain constant region sequence. In some embodiments, the light chain constant region sequence operably linked to the rearranged light chain variable region nucleotide sequence (limited number of human VL gene segments) is a rat κ light chain constant region sequence. In some embodiments, the light chain constant region sequence operably linked to the rearranged light chain variable region nucleotide sequence (limited number of human VL gene segments) is a human λ light chain constant region sequence. In some embodiments, the light chain constant region sequence operably linked to the rearranged light chain variable region nucleotide sequence (limited number of human VL gene segments) is a mouse light chain constant region sequence. In some embodiments, the light chain constant region sequence operably linked to the rearranged light chain variable region nucleotide sequence (limited number of human VL gene segments) is a rat λ light chain constant region sequence.
In various aspects, non-human animals are provided comprising a genetically modified immunoglobulin locus that encodes a rearranged light chain variable domain (e.g., where an immunoglobulin locus comprises a rearranged human immunoglobulin light chain variable region nucleotide sequence or a restricted (limited) number of human VL gene segments), wherein the rearranged light chain variable domain comprises a light chain variable (VL) sequence that is operably linked to a light chain J segment (JL) sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or limited number of human VL gene segments) is operably linked to a non-human light chain constant region gene sequence. In some embodiments, the non-human light chain constant region gene sequence is a mouse or a rat constant region gene sequence. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or limited number of human VL gene segments) is operably linked to a human light chain constant region gene sequence.
In another aspect, genetically modified non-human animals and methods for making said animals are provided in which the animals comprise a functional universal light chain (“ULC”) immunoglobulin locus (see, e.g., 2011-0195454 A1, US 2012-0021409A1, US 2012-0192300A1, US 2013-0045492A1, US 2013-0185821A1 and US 2013-0302836A1, incorporated by reference herein in their entireties) or a functional dual light chain (“DLC”) immunoglobulin locus (see, e.g., U.S. Patent Application Publication No. US-2013-0198880-A1, incorporated by reference herein in its entirety). In some embodiments, such animals further comprise unrearranged light chain variable region gene segments operably linked to a human or non-human heavy chain constant region gene sequence (i.e., human VL and JL gene segments operably linked to an IgM, IgG, etc.). A ULC or DLC as used in the embodiments described herein can also be used to generate antibody variable chain sequences whose diversity results primarily from the processes of somatic mutation, thereby elucidating antibody variable chain sequences whose antigen-binding capacity benefits from post-genomic events.
Methods of Making and Using Non-Human Animals Comprising a High Diversity Hybrid Chain Locus Containing Unrearranged Light Chain Variable Region Gene Segments and a Low Diversity Light Chain Locus Containing a Rearranged Light Chain Variable Region Sequence
Methods of making and using the genetically modified non-human animals described herein are provided. Methods are provided for placing a rearranged human light chain variable region nucleic acid sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) in operable linkage with an immunoglobulin light chain constant region nucleic acid sequence in the genome of a non-human animal. In various embodiments, the constant region nucleic acid sequence is human or non-human, and the non-human animal is a rodent. In various embodiments, the methods comprise making a non-human animal that further comprises a hybrid immunoglobulin chain locus, e.g., an immunoglobuliln locus comprising one or more human light chain variable region gene segments, e.g., 40 human Vκ gene segments and five human Jκ gene segments, operably linked to a human or non-human heavy chain constant region nucleic acid sequence. In various aspects, the methods comprise placing the aforementioned sequences in the germ line of a non-human animal, e.g., a rodent, employing, e.g., transgenic technology including, e.g., employing modified pluripotent or totipotent donor cells (e.g., ES cells or iPS cells) with host embryos, germ cells (e.g., oocytes), etc. Thus, embodiments include a non-human hybrid immunoglobulin chain locus., e.g., an immunoglobulin chain locus in a genome of a non-human germ cell comprising unrearranged human immunoglobulin light chain variable region gene segments operably linked to a heavy chain constant region gene sequence, wherein the constant region gene sequence comprises a non-human sequence, a human sequence, or a combination thereof. In some embodiments, the rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments) is operably linked to an endogenous non-human immunoglobulin constant region gene sequence. In some embodiments, the endogenous non-human immunoglobulin constant region gene sequence is a mouse or a rat light chain constant region gene sequence.
In various aspects, a method of making a non-human animal that comprises a genetically modified immunoglobulin locus is provided, wherein the method comprises: (a) modifying a genome of a non-human animal to delete or render non-functional endogenous functional immunoglobulin heavy chain V, D, and J gene segments; and (b) placing in the genome unrearranged human immunoglobulin light chain variable region gene segments. In one such aspect, a method is provided for making a non-human animal that expresses a single immunoglobulin light chain from a rearranged light chain gene sequence in the germ line of the non-human animal (or expressing an immunoglobulin light chain from a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments), the method comprising a step of genetically modifying a non-human animal such that its entire antibody-expressing mature B cell population expresses a light chain derived from (i) a single VL gene segment, and (ii) a single JL gene segment or from (iii) a limited number of human VL gene segments (e.g., two or more but less than the wild type number of human VL gene segments). In some aspects, the method comprises inactivating or replacing an endogenous light chain immunoglobulin variable locus with a single rearranged light chain gene (or limited number of human VL gene segments) as described herein.
In another aspect, methods of making a non-human animal that comprises a genetically modified immunoglobulin heavy chain locus are provided, such methods comprising: (a) modifying a genome of a non-human animal to delete or render non-functional endogenous functional immunoglobulin heavy chain V, D, and J gene segments; and (b) placing in the genome unrearranged human immunoglobulin light chain variable region gene segments. In some embodiments, substantially all endogenous functional VH, D, and JH gene segments are deleted from the immunoglobulin heavy chain locus of the non-human animal or rendered non-functional (e.g., via insertion of a nucleotide sequence (e.g., an exogenous nucleotide sequence in the immunoglobulin locus) or via non-functional rearrangement, or inversion of, endogenous VH, D, JH segments). In some embodiments, the method comprises inserting unrearranged human immunoglobulin light chain variable region gene segments into an endogenous location (e.g., an endogenous immunoglobulin heavy chain locus). In some embodiments, the unrearranged human immunoglobulin light chain variable region gene segments are present elsewhere in the genome (e.g., at a locus different from the endogenous immunoglobulin chain locus in its genome, or within its endogenous locus, e.g., within an immunoglobulin variable locus, wherein the endogenous locus is placed or moved to a different location in the genome). In some embodiments, e.g., about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of all endogenous functional V, D, or J gene segments are deleted or rendered non-functional. In some embodiments, e.g., at least 95%, 96%, 97%, 98%, or 99% of endogenous functional heavy chain V, D, or J gene segments are deleted or rendered non-functional.
In another aspect, methods are provided for making a non-human animal that comprises a genetically modified immunoglobulin locus, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional endogenous functional immunoglobulin light chain V and J gene segments; and (b) placing in an endogenous immunoglobulin light chain locus a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (i.e., a nucleotide sequence that encodes a rearranged light chain variable domain) or a limited number of human or non-human VL gene segments (e.g., two or more but less than the wild type number of human VL gene segments), wherein the nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a light chain constant region gene sequence. In some embodiments, the genetically engineered immunoglobulin locus is present in the germ line genome of the non-human animal. In some embodiments, the rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a κ light chain constant region gene sequence. In some embodiments, the rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a mouse or rat κ light chain constant region gene sequence. In some embodiments, the rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a human κ light chain constant region gene sequence. In some embodiments, the rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a λ light chain constant region gene sequence. In some embodiments, rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a mouse or rat λ light chain constant region gene sequence. In some embodiments, the rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or limited number of human or non-human VL gene segments) is operably linked to a human λ light chain constant region gene sequence.
In some embodiments, the limited number of human or non-human VL gene segments are operably linked to one or more human or non-human JL gene segments.
In another aspect, methods are provided for making a non-human animal that comprises a genetically modified immunoglobulin locus, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional: (i) endogenous functional immunoglobulin heavy chain V, D, and J gene segments, and (ii) endogenous functional immunoglobulin light chain V and J gene segments; and (b) placing in the genome: (i) a first nucleotide sequence that encodes a rearranged light chain variable domain (e.g., where the first nucleotide sequence is a rearranged human immunoglobulin light chain variable region nucleotide sequence or where the first nucleotide sequence contains a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments), wherein the first nucleotide sequence is operably linked to a light chain constant region gene sequence, and (ii) a second nucleotide sequence that encodes a human immunoglobulin light chain variable domain (i.e., where the second nucleotide sequence is an unrearranged human immunoglobulin light chain variable region nucleotide sequence), wherein the second nucleotide sequence is operably linked to a heavy chain constant region gene sequence comprising one or more heavy chain constant region genes each one comprising a sequence encoding a functional CH1 domain, e.g., comprising at least an intact Igμ gene and at least one of an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and an intact Igα gene. In some embodiments, the genetically engineered immunoglobulin locus is present in the germ line genome of the non-human animal. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a κ light chain constant region gene sequence. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a mouse or rat κ light chain constant region gene sequence. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a human κ light chain constant region gene sequence. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a λ light chain constant region gene sequence. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a mouse or rat λ light chain constant region gene sequence. In some embodiments, the first nucleotide sequence that encodes the rearranged light chain variable domain (or contains a limited number of human VL gene segments) is operably linked to a human λ light chain constant region gene sequence. In some embodiments, the human immunoglobulin light chain variable domain is a κ light chain variable domain. Thus, in some embodiments, the second nucleotide sequence is a human kappa light chain variable region nucleotide sequence. In some embodiments, the human immunoglobulin light chain variable domain is a λ light chain variable domain. Thus, in some embodiments, the second nucleotide sequence is a human lambda light chain variable region nucleotide sequence. In some embodiments, the heavy chain constant region gene sequence is a non-human immunoglobulin heavy chain constant region gene sequence. In some embodiments, the non-human immunoglobulin heavy chain constant region gene sequence is a mouse or a rat heavy chain constant region gene sequence. In some embodiments, the non-human immunoglobulin heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene.
Methods are provided for making a non-human animal, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional (i) endogenous immunoglobulin heavy chain VH, D, and and/or JH gene segments, and (ii) endogenous immunoglobulin light chain V and J gene segments; and (b) placing (i) a rearranged light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) at a light chain locus, wherein the rearranged light chain variable region nucleotide sequence (or limited number of human VL gene segments) comprises a light chain V gene segment (VL) sequence that is operably linked to a light chain J gene segment (JL) sequence; and (ii) one or more unrearranged human immunoglobulin light chain variable region gene segments (e.g., 40 human Vκ gene segments and at least one human Jκ gene segments) at a heavy chain locus so that the gene segments are operably linked to a human or non-human heavy chain constant region nucleotide sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the rearranged light chain variable region nucleotide sequence encodes one or more histidine codons that are not encoded by a corresponding human germ line light chain variable gene segment.
In some aspects, a method for making a non-human animal comprising a genetically modified immunoglobulin locus is provided, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional endogenous immunoglobulin light chain V and J gene segments; and (b) placing in the genome of the non-human animal a rearranged human or non-human light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) in operable linkage to a light chain constant region nucleotide sequence.
In various embodiments, the non-human animal is a rodent, e.g., a mouse, a rat, or a hamster. In some embodiments, the rodent is a mouse. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region.
In another aspect, a method for making a non-human animal comprising a genetically modified immunoglobulin locus is provided, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional: (i) endogenous immunoglobulin heavy chain V, D, and/or J gene segments, and (ii) endogenous immunoglobulin light chain V and J gene segments; and (b) placing in the genome of the non-human animal: (i) a first nucleotide sequence that encodes a rearranged light chain variable domain (e.g., where the first nucleotide sequence is a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence or where the first nucleotide sequence contains a limited number of human VL gene segments; e.g., two or more but less than the wild type number of human VL gene segments), wherein the first nucleotide sequence is operably linked to a light chain constant region gene sequence, and (ii) a second nucleotide sequence that encodes a human or non-human light chain variable domain (i.e., where the second nucleotide sequence is an unrearranged human immunoglobulin light chain variable region nucleotide sequence), wherein the second nucleotide sequence is operably linked to a heavy chain constant region gene sequence. In some embodiments, the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene.
In various embodiments, the non-human animal is a rodent, e.g., a mouse, a rat, or a hamster. In some embodiments, the rodent is a mouse. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region. In some embodiments, the second nucleotide sequence is operably linked to a mouse or rat heavy chain constant region gene sequence comprising a nucleotide sequence encoding a CH1, a hinge, a CH2, a CH3, or a combination thereof. In some embodiments, the second nucleotide sequence is operably linked to a human heavy chain constant region gene sequence comprising a nucleotide sequence encoding a CH1, a hinge, a CH2, a CH3, or a combination thereof.
In another aspect, a method is provided for making a non-human animal that comprises a genetically modified immunoglobulin locus, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional: (i) endogenous immunoglobulin heavy chain V, D, and/or J gene segments, and (ii) endogenous immunoglobulin light chain V and J gene segments; and (b) placing in the genome of the non-human animal: (i) a first allele comprising a first nucleotide sequence that encodes a rearranged light chain variable domain (e.g., where the first nucleotide sequence is a rearranged human immunoglobulin light chain variable region nucleotide sequence or where the first nucleotide sequence contains a limited number of human VL gene segments; e.g., two or more but less than the wild type number of human VL gene segments) operably linked to a light chain constant region gene sequence, and (ii) a second allele comprising a second nucleotide sequence that encodes a light chain variable domain (i.e., where the second nucleotide sequence is an unrearranged human immunoglobulin light chain variable region nucleotide sequence) operably linked to a heavy chain constant region gene sequence.
In another aspect, a method of making a non-human animal that comprises a genetically modified immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) and a modified immunoglobulin light chain locus is provided comprising: (a) modifying a genome of a non-human animal to delete or render non-functional endogenous immunoglobulin heavy chain V, D, and and/or J gene segments; (b) placing in an endogenous heavy chain locus of the non-human animal unrearranged human immunoglobulin light chain variable region gene segments in operable linkage with a heavy chain constant region, wherein the unrearranged human immunoglobulin light chain variable region gene segments comprise human Vκ and human Jκ gene segments; (c) modifying a genome of a non-human animal to delete or render non-functional endogenous immunoglobulin light chain V and and/or J gene segments; and (d) placing in an endogenous light chain locus of the non-human animal a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) in operable linkage with a light chain constant region, wherein the rearranged human immunoglobulin light chain variable region nucleotide sequence (or limited number of human VL gene segments) comprises a rearranged human VκCκ sequence (or 2, 3, or 4 human VL gene segments). In some embodiments, the rearranged human VκJκ sequence is a human Vκ1-39Jκ5 sequence (e.g., set forth in SEQ ID NO: 1). In some embodiment, the rearranged human VκJκ sequence is a human Vκ3-20Jκ1 sequence (e.g., set forth in SEQ ID NO:2). In some embodiments, the limited number of human VL gene segments includes a human Vκ1-39 gene segment and a human Vκ3-20 gene segment. In some embodiments, the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene.
In various embodiments, the non-human animal is a rodent, e.g., a mouse, a rat, or a hamster. In some embodiments, the rodent is a mouse. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region. In some embodiments, the unrearranged human light chain variable region gene segments are operably linked to a mouse or rat heavy chain constant region gene sequence comprising a nucleotide sequence encoding CH1, a hinge, a CH2, a CH3, or a combination thereof. In some embodiments, the unrearranged light chain variable region gene segments are operably linked to a human heavy chain constant region gene sequence comprising a nucleotide sequence encoding a CH1, a hinge, a CH2, a CH3, or a combination thereof. In some embodiments, the unrearranged light chain variable region gene segments are operably linked to a human heavy chain constant region gene sequence comprising a nucleotide sequence encoding each of a CH1, a hinge, a CH2, and a CH3 domain.
In another aspect, a method for making a non-human animal comprising a genetically modified immunoglobulin locus is provided, comprising: (a) modifying a genome of a non-human animal to delete or render non-functional: (i) endogenous immunoglobulin heavy chain V, D, and/or J gene segments, and (ii) endogenous immunoglobulin light chain V and J gene segments; and (b) placing in the genome of the non-human animal: (i) a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) in operable linkage to a light chain constant region nucleotide sequence; and (ii) one or more human immunoglobulin light chain variable VL and JL gene segments in operable linkage to a heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene.
In various embodiments, the non-human animal is a rodent, e.g., a mouse, a rat, or a hamster. In some embodiments, the rodent is a mouse. In some embodiments, the light chain constant region is a rat or a mouse constant region, e.g., a rat or a mouse Cκ constant region.
In another aspect, nucleic acid sequences encoding rearranged light chain variable domains are provided. In some embodiments, the nucleic acid sequence is derived from a human Vκ and Jκ gene segments. In some embodiments, the nucleic acid sequence is derived from a human germ line Vκ segment and a human germ line Jκ segment. In some embodiments, the human Vκ segment corresponds to observed variants in the human population. In various embodiments, the nucleic acid sequence comprises a human Vκ gene selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1D-27, Vκ1D-32, Vκ1D-33, Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3, and a polymorphic variant thereof. In some embodiments, the nucleic acid sequence further comprises a human or non-human animal heavy chain constant region gene sequence selected from a nucleotide sequence encoding a CH1, a hinge, a CH2, a CH3, and a combination thereof. In specific embodiments, the nucleic acid comprises a constant region gene sequence comprising a nucleotide sequence encoding a CH1, a hinge, a CH2, and a CH3. In various embodiments, the nucleic acid sequence comprises a human Jκ gene segment is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a polymorphic variant thereof.
In another aspect, a nucleic acid construct is provided comprising an unrearranged human immunoglobulin light chain variable region nucleotide sequence (e.g., a nucleotide sequence that contains unrearranged human VL and JL gene segments) as described herein. In some embodiments, the nucleic acid construct is designed in such a way that the unrearranged human immunoglobulin light chain variable region nucleotide sequence is operably linked to a human or non-human animal heavy chain constant region gene sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the nucleic acid construct contains two, three, four, or more unrearranged human immunoglobulin light chain variable region gene segments operably linked to a heavy chain constant region gene sequence. In some embodiments, the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene. In some embodiments, the nucleic acid construct is a targeting vector. In some embodiments, the targeting vector comprises an Adam6a gene, an Adam6b gene, or both, in order to prevent fertility problems associated with the deletion of the Adam6a/6b genes (see, for example, U.S. Pat. No. 8,642,835, incorporated by reference in its entirety). In some embodiments, the Adam6a and the Adam6b genes are placed at 5′ upstream of the transcriptional unit of the unrearranged human light chain gene segments. In some embodiments, the targeting vector comprises a selection cassette flanked by recombination sites. In some embodiments, the targeting vector comprises one or more site-specific recombination sites (e.g., a loxP or a FRT site).
In another aspect, methods are provided for obtaining a light chain variable region (VL/CHxULC) amino acid sequence capable of binding an antigen independently from a heavy chain variable region amino acid sequence, comprising: (a) immunizing a genetically modified non-human animal as described herein (e.g., a genetically modified animal whose genome comprises unrearranged human light chain variable region gene segments in operable linkage with a heavy chain constant region gene and a rearranged human or non-human light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) in operable linkage with a light chain constant region gene) with an antigen of interest, wherein the non-human animal mounts an immune response to the antigen; and (b) obtaining a rearranged light chain (VJ) nucleic acid sequence of a light chain variable domain that specifically binds the antigen from a cell (e.g., a B cell) of the genetically modified non-human animal. In various embodiments, the light chain variable regions produced by such methods are provided.
In some aspects, methods for obtaining a nucleic acid sequence that encodes an immunoglobulin light chain variable region (VL/CHxULC) domain, comprise: (a) optionally immunizing a non-human animal with an antigen of interest or an immunogen thereof, wherein the non-human animal comprises in its genome (i) unrearranged human light chain variable region gene segments operably linked to a heavy chain constant region gene comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Ig gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene, and (ii) a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) operably linked to a light chain constant region gene, (b) allowing the non-human animal to mount an immune response, (c) isolating from the immunized non-human animal a cell comprising a nucleic acid sequence that encodes a light chain variable domain that binds the antigen of interest, and (d) obtaining from the cell a nucleic acid sequence that encodes the light chain variable domain (VL/CHxULC domain) that binds the antigen of interest. In some embodiments, the heavy chain constant region gene sequence is a mouse or rat heavy chain constant region gene sequence. In some embodiments, the heavy chain constant region gene sequence is a human heavy chain constant region gene sequence. In some embodiments, the rearranged light chain variable domain expressed by the genetically modified locus is not autoreactive, i.e., non-immunogenic to the non-human animal. In some embodiments, the non-human animal comprises in its genome one or more (e.g., 6, 16, 30 or 40) unrearranged human VL gene segments and one or more (e.g., 5) human JL gene segments. In some certain embodiments, the unrearranged human VL and JL gene segments are Vκ and Jκ gene segments. In some embodiments, the isolating step (c) is carried out via fluorescence-activated cell sorting (FACS) or flow cytometry. In some embodiments, the cell comprising the nucleic acid sequence that encodes the light chain variable domain that binds the antigen of interest is a lymphocyte. In some embodiments, the lymphocyte comprises natural killer cells, T cells, or B cells. In some embodiments, the method further comprises a step of (c)′ fusing the lymphocyte with a cancer cell. In certain embodiments, the cancer cell is a myeloma cell.
Thus, in various aspects, methods are provided for obtaining a nucleic acid sequence that encodes an immunoglobulin light chain variable domain (VL/CHxULC) capable of binding an antigen independently from a heavy chain variable domain, comprising: (a) optionally immunizing a non-human animal with an antigen of interest or an immunogen thereof, wherein the non-human animal comprises in its genome (i) a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) operably linked to a light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to a heavy chain constant region nucleotide sequence; (b) allowing the non-human animal to mount an immune response; (c) isolating from the immunized non-human animal a cell comprising a nucleic acid sequence that encodes a light chain variable domain that can bind the antigen; and (d) obtaining from the cell a nucleic acid sequence that encodes the light chain variable domain (VL/CHxULC domain) that can bind the antigen.
In some embodiments, the isolating step (c) is carried out via fluorescence-activated cell sorting (FACS) or flow cytometry. In some embodiments, the cell comprising the nucleic acid sequence that encodes the light chain variable domain that binds the antigen is a lymphocyte. In particular embodiments, the lymphocyte comprise natural killer cells, T cells, or B cells. In some embodiments, the methods further comprise a step of (c)′ fusing the lymphocyte with a cancer cell. In particular embodiments, the cancer cell is a myeloma cell. In some embodiments, the nucleic acid sequence of (d) is fused with a nucleic acid sequence encoding an immunoglobulin constant region nucleic acid sequence. In some embodiments, the light chain constant region nucleic acid sequence is a human kappa sequence or a human lambda sequence. In some embodiments, the light chain constant region nucleic acid sequence is a mouse kappa sequence or a mouse lambda sequence. In some embodiments, the light chain constant region nucleic acid sequence is a rat kappa sequence or a rat lambda sequence. In some embodiments, the heavy chain constant region nucleic acid sequence is a human sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Ig gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the heavy chain constant region nucleic acid sequence is a mouse or rat sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Ig gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the nucleic acid sequence of (d) comprises one or more histidine codon substitutions or insertions that are derived from the unrearranged VL gene segment in the genome of the animal.
In some aspects, methods are provided for obtaining a nucleic acid sequence that encodes an immunoglobulin light chain variable domain (VL/CHxULC), comprising: (a) optionally immunizing a non-human animal containing a genetically modified immunoglobulin loci as described herein with an antigen of interest, wherein the non-human animal comprises in its genome a rearranged human immunoglobulin light chain variable region nucleic acid sequence (or a limited number of human VL gene segments) operably linked to a light chain constant region nucleic acid sequence and unrearranged human immunoglobulin light chain variable region gene segments operably linked to a heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Ig gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene; (b) allowing the non-human animal to mount an immune response; (c) harvesting a lymphocyte (e.g., a B cell) from the immunized non-human animal; (d) fusing the lymphocyte with a myeloma cell to form a hybridoma cell; and (e) obtaining from the hybridoma cell a nucleic acid sequence that encodes a light chain variable domain (VL/CHxULC domain) that can bind the antigen.
In another aspect, methods are provided for obtaining an immunoglobulin light chain variable region (VL/CHxULC) amino acid sequence, comprising: (a) optionally immunizing a non-human animal containing genetically modified immunoglobulin loci as described herein with an antigen of interest, wherein the non-human animal comprises in its genome (i) a first nucleotide sequence that encodes a rearranged light chain variable domain (i.e., where the first nucleotide sequence is a rearranged human immunoglobulin light chain variable region nucleotide sequence or where the first nucleotide sequence contains a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments), wherein the first nucleotide sequence is operably linked to a light chain constant region gene sequence; and (ii) a second nucleotide sequence that encodes a human or non-human light chain variable domain (i.e., where the second nucleotide sequence is an unrearranged human immunoglobulin light chain variable nucleotide sequence), wherein the second nucleotide sequence is operably linked to a heavy chain constant region gene sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene; (b) allowing the non-human animal to mount an immune response; (c) harvesting a lymphocyte (e.g., a B cell) from the immunized non-human animal; (d) fusing the lymphocyte with a myeloma cell to form a hybridoma cell; and (e) obtaining from the hybridoma cell a nucleic acid sequence that encodes a light chain variable domain (VL domain) that can bind the antigen.
In another aspect, methods are provided for obtaining an immunoglobulin light chain variable region (VL/CHxULC) nucleic acid sequence of an immunoglobulin hybrid chain, comprising: (a) optionally immunizing a non-human animal containing genetically modified immunoglobulin loci as described herein with an antigen of interest, wherein the non-human animal comprises in its genome (i) a rearranged human immunoglobulin light chain variable region nucleic acid sequence (or a limited number of human VL gene segments, e.g., two or more but less than the number of wild type number of human VL gene segments) operably linked to a light chain constant region nucleic acid sequence; and (ii) one or more (e.g., 6, 16, 30, 40 or more) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL); (b) allowing the non-human animal to mount an immune response; (c) identifying a lymphocyte (e.g., a B cell) from the immunized non-human animal that expresses a VL/CHxULC amino acid sequence that binds the antigen independently from a heavy chain variable region; and, (d) cloning a nucleic acid sequence encoding the VL/CHxULC amino acid sequence of (c) from the lymphocyte of (c).
In additional aspects, a genetically modified immunoglobulin locus obtainable by any of the methods as described herein is provided. In various embodiments, the light chain variable regions produced by the methods as described herein and the nucleic acid sequence encoding such light chain variable regions are also provided.
In some aspects, an immunoglobulin heavy chain (e.g., hybrid immunoglobulin chain locus) and light chain locus in a germline genome of a non-human animal are provided, said light chain locus comprising (1) a rearranged human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human VL gene segments) that is operably linked to a light chain constant region gene sequence, and said heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising (2) an unrearranged human immunoglobulin light chain variable region nucleotide sequence that is operably linked to a heavy chain constant region gene sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the light chain constant region gene sequence is a κ light chain constant region gene sequence. In some embodiments, the light chain constant region gene sequence is a λ light chain constant region gene sequence. In some embodiments, the light chain constant region gene sequence is a mouse or rat light chain constant region gene sequence. In some embodiments, the rearranged light chain variable region nucleotide sequence is a κ light chain variable region gene sequence. In some embodiments, the rearranged light chain variable region nucleotide sequence is a light chain variable region gene sequence. In some embodiments, the rearranged light chain variable region nucleotide sequence is a mouse or rat light chain variable region gene sequence. In some embodiments, the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene.
In various embodiments, a limited number of human or non-human VL gene segments includes two human or non human VL gene segments. In some embodiments, the two human or non-human VL gene segments are operably linked to one or more, or five, human or non-human JL gene segments. In some certain embodiments, a limited number of human or non-human VL gene segments include two Vκ gene segments. In some certain embodiments, the two Vκ gene segments are operably linked to one or more, or five, Jκ gene segments.
Antigen-Binding Proteins
Additional aspects include antigen-binding proteins (e.g. antibodies) made by the genetically modified non-human animals described herein. Likewise, antigen-binding proteins (e.g., recombinant antibodies) with light chain variable region (VL/CHxULC) sequences derived from or produced by (i.e., expressed from the unrearranged human immunoglobulin light chain variable region gene segments) the genetically modified non-human animals described herein are also provided. In some embodiments, the antigen-binding proteins as described herein include an immunoglobulin light chain that can specifically bind an antigen of interest with an affinity (KD) lower than 10−6, 10−7, 10−8, 10−9 or 10−10. In some embodiments, the immunoglobulin light chain produced by the methods are capable of specifically binding an antigen of interest in the absence of a heavy chain variable region with an affinity (KD) lower than 10−6, 10−7, 10−8, 10−9, or 10−10.
In various embodiments, the light chain variable domains generated as described herein specifically bind a target molecule (“T”). In one embodiment, a target molecule is any protein, polypeptide, or other macromolecule whose activity or extracellular concentration is desired to be attenuated, reduced or eliminated. In many instances, the target molecule to which a light chain variable region binds is a protein or polypeptide (i.e., a “target protein”); however, also provided are embodiments wherein the target molecule (“T”) is a carbohydrate, glycoprotein, lipid, lipoprotein, lipopolysaccharide, or other non-protein polymer or molecule to which a light chain variable region binds. In various embodiments, T can be a cell surface-expressed target protein or a soluble target protein. Target binding by the antigen-binding molecule may take place in an extracellular or cell surface context. In certain embodiments, however, the antigen-binding molecule binds a target molecule inside the cell, for example within an intracellular component such as the endoplasmic reticulum, Golgi, endosome, lysosome, etc. Examples of cell surface-expressed target molecules include cell surface-expressed receptors, membrane-bound ligands, ion channels, and any other monomeric or multimeric polypeptide component with an extracellular portion that is attached to or associated with a cell membrane.
In another aspect, methods are provided for making an antigen-binding protein that comprises an immunoglobulin light chain variable VL/CHxULC domain that can bind an antigen independently from a heavy chain variable domain. Such methods comprise (a) optionally immunizing a genetically modified non-human animal with an antigen that comprises an epitope or immunogenic portion thereof, wherein the non-human animal comprises in its genome: (i) a rearranged human light chain variable region nucleic acid sequence (or a limited number of human VL gene segments, e.g., two or more but less than the wild type number of human VL gene segments) operably linked to a light chain constant region nucleic acid sequence; and (ii) unrearranged human immunoglobulin light chain variable region gene segments (VL and JL) operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence; (b) allowing the non-human animal to mount an immune response to the epitope or immunogenic portion thereof; (c) isolating from the non-human animal a cell comprising a nucleic acid sequence that encodes a light chain variable domain that specifically binds the epitope or immunogenic portion thereof and/or (d) obtaining from the cell of (c) the nucleic acid sequence that encodes the light chain variable domain that specifically binds the epitope or immunogenic portion thereof; and (e) employing the nucleic acid sequence of (d) in an expression construct, fused to a human immunoglobulin constant region nucleic acid sequence. e.g., a human heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene.
In some embodiments, at least one of the unrearranged human light chain VL or JL gene segments encode one or more histidine codons that are not encoded by a corresponding human germline light chain variable gene segment. In some embodiments, rearranged human light chain variable region nucleic acid sequence (or at least one of the limited number of human VL gene segments) encodes one or more histidine codons that are not encoded by a corresponding human germ line light chain variable gene segment. In some embodiments, the epitope is derived from a cell surface receptor.
In some embodiments, at least one of the human light chain VL or JL gene segments encode one or more histidine codons that are not encoded by a corresponding human germline light chain variable gene segment.
As will be clear throughout the specification, in some embodiments, provided protein variable domains are or comprise immunoglobulin-type variable domains (e.g., are or comprise immunoglobulin variable domains). In some embodiments, provided protein variable domains are or comprise heavy chain variable domains; in some embodiments, provided protein variable domains are or comprise light chain variable domains.
Those skilled in the art, reading the present specification, will readily appreciate that any of a variety of technologies can be utilized to produce, generate, and/or assemble antigen-binding proteins comprising light chain that can bind antigen independently from heavy chain variable domain. In some embodiments described herein, the antigen-binding proteins that are produced include antigen-binding proteins depicted in FIG. 19. These antigen-binding proteins comprise variable domains that are generated in non-human animals described herein, and nucleic acid sequences comprising sequences that encode these variable domains are co-expressed in a cell line to produce the antigen-binding proteins.
Genetically Modified Non-Human Cells and Embryos
In various aspects, a pluripotent cell, induced pluripotent, or totipotent stem cells derived from a non-human animal comprising the various genomic modifications herein are provided. In some embodiments, the pluripotent or totipotent cell is derived from a non-human animal. In some embodiments, the non-human animal is a rodent, e.g., a mouse, a rat, or a hamster. In some embodiments, the rodent is a mouse. In specific embodiments, the pluripotent cell is an embryonic stem (ES) cell. In some embodiments, the pluripotent cell comprises in its genome: (i) an immunoglobulin light chain locus that comprises a rearranged human or non-human light chain variable region nucleic acid sequence (or a limited number of human or non-human VL gene segments, e.g., two or more but less than the number of wild type human or non-human VL gene segments) operably linked to a light chain constant region nucleic acid sequence; and (ii) an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising one or more unrearranged human immunoglobulin VL and JL gene segments, operably linked to a heavy chain constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene. In specific embodiments, the pluripotent, induced pluripotent, or totipotent stem cells are mouse or rat embryonic stem (ES) cells. In some embodiments, the pluripotent, induced pluripotent, or totipotent stem cells have an XX karyotype or an XY karyotype.
Cells that comprise a nucleus containing a genetic modification as described herein are also provided, e.g., a modification introduced into a cell by pronuclear injection. In another aspect, a hybridoma or quadroma is provided, derived from a cell of the non-human animal as described herein. In some embodiments, the non-human animal is a rodent, such as a mouse, a rat, or a hamster.
In another aspect, a lymphocyte isolated from a genetically modified non-human animal as described herein is provided. In some embodiments, the lymphocyte is a B cell, wherein the B cell comprises an immunoglobulin genomic locus comprising a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) operably linked to a human or a non-human animal (e.g., mouse or rat) light chain constant region gene sequence. In some embodiments, the B cell further comprises an immunoglobulin genomic locus comprising a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to a human or non-human animal (e.g., mouse or rat) heavy chain constant region gene sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene. In some embodiments, the B cell is capable of producing antibodies wherein the rearranged light chain variable domain as described herein is operably linked to a heavy chain or light chain constant domain.
In another aspect, a non-human animal embryo comprises a cell whose genome comprises: (i) an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising unrearranged human light chain variable region gene segments operably linked to a constant region nucleic acid sequence comprising one or more heavy chain constant region genes, each one comprising a sequence encoding a functional CH1 domain, e.g., comprising an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igε gene, and/or an intact Igα gene; and (ii) an immunoglobulin light chain locus comprising a rearranged human or non-human immunoglobulin light chain variable region nucleotide sequence (or a limited number of human or non-human VL gene segments) operably linked to a light chain constant region nucleic acid sequence. In some embodiments, the hybrid immunoglobulin locus comprising unrearranged human light chain variable region gene segments operably linked to a constant region nucleic acid sequence is operably linked to a heavy chain constant region nucleic acid sequence, and the heavy chain constant region gene sequence comprises an intact Igμ gene, an intact Igδ gene, an intact Igγ gene, an intact Igα gene, and/or an intact Igε gene.
In various embodiments, the genetically modified non-human animals express an antibody repertoire (e.g., an IgG repertoire) that is derived from the nucleotide sequence that encodes the rearranged light chain variable domain (or the nucleotide sequence that contains a limited number of human VL gene segments), and a plurality of light chain V segments (and a plurality of light chain J segments). In some embodiments, the genetically modified locus produces an antibody population that comprises an immunoglobulin light chain that is capable of specifically binding an antigen of interest with an affinity (KD) lower than 10−6, 10−7, 10−, 10−9 or 10−10. In some embodiments, the immunoglobulin light chain expressed by the genetically modified locus is capable of specifically binding an antigen of interest in the absence of a heavy chain variable region with an affinity (KD) lower than 10−610−7, 10−8, 10−9, or 10−10.
In various embodiments, the genetic modifications described herein do not affect fertility of the non-human animal (see, for example, U.S. Pat. No. 8,642,835, incorporated by reference in its entirety). In some embodiments, the heavy chain locus, e.g., hybrid chain locus, comprises an endogenous Adam6a gene, Adam6b gene, or both, and the genetic modification does not affect the expression and/or function of the endogenous Adam6a gene, Adam6b gene, or both. In some embodiments, the genome of the genetically modified non-human animal comprises an Adam6a gene, Adam6b gene, or both integrated in the genome at location outside the heavy chain locus or hybrid chain locus. In some embodiments, an Adam6a and/or Adam6b gene is placed 5′ upstream of the unrearranged light chain variable region gene segments. In some embodiments, the Adam6a and/or the Adam6b gene is placed 3′ downstream of the unrearranged light chain variable region gene segments. In some embodiments, the heavy chain locus comprises a functional ectopic mouse Adam6 gene.
The capabilities of the genetically modified non-human animals described herein to apply selective pressure to genes or polynucleotides encoding light chain variable regions or domains (e.g., light chain CDR3s) can be applied to a variety of variable light chain gene sequences. In other words, the rearranged light chain variable region nucleotide sequences disclosed herein can be paired with one or more genetic modifications of a heavy chain locus and/or the insertion of nucleotide sequences encoding light chain variable domains into a heavy chain locus. This can be accomplished by, for example, mating (i.e., cross-breeding or intercrossing of animals with single modification) the non-human animals described herein (restricted to a common or universal light chain variable domain) with non-human animals comprising genetic modifications within one or more heavy chain-encoding loci. Genetically modified non-human animals comprising immunoglobulin light chain loci with a rearranged light chain variable region nucleotide sequence (or a limited number of human VL gene segments) and one or more heavy chain loci modifications can also be generated by targeted gene replacement of multiple loci, either simultaneously or sequentially (e.g., by sequential recombination in embryonic stem cells). Neither the type nor method of modification at the heavy chain loci limits embodiments described herein unless specifically noted. Rather, the selective pressure facilitated by embodiments described herein can be applied to virtually any polynucleotide sequence capable of being expressed and functioning as a heavy chain antigen-binding sequence, thereby driving the evolution of fitter antibody variable regions.
For example, as described herein, genetically modified non-human animals comprising an immunoglobulin locus with a rearranged light chain variable region nucleotide sequence (or a limited number of human VL gene segments) may further comprise (e.g., via cross-breeding or multiple gene targeting strategies) one or more modifications as described in WO 2011/072204, WO 2011/163311, WO 2011/163314, WO 2012/018764, WO 2012/141798, WO 2013/022782, WO 2013/059230, WO 2013/096142, WO 2013/116609, WO 2013/187953; these publications are incorporated herein by reference in their entirety. In particular embodiments, a genetically modified mouse comprising a rearranged light chain variable region nucleic acid sequence, or a limited number of VL gene segments, in a light chain locus (e.g., a rearranged light chain variable domain gene sequence, or two VL gene segments, operably linked to a human or non-human κ light chain constant region gene sequence) is crossed to a genetically modified mouse comprising an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising human light chain variable region gene segments (e.g., 40 human Vκ genes and all human Jκ genes inserted into a mouse heavy chain locus; see, e.g., U.S. Patent Application Publication no. 2012-0096572 A1, incorporated herein by reference). In specific embodiments, a genetically modified mouse comprising a rearranged light chain variable region nucleic acid sequence, or a limited number of VL gene segments, in a light chain locus (e.g., a rearranged light chain variable region nucleotide sequence, or two VL gene segments, operably linked to a human or non-human κ light chain constant region gene sequence) is crossed to a genetically modified mouse comprising an immunoglobulin heavy chain locus (e.g., hybrid immunoglobulin chain locus) comprising one or more human light chain variable region gene segments. The resulting mice are able to produce Igκ+B cells with variable heavy chains derived from genomic light chain variable sequences, thus facilitating the identification of Vκ domains that bind to specific targets.
EXAMPLES
The following non-limiting examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use non-human animals described herein and aid in the understanding thereof, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1. Generation of Non-Human Animals Having Modified Immunoglobulin Loci
This example illustrates exemplary methods of engineering immunoglobulin heavy chain loci of non-human animals to contain (a) an immunoglobulin heavy chain locus comprising unrearranged human immunoglobulin light chain VL and JL gene segments operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence (e.g., hybrid immunoglobulin chain locus); and (b) an immunoglobulin light chain locus comprising a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence.
Construction of Immunoglobulin Heavy Chain Loci with Light Chain Gene Segments
Wild type mouse heavy chain and human κ light chain loci are depicted in FIG. 1. Construction of exemplary targeting vectors for the insertion of human light chain V and J gene segments (e.g., Vκ and Jκ) into a murine immunoglobulin heavy chain locus is described below. FIG. 2 illustrates four exemplary targeting vectors that contain a plurality of human κ light chain gene segments for insertion into a murine immunoglobulin heavy chain locus using homologous recombination.
Various targeting constructs were made using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W., Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C., Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, A. L., Esau, L., Zheng, J., Griffiths, J. A., Wang, X., Su, H., Xue, Y., Dominguez, M. G., Noguera, I., Torres, R., Macdonald, L. E., Stewart, A. F., DeChiara, T. M., Yancopoulos, G. D. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652-659) to modify mouse genomic Bacterial Artificial Chromosome (BAC) libraries. Mouse BAC DNA may be modified by homologous recombination to deletion the endogenous VH, DH and JH gene segments for the subsequent insertion of unrearranged human VL and JL gene segments. Alternatively, the endogenous VH, DH and JH gene segments may be left intact and inactivated so that recombination of endogenous gene segments to form a functional variable region is inhibited (e.g., by inversion or disruption of gene segments).
Genetically modified mice, and methods of making the same, whose genome contains an immunoglobulin hybrid chain locus comprising unrearranged human immunoglobulin light chain VL and JL gene segments operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence are described in U.S. Patent Application Publication No. 2012-0096572 A1, incorporated herein by reference in its entirety. As shown in FIG. 2, four targeting vectors were engineered to progressively insert 40 human Vκ gene segments and five human Jκ gene segments into an non-human ES cell comprising an inactivated heavy chain locus (e.g., deleted endogenous VH, DH and JH gene segments) and/or a light chain locus comprising a single rearranged human VL/JL gene sequence operably linked to a light chain constant region, e.g., a non-human light chain constant region, e.g., at an endogenous non-human light chain locus, using standard molecular techniques recognized in the art. Table 1 sets forth the size of human DNA included in each targeting vector which contains various human κ light chain gene segments for insertion into a mouse immunoglobulin heavy chain locus. Any number of human Vκ and Jκ gene segments may be included in the targeting vectors. The exemplary targeting vectors set forth in FIG. 2 include human κ light chain gene segments that are naturally found in the proximal contig of the germ line human κ light chain locus (FIG. 1). The resulting endogenous heavy chain locus after successive insertion of all four targeting vectors is shown in the bottom of FIG. 2.
TABLE 1
Targeting
Size
Human κ Gene Segments Added
Vector
of Human κ Sequence
Vκ
Jκ
1
~110.5 kb
4-1, 5-2, 7-3, 2-4, 1-5, 1-6
1-5
2
~140 kb
3-7, 1-8, 1-9, 2-10, 3-11,
—
1-12, 1-13, 2-14, 3-15, 1-16
3
~161 kb
1-17, 2-18, 2-19, 3-20, 6-21,
—
1-22, 1-23, 2-24, 3-25, 2-26,
1-27, 2-28, 2-29, 2-30
4
~90 kb
3-31, 1-32, 1-33, 3-34, 1-35,
—
2-36, 1-37, 2-38, 1-39, 2-40
Using a similar approach, other combinations of human light chain variable domains in the context of murine heavy chain constant regions may be constructed. Additional light chain variable domains may be derived from human Vλ and Jλ gene segments. Exemplary targeting vectors that include human DNA that include various numbers of human Vλ and Jλ gene segments are set forth in FIG. 3.
The human λ light chain locus extends over 1,000 kb and contains over 80 genes that encode variable (V) or joining (J) segments. Among the 70 Vλ gene segments of the human λ light chain locus, anywhere from 30-38 appear to be functional gene segments according to published reports. The 70 Vλ sequences are arranged in three clusters, all of which contain different members of distinct V gene family groups (clusters A, B and C). Within the human λ light chain locus, over half of all observed Vλ domains are encoded by the gene segments 1-40, 1-44, 2-8, 2-14, and 3-21. There are seven Jλ gene segments, only four of which are regarded as generally functional Jλ gene segments Jλ1, Jλ2, Jλ3, and Jλ7. In some alleles, a fifth Jλ-Cλ gene segment pair is reportedly a pseudo gene (Cλ6). Incorporation of multiple human Jλ gene segments into a hybrid heavy chain locus, as described herein, may be constructed by de novo synthesis. In this way, a genomic fragment containing multiple human Jλ gene segments in germline configuration is engineered with multiple human Vλ gene segments and allows for normal V-J recombination in the context of a heavy chain constant region. An exemplary targeting vector that includes multiple Jλ gene segments is shown in FIG. 3 (Targeting Vector 1′).
Coupling light chain variable domains with heavy chain constant regions represents a potentially rich source of diversity for generating unique VL binding proteins with human VL regions in non-human animals. Exploiting this diversity of the human λ light chain locus (or human κ locus as described above) in mice results in the engineering of unique hybrid heavy chains and gives rise to another dimension of binding proteins to the immune repertoire of genetically modified animals and their subsequent use as a next generation platform for the generation of therapeutics.
The targeting vectors described above are used to electroporate mouse embryonic stem (ES) cells to created modified ES cells for generating chimeric mice that express VL binding proteins (i.e., human light chain gene segments operably linked to mouse heavy chain constant regions). ES cells containing an insertion of unrearranged human light chain gene segments are identified by the quantitative PCR assay, TAQMAN® (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). Specific primers sets and probes are designed for insertion of human sequences and associated selection cassettes, loss of mouse heavy chain sequences and retention of mouse sequences flanking the endogenous heavy chain locus.
ES cells bearing the human light chain gene segments (e.g., Vκ and Jκ) operably linked to a heavy chain constant region sequence can be transfected with a construct that expresses a recombinase in order to remove any undesired selection cassette introduced by the insertion of the human light chain gene segments. Optionally, the selection cassette may be removed by breeding to mice that express the recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, the selection cassette is retained in the mice.
Targeted ES cells described above are used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou, W. T., Auerbach, W., Frendewey, D., Hickey, J. F., Escaravage, J. M., Esau, L., Dore, A. T., Stevens, S., Adams, N. C., Dominguez, M. G., Gale, N. W., Yancopoulos, G. D., DeChiara, T. M., Valenzuela, D. M. (2007). F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25, 91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing human light chain gene segments at a mouse immunoglobulin heavy chain locus are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human light chain gene segments at an endogenous immunoglobulin heavy chain locus. Pups are genotyped and a pup heterozygous or homozygous for the genetically modified immunoglobulin heavy chain locus is selected for characterizing expression of VL-containing heavy chains.
Mice whose genome comprises an immunoglobulin heavy chain allele that contains an insertion of forty (40) unrearranged human Vκ and five (5) Jκ gene segments into an endogenous locus so that said human Vκ and Jκ gene segments are operably linked to endogenous heavy chain constant regions are referred to as MAID1713 (see U.S. Patent Application Publication no. 2012-0096572 A1, incorporated herein by reference in its entirety). Mice having the same and also an integrated mouse Adam6 gene are referred to as MAID1994 (see U.S. Patent Application Publication no. 2013-0212719 A1, herein incorporated by reference in its entirety).
Construction of Immunoglobulin Light Chain Loci with a Rearranged Human Light Chain Nucleotide Sequence
Construction of exemplary targeting vectors for the insertion of a single rearranged human light chain nucleotide sequence (e.g., a single human rearranged VL/JL nucleotide sequence) into a murine immunoglobulin light chain locus are described below. FIG. 4 illustrates a targeting vector that contains a single rearranged human light chain nucleotide sequence for insertion into a murine immunoglobulin light chain locus using homologous recombination.
Genetically modified mice, and methods of making the same, whose genome contains an immunoglobulin light chain locus comprising a rearranged human immunoglobulin light chain variable region nucleotide sequence operably linked to an immunoglobulin light chain constant region nucleic acid sequence are described in U.S. Patent Application Publication No. US 2011-0195454A1, incorporated herein by reference in its entirety. As shown in FIG. 4, a targeting vector was engineered to contain a single rearranged human light chain (i.e., a rearranged human VL/JL) nucleotide sequence for insertion into an ES cell comprising an inactivated mouse κ light chain locus (e.g., deleted endogenous Vκ and Jκ gene segments) and, optionally, a hybrid immunoglobulin locus, using standard molecular techniques recognized in the art. The single rearranged human light chain nucleotide sequence may include any human VL and human JL sequence. Suitable exemplary rearranged human light chain nucleotide sequences that can be employed include those derived from a rearranged human Vκ1-39Jκ5 nucleotide sequence (MAID1633, FIG. 5), a rearranged human Vκ3-20Jκ1 nucleotide sequence (MAID1635, FIG. 5).
Alternatively, as described above, in some embodiments, a mouse may also be engineered to comprise an insertion of human Vλ and Jλ gene segments into an endogenous immunoglobulin heavy chain locus so that said human Vλ and Jλ gene segments are operably linked to heavy chain constant regions. In such embodiments, to achieve optimal expression and usage of the inserted human Vλ and Jλ gene segments, those skilled in the art are aware that one might use a rearranged sequence such as a rearranged human VλJλ nucleotide sequence. Such rearranged human VλJλ nucleotide sequence would provide a better ability of the rearranged human VλJλ sequences in the context of a heavy chain constant region to pair with the rearranged human VλJλ sequence in the context of a light chain constant region. Rearranged human VκJκ sequences in the context of heavy chain constant regions may not be able to effectively associate with rearranged VλJλ sequences in the context of light chain constant regions (see US 2012-0096572 A1). Therefore, an exemplary rearranged human VλJλ sequence includes a rearranged human Vλ2-14Jλ1 nucleotide sequence.
The targeting vector described above is used to electroporate mouse embryonic stem (ES) cells, which may optionally comprise a hybrid immunoglobulin locus, to create modified ES cells for generating chimeric mice that express light chains encoded by a single rearranged human light chain nucleotide sequence (i.e., a single human VL/JL nucleotide sequence operably linked to mouse light chain constant regions). ES cells containing an insertion of a single rearranged human light chain nucleotide sequence is identified by the quantitative PCR assay, TAQMAN® (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). Specific primers sets and probes are designed for insertion of the single rearranged human light chain nucleotide sequence and associated selection cassettes, loss of mouse light chain sequences and retention of mouse sequences flanking an endogenous light chain locus.
ES cells bearing the single rearranged human light chain nucleotide sequence can be transfected with a construct that expresses a recombinase in order to remove any undesired selection cassette introduced by the insertion of the single rearranged human light chain nucleotide sequence. Optionally, the selection cassette may be removed by breeding to mice that express the recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, the selection cassette is retained in the mice.
Targeted ES cells described above are used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou, W. T., Auerbach, W., Frendewey, D., Hickey, J. F., Escaravage, J. M., Esau, L., Dore, A. T., Stevens, S., Adams, N. C., Dominguez, M. G., Gale, N. W., Yancopoulos, G. D., DeChiara, T. M., Valenzuela, D. M. (2007). F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25, 91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a single rearranged human light chain nucleotide sequence at a mouse immunoglobulin light chain locus are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique rearranged human light chain nucleotide sequence at an endogenous immunoglobulin light chain locus. Pups are genotyped and a pup heterozygous or homozygous for the genetically modified immunoglobulin light chain locus is selected for characterizing expression of the single human light chain.
Example 2. Characterization of Mice Comprising a Single Rearranged Human Immunoglobulin Light Chain Nucleotide Sequence and a Plurality of Human κ Light Chain Gene Segments
Mice comprising a rearranged light chain variable region nucleic acid sequence in a light chain locus (ULC Mouse: MAID1633, single rearranged human Vκ1-39/Jκ5 or MAID1635, single rearranged human Vκ3-20Jκ1) were generated as described above. Briefly, in the ULC mouse, all endogenous functional light chain variable gene segments were deleted and replaced with a single rearranged light chain variable region nucleic acid sequence (e.g., a sequence that encodes a human Vκ1-39/Jκ5 or a human Vκ3-20Jκ1), which is operably linked to an endogenous light chain constant region nucleic acid sequence.
Mice comprising genetically engineered heavy chain loci containing unrearranged human immunoglobulin light chain VL and JL gene segments in a heavy chain locus (KOH Mouse: MAID1713: 40 human Vκ gene segments and five human Jκ gene segments; MAID1994: 40 human Vκ gene segments and five human Jκ gene segments, and an integrated Adam6 gene) were generated as described above. Briefly, in the KOH Mouse, all endogenous functional heavy chain variable gene segments were deleted and replaced with 40 unrearranged human Vκ gene segments and five (5) unrearranged human Jκ gene segments, which are operably linked to an immunoglobulin heavy chain constant region nucleic acid sequence.
Homozygous ULC mice (MAID1633 or MAID 1635) described above were bred to homozygous KOH mice (MAID1713 or MAID 1994) mice to produce a mouse heterozygous for the ULC allele and the KOH allele. F1 heterozygous mice generated from this cross were bred to each other to obtain mice homozygous for each allele (MAID1713HO 1633HO, MAID1713HO 1635HO, MAID1994HO 1633HO, or MAID1994HO 1635HO; “KOH×ULC”). Such mice express VL binding proteins that have a structure that resembles that of immunoglobulins, but yet are distinct in that such binding proteins lack heavy chain variable domains. The presence of the genetically modified alleles in the immunoglobulin heavy chain and light chain loci was confirmed by TAQMAN™ screening and karyotyping using specific probes and primers described above. The homozygous KOH×ULC mice comprise an insertion of unrearranged human light chain gene segments as described herein (e.g., human Vκ and Jκ) into the mouse heavy chain locus in which all endogenous variable heavy chain VDJ gene segments have been deleted and an insertion of a single rearranged human light chain variable region nucleotide sequence (MAID1633: rearranged human Vκ1-39Jκ5; MAID1635: rearranged human Vκ3-20Jκ1) into the mouse kappa (κ) light chain locus in which all mouse Vκ and Jκ genes have been deleted (FIG. 6). In some embodiments, KOH×ULC mouse further comprise an integrated Adam6 gene.
Alternatively, to generate mice comprising both ULC allele and KOH allele, ES cells harboring a ULC modification or ES cells harboring a KOH modification are targeted with KOH or ULC targeting vector, respectively. Mice are generated from ES cells harboring both modifications by introducing ES cells into an 8 stage mouse embryo by VELOCIMMUNE® method and screening as described above in Example 3. F1 heterozygous mice are bred to obtain homozygous mice.
All mice were housed and bred in specific pathogen-free conditions at Regeneron Pharmaceuticals, Inc. Three KOH (MAID1994HO 1242HO; see U.S. Patent Application Publication No. US 2013-0212719 A1, incorporated by reference herein) mice (˜11 weeks old, male) and two groups of three KOH×ULC (MAID1994HO 1633HO, ˜12 weeks old, female; MAID1994HO 1635HO, ˜11 weeks old, 2 male and 1 female) mice were sacrificed, and spleens and bone marrow were harvested from the animals. Bone marrow was collected from femurs by flushing with complete RPMI medium (RPMI medium supplemented with fetal calf serum, sodium pyruvate, Hepes, 2-mercaptoethanol, non-essential amino acids, and gentamycin). Red blood cells from spleen and bone marrow preparations were lysed with ACK lysis buffer and washed with complete RPMI medium.
Flow Cytometry
In order to examine the ability of the genetically modified homozygous “KOH×ULC” (MAID1994HO 1633HO and MAID1994HO 1635HO) mice described herein to produce VL binding proteins derived from the genetically modified alleles (e.g., from the allele that contains a single copy of the rearranged human light chain nucleotide sequence in the light chain locus and the allele that contains unrearranged human Vκ and Jκ gene segments in the heavy chain locus), fluorescence-activated cell sorting (FACS) analysis was performed. KOH mice comprising an unrearranged light chain locus comprising unrearranged human VL and JL gene segments (1994 HO 1242 HO), as well as VELOCIMMUNE® mice comprising unrearranged human heavy and light chain gene segments on mouse heavy and light chain loci, respectively (VI3) were used as controls.
Briefly, 1×106 cells were incubated with anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling with the following antibody cocktail for 30 minutes on ice: APC-H7 conjugated anti-mouse CD19 (clone 1D3, BD Pharmigen), Pacific Blue conjugated anti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ (clone 187.1, BD Pharmigen) or anti-mouse CD43 (clone 1B11, BioLegend), PE conjugated anti-mouse Igλ (clone RML-42, BioLegend) or anti-mouse c-kit (clone 2B8, BioLegend), PerCP-Cy5.5 conjugated anti-mouse IgD (BioLegend), PE-Cy7 conjugated anti-mouse IgM (clone II/41, eBioscience), APC conjugated anti-mouse B220 (clone RA3-6B2, eBioscience). Following staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRII flow cytometer and analyzed with FlowJo (Tree Star, Inc.). Gating: total B cells (CD19+CD3−), Igκ+B cells (Igκ+Igλ−CD19+CD3−), Igλ+B cells (Igκ−Igλ+CD19+CD3−). Results for the bone marrow compartment are shown in FIGS. 7-10. Results for the splenic compartment are shown in FIGS. 11-15.
Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. Selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells have been identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. Loder, F. et al., J. Exp. Med., 190(1): 75-89, 1999.
The FACS analysis (FIGS. 7-15) suggested that the KOH×ULC mice were able to produce nearly normal B cell populations in the bone marrow compartment (FIGS. 7-8). Interestingly, KOH×ULC mice demonstrate a lack of lambda (λ) expression in the bone marrow (FIG. 10).
In the splenic compartment, KOH×ULC mice produced nearly normal B cell populations (FIGS. 11, 12, and 14). As in the bone marrow compartment, KOH×ULC mice demonstrated a lack of lambda (λ) expression in the spleen (FIGS. 11 and 12). Also in the splenic compartment, KOH×ULC mice demonstrated nearly normal transitional and mature B cell populations as compared to VELOCIMMUNE® (VI3) mice (FIGS. 13-15).
Taken together, these data show that the KOH×ULC mice provided by the present invention, such as those with the genetic modifications described in Example 1, are healthy and demonstrate a near wild-type B cell development. Moreover, such mice express binding proteins that resemble natural antibodies in structure, but yet lack heavy chain variable region sequences.
Finally, as depicted in FIG. 16, mice comprising the genetic modifications described herein were capable of generating antigen-specific titers when immunized with Antigen 1 (a cell surface receptor).
Example 3. Antigen-Binding Characterization of VL/CHxULC Domains from KOH×ULC Mice
This example illustrates exemplary methods of obtaining nucleic acid sequences that encode an immunoglobulin light chain variable domain (VL/CHxULC) that can detectably bind an antigen independently from a cognate variable domain, e.g., a cognate universal light chain variable domain. Exemplary VL/CHxULC domains that detectably bind an antigen independently from a cognate variable domain are obtained from genetically modified non-human animals (e.g., mice) whose genome includes an immunoglobulin heavy chain locus (hybrid immunoglobulin chain locus) containing unrearranged human light chain gene segments (e.g., VL and JL gene segments) operably linked to a heavy chain constant region sequence and an immunoglobulin light chain locus containing a rearranged immunoglobulin light chain variable sequence (i.e., a universal or common light chain variable region) operably linked to a light chain constant region sequence. Such non-human animals express binding proteins that contain immunoglobulin light chain VL/CHxULC variable domains operably linked to a heavy chain constant regions and common immunoglobulin light chain variable domains operably linked to a light chain constant regions, wherein the VL/CHxULC light chains are derived from the unrearranged human light chain gene segments, and wherein the common light chain variable domains are encoded by the single rearranged light chain variable gene sequence.
Preparation of a VL/CHxULC, specifically a VκOHxULC immunoglobulin light chain variable domain, that retains antigen binding when paired with an unrelated, e.g., noncognate, human VH domain was performed. KOH×ULC mice were immunized with a cell surface protein (Antigen 2). Antigen positive B-cells were sorted from two KOH×ULC mice; MAID1712 1635 (KOH×ULC:Vκ3-20Jκ1). Cells were sorted based on Antigen 2 and 1536 B-cells were collected. 384 B-cells were processed from the “best” mouse as judged during sorting. 176 KOH VL domains, e.g., Vκ/CHxULC domains were cloned into Fab plasmids. Individual sequences encoding one of 176 KOH VL domains were cloned into Fab plasmids along with a sequence encoding a human Vκ3-20 germline ULC sequence. Each sequence encoding a KOH VL (VκOHxULC) domain was cloned operably linked with a heavy chain constant region sequence (i.e. CH1) and the ULC sequence was closed operably linked with a light chain constant κ gene sequence. Transient transfections were carried out to produce protein for Ag+ screening. Screening for Antigen 2 binding was assayed by ELISA and BIACORE™. Fourteen (14) samples bound Antigen 2 at neutral pH as determined by ELISA, as shown in FIG. 17. Binding was confirmed by BIACORE™ for 13 of the 14 ELISA binders.
Subsequently, two KOH derived VL (VκOHxULC) domains were chosen and independently cloned and reformatted with light chain constant regions (i.e., Cκ). Each of the reformatted KOH VL/Cκ chains were independently paired with non-cognate VH domain formatted with a heavy chain CH to form a typical antibody structure. Notably, the non-cognate VH domain was generated in a mouse that was genetically modified to generate all VH domains from a single rearranged heavy chain variable region sequence, see, e.g., U.S. Patent Publication No. 20140245468, incorporated herein in its entirety by reference, and immunized with an unrelated enzyme (Antigen 3). The reformatted VL domains were tested for Antigen 2 binding by BIACORE™. Results are shown in FIG. 18.
In FIG. 18, antibodies A and B each comprise an immunoglobulin light chain comprising a distinct KOH VL (VκOHxULC) fused with a Cκ constant domain and an immunoglobulin heavy chain comprising a VH domain fused with an intact CH domain. In contrast, while antibody C comprises the same immunoglobulin heavy chain as antibodies A and B, antibody C comprises an immunoglobulin light chain comprising a VL domain that is cognate to the VH domain fused with a Cκ domain.
The results show that when a VH domain derived from a single rearranged heavy chain variable region and raised against Antigen 3 is paired with cognate light chain variable domains, antigen binding to Antigen 3 is maintained (see, FIG. 18; showing antibody C binds to Antigen 3 as expected). Not surprising, when the same VH domain was paired with a noncognate KOH VL domain, Antigen 3 binding was undetectable (FIG. 18).
In contrast, Antigen 2 binding was maintained for both KOH VL (VκOHxULC) domains (i.e., antibody A and B VL domains) despite being (1) reformatted onto a Cκ domain and (2) paired with a non-cognate VH domain.
The results suggest that KOH antibodies isolated from KOH×ULC mice can bind antigen solely through one VL domain (i.e., a Vκ domain). This is confirmed by reformatting a KOH VL (VκOHxULC) domain onto a light chain backbone (i.e., a Cκ region) and pairing with a VH domain raised against a different antigen. Such a molecule was shown to retain binding to the antigen to which the parental KOH antibody (i.e., VκOHxULC domain) was raised.
Taken together, this Example demonstrates that KOH×ULC mice provide a robust in vivo system to select for “antibody-like” molecules that bind antigen solely through a VL/CHxULC domain (e.g., Vκ/CHxULC), i.e., independent of a cognate variable domain. Such mice provide the opportunity to select VL domains (Vκ/CHxULC or Vλ/CHxULC) that bind antigen in the absence of a cognate variable domain and/or when paired with a noncognate variable domain. The VL binding proteins expressed by the mice described herein may provide a novel paratope or binding surface to targets that evolve to avoid conventional antibodies (e.g., HIV and influenza).
Example 4. Making a Multi-Specific Antigen Binding Protein Comprising a VL/CHxULC Domain
This example illustrates an exemplary method of making a multi-specific antigen-binding protein comprising a light chain variable VL/CHxULC domain derived from an immunoglobulin hybrid chain that is cognate with a universal light chain. As described in Example 3, a first nucleic acid sequence encoding a KOH VL domain, e.g., a Vκ/CHxULC, is isolated from a non-human animal genetically modified to comprise in its genome an immunoglobulin hybrid chain locus containing unrearranged human light chain gene segments (e.g., Vκ and Jκ gene segments) operably linked to a heavy chain constant region sequence and an immunoglobulin light chain locus containing a rearranged immunoglobulin light chain variable sequence (i.e., a universal or common light chain variable region) operably linked to a light chain constant region sequence. A second nucleic acid encoding a second VL/CHxULC domain may also be isolated from a non-human animal genetically modified to comprise in its genome an immunoglobulin hybrid chain locus containing unrearranged human light chain gene segments (e.g., VL and JL gene segments) operably linked to a heavy chain constant region sequence and an immunoglobulin light chain locus containing a rearranged immunoglobulin light chain variable sequence (i.e., a universal or common light chain variable region) operably linked to a light chain constant region sequence. Alternatively, a second nucleic acid encoding a heavy chain variable VHxULC domain that binds the second antigen and is cognate to a universal light chain may be isolated from a non-human animal genetically modified with a universal light chain (“ULC”), see, e.g., 2011-0195454 A1, US 2012-0021409A1, US 2012-0192300A1, US 2013-0045492A1, US 2013-0185821A1 and US 2013-0302836A1, incorporated by reference herein in their entireties) or a restricted (limited) immunoglobulin light chain variable region gene segment repertoire (e.g., a restricted immunoglobulin light chain variable segment repertoire comprising two or more but less than the wild type number of human VL gene segments; for example, a dual light chain, or “DLC”, U.S. Patent Application Publication No. US-2013-0198880-A1, incorporated by reference herein in its entirety).
A first binding component encoded by a nucleic acid comprising the first nucleic acid sequence encoding the first a VL/CHxULC domain that binds the first antigen may be co-expressed in a cell with a second binding component encoded by the second nucleic acid comprising a nucleic acid sequence encoding the second variable VL/CHxULC domain or VH/CHxULC domain that binds the second antigen such that the first and second binding components are expressed as a multi-specific, e.g., a bi-specific antigen-binding protein. Exemplary pairing formats include the first and second binding components respectively pairing in an Fv format, an scFv format, a Fab format, an scFab format, a tetrameric antibody format wherein the first and second binding components are each heavy chains comprising a functional CH1 domain associated with a universal light chain, or a tetrameric antibody format wherein one of the first or second binding components is a heavy chains comprising a functional CH1 domain and is associated with the other of the first or second binding component as a light chain
KOH×ULC mice comprising unrearranged human light chain variable region gene segments were immunized with Antigen A, a multivalent high molecular weight protein, to form Vκ/CHxULC variable domains specific for Antigen A. ULC mice comprising unrearranged human heavy chain variable region gene segments as described in e.g., 2011-0195454 A1, US 2012-0021409A1, US 2012-0192300A1, US 2013-0045492A1, US 2013-0185821A1 and US 2013-0302836A1, incorporated by reference herein in their entireties, were immunized with Antigen B, a monomeric lower molecular weight protein, to form VHxULC variable domains specific for antigen B.
B-cells expressing antigen-binding proteins capable of binding Antigen A or Antigen B were respectively sorted from KOH×ULC or ULC mice as described in U.S. Pat. No. 7,582,298, incorporated herein by reference. Both KOH×ULC and ULC mice used in this study were genetically modified with a ULC encoded by a rearranged immunoglobulin light chain comprising a human Vκ3-20 gene segment rearranged with a human Jκ1 gene segment.
Briefly, red blood cells were removed by lysis followed by pelleting the harvested splenocytes. Resuspended splenocytes were first incubated with a cocktail of human IgG, FITC-anti-mFc, and Antigen A labeled with biotin or Antigen B labeled with biotin (as appropriate) for 1 hour. The stained cells were washed twice with PBS, then stained with a cocktail of human and rat IgG, APC-anti-mIgM, and SA-PE for one hour. The stained cells were washed once with PBS and were analyzed by flow cytometry on a Reflection (Sony). Each IgG positive, IgM negative, and antigen positive B cell was sorted and plated into a separate well on a 384-well plate. RT-PCR of antibody genes from these B cells was performed according to a method described by Wang et al. (2000) (J Immunol Methods 244:217-225).
Briefly, cDNAs for each single B cell were synthesized via reverse transcription (RT). The VκOHxULC region DNA sequences from Antigen A immunized κOHxULC mice were amplified by PCR using a 5′ degenerate primer specific for human kappa chain variable region leader sequence and a 3′ primer specific for mouse heavy chain constant region, to form an amplicon. The amplicon was then amplified again by PCR using a 5′ degenerate primer set specific for framework 1 of human kappa variable region sequence and a nested 3′ primer specific for mouse heavy chain constant region. The VKOHXULC PCR product was cloned into a first Sap I-linearized antibody vector containing human IgG1 heavy chain constant region and an expression cassette for the universal light chain derived from the rearranged Vκ3-20Jκ1. The heavy chain variable region DNA sequences from Antigen B immunized ULC mice were amplified by PCR using a 5′ degenerate primer specific for human IgG heavy chain variable region leader sequence and a 3′ primer specific for mouse heavy chain constant region, to form an amplicon. The amplicon was then amplified again by PCR using a 5′ degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence and a nested 3′ primer specific for mouse heavy chain constant region. The VH/CHxULC PCR products were cloned into a second Sap I-linearized antibody vectors containing a human IgG1 heavy chain constant region.
Purified recombinant plasmid having a rearranged gene encoding the universal light chain derived from the rearranged Vκ3-20Jκ1 sequence operably linked to a human κ constant gene and a VL/CHxULC sequence operably linked with the human IgG1 constant region sequence, and a purified plasmid having a VH/CHxULC sequence operably linked with the human IgG1 constant region sequence were combined and transfected into a CHO host cell line. Stably transfected CHO cell pools were isolated after selection with 400 μg/ml hygromycin for 12 days. The CHO cell pools were used to produce the antigen-binding proteins as shown in FIG. 19A.
Equilibrium dissociation constants (KD) for selected antibody supernatants or purified antibodies were determined by SPR (Surface Plasmon Resonance) using a Biacore T200 or 4000 instrument (GE Healthcare). All data was obtained using HBS-EP (10 mM Hepes, 150 mM NaCl, 0.3 mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the running and sample buffers, at 25° C. or 37° C. Antibodies were captured from crude supernatant samples or purified mAbs on a CM4 or CM5 sensor chip surface previously derivatized with a high density of anti-human Fc antibodies using standard amine coupling chemistry. During the capture step, supernatants or purified mAbs were injected across the anti-human Fc surface at a flow rate of 10 μL/min, for a total of 0.5-2.0 minutes. The capture step was followed by an injection of either running buffer or Antigen A at a concentration range from 3.125 nM-100 nM for 1.5-3.0 minutes at a flow rate of 30 μL/min or Antigen B at a concentration range from 0.37 nM-90 nM for for 3.0 minutes. Dissociation of antigens from the captured antibody was monitored for 3.0-5.0 minutes. The captured antibody was removed by a brief injection of 10 mM glycine, pH 1.5. All sensorgrams were double referenced by subtracting sensorgrams from buffer injections from the analyte sensorgrams, thereby removing artifacts caused by dissociation of the antibody from the capture surface. Binding data for each antibody was fitted to a 1:1 binding model with mass transport limitation.
The binding affinities of universal light chain antibodies are shown in FIG. 20, which exhibits KD values in the nanomolar range. Specifically, all bispecific antibodies (B1-B3) comprising a Vκ/CHxULC binding component, a VHxULC binding component, and universal light chain bound to Antigen A with affinities ranging from 6.8 to 9.6 nM at 25° C. (FIG. 20) and with affinities ranging from 100-140 nM and t1/2 values of less than about 1 min at 37° C. (data not shown). The bispecific antibodies also bound to Antigen B with affinities ranging from about 5-100 nM at 25° C. (FIG. 20) and with affinities ranging from 174-178 nM at 37° C. (data not shown).
Control monospecific antibodies (antibodies CKOH1-CKOH3), which were raised against Antigen A and included universal light chain variable domains paired with bivalent Vκ/CHxULC domains, which were respectively cloned to produce the bispecific antibodies B1-B3, bound to Antigen A with affinities ranging from 2-8 nM at 25° C., but not Antigen B (FIG. 20). Without wishing to be bound by theory, it is possible that the differences in the t1/2 values at 25° C. observed for Antigen A interactions with the bivalent antibodies (CKOH1-CKOH3) compared to the bispecific antibodies (B1-B3) may be due to the multivalent nature of Antigen A, which may contribute to a predominantly avidity driven interaction. Dissociation constants (t1/2) were not determined for CKOH1-CKOH3 antibodies at 37° C.
A control monospecific antibody (antibody CVH), which was raised against antigen B and included universal light chain variable domains paired with a bivalent hVHxULC domain, which was cloned to produce each of bispecific antibodies B1-B3, bound antigen B with an affinity of 5.2 nM at 25° C. and a t1/2 value (41.1 min) that was similar to t1/2 values (23-31 min) with which Antigen B dissociated from the bispecific antibodies (FIG. 20). Binding of CVH to Antigen B at 37° C. was not tested.
An isotype control antibody (C1) did not bind to either antigen A or antigen B (FIG. 20). Another control anti-B antibody (C) in typical antibody format, e.g., having two heavy chains, each comprising a VH domain fused with a CH domain, and two light chains, each having a VL domain fused with a CL domain, bound to antigen B with affinity of 1.4 nM and did not bind to antigen A (FIG. 20).
Taken together, this Example demonstrates that a VL/CHxULC domain generated in a KOH×ULC non-human animal is capable of binding antigen in a multi-specific format with another variable domain specific for a second distinct epitope.
EQUIVALENTS
Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated by those skilled in the art that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and the invention is described in detail by the claims that follow.
It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes described herein.
The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
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15559358
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Mar 9th, 2021 12:00AM
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Jan 24th, 2019 12:00AM
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https://www.uspto.gov?id=US10941213-20210309
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Anti-TMPRSS2 antibodies and antigen-binding fragments
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The present invention includes an antibody or antigen-binding fragment thereof that binds specifically to TMPRSS2 and methods of using such antibodies and fragments for treating or preventing viral infections (e.g., influenza virus infections).
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10941213
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1. A human antigen-binding protein that specifically binds to human TMPRSS2, comprising:
(a) an immunoglobulin heavy chain variable region comprising the CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain that comprises the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19; and/or
(b) an immunoglobulin light chain variable region comprising the CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin light chain that comprises the amino acid sequence set forth in SEQ ID NO: 4 or 18.
2. The antigen-binding protein of claim 1 which is an antibody or antigen-binding fragment thereof.
3. A human antigen-binding protein that specifically binds to human TMPRSS2, comprising: a light chain immunoglobulin variable region that comprises:
(a) a CDR-L1 comprising the amino acid sequence: QSISSW (SEQ ID NO: 12),
(b) a CDR-L2 comprising the amino acid sequence: K A S (SEQ ID NO: 14),
(c) a CDR-L3 comprising the amino acid sequence: QQYNSYSYT (SEQ ID NO: 16); and
a heavy chain immunoglobulin variable region that comprises:
(a) a CDR-H1 comprising the amino acid sequence: GFTFSSYG (SEQ ID NO: 6),
(b) a CDR-H2 comprising the amino acid sequence: IWNDGSYV (SEQ ID NO: 8),
(c) a CDR-H3 comprising the amino acid sequence: AREGEWVLYYFD Y (SEQ ID NO: 10).
4. A human antigen-binding protein that specifically binds to human TMPRSS2, comprising:
(a) a heavy chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 17 or 19, or an immunoglobulin heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 2;
and/or
(b) a light chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 18, or an immunoglobulin light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 4.
5. An antigen-binding protein that competes with an antigen-binding protein of claim 1 for binding to TMPRSS2 and/or binds to the same or an overlapping epitope on TMPRSS2.
6. The antigen-binding protein of claim 1 which is multispecific.
7. The antigen-binding protein of claim 1 which comprises one or more of the following properties:
Inhibits growth of influenza virus in TMPRSS2-expressing cells;
Binds to the surface of TMPRSS-expressing cells;
Does not significantly bind to MDCK/Tet-on cells which do not express TMPRSS2;
Binds to human TMPRSS2 with a KD of about 2.81×10−9M at about 25° C.;
Binds to human TMPRSS2 with a KD of about 9.31×10−9M at about 37° C.;
Binds to cynomolgus TMPRSS2 with a KD of about 5.60×10−8M at about 25° C.;
Binds to cynomolgus TMPRSS2 with a KD of about 1.40×10−7M at about 37° C.;
Limits spread of influenza virus infection of cells in vitro; and/or
Protects mice engineered to express the human TMPRSS2 protein from death caused by influenza virus infection.
8. A complex comprising an antigen-binding protein of claim 1 bound to a TMPRSS2 polypeptide.
9. A method for making an antigen-binding protein of claim 1 or immunoglobulin chain thereof comprising:
(a) introducing one or more polynucleotides encoding an immunoglobulin chain of said antigen-binding protein;
(b) culturing the host cell under conditions favorable to expression of the polynucleotide; and
(c) optionally, isolating the antigen-binding protein or immunoglobulin chain from the host cell and/or medium in which the host cell is grown.
10. The method of claim 9 wherein the host cell is a Chinese hamster ovary cell.
11. A polypeptide comprising:
(a) CDR1, CDR2, and CDR3 of a VH domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 2; or
(b) CDR1, CDR2, and CDR3 of a VL domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 4.
12. A polynucleotide encoding the polypeptide of claim 11.
13. A vector comprising the polynucleotide of claim 12.
14. A host cell comprising the vector of claim 12.
15. A composition or kit comprising the antigen-binding protein of claim 1 in association with a further therapeutic agent.
16. A pharmaceutical composition comprising the antigen-binding protein of claim 1 and pharmaceutically acceptable carrier and, optionally, a further therapeutic agent.
17. The composition or kit of claim 15 in association with a further therapeutic agent which is an anti-viral drug or a vaccine.
18. The composition or kit of claim 17 wherein the further therapeutic agent is a member selected from the group consisting of: ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin and interferon-alpha2b, interferon-alpha2a, an anti-cancer agent and an antibody or antigen-binding fragment thereof that specifically binds to influenza HA; and/or
an antibody or antigen binding fragment thereof selected from the group consisting of H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.
19. A vessel or injection device comprising the antigen-binding protein of claim 1.
20. A human antigen-binding protein that specifically binds to human TMPRSS2, comprising:
(a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and
(b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 or 18.
21. The human antigen-binding protein of claim 20, comprising:
(a) the immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; and
(b) the immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4.
22. The human antigen-binding protein of claim 21 which is an antibody or antigen-binding fragment thereof.
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This application claims the benefit of U.S. provisional patent application No. 62/622,292, filed Jan. 26, 2018; which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to antibodies and antigen-binding fragments that bind specifically to TMPRSS2 and methods for treating or preventing viral infections with said antibodies and fragments.
BACKGROUND OF THE INVENTION
Influenza viruses have acquired resistance to currently used drugs that target the viral neuraminidase (NA) or the ion channel protein, matrix protein 2 (M2). The emergence of drug resistance highlights the need for the development of novel antiviral strategies. Host cell targeting may reduce or avoid the emergence of escape mutants, but could create a “sink” due to widespread expression and raise the concern for toxicity. A number of respiratory virus fusion proteins have been shown to require cleavage by host protease(s) for activation (Shirato et al. Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017); Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017); Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015); Zmora et al. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015)), including influenza (Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017); Böttcher-Friebertshäuser et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011); Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010); Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), May; 88(9):4744-51).
Influenza A hemagglutinin precursor (HA0) requires cleavage by a host serine protease, to HA1 and HA2, for activation. For example, transmembrane protease, serine 2; TMPRSS2, TMPRSS4 and TMPRSS11D as well as human airway trypsin-like protease (HAT) have been implicated in HA cleavage (Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010); Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 2006 October; 80(19):9896-8; International patent application publication no. WO2017/151453). Also, TMPRSS2 is a target for anti-cancer therapy. See e.g., WO2008127347 and WO2002004953. A fusion between TMPRSS2 and ERG (TMPRSS2:ERG) is a gene fusion known to be a major driver of prostate carcinogenesis which is triggered by the ERα and repressed by the ERβ. Bonkhoff, Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression, Prostate 78(1): 2-10 (2018).
SUMMARY OF THE INVENTION
Although there are small molecule inhibitors of TMPRSS2 and research antibodies, useful, for example, for immunohistochemistry, there is a need in the art for neutralizing therapeutic anti-TMPRSS2 antibodies and their use for treating or preventing viral infection. See e.g., Shen et al. Biochimie 142: 1-10 (2017), WO2008127347; WO2002004953; U.S. Pat. No. 9,498,529; antibody ab92323, available from Abcam (Cambridge, Mass.) or antibodies sc-515727 and sc-101847 available from Santa Cruz Biotech (Dallas, Tex.). The present invention addresses this need, in part, by providing human anti-human TMPRSS2 antibodies, such as H1H7017N, and combinations thereof including, for example, anti-influenza HA antibodies (e.g., Group I HA or Group II HA) and methods of use thereof for treating viral infections.
The present invention provides a neutralizing human antigen-binding protein that specifically binds to human TMPRSS2, for example, an antibody or antigen-binding fragment thereof. For example, in an embodiment of the invention, the antigen-binding protein comprises: (a) the CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain that comprises the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19; and/or (b) the CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin light chain that comprises the amino acid sequence set forth in SEQ ID NO: 4 or 18. In an embodiment of the invention, the antigen-binding protein comprises: (a) a light chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 or 18; and/or (b) a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19. In an embodiment of the invention, the present invention provides antigen-binding protein comprising: (a) CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin comprising an amino acid sequence set forth in SEQ ID NO: 4 or 18 and at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 or 18; and/or (b) CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin comprising an amino acid sequence set forth in SEQ ID NO: 2, 17 or 19 and at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19. For example, in an embodiment of the invention, the antigen-binding protein comprises a light chain immunoglobulin variable region that comprises (a) a CDR-H1 comprising the amino acid sequence: G F T F S S Y G (SEQ ID NO: 6); (b) a CDR-H2 comprising the amino acid sequence: I W N D G S Y V (SEQ ID NO: 8); (c) a CDR-H3 comprising the amino acid sequence: A R E G E W V L Y Y F D Y (SEQ ID NO: 10); and a heavy chain immunoglobulin variable region that comprises (a) a CDR-L1 comprising the amino acid sequence: Q S I S S W (SEQ ID NO: 12); (b) a CDR-L2 comprising the amino acid sequence: K A S (SEQ ID NO: 14); and/or (c) a CDR-L3 comprising the amino acid sequence: Q Q Y N S Y S Y T (SEQ ID NO: 16). The present invention also provides an antigen-binding protein comprising: (a) a heavy chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 17 or 19; and/or (b) a light chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 18.
The present invention also provides any anti-TMPRSS2 antigen-binding protein that competes with any antigen-binding protein that is set forth herein for binding to TMPRSS2 (e.g., as determined by use of using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.)); or which binds to the same or an overlapping epitope on TMPRSS2 (or a fragment thereof) as any antigen-binding protein that is set forth herein.
The present invention also provides multispecific antigen-binding proteins that bind to TMPRSS2 and another antigen or to TMPRSS2 at a different epitope. For example, the multispecific molecule comprises (a) a first antigen-binding domain that binds specifically to TMPRSS2; and (b) a second antigen-binding domain that binds specifically to another antigen or to TMPRSS2 or to an epitope which differs from that of the first antigen-binding domain.
The present invention also provides any anti-TMPRSS2 antigen-binding protein (e.g., an antibody or antigen-binding fragment, e.g., comprising a sequence set forth herein) that comprises one or more of the following properties:
Inhibits growth of influenza virus (e.g., A/Puerto Rico/08/1934 (H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells);
Binds to the surface of TMPRSS-expressing cells (e.g., MDCK/Tet-on), e.g., with an EC50 value of 440 pM or 1.06 nM;
Does not significantly bind to MDCK/Tet-on cells which do not express TMPRSS2;
Binds to human TMPRSS2 with a KD of about 2.81×10−9M at about 25° C.;
Binds to human TMPRSS2 with a KD of about 9.31×10−9M at about 37° C.;
Binds to cynomolgous TMPRSS2 with a KD of about 5.60×10−8M at about 25° C.;
Binds to cynomolgous TMPRSS2 with a KD of about 1.40×10−7M at about 37° C.;
Limits spread of influenza virus infection of cells in vitro; and/or
Protects a mouse engineered to express the human TMPRSS2 protein from death caused by influenza virus infection.
The present invention also provides a complex comprising any antigen-binding protein set forth herein bound to a TMPRSS2 polypeptide, e.g., in vitro or in the body of a subject.
The present invention also provides a method for making an anti-TMPRSS2 antigen-binding protein set forth herein (e.g., H1H7017N) or immunoglobulin chain thereof comprising: (a) introducing one or more polynucleotides encoding a light and/or a heavy immunoglobulin chain of the said antigen-binding protein; (b) culturing the host cell (e.g., CHO cell, Pichia cell or Pichia pastoris cell) under conditions favorable to expression of the polynucleotide; and (c) optionally, isolating the antigen-binding protein or immunoglobulin chain from the host cell and/or medium in which the host cell is grown. An antigen-binding protein or immunoglobulin chain which is a product of such a method is part of the present invention.
A polypeptide (e.g., an immunoglobulin) comprising: (a) CDR1, CDR2, and CDR3 of a VH domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 2; or (b) CDR1, CDR2, and CDR3 of a VL domain of an immunoglobulin chain that comprises the amino acid sequence set forth in SEQ ID NO: 4 (e.g., wherein the polypeptide is in a host cell) also forms part of the present invention.
The present invention also provides a polynucleotide (e.g., DNA or RNA) that encoded a polypeptide of the present invention. In an embodiment of the invention, the polynucleotide encodes two different immunoglobulin chains (e.g., heavy and light). In an embodiment of the invention, one polynucleotide encodes a light immunoglobulin chain and another polynucleotide encodes a heavy immunoglobulin chain, e.g., wherein the chains are in a host cell or are in a vessel. For example, the polynucleotide is in a vector (e.g., a plasmid) and/or is integrated into a host cell chromosome.
Host cells (e.g., CHO cell, Pichia cell or Pichia pastoris cell) of the present invention may include an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N), polypeptide thereof or polynucleotide encoding such a polypeptide and/or a vector including such a polynucleotide.
The present invention also provides a composition or kit comprising an anti-TMPRSS2 antigen-binding protein set forth herein (e.g., H1H7017N) in association with a further therapeutic agent (e.g., an anti-viral drug and/or a vaccine). For example, the composition may be a pharmaceutical composition comprising the antigen-binding protein and pharmaceutically acceptable carrier and, optionally, a further therapeutic agent. The further therapeutic agent may be ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin and interferon-alpha2b, interferon-alpha2a and/or an antibody or antigen-binding fragment thereof that specifically binds to influenza HA. In an embodiment of the invention, the further therapeutic agent is an antibody or antigen binding fragment thereof selected from the group consisting of H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.
In an embodiment of the invention, a further therapeutic agent which is provided in association with an anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to influenza Group II HA protein, such as H1H14611N2; or an antibody or fragment that comprises VH and VL of H1H14611N2; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).
In an embodiment of the invention, a further therapeutic agent which is provided in association with an anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to influenza Group II HA protein, such as H1H14612N2; or an antibody or fragment that comprises VH and VL of H1H14612N2; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).
In an embodiment of the invention, a further therapeutic agent which is provided in association with an anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to influenza Group I HA protein, such as H1H11729P; or an antibody or fragment that comprises VH and VL of H1H11729P; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39).
The present invention also provides a vessel or injection device that comprises an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) or composition thereof (e.g., pharmaceutical composition).
The present invention also provides a method for treating or preventing a viral infection other than an influenza virus infection, in a subject (e.g., a human) in need thereof, comprising administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein set forth herein (e.g., H1H7017N).
The present invention also provides a method for treating or preventing cancer (e.g., prostate cancer) or infection, e.g., a viral infection, e.g., an infection with an influenza virus, coronavirus, SARS-Co virus, MERS-Co virus, parainfluenza virus, human metapneumovirus or hepatitis C virus (HCV), in a subject (e.g., a human) in need thereof, comprising administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein set forth herein (e.g., H1H7017N). For example, the antigen-binding protein is administered in association with one or more further therapeutic agents (e.g., anti-viral drug and/or a vaccine). In an embodiment of the invention, a further therapeutic agent is a member selected from the group consisting of: ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin and interferon-alpha2b, interferon-alpha2a and an antibody or antigen-binding fragment thereof that specifically binds to influenza HA. In an embodiment of the invention, a further therapeutic agent is an antibody or antigen binding fragment thereof selected from the group consisting of H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.
The present invention also provides a method for administering an anti-TMRPSS2 antigen-binding protein (e.g., H1H7017N) set forth herein into the body of a subject (e.g., a human) comprising injecting the antigen-binding protein into the body of the subject parenterally (e.g., subcutaneously, intravenously or intramuscularly).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the progression of the A/Puerto Rico/08/1934 (H1N1)-GFP virus spreading in different cell lines with an initial multiplicity of infection of 0.01 in absence of exogenous trypsin. Calu3 (circle), A549 (square), MDCK (triangle) and HepG2 (inverted triangle) cells.
FIG. 1B shows the progression of the A/Puerto Rico/08/1934 (H1N1)-GFP virus spreading in different cell lines with an initial multiplicity of infection of 0.001 in absence of exogenous trypsin. Calu3 (circle), A549 (square), MDCK (triangle) and HepG2 (inverted triangle) cells.
FIG. 2. shows application of H1H7017N during the infection cycle decreases the number of Fluorescent Focus Units (FFU) of A/Puerto Rico/08/1934 (H1N1) at 72 hours post-infection compared to isotype control antibody, no antibody, anti-HA antibody and uninfected controls.
FIG. 3A shows anti-TMPRSS2, H1H7017N, binds to human and cynomolgous monkey TMPRSS2 expressed on cells. H1H7017N, bound to MDCK/Tet-on/hTMPRSS2 and MDCK/Tet-on/mfTMPRSS2 with EC50 values of 460 pM and 1.06 nM respectively and did not show significant binding to MDCK/Tet-on cells.
FIG. 3B shows anti-TMPRSS2, H1H7017N, binds to human and cynomolgous monkey TMPRSS2 expressed on cells. Control mAb1, an irrelevant isotype control antibody, did not show binding to any of the cell lines tested.
FIG. 4. shows a survival curve of a mouse engineered to express the human TMPRSS2 protein treated with 5 mg/kg of H1H7017N on day −1 PI (inverted triangle, dashed line) or day 0 PI (circle, solid line) showing protection against H1N1 in a prophylactic model. Mice treated with the isotype control H1H1238N (triangle, solid line) showed no protection.
FIG. 5 shows a survival curve of a mouse engineered to express the human TMPRSS2 protein infected with H1N1, treated with 10 mg/kg H1H7017N demonstrating protection. Mice were treated on day 0 (diamond, dotted line), day 1 (circle, solid line), day 2 (inverted triangle, solid line), or day 3 PI (square, dashed line). The isotype control H1H1238N (triangle, solid line) had partial protection with a 25% survival rate.
FIG. 6 shows a survival curve of hTPMRSS2 mice treated with 10 mg/kg of H1H7017N on day 1 PI (triangle) or day 2 PI (circle) showing protection against H3N2. Untreated mice (square) showed no protection.
FIG. 7A shows a survival curve of wild-type mice infected with 150 PFUs (triangle), 750 PFUs (square), or 1,500 PFUs (circle) of A/Puerto Rico/08/1934 (H1N1). Mice were weighed daily until day 14 PI.
FIG. 7B shows a survival curve of mice engineered to express the human TMPRSS2 protein infected with 150 PFUs (triangle), 750 PFUs (square), or 1,500 PFUs (circle) of A/Puerto Rico/08/1934 (H1N1). Mice were weighed daily until day 14 PI.
FIG. 8 shows a survival curve of a mouse engineered to express the human TMPRSS2 protein infected with A/Aichi/2/68 (HA, NA)×A/PR/8/34 (H3N2) on day 0 and treated with a combination of 2.5 mg/kg each of H1H7017N and H1H14611N2 (diamond), 10 mg/kg H1H7017N (triangle), 10 mg/kg H1H14611N2 (square), 5 mg/kg each of H1H7017N and H1H14611N2, or 10 mg/kg hIgG1 isotype control (circle). Mice were weighed daily until day 14 PI.
FIG. 9 shows a survival curve of a mouse engineered to express the human TMPRSS2 protein infected with A/Puerto Rico/08/1934 (H1N1) on day 1 PI and treated with a combination of 1 mg/kg of H1H7017N and 2 mg/kg of H1H11729P (circle), 2.5 mg/kg each of H1H7017N and H1H11729P (inverted triangle), 5 mg/kg H1H11729P (diamond), 5 mg/kg H1H7017N (square), or 5 mg/kg hIgG1 isotype control (triangle). Mice were weighed daily until day 14 PI.
DETAILED DESCRIPTION OF THE INVENTION
Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
The term “influenza hemagglutinin”, also called “influenza HA” is a trimeric glycoprotein found on the surface of influenza virions, which mediates viral attachment (via HA1 binding to α-2,3- and α-2,6-sialic acids) and entry (through conformational change) into host cells. The HA is comprised of two structural domains: a globular head domain containing the receptor binding site (subject to high frequency of antigenic mutations) and the stem region (more conserved among various strains of influenza virus). The influenza HA is synthesized as a precursor (HA0) that undergoes proteolytic processing to produce two subunits (HA1 and HA2) which associate with one another to form the stem/globular head structure. The viral HA is the most variable antigen on the virus and the stem (HA2) is highly conserved within each group.
The term “influenza neuraminidase”, also called “influenza NA” is an exosialidase (EC 3.2.1.18) which cleaves α-ketosidic linkage between the sialic (N-acetylneuraminic) acid and an adjacent sugar residue.
The amino acid sequence of full-length Influenza HA is exemplified by the amino acid sequence of influenza isolate H1N1 A/California/04/2009 provided in GenBank as accession number FJ966082.1. The term “influenza-HA” also includes protein variants of influenza HA isolated from different influenza isolates, e.g., GQ149237.1, NC_002017, KM972981.1, etc. The term “influenza-HA” also includes recombinant influenza HA or a fragment thereof. The term also encompasses influenza HA or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence.
An anti-TMPRSS2 “antigen-binding protein” is a polypeptide or complex of more than one polypeptide (e.g., a tetrameric IgG antibody) that binds specifically to TMPRSS2 polypeptide, for example, an anti-TMPRSS2 antibody or antigen-binding fragment whether monospecific or multispecific.
TMPRSS2
TMPRSS2 (Transmembrane protease serine 2) is a protein, located on human chromosome 21, that belongs to the serine protease family (type II transmembrane serine proteases (TTSPs)) which is important for influenza virus infectivity. TMPRSS2 has been demonstrated to mediate cleavage of influenza virus HA0 to HA1 and HA2.
The human TMPRSS2 gene encodes a predicted protein of 492 amino acids which anchors to the plasma membrane. The protein converts to its mature form through autocatalytic cleavage between Arg255 and Ile256. After cleavage, the mature proteases are mostly membrane bound, yet a portion of them may be liberated into the extracellular milieu.
In an embodiment of the invention, human TMPRSS2 (V160M) comprises the amino acid sequence:
(SEQ ID NO: 22; methionine 160 in bold font)
MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVP
QYAPRVLTQASNPVVCTQPKSPSGTVCTSKTKKALCITLTLGTFLVGAAL
AAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVR
LYGPNFILQMYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGI
VDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNS
SRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEK
PLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMK
LQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAA
KVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKN
NIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADG.
In an embodiment of the invention, the TMPRSS2 polypeptide does not comprise the V160M mutation. See also NM_005656.3.
In an embodiment of the invention, Macaca mulatta TMPRSS2 (S129L, N251S, I415V, R431Q, D492G) comprises the amino acid sequence:
(SEQ ID NO: 23)
MALNSGSPPGVGPYYENHGYQPENPYPAQPTVAPNVYEVHPAQYYPSPVP
QYTPRVLTHASNPAVCRQPKSPSGTVCTSKTKKALCVTMTLGAVLVGAAL
AAGLLWKFMGSKCSDSGIECDSSGTCISLSNWCDGVSHCPNGEDENRCVR
LYGPNFILQVYSSQRKSWHPVCRDDWNENYARAACRDMGYKNSFYSSQGI
VDNSGATSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVRSNL
SRQSRIVGGQNALLGAWPWQVSLHVQNIHVCGGSIITPEWIVTAAHCVEK
PLNSPWQWTAFVGTLRQSSMFYEKGHRVEKVISHPNYDSKTKNNDIALMK
LHTPLTFNEVVKPVCLPNPGMMLEPEQHCWISGWGATQEKGKTSDVLNAA
MVPLIEPRRCNNKYVYDGLITPAMICAGFLQGTVDSCQGDSGGPLVTLKN
DVWWLIGDTSWGSGCAQANRPGVYGNVTVFTDWIYRQMRADG.
In an embodiment of the invention, the TMPRSS2 polypeptide does not comprise the S129L, N251S, I415V, R431Q and/or D492G mutation.
In an embodiment of the invention, Mus musculus TMPRSS2 mRNA comprises the nucleotide sequence set forth in NM_015775.2.
Viruses
The present invention includes methods for treating or preventing a viral infection in a subject. The term “virus” includes any virus whose infection in the body of a subject is treatable or preventable by administration of an anti-TMPRSS2 antibody or antigen-binding fragment thereof (e.g., wherein infectivity of the virus is at least partially dependent on TMPRSS2). In an embodiment of the invention, a “virus” is any virus that expresses HA0 or another substrate of TMPRSS2 whose proteolytic cleavage is required for full infectivity of the virus against a cell in a host. The term “virus” also includes a TMPRSS2-dependent respiratory virus which is a virus that infects the respiratory tissue of a subject (e.g., upper and/or lower respiratory tract, trachea, bronchi, lungs) and is treatable or preventable by administration of an anti-TMPRSS2. For example, in an embodiment of the invention, virus includes influenza virus, coronavirus, SARS-Co virus (severe acute respiratory syndrome coronavirus), MERS-Co virus (middle east respiratory syndrome (MERS) CoV), parainfluenza virus, sendai virus (SeV), human metapneumovirus and/or hepatitis C virus (HCV). “Viral infection” refers to the invasion and multiplication of a virus in the body of a subject. The present invention includes embodiments with a proviso that “virus” excludes influenza virus, e.g., wherein viral infection excludes influenza virus infection.
There are now two genera of human parainfluenza virus (HPIV), respirovirus (HPIV-1 and HPIV-3) and rubulavirus (HPIV-2 and HPIV-4). Both genera (paramyxoviruses) can be separated morphologically from influenza virus.
Sendai virus, also known as murine parainfluenza virus, is the type species in the genus respirovirus, which also contains the species human parainfluenza virus 3, bovine parainfluenza virus 3, and human parainfluenza virus 1. TMPRSS2 Is an Activating Protease for Respiratory Parainfluenza Viruses such as parainfluenza viruses and Sendai virus (SeV). See et al. Abe et al., J. Virol. 87(21): 11930-11935 (2013).
Human metapneumovirus (HMPV) is classified as the first human member of the Metapneumovirus genus in the Pneumovirinae subfamily within the Paramyxoviridae family. It is an enveloped negative-sense single-stranded RNA virus. The RNA genome includes 8 genes coding for 9 different proteins. HMPV is identical in gene order to the avian pneumovirus (AMPV), which also belongs to the Metapneumovirus genus. TMPRSS2 is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication and may be involved in the development of lower respiratory tract illness in HMPV-infected patients. See et al. Shirogane et al. J Virol. 82(17): 8942-8946 (2008).
Hepatitis C virus (HCV) is a small, enveloped, positive-sense single-stranded RNA virus of the family Flaviviridae. HCV, with at least 6 genotypes and numerous subtypes, is a member of the hepacivirus genus. TMPRSS2 may activate HCV infection at the post-binding and entry stage. Esumi et al., Hepatology 61(2): 437-446 (2015).
Influenza viruses are members of the family Orthomyxoviridae. This family represents enveloped viruses the genome of which has segmented negative-sense single-strand RNA segments. There are four genera of this family: types A, B, C and Thogotovirus. The Influenza viruses classes, A, B and C, are based on core protein and are further divided into subtypes determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) (e.g., subtype A/H1N1). There are at least 18 influenza hemagglutinin (“HA”) protein subtypes (H1-H18 or HA1-HA18) and at least 11 influenza neuraminidase (NA) protein subtypes (N1-N11 or NA1-NA11) used to define influenza subtypes. Group 1 influenza has H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18 subtypes and NA8, NA5, Na4 and NA1 subtypes. Group 2 has H3, H4, H7, H10, H14 and H15 subtypes and NA6, NA9, NA7, NA2 and NA3 subtypes. Influenza A viruses infect a range of mammalian and avian species, whereas type B and C infections are largely restricted to humans. The eight genome segments of influenza A and B viruses are loosely encapsidated by the nucleoprotein.
Coronavirus virions are spherical with diameters of approximately 125 nm. The most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses. Both MERS-CoV (middle east respiratory syndrome coronavirus) and SARS-CoV (severe acute respiratory syndrome coronavirus) belong to the coronavirus family. The initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of receptor binding domains (RBD) within the 51 region of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of 51. The 5-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a cathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes.
Anti-TMPRSS2 Antibodies and Antigen-Binding Fragments
The present invention provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to TMPRSS2 protein or an antigenic fragment thereof.
The term “antibody”, as used herein, refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM)—for example, H1H7017N. Each heavy chain comprises a heavy chain variable region (“HCVR” or “VH”) (e.g., SEQ ID NO 2) and a heavy chain constant region (comprised of domains CH1, CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) (e.g., SEQ ID NO 4) and a light chain constant region (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 comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) are identical to the human germline sequences, or are naturally or artificially modified.
Typically, the variable domains of both the heavy and light immunoglobulin chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In an embodiment of the invention, the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.
The present invention includes monoclonal anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. The term “monoclonal antibody”, as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. A “plurality” of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts) antibodies and fragments which is above that which would normally occur in nature, e.g., in the blood of a host organism such as a mouse or a human.
In an embodiment of the invention, an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment comprises a heavy chain constant domain, e.g., of the type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4) or IgM. In an embodiment of the invention, an antigen-binding protein, e.g., antibody or antigen-binding fragment comprises a light chain constant domain, e.g., of the type kappa or lambda.
The term “human” antigen-binding protein, such as an antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., U.S. Pat. No. 8,502,018, 6,596,541 or 5,789,215. The human mAbs 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), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject. See below.
The present invention includes anti-TMPRSS2 chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).
The term “recombinant” antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term includes antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.
Recombinant anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments, disclosed herein may also be produced in an E. coli/T7 expression system. In this embodiment, nucleic acids encoding the anti-TMPRSS2 antibody immunoglobulin molecules of the invention (e.g., H1H7017N) may be inserted into a pET-based plasmid and expressed in the E. coli/T7 system. For example, the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E. coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, in an embodiment of the invention, a bacterial host cell, such as an E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). See U.S. Pat. Nos. 4,952,496 and 5,693,489 or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5; 189(1): 113-30.
There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Pat. No. 4,816,567.
Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.
Thus, the present invention includes recombinant methods for making an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides (e.g., including the nucleotide sequence in any one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 15) encoding light and/or heavy immunoglobulin chains of the antigen-binding protein, e.g., H1H7017N or H4H7017N, for example, wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the antigen-binding protein, (e.g., antibody or fragment) or chain from the host cell and/or medium in which the host cell is grown. When making an antigen-binding protein (e.g., antibody or antigen-binding fragment) comprising more than one immunoglobulin chain, e.g., an antibody that comprises two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antigen-binding protein (e.g., antibody or antigen-binding fragment). The methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of an antibody or antigen-binding fragment that includes such a chain. For example, the present invention also includes anti-TMPRSS2 antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising the nucleotide sequences set forth in SEQ ID NO: 1 and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by the nucleotide sequence set forth in SEQ ID NO: 3 which are the product of such production methods, and, optionally, the purification methods set forth herein. For example, in an embodiment of the invention, the product of the method is an anti-TMPRSS2 antigen-binding protein which is an antibody or fragment comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 2 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 4; or comprising a HC comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and a LC comprising the amino acid sequence set forth in SEQ ID NO: 18.
Eukaryotic and prokaryotic host cells, including mammalian cells, may be used as hosts for expression of an anti-TMPRSS2 antigen-binding protein. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. The present invention includes an isolated host cell (e.g., a CHO cell) comprising an antigen-binding protein, such as H1H7017N; or a polynucleotide encoding such a polypeptide thereof.
The term “specifically binds” refers to those antigen-binding proteins (e.g., mAbs) having a binding affinity to an antigen, such as TMPRSS2 protein (e.g., human TMPRSS2), expressed as KD, of at least about 10−8 M (e.g., 2.81×10−9M; 9.31×10−9M; 10−9 M; 10−10M, 10−11 M, or 10−12 M), as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37° C., e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA. The present invention includes antigen-binding proteins that specifically bind to TMPRSS2 protein.
The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody or antigen-binding protein, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., as defined in WO08/020079 or WO09/138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. In an embodiment of the invention, the antigen-binding fragment comprises three or more CDRs of H1H7017N (e.g., CDR-H1, CDR-H2 and CDR-H3; or CDR-L1, CDR-L2 and CDR-L3).
An antigen-binding fragment of an antibody will, in an embodiment of the invention, comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be mono-specific or multi-specific (e.g., bi-specific). Multispecific antigen-binding proteins are discussed further herein.
In specific embodiments, antibody or antibody fragments of the invention may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), such as an anti-viral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating a viral infection, e.g., influenza viral infection. See below.
The present invention also provides a complex comprising an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment, discussed herein complexed with TMPRSS2 polypeptide or an antigenic fragment thereof and/or with a secondary antibody or antigen-binding fragment thereof (e.g., detectably labeled secondary antibody) that binds specifically to the anti-TMPRSS2 antibody or fragment. In an embodiment of the invention, the antibody or fragment is in vitro (e.g., is immobilized to a solid substrate) or is in the body of a subject. In an embodiment of the invention, the TMPRSS2 is in vitro (e.g., is immobilized to a solid substrate) or is on the surface of a cell or is in the body of a subject. Immobilized anti-TMRPSS2 antibodies and antigen-binding fragments thereof which are covalently linked to an insoluble matrix material (e.g., glass or polysaccharide such as agarose or sepharose, e.g., a bead or other particle thereof) are also part of the present invention; optionally, wherein the immobilized antibody is complexed with TMPRSS2 or antigenic fragment thereof or a secondary antibody or fragment thereof.
“Isolated” antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.
The term “epitope” refers to an antigenic determinant (e.g., on TMPRSS2 polypeptide) that interacts with a specific antigen-binding site of an antigen-binding protein, e.g., a variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
Methods for determining the epitope of an antigen-binding protein, e.g., antibody or fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., antibody or fragment or polypeptide) (e.g., coversin) interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding protein, e.g., antibody or fragment or polypeptide, to the deuterium-labeled protein. Next, the TMPRSS2 protein/antigen-binding protein complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antigen-binding protein interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antigen-binding protein (e.g., antibody or fragment or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding protein interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
The term “competes” as used herein, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., TMPRSS2) and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) to the antigen. The term also includes competition between two antigen-binding proteins e.g., antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa. In certain embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. In an embodiment of the invention, competition between a first and second anti-TMPRSS2 antigen-binding protein (e.g., antibody) is determined by measuring the ability of an immobilized first anti-TMPRSS2 antigen-binding protein (e.g., antibody) (not initially complexed with TMPRSS2 protein) to bind to soluble TMPRSS2 protein complexed with a second anti-TMPRSS2 antigen-binding protein (e.g., antibody). A reduction in the ability of the first anti-TMPRSS2 antigen-binding protein (e.g., antibody) to bind to the complexed TMPRSS2 protein, relative to uncomplexed TMPRSS2 protein, indicates that the first and second anti-TMPRSS2 antigen-binding proteins (e.g., antibodies) compete. The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).
Binding competition between anti-TMPRSS2 antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine competition between two anti-human TMPRSS2 monoclonal antibodies, the anti-TMPRSS2 mAb can be first captured onto anti-hFc antibody coated Octet biosensor tips (Pall ForteBio Corp., #18-5060) by submerging the tips into a solution of anti-human TMPRSS2 mAb (subsequently referred to as “mAb1”). As a positive-control for blocking, the antibody captured biosensor tips can then be saturated with a known blocking isotype control mAb (subsequently referred to as “blocking mAb”) by dipping into a solution of blocking mAb. To determine if mAb2 competes with mAb1, the biosensor tips can then be subsequently dipped into a co-complexed solution of human TMPRSS2 polypeptide and a second anti-human TMPRSS2 mAb (subsequently referred to as “mAb2”), that had been pre-incubated for a period of time and binding of mAb1 to the TMPRSS2 polypeptide can be determined. The biosensor tips can be washed in buffer in between every step of the experiment. The real-time binding response can be monitored during the course of the experiment and the binding response at the end of every step can be recorded.
For example, in an embodiment of the invention, the competition assay is conducted at 25° C. and pH about 7, e.g., 7.4, e.g., in the presence of buffer, salt, surfactant and a non-specific protein (e.g., bovine serum albumin).
Typically, an antibody or antigen-binding fragment of the invention which is modified in some way retains the ability to specifically bind to TMPRSS2, e.g., retains at least 10% of its TMPRSS2 binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the TMPRSS2 binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
A “variant” of a polypeptide, such as an immunoglobulin chain (e.g., H1H7017N VH, VL, HC or LC), refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein (e.g., SEQ ID NO: 2, 4, 17, 18 or 19); when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment).
A “variant” of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to a referenced nucleotide sequence that is set forth herein (e.g., SEQ ID NO: 1 or 3); when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 28; max matches in a query range: 0; match/mismatch scores: 1, −2; gap costs: linear).
Anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof of the present invention, in an embodiment of the invention, include a heavy chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids set forth in SEQ ID NO: 2, 17 or 19; and/or a light chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids set forth in SEQ ID NO: 4 or 18.
In addition, a variant anti-TMPRSS2 antigen-binding protein may include a polypeptide comprising an amino acid sequence that is set forth herein except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present invention includes antigen-binding proteins which include an immunoglobulin light chain variant comprising the amino acid sequence set forth in SEQ ID NO: 4 or 18 but having one or more of such mutations and/or an immunoglobulin heavy chain variant comprising the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19 but having one or more of such mutations. In an embodiment of the invention, a variant anti-TMPRSS2 antigen-binding protein includes an immunoglobulin light chain variant comprising CDR-L1, CDR-L2 and CDR-L3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and/or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2 and CDR-H3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions).
The invention further provides variant anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more variant CDRs (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3) that are set forth herein with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% sequence identity or similarity to, e.g., SEQ ID NO: 12, 14, 16, 6, 8 and/or 10.
Embodiments of the present invention also include variant antigen-binding proteins, e.g., anti-TMPRSS2 antibodies and antigen-binding fragments thereof, that comprise immunoglobulin VHS and VLs; or HCs and LCs, which comprise an amino acid sequence having 70% or more (e.g., 80%, 85%, 90%, 95%, 97% or 99%) overall amino acid sequence identity or similarity to the amino acid sequences of the corresponding VHS, VLs, HCs or LCs specifically set forth herein, but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulins are not variants and comprise the amino acid sequence set forth in SEQ ID NOs: 12, 14, 16, 6, 8 and 10, respectively. Thus, in such embodiments, the CDRs within variant antigen-binding proteins are not, themselves, variants.
Conservatively modified variant anti-TMPRSS2 antibodies and antigen-binding fragments thereof are also part of the present invention. A “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.
Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45.
Function-conservative variants of the anti-TMPRSS2 antibodies and antigen-binding fragments thereof are also part of the present invention. Any of the variants of the anti-TMPRSS2 antibodies and antigen-binding fragments thereof (as discussed herein) may be “function-conservative variants”. Such function-conservative variants may, in some cases, also be characterized as conservatively modified variants. “Function-conservative variants,” as used herein, refers to variants of the anti-TMPRSS2 antibodies or antigen-binding fragments thereof in which one or more amino acid residues have been changed without significantly altering one or more functional properties of the antibody or fragment. In an embodiment of the invention, a function-conservative variant anti-TMPRSS2 antibody or antigen-binding fragment thereof of the present invention comprises a variant amino acid sequence and exhibits one or more of the following functional properties:
Inhibits growth of influenza virus (e.g., A/Puerto Rico/08/1934 (H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells);
Binds to the surface of TMPRSS-expressing cells (e.g., MDCK/Tet-on), e.g., with an EC50 value of 440 pM or 1.06 nM, respectively;
Does not significantly bind to MDCK/Tet-on cells which do not express TMPRSS2;
Binds to human TMPRSS2 with a KD of about 2.81×10−9M at about 25° C.;
Binds to human TMPRSS2 with a KD of about 9.31×10−9M at about 37° C.;
Binds to cynomolgous TMPRSS2 with a KD of about 5.60×10−8M at about 25° C.;
Binds to cynomolgous TMPRSS2 with a KD of about 1.40×10−7M at about 37° C.;
Limits spread of influenza virus infection (e.g., by H1_PR34; H1_CA09; H1_Bris; H9N2 or H3N2 influenza virus) of cells, e.g., Calu-3, in vitro; and/or
Protects a mouse engineered to express the human TMPRSS2 protein from death caused by influenza virus infection, e.g., H1N1, or H3N2, for example, wherein the mice are infected with an otherwise lethal dose of the virus, optionally when combined with an anti-HA antibody.
The present invention includes a mouse engineered to express the human TMPRSS2 protein which includes, within the mouse's body, an anti-TMPRSS2 antigen-binding protein (e.g., antibody or antigen-binding fragment) such as H1H7017N and H4H7017N. See International patent application publication no. WO2017/151453.
A “neutralizing” or “antagonist” anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment, refers to a molecule that inhibits an activity of TMPRSS2 to any detectable degree, e.g., inhibits protease activity of TMPRSS2, for example, of a substrate such as HA; Cbz-Gly-Gly-Arg-AMC (Sigma), where Cbz is benzyloxycarbonyl and AMC is 7-amino-4-methylcoumarin; influenza virus HA0; coronavirus S protein; or precursor TMPRSS2 which is autocatalytically cleaved between Arg255 and Ile256 and/or inhibits influenza virus entry into a cell and/or inhibits influenza virus reproduction in the body of a subject.
“H1H7017N” and “H4H7017N” refer to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that comprise the heavy chain or VH (or a variant thereof) and light chain or VL (or a variant thereof) as set forth below; or that comprise a VH that comprises the CDRs thereof (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variant thereof)) and a VL that comprises the CDRs thereof (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variant thereof)), e.g., wherein the immunoglobulin chains, variable regions and/or CDRs comprise the specific amino acid sequences described below.
In an embodiment of the invention, “H1H7017N” or “H4H7017N” refers to an antibody or antigen-binding fragment thereof comprising CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain that comprises the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19 and CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin light chain that comprises the amino acid sequence set forth in SEQ ID NO: 4 or 18.
In an embodiment of the invention, “H1H7017N” or “H4H7017N” refers to an antibody or antigen-binding fragment thereof comprising a VH that comprises the amino acid sequence set forth in SEQ ID NO: 2; and a VL that comprises the amino acid sequence set forth in SEQ ID NO: 4.
In an embodiment of the invention, “H1H7017N” refers to an antibody or antigen-binding fragment comprising a heavy chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 17; and a light chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 18.
In an embodiment of the invention, “H4H7017N” refers to an antibody or antigen-binding fragment comprising a heavy chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 19; and a light chain immunoglobulin that comprises the amino acid sequence set forth in SEQ ID NO: 18. The term “H4H7017N” also includes embodiments wherein the VH is fused to a wild-type IgG4, e.g., wherein residue 108 is S.
Anti-TMRPS22 Antibody or Antigen-Binding Fragment
H1H7017N and H4H7017NH1H7017N and H4H7017N Heavy
Chain Variable Region (DNA)
(SEQ ID NO: 1)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTCCTATGGCA
TGCACTGGGTCCGCCAGTCTCCAGGCAAGGGGCTCGAGTGGGTGGCAGTT
ATATGGAATGATGGAAGTTATGTATACTATGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTTTCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGG
GAGTGGGTACTTTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCAC
CGTCTCCTCA
H1H7017N and H4H7017N Heavy Chain Variable Region
(Polypeptide)
(SEQ ID NO: 2)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAV
IWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREG
EWVLYYFDYWGQGTLVTVSS
H1H7017N and H4H7017N Light Chain Variable Region
(DNA)
(SEQ ID NO: 3)
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTTGGAGA
CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGG
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAG
GCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGTATAATAGTTATTCGTACACTTTTGGCCAG
GGGACCAAGCTGGAGATCAAA
H1H7017N and H4H7017N Light Chain Variable Region
(Polypeptide)
(SEQ ID NO: 4)
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQ
GTKLEIK
H1H7017N and H4H7017N CDR-H1 (DNA)
(SEQ ID NO: 5)
GGA TTC ACC TTC AGT TCC TAT GGC
H1H7017N and H4H7017N CDR-H1 (Polypeptide)
(SEQ ID NO: 6)
G F T F S S Y G
(or a variant thereof having 1, 2, 3 or 4 point
mutations and/or point deletions)
H1H7017N and H4H7017N CDR-H2 (DNA)
(SEQ ID NO: 7)
ATA TGG AAT GAT GGA AGT TAT GTA
H1H7017N and H4H7017N CDR-H2 (Polypeptide)
(SEQ ID NO: 8)
I W N D G S Y V
(or a variant thereof having 1, 2, 3 or 4 point
mutations and/or point deletions)
H1H7017N and H4H7017N CDR-H3 (DNA)
(SEQ ID NO: 9)
GCG AGA GAG GGG GAG TGG GTA CTT TAC TAC TTT GAC
TAC
H1H7017N and H4H7017N CDR-H3 (Polypeptide)
(SEQ ID NO: 10)
A R E G E W V L Y Y F D Y
(or a variant thereof having 1, 2, 3 or 4 point
mutations and/or point deletions)
H1H7017N and H4H7017N CDR-L1 (DNA)
(SEQ ID NO: 11)
CAG AGT ATT AGT AGC TGG
H1H7017N and H4H7017N CDR-L1 (Polypeptide)
(SEQ ID NO: 12)
Q S I S S W
(or a variant thereof having 1, 2, 3 or 4 point
mutations and/or point deletions)
H1H7017N and H4H7017N CDR-L2 (DNA)
(SEQ ID NO: 13)
AAG GCG TCT
H1H7017N and H4H7017N CDR-L2 (Polypeptide)
(SEQ ID NO: 14)
K A S
(or a variant thereof having a point mutation and/
or point deletion)
H1H7017N and H4H7017N CDR-L3 (DNA)
(SEQ ID NO: 15)
CAA CAG TAT AAT AGT TAT TCG TAC ACT
H1H7017N and H4H7017N CDR-L3 (Polypeptide)
(SEQ ID NO: 16)
Q Q Y N S Y S Y T
(or a variant thereof having 1, 2, 3 or 4 point
mutations and/or point deletions)
H1H7017N
Full length heavy chain-human IgG1
(SEQ ID NO: 17)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAV
IWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREG
EWVLYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Full length light chain-human Kappa
(SEQ ID NO: 18)
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
H4H7017N
Full length heavy chain-human IgG4 (S108P)
(SEQ ID NO: 19)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAV
IWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREG
EWVLYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT
YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Full length light chain-human Kappa
(SEQ ID NO: 18)
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
Antibodies and antigen-binding fragments of the present invention comprise immunoglobulin chains including the amino acid sequences set forth herein as well as cellular and in vitro post-translational modifications to the antibody. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to TMPRSS2 comprising heavy and/or light chain amino acid sequences set forth herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal Gln is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing.
The present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising an anti-TMPRSS2 antigen-binding protein of the present invention, e.g., H1H7017N or H4H7017N.
The present invention also provides an injection device comprising one or more antigen-binding proteins (e.g., antibody or antigen-binding fragment) that bind specifically to TMPRSS2, e.g., H4H7017N or H1H7017N, or a pharmaceutical composition thereof. The injection device may be packaged into a kit. An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the antibody or fragment or a pharmaceutical composition thereof), a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the invention, an injection device that comprises an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, from a combination of the present invention, or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device can include the antigen-binding protein or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline) introduced into the body of the subject through the cannula or trocar/needle. The antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the invention, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a subject's body. External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a subject's body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.
The present invention further provides methods for administering an anti-TMPRSS2 antigen-binding protein of the present invention, e.g., H4H7017N or H1H7017N, comprising introducing the antigen-binding protein into the body of a subject (e.g., a human). For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
Preparation of Human Antibodies
Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to TMPRSS2. An immunogen comprising any one of the following can be used to generate antibodies to TMPRSS2. In certain embodiments of the invention, the antibodies of the invention are obtained from mice immunized with a full length, native TMPRSS2, or with a live attenuated or inactivated virus, or with DNA encoding the protein or fragment thereof. Alternatively, the TMPRSS2 protein or a fragment thereof may be produced using standard biochemical techniques and modified and used as immunogen. In one embodiment of the invention, the immunogen is a recombinantly produced TMPRSS2 protein or fragment thereof. In certain embodiments of the invention, the immunogen may be a TMPRSS2 polypeptide vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the immunogen may be a recombinant TMPRSS2 polypeptide expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.
Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to TMPRSS2 can be initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.
Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.
Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
Anti-TMPRSS2 Antibodies Comprising Fc Variants
According to certain embodiments of the present invention, anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments, are provided comprising an Fc domain comprising one or more mutations, which, for example, enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes anti-TMPRSS2 antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A) modification.
For example, the present invention includes anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments, comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).
Anti-TMPRSS antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, that comprise a VH and/or VL as set forth herein comprising any possible combinations of the foregoing Fc domain mutations, are contemplated within the scope of the present invention.
The present invention also includes anti-TMPRSS2 antigen-binding proteins, antibodies or antigen-binding fragments, comprising a VH set forth herein and a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO2014/022540).
Immunoconjugates
The invention encompasses an anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments, conjugated to another moiety, e.g., a therapeutic moiety (an “immunoconjugate”), such as a toxoid or an anti-viral drug to treat influenza virus infection. In an embodiment of the invention, an anti-TMPRSS2 antibody or fragment is conjugated to any of the further therapeutic agents set forth herein. As used herein, the term “immunoconjugate” refers to an antigen-binding protein, e.g., an antibody or antigen-binding fragment, which is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein or a therapeutic agent. The antigen-binding protein may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target (TMPRSS2). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the invention, the agent may be a second, different antibody that binds specifically to TMPRSS2. The type of therapeutic moiety that may be conjugated to the anti-TMPRSS2 antigen-binding protein (e.g., antibody or fragment) will take into account the condition to be treated and the desired therapeutic effect to be achieved. See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, 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”, Monoclonal Antibodies 1984: 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”, 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).
Multi-Specific Antibodies
The present invention includes anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as methods of use thereof and methods of making such antigen-binding proteins. The term “anti-TMPRSS2” antigen-binding proteins, e.g., antibodies or antigen-binding fragments, includes multispecific (e.g., bispecific or biparatopic) molecules that include at least one first antigen-binding domain that specifically binds to TMPRSS2 (e.g., an antigen-binding domain from H1H7017N or H4H7017N) and at least one second antigen-binding domain that binds to a different antigen or to an epitope in TMPRSS2 which is different from that of the first antigen-binding domain (e.g., influenza HA such as an antigen-binding domain from H1H14611N2, H1H14612N2 or H1H11729P). In an embodiment of the invention, the first and second epitopes overlap. In another embodiment of the invention, the first and second epitopes do not overlap. For example, in an embodiment of the invention, a multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) that includes a first antigen-binding domain that binds specifically to TMPRSS2 including the heavy and light immunoglobulin chain of H1H7017N or H4H7017N, and a second antigen-binding domain that binds specifically to influenza HA (comprising a different light and heavy immunoglobulin chain such as from H1H14611N2, H1H14612N2 or H1H11729P).
“H1H7017N” includes a multispecific molecules, e.g., antibodies or antigen-binding fragments, that include the HCDRs and LCDRs, VH and VL, or HC and LC of H1H7017N (including variants thereof as set forth herein).
“H4H7017N” includes a multispecific molecules, e.g., antibodies or antigen-binding fragments, that include the HCDRs and LCDRs, VH and VL, or HC and LC of H4H7017N (including variants thereof as set forth herein).
In an embodiment of the invention, an antigen-binding domain that binds specifically to TMPRSS, which may be included in a multispecific molecule, comprises:
(1)
(i) a heavy chain variable domain sequence that comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 6, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 10, and
(ii) a light chain variable domain sequence that comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16;
or,
(2)
(i) a heavy chain variable domain sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, and
(ii) a light chain variable domain sequence comprising the amino acid sequence set forth in SEQ ID NO: 4;
or,
(3)
(i) a heavy chain immunoglobulin sequence comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19, and
(ii) a light chain immunoglobulin sequence comprising the amino acid sequence set forth in SEQ ID NO: 18.
In an embodiment of the invention, the multispecific antibody or fragment includes more than two different binding specificities (e.g., a trispecific molecule), for example, one or more additional antigen-binding domains which are the same or different from the first and/or second antigen-binding domain.
In an embodiment of the invention, a multispecific molecule comprises, in addition to an antigen-binding site that bind specifically to TMPRSS2, an antigen-binding site that binds specifically to influenza HA taken from an antibody selected from the group consisting of:
H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B; as set forth in International patent application publication no. WO2016/100807 (e.g., the CDR-Hs, VH or heavy chain thereof; and the CDR-Ls, VL or light chain thereof).
In an embodiment of the invention, a multispecific molecule comprises, in addition to an antigen-binding site that binds specifically to TMPRSS2, an antigen-binding site that binds specifically to influenza Group II HA protein, e.g., which comprises VH and VL of H1H14611N2 (e.g., SEQ ID Nos: 24 and 28); or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).
In an embodiment of the invention, a multispecific molecule comprises, in addition to an antigen-binding site that bind specifically to TMPRSS2, an antigen-binding site that binds specifically to influenza Group II HA protein, e.g., which comprises VH and VL of H1H14612N2 (e.g., SEQ ID Nos: 40 and 44); or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).
In an embodiment of the invention, a multispecific molecule comprises, in addition to an antigen-binding site that bind specifically to TMPRSS2, an antigen-binding site that binds specifically to influenza Group I HA protein, e.g., which comprises VH and VL of H1H11729P (e.g., SEQ ID Nos: 32 and 36); or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39).
In one embodiment of the invention, a bispecific antigen-binding fragment comprises a first scFv (e.g., comprising VH and VL of H1H7017N or H4H7017N) having binding specificity for a first epitope (e.g., TMPRSS2) and a second scFv (e.g., comprising VH and VL of an anti-influenza HA antibody) having binding specificity for a second, different epitope. For example, in an embodiment of the invention, the first and second scFv are tethered with a linker, e.g., a peptide linker (e.g., a GS linker such as (GGGGS)n (SEQ ID NO: 48) wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Other bispecific antigen-binding fragments include an F(ab)2 of a bispecific IgG antibody which comprises the heavy and light chain CDRs of H1H7017N or H4H7017N and of another antibody that binds to a different epitope.
Therapeutic Methods
The present invention provides methods for treating or preventing viral infection or cancer (e.g., prostate cancer) by administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment, (e.g., H1H7017N or H4H7017N) to a subject (e.g., a human) in need of such treatment or prevention.
Influenza virus infection may be treated or prevented, in a subject, by administering an anti-TMPRSS2 antigen-binding protein of the present invention to a subject. The influenza viruses are classified into types A, B and C on the basis of their core proteins. The subtypes of influenza A viruses are determined by envelope glycoproteins possessing either hemagglutinin (HA) or neuraminidase (NA) activity. There are several HA subtypes (e.g., HAL HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15, HA16, HA17 or HA18—these subtypes may be designated as H1, H2, H3, etc.) and NA subtypes (e.g., NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10 or NA11—these subtypes may be designated as N1, N2, N3, etc.) of influenza A viruses which are used to designate influenza A subtype. For example, Influenza A virus H1N1 and H3N2 are commonly known human pathogens. Humans are commonly infected by viruses of the subtypes H1, H2 or H3, and N1 or N2. The present invention includes methods for treating or preventing infection with an influenza virus subtype discussed herein. Multispecific antibodies and antigen-binding fragments thereof that bind to TMPRSS2, in an embodiment of the invention, also bind to HA/and/or to NA, e.g., of a subtype set forth herein.
An effective or therapeutically effective dose of anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N), for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms. The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In an embodiment of the invention, an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present invention, for treating or preventing viral infection, e.g., in an adult human subject, is about 0.01 to about 200 mg/kg, e.g., up to about 150 mg/kg. In an embodiment of the invention, the dosage is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 grams). Depending on the severity of the infection, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antigen-binding protein of the present invention can be administered at an initial dose, followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, for example, in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, e.g., an influenza infection, or be predisposed to developing an infection. Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection (e.g., of influenza virus), include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g., 5 years of age or younger) or old age (e.g., 65 years of age or older) are at increased risk. Moreover, a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
“Treat” or “treating” means to administer an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment of the present invention (e.g., H1H7017N or H4H7017N), to a subject having one or more signs or symptoms of a disease or infection, e.g., viral infection, for which the antigen-binding protein is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein).
The present invention also encompasses prophylactically administering an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present invention (e.g., H1H7017N or H4H7017N), to a subject who is at risk of viral infection so as to prevent such infection. Passive antibody-based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. See e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat. 2014; 2014: 267594. Epub 2014 Sep. 22; and Jianqiang et al., Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy. 2012 February; 4(2): 175-186; Prabhu et al., Antivir Ther. 2009; 14(7):911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. “Prevent” or “preventing” means to administer an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment of the present invention (e.g., H1H7017N or H4H7017N), to a subject to inhibit the manifestation of a disease or infection (e.g., viral infection) in the body of a subject, for which the antigen-binding protein is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein).
In an embodiment of the invention, a sign or symptom of a viral infection in a subject is survival or proliferation of virus in the body of the subject, e.g., as determined by viral titer assay (e.g., influenza virus propagation in embryonated chicken eggs or influenza virus hemagglutination assay). Other signs and symptoms of viral infection are discussed herein.
The present invention provides a method for treating or preventing viral infection (e.g., influenza virus or corona virus infection) or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of viral infection such as:
Fever or feeling feverish/chills;
Cough;
Sore throat;
Runny or stuffy nose;
Sneezing;
Muscle or body aches;
Headaches;
Fatigue (tiredness);
vomiting;
diarrhea;
respiratory tract infection;
chest discomfort;
shortness of breath;
bronchitis; and/or
pneumonia,
which sign or symptom is secondary to viral infection, in a subject in need thereof (e.g., a human), by administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N) to the subject, for example, by injection of the protein into the body of the subject.
The present invention also includes methods for treating or preventing cancer, e.g., metastatic cancer, e.g., prostate cancer (e.g., which is characterized by expression of a TMPRSS2:ERG fusion), colon cancer, lung cancer, pancreas cancer, urinary tract cancer, breast cancer, ovarian cancer, prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and/or pleural mesothelioma, in a subject, by administering a therapeutically effective amount of TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N) to the subject, for example, by injection of the protein into the body of the subject. In an embodiment of the invention, the subject is also administered the TMPRSS2 antigen-binding protein in association with a further therapeutic agent, for example, an anti-cancer therapeutic agent. In an embodiment of the invention, the cancer is a tumor whose cells express TMPRSS2 or a variant thereof.
Combinations and Pharmaceutical Compositions
To prepare pharmaceutical compositions of the anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof (e.g., H1H7017N or H4H7017N), antigen-binding protein is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. In an embodiment of the invention, the pharmaceutical composition is sterile. Such compositions are part of the present invention.
The scope of the present invention includes desiccated, e.g., freeze-dried, compositions comprising an anti-TMPRSS2 antigen-binding proteins, e.g., antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water.
In a further embodiment of the invention, a further therapeutic agent that is administered to a subject in association with an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), disclosed herein is administered to the subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)).
The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial.
The present invention provides methods for administering an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), comprising introducing the protein into the body of a subject. For example, the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., into the vein, artery, tumor, muscular tissue or subcutis of the subject.
The present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., H1H7017N or H4H7017N), polypeptides (e.g., an HC, LC, VH or VL of H1H7017N or H4H7017N) or polynucleotides or vectors set forth herein or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.
In an embodiment of the invention, an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present invention (e.g., H1H7017N or H4H7017N), is in association with one or more further therapeutic agents. For example, in an embodiment of the invention, the further therapeutic agent is an anti-viral drug and/or a vaccine. As used herein, the term “anti-viral drug” refers to any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. The term “anti-viral drug” includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, amantadine, rimantadine, oseltamivir, zanamivir, ribavirin, or interferon-alpha2b. Methods for treating or preventing virus (e.g., influenza) infection in a subject in need of said treatment or prevention by administering H1H7017N or H4H7017N in association with a further therapeutic agent are part of the present invention.
For example, in an embodiment of the invention, the further therapeutic agent is a vaccine, e.g., an influenza vaccine. In an embodiment of the invention, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.
For example, in an embodiment of the invention, the further therapeutic agent is:
See Shen et al. Biochimie 142: 1-10 (2017).
In an embodiment of the invention, the anti-viral drug is an antibody or antigen-binding fragment that binds specifically to influenza virus, e.g., influenza HA. For example, in an embodiment of the invention, the anti-HA antibody is any one of H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; or H1H18335B; as set forth in International patent application publication no. WO2016/100807; or an antigen-binding fragment thereof, e.g., wherein the antibody or fragment comprises a light chain immunoglobulin that includes CDR-L1, CDR-L2 and CDR-L3 (e.g., the VL or light chain thereof); and a heavy chain that includes CDR-H1, CDR-H2 and CDR-H3 (e.g., the VH or heavy chain thereof) of any of the foregoing anti-influenza HA antibodies.
In an embodiment of the invention, a further therapeutic agent is an antibody or antigen-binding fragment that binds to influenza Group II HA protein such as H1H14611N2; or an antibody or fragment that comprises VH and VL of H1H14611N2; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31). “H1H14611N2” refers to any anti-group II HA antibody comprising such sequences.
H1H14611N2
Heavy chain variable region
(SEQ ID NO: 24)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSGFSMNWVRQVPGKGLEWVSS
ISTSGNYMYYADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYCARGG
GYNWNLFDYWGQGSL
VTVSS
CDR-H1:
(SEQ ID NO: 25)
GFTFSGFS
CDR-H2:
(SEQ ID NO: 26)
ISTSGNYM
CDR-H3:
(SEQ ID NO: 27)
ARGGGYNWNLFDY
Light chain variable region
(SEQ ID NO: 28)
EIVLTQSPGTLSLSPGERATLSCRASQSLNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTITRLESEDFAVYYCQQYGNSPLTFG
GGTKVEIK
CDR-L1:
(SEQ ID NO: 29)
QSLNSNY
CDR-L2:
(SEQ ID NO: 30)
GAS
CDR-L3:
(SEQ ID NO: 31)
QQYGNSPLT
In an embodiment of the invention, a further therapeutic agent is an antibody or antigen-binding fragment that binds to influenza Group II HA protein such as H1H14612N2; or an antibody or fragment that comprises VH and VL of H1H14612N2; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47). “H1H14612N2” refers to any anti-group II HA antibody comprising such sequences.
H1H14612N2
Heavy chain variable region
EVQLVESGGGLVKPGGSLRLSCAASGFSFSGFSMNWVRQAPGKGLEWVSS
ISTSGNYMYY
(SEQ ID NO: 40)
ADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYCARGGGYNWNLFDYW
GQGSLVTVSS
CDR-H1:
(SEQ ID NO: 41)
GFSFSGFS
CDR-H2:
(SEQ ID NO: 42)
ISTSGNYM
CDR-H3:
(SEQ ID NO: 43)
ARGGGYNWNLFDY
Light chain variable region
(SEQ ID NO: 44)
EIVLTQSPGTLSLSPGERATLSCRASQSLNSNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGADFTLTISRLESEDFAVYYCQQYGNSPLTFG
GGTKVEIK
CDR-L1:
(SEQ ID NO: 45)
QSLNSNY
CDR-L2:
(SEQ ID NO: 46)
GAS
CDR-L3:
(SEQ ID NO: 47)
QQYGNSPLT
In an embodiment of the invention, a further therapeutic agent is an antibody or antigen-binding fragment that binds to influenza Group I HA protein such as H1H11729P; or an antibody or fragment that comprises VH and VL of H1H11729P; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2 and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39). “H1H11729P” refers to any anti-group I HA antibody comprising such sequences.
H1H11729P
Heavy chain variable region
(SEQ ID NO: 32)
QVQLVQSGAEVKKSGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIPIFGTPSYAQKFQDRVTITTDESTSTVYMELSSLRSEDTAVYYCARQQ
PVYQYNMDVWGQGTTVTVSS
CDR-H1:
(SEQ ID NO: 33)
GGTFSSYA
CDR-H2:
(SEQ ID NO: 34)
IIPIFGTP
CDR-H3:
(SEQ ID NO: 35)
ARQQPVYQYNMDV
Light chain variable region
(SEQ ID NO: 36)
DIQMTQSPSSLSASVGDRVTITCRASQGIRNNLGWYQQKPLKAPKRLIYA
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNNYPWTFGQ
GTKVEIK
CDR-L1:
(SEQ ID NO: 37)
QURNN
CDR-L2:
(SEQ ID NO: 38)
AAS
CDR-L3:
(SEQ ID NO: 39)
LQYNNYPWT
In a certain embodiment of the invention, the further therapeutic agent is not amantadine, rimantadine, oseltamivir, zanamivir, aprotinin, leupeptin, a cationic steroid antimicrobial, an influenza vaccine (e.g., killed, live, attenuated whole virus or subunit vaccine), or an antibody against influenza virus (e.g., an anti-hemagglutinin antibody).
The term “in association with” indicates that the components, an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present invention, along with another agent such as oseltamivir, can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route (e.g., wherein an anti-TMPRSS2 antibody or antigen-binding fragment thereof.
Kits
Further provided are kits comprising one or more components that include, but are not limited to, an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment as discussed herein (e.g., H1H7017N or H4H7017N), in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein. The antigen-binding protein and/or the further therapeutic agent can be formulated as a single composition or separately in two or more compositions, e.g., with a pharmaceutically acceptable carrier, in a pharmaceutical composition.
In one embodiment of the invention, the kit includes an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N), or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).
In another embodiment, the kit comprises a combination of the invention, including an anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N), or pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.
If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an injection device) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-TMPRSS2 antigen-binding protein, e.g., antibody or antigen-binding fragment thereof of the present invention (e.g., H1H7017N or H4H7017N).
The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
Diagnostic Uses of the Antibodies
The anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof of the present invention (e.g., H1H7017N or H4H7017N), may be used to detect and/or measure TMPRSS2 in a sample. Exemplary assays for TMPRSS2 may include, e.g., contacting a sample with an anti-TMPRSS2 antigen-binding protein of the invention, wherein the anti-TMPRSS2 antigen-binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate TMPRSS2 from samples. The presence of an anti-TMPRSS2 antigen-binding protein complexed with TMPRSS2 indicates the presence of TMRPSS2 in the sample. Alternatively, an unlabeled anti-TMPRSS2 antibody can be used in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure TMPRSS2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Thus, the present invention includes a method for detecting the presence of TMPRSS2 polypeptide in a sample comprising contacting the sample with an anti-TMPRSS2 antigen-binding protein and detecting the presence of a TMPRSS/anti-TMPRSS2 antigen-binding protein wherein the presence of the complex indicates the presence of TMPRSS2.
The present invention includes cell-based ELISA methods using the anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof of the present invention (e.g., H1H7017N), to detect the presence of TMPRSS2 on a cell. In an embodiment of the invention, the method includes the steps:
(i) contacting cells immobilized to a solid surface (e.g., a microplate) to be tested for the presence of TMPRSS2 with an anti-TMPRSS2 antigen-binding protein of the present invention;
(ii) optionally washing the mixture to remove unbound anti-TMPRSS2 antigen-binding protein;
(iii) contacting the anti-TMPRSS2 antigen-binding protein with a labeled secondary antibody or antigen-binding fragment thereof that binds to the anti-TMPRSS2 antigen-binding protein;
(iv) optionally washing the complex to remove unbound antigen-binding protein; and
(v) detecting the presence of the label on the secondary antibody or fragment, wherein detection of the label indicates that the cells contain TMPRSS2. For example, the present invention includes such cell-based ELISA methods for identifying TMPRSS2+ cells in a sample.
An anti-TMPRSS2 antigen-binding protein of the invention (e.g., H1H7017N or H4H7017N) may be used in a Western blot or immune-protein blot procedure for detecting the presence of TMPRSS2 or a fragment thereof in a sample. Such a procedure forms part of the present invention and includes the steps of e.g.:
(1) providing a membrane or other solid substrate comprising a sample to be tested for the presence of TMPRSS2, e.g., optionally including the step of transferring proteins from a sample to be tested for the presence of TMPRSS2 (e.g., from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting); and contacting the membrane or other solid substrate to be tested for the presence of TMPRSS2 or a fragment thereof with an anti-TMPRSS2 antigen-binding protein of the invention.
Such a membrane may take the form, for example, of a nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins to be tested for the presence of TMPRSS2 in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel). Before contacting the membrane with the anti-TMPRSS2 antigen-binding protein, the membrane is optionally blocked, e.g., with non-fat dry milk or the like so as to bind non-specific protein binding sites on the membrane.
(2) washing the membrane one or more times to remove unbound anti-TMPRSS2 antigen-binding protein and other unbound substances; and
(3) detecting the bound anti-TMPRSS2 antigen-binding protein.
Detection of the bound antigen-binding protein indicates that the TMPRSS2 protein is present on the membrane or substrate and in the sample. Detection of the bound antigen-binding protein may be by binding the antigen-binding protein with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody label.
The anti-TMPRSS2 antigen-binding proteins (e.g., antibodies and antigen-binding fragments (e.g., H1H7017N or H4H7017N)) disclosed herein may also be used for immunohistochemistry. Such a method forms part of the present invention and comprises, e.g.,
(1) contacting tissue to be tested for the presence of TMPRSS2 protein with an anti-TMPRSS2 antigen-binding protein of the invention; and
(2) detecting the antigen-binding protein on or in the tissue.
If the antigen-binding protein itself is detectably labeled, it can be detected directly. Alternatively, the antigen-binding protein may be bound by a detectably labeled secondary antibody wherein the label is then detected.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25° C., and pressure is at or near atmospheric.
Example 1: In Vitro Multicycle Replication
The ability of the influenza virus, A/Puerto Rico/08/1934 (H1N1)-GFP, to replicate in Calu3, A549, MDCK and HepG2 cells was assessed.
TABLE 1
Reagents used.
Description
Vendor
Calu-3 cells
American Type Culture
Collection (ATCC)
A549 cells
American Type Culture
Collection (ATCC)
MDCK (London) cells
IRR
HepG2 cells
American Type Culture
Collection (ATCC)
A/Puerto Rico/08/1934 (H1N1)-GFP
N/A
DMEM
Gibco
F12
Gibco
Pen/Strep
Gibco
Low IgG BSA
Sigma
PBS
Life Technologies
Fetal Bovine Serum
Life Technologies
Experimental Procedure
Calu-3 cells (ATCC HTB55), A549 cells (ATCC CCL-185), MDCK cells (IRR FR-58) and HepG2 cells (ATCC HB-8065) were diluted to 40,000 cells/well in a 96-well plate in DMEM:F12 medium with 5% FBS. The next day, A/Puerto Rico/08/1934 (H1N1) carrying a GFP reporter gene in the NS segment (B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6) was prepared at an MOI (multiplicity of infection) of 0.1 and 0.01 in DMEM:F12 with low IgG BSA after three washes. The virus was incubated on the cells for 1 h at 37° C. after which the virus was removed and the wells washed three more times. The number of infected cells was quantified at 24, 48, 72 and 142 h post-infection on a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, Ohio).
Results Summary and Conclusions
Calu-3 is an immortalized human airway epithelial cell line which has been shown to allow multi-cycle replication of human influenza viruses in the absence of exogenous trypsin (Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007)). In addition, Calu-3 cells have been shown to express both TMPRSS2 and TMPRSS4, but not TMPRSS11D (HAT) at least at the mRNA level (Böttcher-Friebertshäuser et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011)). To confirm that Calu-3 cells can support the proteolytic activation of influenza virus possessing hemagglutinin with a monobasic cleavage site, the growth of an H1N1 GFP reporter virus in Calu-3 cells was analyzed and replication over time with A549 (human alveolar basal epithelial), MDCK (Madin Darby canine kidney) and HepG2 (human liver carcinoma) cells in the absence of trypsin was compared. The cells were infected at a low MOI and, at the indicated timepoint, viral titers were determined by counting fluorescent focus spots. Table 2 and FIG. 1 show low levels of infection in A549, MDCK and HepG2 cells, while Calu-3 cells show significantly increased titers at every timepoint. Although Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at the mRNA level, knockdown of TMPRSS2 reduced influenza virus titers by 100- to 1,000-fold (Böttcher-Friebertshäuser et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011)). The low level of viral titers in A549, MDCK and HepG2 cells in the absence of trypsin are probably due to the addition of cleaved virus (harvested from embryonated chicken eggs or from MDCK culture with trypsin), but the presence of another HA-activating protease could be an explanation.
TABLE 2
Number of infected cells represented by Fluorescent
Focus Units (FFU) on different days post-infection with
a MOI of 0.1 or 0.01 in different cell types after infection
with A/Puerto Rico/08/1934 (H1N1)-GFP.
Cell
Day(s)
MOI 0.1
MOI 0.01
line
post-infection
FFU
FFU
Calu3
1
697
54
2
1167
201
3
1644
376
4
1530
500
A549
1
238
35
2
238
46
3
258
53
4
228
52
MDCK
1
740
77
2
750
60
3
879
58
4
796
53
HepG2
1
3
1
2
14
9
3
20
13
4
21
20
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), May; 88(9):4744-51.doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Böttcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 2006 October; 80(19):9896-8. PMID: 16973594.
10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 2: Anti-TMPRSS2 Antibody H1H7017N Blocks Spread of Influenza In Vitro
The ability of various antibodies to reduce the titers of influenza virus A/Puerto Rico/08/1934 (H1N1) in Calu-3 cells was assessed.
TABLE 3
Reagents used.
Description
Vendor
Calu-3 cells
ATCC
F12
Gibco
FBS
Life Technologies
A/Puerto Rico/08/1934 (H1N1)
ATCC
DMEM
Gibco
Pen/Strep
Gibco
Low IgG BSA
Sigma
PBS
Life Technologies
Paraformaldehyde (16% w/v aq.)
Alfa Aesar
Triton X-100
EMD
Anti-NP antibody
Millipore
Anti-Influenza A Antibody,
nucleoprotein, clones A1, A3 Blend
Goat anti-mouse IgG AF488 conjugated
Life Technologies
Experimental Procedure
Calu-3 cells (ATCC HTB55) were diluted to 40,000 cells/well in a 96-well plate in DMEM:F12 medium with 5% FBS. The next day, the monoclonal antibodies were diluted to 166.7 nM in DMEM:F12 with low IgG BSA and added to the cells for 3 h at 37° C. and 5% CO2. The mAb solution was removed and the cells were infected with A/Puerto Rico/08/1934 (H1N1) at an MOI of 0.001. The virus was incubated on the cells for 1 h at 37° C. in 5% CO2 after which the virus was removed and the medium replaced with DMEM:F12 containing 166.7 nM mAbs. After 24 h and 48 h, the medium was replaced with fresh medium containing mAb and the cells were washed twice with PBS at 72 h. The cells were then fixed with 4% paraformaldehyde in PBS and virus detected using the anti-NP primary antibody at a 1:1000 dilution. The cells were incubated for 1 h and then washed and the secondary at 1:2000 dilution was added. The number of infected cells was quantified at on a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, Ohio).
Results Summary and Conclusions
Calu-3 is an immortalized human airway epithelial cell line which has been shown to allow multicycle replication of human influenza viruses in the absence of exogenous trypsin (Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 2007 November; 81(22):12439-49). In addition, Calu-3 cells have been shown to express both TMPRSS2 and TMPRSS4, but not TMPRSS11D (HAT) at least at the mRNA level (Böttcher-Friebertshäuser et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011)). It has been previously shown that Calu-3 cells supported the proteolytic activation of influenza virus—but inhibition of TMPRSS2 using the TMPRSS2-specific monoclonal antibody, H1H7017N was tested herein. The growth of A/Puerto Rico/08/1934 (H1N1) over 72 h after treating the cells with 166.7 nM of H1H7017N was analyzed. Viral titers were determined by counting fluorescent focus spots. Table 4 and FIG. 2 show decreased titers after treatment with antibody H1H7017N. Although Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at the mRNA level, knockdown of TMPRSS2 reduced influenza virus titers by 100- to 1,000-fold (Böttcher-Friebertshäuser et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011)). The low level of existing viral titers in the absence of mAb were probably due to the addition of cleaved virus (harvested from embryonated chicken eggs or from MDCK culture with trypsin), but the presence of another HA-activating protease could also account for the presence of virus despite treatment with anti-TMPRSS2 mAb.
TABLE 4
Application of H1H7017N during the infection cycle decreases
the number of Fluorescent Focus Units (FFU) of A/Puerto
Rico/08/1934 (H1N1) at 72 hours post-infection.
Treatment
Description
FFU
H1H7017N
Anti-TMPRSS2 mAb
259
H1H11729P
Anti-influenza A group
18
1 positive control
anti-hIgG4 with
IgG1 isotype control
2338
a mouse IgG2a
Fc
No mAb
Infection control
2656
Uninfected
Background control
6
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), May; 88(9):4744-51.doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 2006 October; 80(19):9896-8. PMID: 16973594.
10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 3: FACS Analysis with MDCK/Tet-on, MDCK/Tet-on/hTMPRSS2, and MDCK/Tet-on/MfTMPRSS2 Cells
The ability of anti-TMPRSS2 antibody, H1H7017N, to bind to MDCK cells expressing TMPRSS2 or not expressing TMPRSS2 was assessed.
TABLE 5
Reagents used.
Reagent
Source
MDCK
ATCC
pLVX-EF1α-Tet3G
Clontech
pLVX Tight hTMPRSS2 Puro
pLVX Tight MfTMPRSS2 Puro
DMEM
Irvine Scientific
FBS
Seradigm
Pen/strep/glut
Invitrogen
Sodium Pyruvate 100 mM (100X)
Specialty Media
Geneticin ™ Selective
Invitrogen
Antibiotic (G418 Sulfate)
Puromycin
Sigma
Doxycycline
Sigma
PBS without Ca++/Mg++
Irvine Scientific
Accutase
Millipore
96-well filter plates
Pall
BD CytoFix ™
Becton Dickinson
Allophycocyanin (APC)
Jackson Immuno
AffiniPure F(ab′)2 Fragment Goat
Anti-Human IgG, Fcγ Fragment
Specific
Control mAb1 (hIgG1 isotype
control)
Cytoflex
Beckman Coulter
FlowJo 10.1r5
FlowJo
Prism 7
Graphpad
Experimental Procedure
Cell lines were developed to express human and cynomolgous monkey TMPRSS2 (hTMPRSS2 and mfTMPRSS2) in MDCK (Madin Darby Canine Kidney) cells upon induction with doxycycline. MDCK cells were transduced to stably express a modified tetracycline-controlled transactivator protein (Clontech) and the resulting cell line was termed MDCK/Tet-on cell line. MDCK/Tet-on cell line was transduced with a construct containing hTMPRSS2 (NP_005647.3 with a V160M) or mfTMPRSS2 (Ref seq XP_015302311.1 with S129L, N251S, I415V, R431Q, D492G) under the control of inducible promoter and the cell lines were termed MDCK/Tet-on/hTMPRSS2 and MDCK/Tet-on/mfTMPRSS2. The stable cell lines were maintained in growth media containing DMEM supplemented with 10% FBS, sodium pyruvate, penicillin/streptomycin/glutamine, 500 □g/mL G418 with or without 2 □g/mL puromycin.
For cell binding analysis by flow cytometry, cells were plated in growth media and incubated with doxycycline at 1 □g/mL for 16 hours to induce expression of TMPRSS2. Cells are detached using Accutase and resuspended in 1% FBS in PBS. Antibodies were serially diluted from 500 nM to 25 pM and each concentration of antibody was incubated with 1×106 cells at 4° C. for 30 minutes. A condition was included where no antibody was added to the cells. After incubation with primary antibodies, the cells were stained with allophycocyanin conjugated anti-human IgG secondary antibody at 1:1000 at 4° C. for 30 minutes. Cells were fixed using BD CytoFix™ and analyzed using CytoFLEX flow cytometer. Unstained and secondary antibody alone controls were also included for all cell lines. Geometric mean values of fluorescence for viable cells were determined using FlowJo software and the results were analyzed using nonlinear regression (4-parameter logistics) with Prism 7 software (GraphPad) to obtain EC50 values of cell binding by the antibodies.
As shown in FIG. 3, the anti-hTMPRSS2 antibody of the invention, H1H7017N, bound to MDCK/Tet-on/hTMPRSS2 and MDCK/Tet-on/mfTMPRSS2 with EC50 values of 460 pM and 1.06 nM respectively. H1H7017N did not show significant binding to MDCK/Tet-on cells. Control mAb1, an irrelevant isotype control antibody, did not show binding to any of the cell lines tested.
Example 4: Biacore Binding Kinetics of Anti-TMPRSS2 Monoclonal Antibodies Binding to Different TMPRSS2 Reagents Measured at 25° C. and 37° C.
Equilibrium dissociation constant (KD) for different TMPRSS2 reagents binding to purified anti-TMPRSS2 monoclonal antibodies were determined using a real-time surface plasmon resonance based Biacore 4000 biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25° C. and 37° C. The Biacore CMS sensor chip surface was first derivatized by amine coupling with the rabbit anti-mouse Fc specific polyclonal antibody (GE Healthcare Cat #BR100838) to capture anti-TMPRSS2 monoclonal antibodies. Binding studies were performed on human TMPRSS2 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hTMPRSS2.mmh), and monkey TMPRSS2 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfTMPRSS2.mmh). Different concentrations of HMM-hTMPRSS2 and HMM-mfTMPRSS2 (100 nM-6.25 nM; 4-fold serial dilution) were first prepared in HBS-ET running buffer and were injected over anti-mouse Fc captured anti-TMPRSS2 monoclonal antibody surface for 2.5 minutes at a flow rate of 30 μL/minute, while the dissociation of monoclonal antibody bound TMPRSS2 reagent was monitored for 7 minutes in HBS-ET running buffer. The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constant (KD) and dissociative half-life (t½) were calculated from the kinetic rates as:
K
D
=
(
M
)
=
kd
ka
,
and
t
1
/
2
(
min
)
=
ln
(
2
)
60
*
kd
Binding kinetics parameters for HMM-hTMPRSS2 or HMM-mfTMPRSS2 binding to different anti-TMPRSS2 monoclonal antibodies of the invention at 25° C. and 37° C. are shown in Tables 6 through 9.
At 25° C., anti-TMPRSS2 monoclonal antibodies bound to HMM-hTMPRSS2 with KD value 2.81 nM, as shown in Table 6. At 37° C., anti-HMM-hTMPRSS2 monoclonal antibodies bound to HMM-hTMPRSS2 with KD value 9.31 nM, as shown in Table 7.
At 25° C., anti-TMPRSS2 monoclonal antibodies bound to HMM-mfTMPRSS2 with KD value 56.0 nM, as shown in Table 8. At 37° C., anti-TMPRSS2 monoclonal antibodies bound to HMM-mfTMPRSS2 with KD value 140 nM, as shown in Table 9.
TMPRSS2 Proteins
hTMPRSS2 knob_mmh (W106-R255).mmh:
amino acids 1-150: amino acids 106 through 255 of human TMPRSS2 (accession number NP_005647.3 with a V160M)
Amino acids: 151-178: myc-myc-hexahistidine tag
(SEQ ID NO: 20;
WKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYG
PNFILQMYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVD
DSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSS
RQSREQKLISEEDLGGEQKLISEEDLHHHHHH
myc tags underscored, His6 tag doubly underscored)
mfTMPRSS2 knob_mmh (W106-R255).mmh:
Amino acids 1-150: amino acids 106-255 of monkey TMPRSS2 (accession number XP_005548700.1 with S129L, N251S)
Amino acids 151-178: myc-myc-hexahistidine tag
(SEQ ID NO: 21;
WKFMGSKCSDSGIECDSSGTCISLSNWCDGVSHCPNGEDENRCVRLYGPN
FILQVYSSQRKSWHPVCRDDWNENYARAACRDMGYKNSFYSSQGIVDNSG
ATSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVRSNLSRQSR
EQKLISEEDLGGEQKLISEEDLHHHHHH
myc tags underscored, His6 tag doubly underscored)
Results
TABLE 6
Binding kinetics parameters of HMM-hTMPRSS2 binding
to TMPRSS2 monoclonal antibodies at 25° C.
mAb
100 nM
Capture
Ag
mAb
Level
Bound
ka
kd
KD
t½
Captured
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H2aM7017N
510 ± 5.3
103
2.65E+05
7.45E−
2.81E−
15.5
04
09
* H2aM7017N is an antibody with the H1H7017N variable domains set forth herein and a mouse IgG2a Fc.
TABLE 7
Binding kinetics parameters of HMM-hTMPRSS2 binding to
TMPRSS2 monoclonal antibodies at 37° C.
mAb
100 nM
Capture
Ag
mAb
Level
Bound
ka
kd
KD
t½
Captured
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H2aM7017N
587 ± 4.5
117
3.47E+05
3.23E−
9.31E−
3.6
03
09
TABLE 8
Binding kinetics parameters of HMM-mfTMPRSS2 binding to
TMPRSS2 monoclonal antibodies at 25° C.
mAb
100 nM
Capture
Ag
mAb
Level
Bound
ka
kd
KD
t½
Captured
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H2aM7017N
484 ± 1.8
67
2.80E+05
1.57E−
5.60E−
0.7
02
08
TABLE 9
Binding kinetics parameters of HMM-mfTMPRSS2 binding to
MSR1 monoclonal antibodies at 37° C.
mAb
100 nM
Capture
Ag
mAb
Level
Bound
ka
kd
KD
t½
Captured
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H2aM7017N
569 ± 1.6
48
3.66E+05
5.12E−
1.40E−
0.2
02
07
Example 5: In Vitro Influenza Spread of Influenza H1, H3, and FluB Strains
In this example, the ability of various types of influenza to spread across an in vitro culture of Calu-3 cells and the effect of anti-TMPRSS2 antibodies on this spread was determined.
TABLE 10
Reagents used and lot numbers.
Cat#
Description
Vendor
HTB55
Calu-3 cells
ATCC (American Type
Culture Collection)
11995-073
DMEM
Gibco
211703
F12
Gibco
15140-122
Pen/Strep
Gibco
A033650ML
Low IgG BSA
Sigma
10010-023
PBS
Life Technologies
26140079
Fetal Bovine Serum
Life Technologies
VR-1469
Influenza A A/Puerto
ATCC
Rico/08/1934 (H1_PR34)
NR-13658
H1N1 A/CaliforniaA/04/2009
BEI Resources
(H1_CA09)
FR-28
Influenza A/Brisbane/59/2007
Influenza Reagent
(H1_Bris)
Resource
FR-1068
Influenza A/Hong
Influenza Reagent
Kong/38982/2009 (H9N2)
Resource
3483
Influenza A H3N2 Kilbourne
BEI Resources
F108 A/Aichi/2/68 (HA, NA) ×
A/PR/8/34, Re-assorted X-31
NR-41795
Influenza B/Florida/04/2006
ATCC
(Florida)
NR-12280
Influenza B Malaysia (Malaysia)
ATCC
MAB8251
Anti-Influenza A Antibody,
Millipore
nucleoprotein, clones A1, A3
Blend
Ab20711
Anti-Influenza B Virus
Abcam
Nucleoprotein antibody [B017]
A-11001
Goat anti-Mouse IgG (H + L)
ThermoFisher Scientific
Cross-Absorbed Secondary
Antibody, Alexa Fluor 488
Experimental Procedure
Calu-3 cells were seeded at 40,000 cells/well in a 96-well plate in DMEM:F12 medium with 5% FBS. The next day, influenza virus strains were diluted to a previously determined MOI (see Table 11) and antibodies were diluted to 100 μg/mL. In these experiments, the anti-HA and anti-TMPRSS2 antibodies had different mechanisms of action, therefore, the experimental procedure was different for these antibodies in order to appropriately test them. The anti-HA antibodies were pre-incubated with an individual influenza virus strain for one hour at 37° C. in a separate plate. After the preincubation period, the antibody/virus mixture was added to Calu-3 cells for one hour. The anti-TMPRSS2 antibody was preincubated with uninfected Calu-3 cells for three hours at 37° C. After the preincubation period, virus was added to the Calu-3 cells pre-incubated with anti-TMPRSS2 antibodies for one hour. After the hour-long infection, the cells were washed three times with PBS and fresh antibody was, added along with new medium, to each well. Additional antibody was added at 24 and 48 hours post-infection. At 72 hours post-infection, the cells were stained with an anti-NP and quantified on a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, Ohio).
TABLE 11
Influenza
Strain
Final MOI
A. Experiment 1.
H1_PR34
0.001
H1_CA09
0.001
H1_Bris
0.001
H9N2
0.01
H3N2
0.001
B. Experiment 2.
H1_PR34
0.01
Florida
0.01
Malaysia
0.001
Results Summary and Conclusions
Calu-3 is an immortalized human airway epithelial cell line which has been shown to allow multicycle replication of human influenza viruses in the absence of exogenous trypsin (Zeng et al., Journal of Virology 81: 12439-12449 (2007)). In addition, Calu-3 cells have been shown to express TMPRSS2 (Böttcher-Friebertshäuser et al., Journal of Virology 85: 1554-1562 (2011)) which is essential for these experiments as an anti-TMPRSS2 antibody is being tested. In these experiments, whether or not H1H7017N, an anti-TMPRSS2 antibody, can prevent the spread in different strains of influenza was examined. In addition, the corresponding anti-HA antibody for the different strains as a positive control was run. As expected, there was an initial infection in the presence of the anti-TMPRSS2 antibody but H1H7017N successfully prevented the spread of infection of H1_PR34, H1_CA09, H1_Bris, H9N2, and H3N2. This can be observed by examining the differences in the number of infected cells between the anti-TMPRSS2-treated cells and the infected controls (Table 12). It was concluded that the anti-TMPRSS2 antibody was not able to prevent spread in either of the influenza B strains because the number of infected cells in the control and treated wells were the same. In comparison, the anti-HA antibodies were pre-incubated with the virus and prevented the initial infection. This can also be seen by comparing the number of infected cells. Counting of the infected cells was performed on the CTL machine and are reported in the tables below.
TABLE 12
Infected cells
treated with
Infected cells
H1H7017N
treated with
anti-
Influenza
Uninfected
Infected
group specific
TMPRSS2
Strain
Control
Control
HA antibody
antibody
A. Experiment 1.
H1_PR34
10
3847.5
3
1496
H1_CA09
3.5
4645.4
1.5
17
H1_Bris
15.5
3882
0.5
1005
H9N2
4.5
4172
4.5
196.5
H3N2
7.5
3922
9
754.5
B. Experiment 2.
H1_PR34
1
2848
18
60
Florida
4
1339
229
1234
Malaysia
10
1184
758
1451
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 6: The Effect of Treatment with H1H7017N Alone in TMPRS22 Humanized Mice
The ability of anti-TMPRSS2 antibodies to protect mice engineered to express the human TMPRSS2 protein from infection with H1N1 influenza virus was assessed.
TABLE 13
Reagents used and lot numbers.
Cat#
Description
Vendor
VR-1469
Influenza A A/Puerto
ATCC
Rico/08/1934 (H1N1)
20012-043
PBS
Gibco
Ketamine:Xylazine
TABLE 14
mAb Clone IDs.
AbPID
Description
H1H7017N
anti-TMPRSS2 mAb
H1H1238N
IgG1 isotype control
Experimental Procedure
These experiments were performed in 5-8 week-old male and female mice engineered to express the human TMPRSS2 protein. Mice were challenged with 150 plaque-forming units (PFUs) of H1N1. The mice were sedated with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitoneal injection and then infected with 24 μL of virus intranasally. Antibodies were delivered either subcutaneously (SC) one day before infection or intravenously (IV) on various days post infection (PI). The antibody dosing schedule varied between experiments (Table 15). Body weights were collected daily up to day 14 PI and mice were sacrificed when they lost 20% of their starting body weight. Results are reported as percent survival.
TABLE 15
Antibody
Days PI
Dose
Delivery
A. Antibody Dosing (Experiment 1).
H1H1238N
−1
5
mg/kg
SC
H1H7017N
−1, 0
5
mg/kg
SC, IV
B. Antibody Dosing (Experiment 2).
H1H1238N
0
10
mg/kg
IV
H1H7017N
0, 1, 2, 3
10
mg/kg
IV
Results Summary and Conclusions
It has been shown that mice engineered to express the human TMPRSS2 protein can be infected with a lethal dose of influenza. The aim of these experiments was to demonstrate that H1H7017N can protect mice engineered to express the human TMPRSS2 protein against influenza A group 1. The antibody was tested in prophylactic and therapeutic models. Treatment with H1H7017N resulted in higher survival than the isotype control (H1H1238N) treated mice in both experiments (FIGS. 4 and 5). In the prophylactic experiment, the survival was 0% for mice treated with H1H1238N, 85.7% for mice treated on day −1 PI, and 100% for mice treated on day 0 PI with H1H7017N. For the therapeutic model, the H1H1238N-treated group resulted in 25% survival while the groups treated with H1H7017N on day 0-3 PI resulted in 100% survival. Data are summarized in Table 16. H1H7017N shows efficacy in mice engineered to express the human TMPRSS2 protein.
TABLE 16
Percent survival
Number
(no. of surviving
of mice
mice/total no. of
Group ID
per group
mice in the group)
A. Tabulated Data Summary (Experiment 1).
H1H1238N, Day −1 PI, SC
4
0
(0/4)
H1H7017N, Day −1 PI, SC
7
85.7
(6/7)
H1H7017N, Day 0 PI, IV
6
100
(6/6)
B. Tabulated Data Summary (Experiment 2).
H1H1238N, Day 0 PI, IV
4
25
(1/4)
H1H7017N, Day 0 PI, IV
5
100
(5/5)
H1H7017N, Day 1 PI, IV
5
100
(5/5)
H1H7017N, Day 2 PI, IV
5
100
(5/5)
H1H7017N, Day 3 PI, IV
5
100
(5/5)
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 7: Anti-TMPRSS2 mAb, H1H7017N, Activity in TMPRSS2 Humanized Mouse Model
The ability of anti-TMPRSS2 antibodies to protect a mouse engineered to express the human TMPRSS2 protein from infection with H3N2 influenza virus was assessed.
TABLE 17
mAb Clone IDs.
AbPID
Description
H1H7017N
Anti-TMPRSS2 antibody
TABLE 18
Reagents used and lot numbers.
Cat#
Description
Vendor
3483
Influenza A H3N2 Kilbourne F108
BEI Resources
A/Aichi/2/68 (HA, NA) × A/PR/8/34,
Reassorted X-31
20012-043
PBS
Gibco
Ketamine:Xylazine
Experimental Procedure
Eleven week-old male and female mice engineered to express the human TMPRSS2 protein were challenged with 20,000 plaque-forming units (PFUs) of H3N2. The mice were sedated with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitoneal injection and then infected with 20 μL of virus intranasally. On day 1 or day 2 post-infection (PI), mice were intravenously injected with antibody. Mice were weighed and observed daily up to day 14 post-infection (PI). They were sacrificed when they lost 25% of their starting body weight.
Results Summary and Conclusions
Breadth is an important quality when considering an influenza therapy. It has already been demonstrated that anti-TMPRSS2 antibody H1H7017N was efficacious against influenza A group 1. The aim of this experiment was to demonstrate that H1H7017N can protect mice engineered to express the human TMPRSS2 protein against influenza A group 2. Mice engineered to express the human TMPRSS2 protein were infected with a lethal dose of H3N2 and treated on day 1 or day 2 PI. Both treatment groups had higher survival rates than the infected control. Mice treated on day 1 PI had a survival rate of 100% which was higher than the group treated on day 2 PI which had a 50% survival, while untreated mice had 0% survival. All mice died between days 5-6 PI. The survival graph is shown in FIG. 6 and % survival is summarized in Table 19. These results demonstrated that H1H7017 improved outcomes in an H3N2-lethal model.
TABLE 19
Tabulated Data Summary.
Percent survival
Number
(no. of surviving
of mice
mice/total no. of
Group ID
per group
mice in the group)
Untreated
5
0
(0/5)
H1H7017N, Day 1 PI
5
100
(5/5)
H1H7017N, Day 0 PI, IV
4
50
(2/4)
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 8: Infection of Mice Engineered to Express the Human TMPRSS2 Protein (Versus WT)
The survival of mice engineered to express the human TMPRSS2 protein infected with H1N1 influenza virus was assessed and compared with that of wild-type (WT) mice.
TABLE 20
Reagents used and lot numbers.
Cat#
Description
Vendor
VR-1469
Influenza A A/Puerto
ATCC
Rico/08/1934 (H1N1)
20012-043
PBS
Gibco
Ketamine:Xylazine
Experimental Procedure
The experiment was performed in 7.5-8 week-old male and female mice engineered to express the human TMPRSS2 protein or wild-type littermates. Mice were challenged with 150, 750, or 1,500 plaque-forming units (PFUs) of A/Puerto Rico/08/1934 (H1N1). The mice were sedated with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitoneal injection and then infected with 20 μL of virus intranasally. Body weights were collected daily up to day 14 PI and mice were sacrificed when they lost 20% of their starting body weight. Results are reported as percent survival (FIG. 7).
Results Summary and Conclusions
Mice engineered to express the human TMPRSS2 protein were generated in order to test the therapeutic efficacy of the anti-TMPRSS2 antibodies in an influenza in vivo model. In this experiment, the survival rates of mice engineered to express the human TMPRSS2 protein and wild-type mice infected with 150, 750 or 1,500 PFUs of a historical strain of H1N1 was compared. There was 0% survival for mice engineered to express the human TMPRSS2 protein and wild-type mice in all three infection groups. All mice died between day 5 and day 8 PI, with those receiving a higher virus dose dying sooner than those who received a lower virus dose. The survival patterns of mice engineered to express the human TMPRSS2 protein were similar to the wild-type mice. This shows that mice engineered to express the human TMPRSS2 protein can be used as an influenza in vivo model to assess the effectiveness of TMPRSS2-specific antibodies. See Table 21.
TABLE 21
Tabulated Data Summary.
Percent survival
Number
(no. of surviving
of mice
mice/total no. of
Group ID
per group
mice in the group)
Wild-type; 150 PFUs H1N1
4
0 (0/4)
Wild-type; 750 PFUs H1N1
4
0 (0/4)
Wild-type; 1,500 PFUs H1N1
3
0 (0/3)
Mice engineered to express the human
4
0 (0/4)
TMPRSS2 protein; 150 PFUs H1N1
Mice engineered to express the human
3
0 (0/3)
TMPRSS2 protein; 750 PFUs H1N1
Mice engineered to express the human
3
0 (0/3)
TMPRSS2 protein; 1,500 PFUs H1N1
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 9: The Effect of Treatment with the Combination of H1H14611N2 and H1H7017N in Mice after Infection with H3N2
The ability of a combination of anti-TMPRSS2 and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from infection with H3N2 influenza virus was assessed.
TABLE 22
mAb Clone IDs.
AbPID
Description
H1H7017N
Anti-TMPRSS2 antibody
H1H14611N2
Anti-influenza A group 2
antibody
H1H1238N
IgG1 isotype control
TABLE 23
Reagents used and lot numbers.
Description
Vendor
Influenza A H3N2 Kilbourne
BEI Resources
F108 A/Aichi/2/68 (HA, NA) ×
A/PR/8/34, Reassorted X-31
PBS
Gibco
Ketamine:Xylazine
Experimental Procedure
Eight week-old male and female mice engineered to express the human TMPRSS2 protein were challenged with 20,000 plaque-forming units (PFUs) of A/Aichi/2/68 (HA, NA)×A/PR/8/34, Re*assorted X-31 (H3N2). The mice were sedated with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) via intraperitoneal injection and then infected with 20 μL of virus intranasally. On day 4 post-infection (PI), mice were intravenously injected with antibody. Body weights were collected daily up to day 14 PI and mice were sacrificed when they lost 25% of their starting body weight. Results are reported as percent survival.
Results Summary and Conclusions
It has been shown that, individually, the TMPRSS2 antibody, H1H7017N, and the broad influenza A group 2 antibody, H1H14611N2, have therapeutic efficacy against a lethal mouse challenge with a historical strain of H3N2. It has also been shown that survival of mice infected with a lethal H1N1 challenge can be significantly increased after treatment with less total antibody than either alone through the combination of H1H7017N and the broad influenza A group 1 antibody, H1H11729P. The aim for this experiment was to evaluate the synergistic effect of H1H7017N and H1H14611N2 in combination. As shown in FIG. 8, 3 of 4 mice treated with the hIgG1 isotype control antibody at day 4 PI died by day 7 PI. 3 of 5 animals survived when dosed with 10 mg/kg of H1H14611N2 and 4 of 5 animals survived when dosed with 10 mg/kg of H1H7017N. When dosed in a combination of 5 mg/kg of each antibody, H1H14611N2 and H1H7017N, there was 40% survival. One hundred percent of mice treated with the combination of 2.5 mg/kg of each antibody, H1H14611N2 and H1H7017N, survived the challenge. Survival of mice infected with a lethal H3N2 challenge was increased through the combination of lower concentrations of H1H7017N and H1H14611N2 compared to higher concentrations of combined antibodies or either antibody alone. Percent survival is summarized in Table 24.
TABLE 24
Tabulated Data Summary.
Percent survival
Number
(no. of surviving
of mice
mice/total no. of
Group ID
per group
mice in the group)
10 mg/kg hIgG1 isotype control
5
20
(1/5)
10 mg/kg H1H14611N2
5
60
(3/5)
10 mg/kg H1H7017N
5
80
(4/5)
5 mg/kg H1H7017N + 5 mg/kg
5
40
(2/5)
H1H14611N2
2.5 mg/kg H1H7017N + 2.5 mg/kg
5
100
(5/5)
H1H14611N2
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
Example 10: The Effect of Treatment with the Combination of H1H11729P and H1H7017N in Mice after Infection with H1N1
The ability of a combination of anti-TMPRSS2 and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from infection with H1N1 influenza virus was assessed.
TABLE 25
mAb Clone IDs.
AbPID
Description
H1H7017N
Anti-TMPRSS2 antibody
H1H11729P
Anti-influenza A group 1
antibody
H1H1238N
IgG1 isotype control
TABLE 26
Reagents used and lot numbers.
Cat#
Description
Vendor
VR-1469
Influenza A A/Puerto
ATCC
Rico/08/1934 (H1N1)
20012-043
PBS
Gibco
Ketamine:Xylazine
Experimental Procedure
Five week-old male and female mice engineered to express the human TMPRSS2 protein were challenged with 1,500 plaque-forming units (PFUs) of H1N1. The virus was delivered by sedating the mice with 200 μL of Ketamine:Xylazine (12 mg/ml:0.5 mg/ml) and delivering 20 μL of virus intranasally. On day 3 post-infection (PI), mice were intravenously injected with antibody. Body weights were collected daily up to day 14 PI and mice were sacrificed when they lost 25% of their starting body weight.
Results Summary and Conclusions
It has been shown that, individually, the TMPRSS2 antibody, H1H7017N, and the broad influenza A group 1 antibody, H1H11729P, have therapeutic efficacy against a lethal mouse challenge with a historical strain of H1N1. However, the aim of this experiment was to evaluate the synergistic effect of the antibodies in combination. All mice treated with hIgG1 isotype control antibody at day 3 PI died by day 6 PI. When animals received 5 mg/kg of H1H11729P or H1H7017N, 40% and 0% of animals survived the infection, respectively. However, the combination of 2.5 mg/kg of each antibody, H1H11729P and H1H7017N, resulted in 60% survival. Eighty percent of mice treated with the combination of 1 mg/kg of H1H7017N and 2 mg/kg of H1H11729P (3 mg/kg total) survived the challenge. Survival of mice infected with a lethal H1N1 challenge was significantly increased after treatment with less total antibody than either alone through the combination H1H7017N and H1H11729P (See FIG. 9 and Table 27).
TABLE 27
Tabulated Data Summary.
Percent survival
Number
(no. of surviving
of mice
mice/total no. of
Group ID
per group
mice in the group)
5 mg/kg hIgG1 isotype control
3
0
(0/3)
5 mg/kg H1H11729P
5
40
(2/5)
5 mg/kg H1H7017N
5
0
(0/5)
2.5 mg/kg H1H7017N + 2.5 mg/kg
5
60
(3/5)
H1H11729P
1 mg/kg H1H7017N + 2 mg/kg
5
80
(4/5)
H1H11729P
REFERENCES
1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. Journal of Virology. 91, e01387-16 (2017). PMID: 27733646.
2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671.
3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761.
4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044.
5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS ONE. 12, e0176597 (2017). PMID: 28493964.
6. E. Böttcher-Friebertshäuser, D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554-1562 (2011). PMID: 21123387.
7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology. 84, 10016-10025 (2010). PMID: 20631123.
8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. Journal of Virology (2014), doi:10.1128/JVI.03799-13. PMID: 24522916.
9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium. Journal of Virology. 80, 9896-9898 (2006). PMID: 16973594.
10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci USA. 2010 Jun. 22; 107(25):11531-6. doi: 10.1073/pnas.0914994107. Epub 2010 Jun. 7. PMID: 20534532.
11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549.
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent identified even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
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16256560
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Feb 2nd, 2021 12:00AM
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Dec 4th, 2019 12:00AM
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https://www.uspto.gov?id=US10905786-20210202
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Sterilisation method
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Embodiments of the present disclosure relate to systems and methods for the application of vaporized chemicals in the sterilization of medical products. For example, embodiments of the present disclosure may relate to systems and methods for the terminal sterilization of medical products using vaporized hydrogen peroxide (VHP). Embodiments of the present disclosure may relate to, e.g., systems and methods for the terminal sterilization of medical products, such as pre-filled syringes (PFS).
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10905786
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1. A sterilization method comprising:
creating a turbulent flow within a chamber; and
while maintaining the turbulent flow, performing a sterilization pulse comprising:
maintaining a sterilization pressure within the chamber for at least 5 minutes;
introducing vaporized hydrogen peroxide (VHP) into the chamber;
allowing the VHP to circulate within the chamber for at least 5 minutes, wherein allowing the VHP to circulate within the chamber includes removing the VHP from a lower interior of the chamber and re-introducing the VHP into an upper interior of the chamber; and
introducing dry gas into the chamber, wherein the dry gas has a dew point of −10° C. or lower
wherein, during the sterilization pulse, a condensation layer forms on a primary packaging component.
2. The method of claim 1, wherein the sterilization pressure is between about 400 millibars and about 800 millibars.
3. The method of claim 1, further comprising performing between 2 and 5 sterilization pulses.
4. The method of claim 1, wherein the dry gas includes nitrogen.
5. The method of claim 1, wherein the turbulent flow is maintained using a blower external to the chamber.
6. The method of claim 1, further comprising performing at least 2 sterilization pulses, and wherein each sterilization pulse further comprises creating the sterilization pressure within the chamber prior to introducing VHP into the chamber.
7. The method of claim 1, further comprising positioning a primary packaging component within the chamber, wherein the primary packaging component is configured for receiving a formulated drug substance including an antibody.
8. The method of claim 1, wherein the dry gas is a first dry gas having a dew point of −10° C. or lower, and further comprising performing an aeration pulse comprising:
achieving a first aeration pressure of between about 400 millibars and about 800 millibars in the chamber;
increasing the pressure within the chamber to a second aeration pressure higher than the first aeration pressure; and
simultaneously exhausting the gas from the chamber while introducing room air, air having a dew point of −10° C. or lower, or a second dry gas having a dew point of −10° C. or lower, into the chamber.
9. The method of claim 8, further comprising halting the turbulent flow in the chamber prior to performing the aeration pulse.
10. The method of claim 8, further comprising performing between 2 and 35 aeration pulses.
11. The method of claim 8, wherein the steps of (a) introducing room air, air having a dew point of −10° C. or lower, or a second dry gas having a dew point of −10° C. or lower, into the chamber and (b) achieving the first aeration pressure, are performed simultaneously.
12. The method of claim 8, wherein the second aeration pressure is between about 550 millibars and about 1100 millibars.
13. The method of claim 1, further comprising maintaining a temperature of between about 25° C. and about 60° C. within the chamber while performing the sterilization pulse.
14. The method of claim 1, wherein introducing VHP into the chamber comprises introducing between about 50 g and about 700 g of VHP into the chamber.
15. A sterilization method comprising:
creating a turbulent flow within a chamber;
while maintaining the turbulent flow, performing a sterilization pulse comprising:
maintaining a sterilization pressure within the chamber of between about 400 millibars and about 800 millibars for at least 5 minutes;
introducing vaporized hydrogen peroxide (VHP) into the chamber;
allowing the VHP to circulate within the chamber for at least 5 minutes, wherein allowing the VHP to circulate within the chamber includes removing the VHP from a lower interior of the chamber and re-introducing the VHP into an upper interior of the chamber; and
introducing a first gas into the chamber;
halting the turbulent flow within the chamber; and
performing an aeration pulse comprising:
introducing a second gas into the chamber;
maintaining a first aeration pressure in the chamber for at least 5 minutes;
increasing the pressure within the chamber to a second aeration pressure higher than the first aeration pressure; and
exhausting the second gas from the chamber
wherein, during the sterilization pulse, condensation comprising hydrogen peroxide forms in the chamber.
16. The method of claim 15, wherein the first aeration pressure is between about 400 millibars and about 800 millibars, and wherein the second aeration pressure is between about 550 millibars and about 1100 millibars.
17. The method of claim 15, wherein the first gas includes one of nitrogen or a gas having a dew point of −10° C. or lower, and wherein the second gas includes one of nitrogen, a gas having a dew point of −10° C. or lower, or air.
18. The method of claim 15, further comprising performing a drying pulse after performing the aeration pulse, wherein the drying pulse comprises:
introducing a third gas into the chamber;
maintaining a first drying pressure in the chamber for at least 1 minute; and
maintaining a second drying pressure in the chamber for at least 1 minute.
19. The method of claim 18, wherein the second gas is air, and the third gas is one of nitrogen or a gas having a dew point of −10° C. or lower.
20. The method of claim 18, wherein at least 99% of the VHP introduced into the chamber during the sterilization pulse is removed from the chamber by the conclusion of the drying pulse.
21. The method of claim 15, wherein introducing VHP into the chamber includes introducing between about 50 g and about 700 g of VHP into the chamber.
22. A sterilization method comprising:
creating a turbulent flow within a chamber;
while maintaining the turbulent flow, performing a plurality of sterilization pulses, wherein each sterilization pulse includes:
maintaining a sterilization pressure within the chamber of between about 400 millibars and about 800 millibars for at least 5 minutes;
introducing vaporized hydrogen peroxide (VHP) into the chamber;
allowing the VHP to circulate within the chamber for at least 5 minutes, wherein allowing the VHP to circulate within the chamber includes removing the VHP from a lower interior of the chamber and re-introducing the VHP into an upper interior of the chamber; and
introducing one of nitrogen or a gas having a dew point of −10° C. or lower into the chamber;
performing a plurality of aeration pulses after performing the plurality of sterilization pulses, wherein each aeration pulse includes:
introducing one of air, nitrogen, or a gas having a dew point of −10° C. or lower into the chamber;
maintaining a first aeration pressure of between about 400 and about 800 millibars in the chamber for at least 5 minutes;
increasing the pressure within the chamber to a second aeration pressure of between about 550 and 1100 millibars; and
exhausting the second gas from the chamber; and
performing a plurality of drying pulses after performing the plurality of aeration pulses, wherein each of the plurality of drying pulses includes:
introducing one of nitrogen or a gas having a dew point of −10° C. or lower into the chamber;
maintaining a first drying pressure of between about 500 and about 850 millibars in the chamber for at least 1 minute; and
maintaining a second drying pressure higher than the first drying pressure in the chamber for at least 1 minute
wherein, during one of the sterilization pulses, VHP condenses on a primary packaging component within the chamber.
23. The method of claim 22, wherein the plurality of sterilization pulses includes between 2 and 5 pulses.
24. The method of claim 22, wherein each drying pulse includes introducing a gas having a dew point of 10° C. or lower into the chamber.
25. The method of claim 22, wherein creating the turbulent flow in the chamber comprises activating a blower external to the chamber.
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25
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 16/498,080, filed Sep. 26, 2019, which is a U.S. national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2018/021013, filed Mar. 6, 2018, which claims priority to U.S. Application No. 62/477,030, filed Mar. 27, 2017, and U.S. Application No. 62/568,850, filed Oct. 6, 2017. All of which are incorporated by reference herein in their entireties.
FIELD OF THE DISCLOSURE
Various embodiments of the present disclosure relate to systems and methods for sterilization of medical products. More specifically, particular embodiments of the present disclosure relate to systems and methods for moist chemical sterilization of medical products, including terminal sterilization of pre-filled syringes (or other pre-filled drug delivery devices) using vaporized chemicals, such as vaporized hydrogen peroxide.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments. The drawings show different aspects of the present disclosure and, where appropriate, reference numerals illustrating like structures, components, materials and/or elements in different figures are labeled similarly. It is understood that various combinations of the structures, components, and/or elements, other than those specifically shown, are contemplated and are within the scope of the present disclosure.
There are many inventions described and illustrated herein. The described inventions are neither limited to any single aspect nor embodiment thereof, nor to any combinations and/or permutations of such aspects and/or embodiments. Moreover, each of the aspects of the described inventions, and/or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the described inventions and/or embodiments thereof. For the sake of brevity, certain permutations and combinations are not discussed and/or illustrated separately herein. Notably, an embodiment or implementation described herein as “exemplary” is not to be construed as preferred or advantageous, for example, over other embodiments or implementations; rather, it is intended reflect or indicate the embodiment(s) is/are “example” embodiment(s).
FIG. 1 is a schematic drawing of an exemplary sterilization system that may be used for sterilization of medical products.
FIG. 2 is a flow diagram of steps in an exemplary method of sterilizing medical products using vaporized chemicals.
FIGS. 3A-3C are additional flow diagrams of steps in an exemplary method of sterilizing medical products using vaporized chemicals.
FIGS. 4A-4C are schematic drawings of an exemplary sterilization system at various stages in an exemplary method of sterilizing medical products using vaporized chemicals.
DETAILED DESCRIPTION
As used herein, the terms “comprises,” “comprising,” “include,” “have,” “with,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements need not include only those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. The term “exemplary” is used in the sense of “example,” rather than “ideal.” Any implementation described herein as exemplary is not to be construed as preferred or advantageous over other implementations. Further, the terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. Similarly, terms of relative orientation, such as “front side, “top side,” “back side,” “bottom side,” “upper,” “lower,” etc. are referenced relative to the described figures.
As used herein, the terms “about” and “approximately” are meant to account for possible variation of ±10% in a stated numeric value. All measurements reported herein are understood to be modified by the term “about,” or the term “approximately,” whether or not those terms are explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Moreover, in the claims, values, limits, and/or ranges means the value, limit, and/or range+/−10%.
As used in the present disclosure, the term “sterilization” refers to achieving a level of sterility appropriate for a formulated drug substance or drug product for commercial distribution and use. Such a level of sterility may be defined in, for example, regulatory guidelines or regulations, such as guidelines released by the U.S. Food and Drug Administration. In some embodiments, such a level of sterility may include, for example, a 6-log reduction in microbial populations of biological indicators placed on an outside or inside surface of a drug product (e.g., an outside surface of a syringe or an inside surface of a blister pack). In other embodiments, such a level of sterility may include, for example, a 9-log or 12-log reduction in microbial populations of biological indicators. Sterilization refers to achieving such an appropriate level of sterility while also achieving a sufficiently low level of residual sterilizing chemicals (e.g., vaporized hydrogen peroxide, ethylene oxide, etc.) for commercial distribution and use. Such a low level of residual sterilizing chemical may also be defined in regulatory guidelines or regulations.
As used in the present disclosure, the term “terminal sterilization” refers to the sterilization of a drug product in a container or packaging, such as in a primary packaging component, or in both primary and secondary packaging components, suitable for commercial distribution and use.
As used in the present disclosure, the term “medical product” refers to a product for medical use on a living animal. The term “medical product” includes, for example, drug products, formulated drug substances, medical implants, medical instruments, or combinations thereof. For example, the term “medical product” may refer to a syringe containing a formulated drug substance, such as a parenteral or an ophthalmic syringe. Other exemplary medical products include, e.g., suppository applicators and medication, transdermal drug delivery devices, medical implants, needles, cannulas, medical instruments, and any other product requiring sterilization prior to an intended medical use.
As used in the present disclosure, the term “formulated drug substance” refers to a composition containing at least one active ingredient (e.g., a small molecule, a protein, a nucleic acid, or a gene therapy medicament) and an excipient, prepared for medical distribution and use. A formulated drug substance may include fillers, coloring agents, and other active or inactive ingredients.
As used in the present disclosure, the term “drug product” refers to a dosage form that contains a formulated drug substance, such as a finished dosage form for an active ingredient. A drug product may include packaging for commercial distribution or use, such as a bottle, vial, or syringe.
As used in the present disclosure, the term “vaporized chemical” refers to a chemical that has been converted into a substance that may be diffused or suspended in air. In some instances, a vaporized chemical may be a chemical that has been combined with water and then converted into a substance that may be diffused or suspended in air.
As used in the present disclosure, the term “fluid” refers to a liquid, semi-liquid, vapor, or gas including oxygen, hydrogen, nitrogen, or a combination thereof.
Embodiments of the present disclosure relate to systems and methods for the application of vaporized chemicals in the sterilization of medical products. For example, embodiments of the present disclosure may relate to systems and methods for the terminal sterilization of medical products using vaporized hydrogen peroxide (VHP). More particularly, embodiments of the present disclosure may relate to, e.g., systems and methods for the terminal sterilization of medical products, such as pre-filled syringes (PFS).
It is generally desired that exposure to sterilization cycles have no adverse impact and minimized risk of damage or alteration to products being sterilized. Medical products that undergo terminal sterilization, such as PFS, may thus require sterilization equipment, machinery, controls, cycle, and methods to conform to certain constraints and requirements in order to achieve appropriate sterilization and/or avoid damage to the medical products and/or devices, formulated drug substances, drug products, or other products. Such constraints and requirements may include, e.g.:
The medical products and/or surrounding packaging may be sensitive to deep vacuum pressures during the sterilization cycle. For example, PFS may include pre-positioned plungers susceptible to becoming dislodged when exposed to deep vacuum environments. Additionally, medical products may include fragile materials, such as glass, which may be affected by deep vacuum environments.
The medical products, compositions contained in medical products, and/or surrounding environment may be adversely affected by extreme temperatures during sterilization cycle. For example, products containing liquid formulations (e.g., liquid medicaments in PFS) may not be stable when heated to the higher temperatures to which they may be exposed during typical sterilization cycles. For example, medicaments in such liquid formulations may become denatured, deactivated, or otherwise altered when exposed to and/or heated to high temperatures.
Medical products may be densely packed; e.g., bulk packaged medical products may contain a large sum of fully assembled, packaged, and labeled medical products. In the case of terminal sterilization, sterilizing agents may need to traverse several layers of packaging materials, container materials, and/or labels.
In the case of some types of sterilization, such as terminal sterilization, sterilizing agents may need to traverse a semi-permeable membrane, either by heat or by mass, to sterilize the exterior of each medical product as well as the interior of packaging elements.
Packaging for medical products may resist penetration of sterilization materials, and/or may be sensitive to temperature and pressure changes caused by sterilization. For example, a syringe may be packaged in a plastic ‘blister’ configured to house the syringe and restrict it from movement. Such a blister may be only somewhat permeable to sterilization materials, and/or may be sensitive to changes in pressure.
Medical products may be sealed using temperature- or pressure-sensitive elements. For example, PFS may be sealed using a semi-permeable gas membrane ‘lidding.’
Chemical sterilization, including moist chemical sterilization, may provide advantages addressing some of the above-described characteristics of medical product sterilization. For example, sterilization using a combination of VHP and vaporized water may advantageously be performed at relatively low temperatures, negating the need to expose medical products to disruptive high temperatures. However, there is limited evidence demonstrating successful application of VHP sterilization technology for terminal sterilization (e.g., for terminal sterilization of PFS), due to, e.g., sterilization cycles achieving incomplete sterilization, sterilization cycles unable to operate within allowable temperature and/or pressure ranges for medical products, difficulties in removing toxic residual VHP from sterilized articles, and/or long sterilization times. Ethylene oxide (“EtO”) is a viable alternative to VHP, and is known to be an effective agent for sterilization of items sensitive to high temperatures and
pressures. However, EtO is more toxic to humans than VHP, and as such presents health and safety issues during and after its use in a sterilization system.
For at least the above reasons, it may be desirable to more successfully apply VHP in terminal sterilization of medical products. It may also be desirable to do so while achieving relative sterilization “cycle efficiency” (e.g., (1) a decrease in overall sterilization cycle time, and/or (2) a decrease in extremity of the temperature at which a sterilization cycle operates). There is potentially significant value associated with successful application of VHP in terminal sterilization (e.g., of PFS), as well as improving cycle efficiency while applying VHP in terminal sterilization of PFS. The potential value may be derived by minimizing risk to product, and to business, by allowing more overall throughput of medical products (e.g., PFS) per unit of time.
Several aspects of VHP sterilization may (positively or negatively) affect the safety, efficacy, efficiency, and other aspects of sterilization processes for medical products. For example:
Vaporized sterilizing chemicals, such as VHP, may be stored as aqueous liquid mixtures, may be vaporized in the presence of water, and/or may otherwise exist in environments with water vapor. Under some sterilization conditions, vaporized sterilizing chemicals may not behave as a dry and/or ideal gas. VHP, for example, may not fully dissociate from water vapor in a sterilization chamber; the VHP may instead behave as a binary mixture of VHP and water vapor.
During some or all of a sterilization cycle, chemical sterilant vapors and water vapors in a sterilization chamber may adsorb to and/or condense on surfaces having cooler temperatures than the environmental temperature in the sterilization chamber. For example, during vapor sterilization of PFS loads, “cold spots” created by aqueous, high heat capacity, liquid product in each PFS, may serve to attract vapor adsorption and promote surface condensation. Upon proximity to a surface, chemical sterilant vapors and water vapors may adsorb to the surfaces due to the chemical properties of the vapors themselves, the operating conditions inside the chamber during sterilization, and the cooler temperatures on the surfaces of the PFS load as compared to the rest of the chamber environment.
During some or all of a sterilization cycle, VHP may preferentially adsorb onto surfaces as compared to water vapor, due to the fact that hydrogen peroxide is more dense and less volatile than water. In some instances, VHP and water vapor may be adsorbing and condensing on surfaces at the same time, with VHP adsorbing and condensing in greater quantities and percentages as compared to the water vapor, and in closer proximity to the surfaces of the sterilization load than the water vapor.
During some or all of a sterilization cycle, multiple layers of adsorption may form on the surfaces of PFS loads. In some instances, each layer of adsorption and/or condensation further away from the surface may contain less VHP and more water vapor, such that a gradient of VHP to water is formed on the surface. VHP may preferentially adsorb and condense closer to the surfaces of the load because of the thermodynamic behavior of binary mixtures of VHP and water vapor close to or at saturation (vapor/liquid equilibrium). Vapor/liquid equilibrium may be analogous to gas/adsorbate equilibrium for binary mixtures of VHP and water vapor in sterilization applications.
During or after a VHP sterilization cycle, condensed/adsorbed hydrogen peroxide may be difficult to remove from surfaces that it has sterilized, due in part to the condensation of water vapor over, and adsorption of water around, the condensed hydrogen peroxide, which may trap the hydrogen peroxide in place on the sterilized surfaces.
Systems and methods disclosed herein may advantageously be used in successfully sterilizing medical products, while decreasing the impact and/or risk of the sterilization process on the products undergoing sterilization. For example, systems and methods disclosed herein may provide for full (e.g., 100%) sterilization of medical products using VHP, followed by full (e.g., 100%) removal of VHP from sterilized products. Systems and methods disclosed herein may, e.g., increase efficiency, safety, and efficacy of sterilization, and/or decrease sterilization cycle time. Additionally, while aspects of the present disclosure may be described with respect to the use of VHP in terminal sterilization of PFS, sterilization of other medical products is contemplated by the present disclosure as well.
The present disclosure also contemplates performance of “moist chemical sterilization,” by which chemical sterilization may be achieved in the presence of water vapor. Comparison of “moist chemical sterilization” to “chemical sterilization” may be analogous, in some cases, to comparison of “moist heat sterilization” to “heat sterilization.” In some instances, moist chemical sterilization may be a more effective and efficient means of achieving sterilization than chemical sterilization technology that currently exists, in the same way that “moist heat sterilization” is considered to be, in some cases, more effective and efficient than only “heat sterilization.”
“Moist chemical sterilization” may take place when environmental conditions of relatively high chemical concentration, high water vapor concentration, and high pressure (e.g., above 400 mbar) act in concert to force the chemical and water vapor to behave as a binary mixture. In order to achieve the desired relatively high chemical concentration, high water vapor concentration, and high pressure, the sterilization chamber (e.g., sterilization chamber 102) may be saturated with a combination of water vapor and sterilizing chemical (e.g., VHP), forcing vapor to condense on surfaces of the “load” or item or items to be sterilized (e.g., products 105). Most commercially available hydrogen peroxide is available and sold as aqueous liquid mixtures in varying concentrations (e.g., 3%, 15%, 35%, 59%), and thus, vaporizing hydrogen peroxide will generally simultaneously include vaporizing water. When VHP is used, because VHP has a higher density than water vapor, VHP may preferentially condense on the surfaces of the item or items to be sterilized over water vapor.
It is recognized herein that a portion of a sterilization load having a lower temperature than the surrounding sterilization environment (e.g., the ambient temperature of sterilization chamber 102), may act as a “cold spot” that attracts vapor to condense on the surface area of the load. If specific “cold spots” within the load are located inside packages which require vapor to travel through a semi-permeable membrane, these “cold spots” can advantageously attract condensation of vaporized VHP to the surface area surrounding the “cold spots,” thus creating a higher density of condensed VHP in areas of the load and promoting diffusion of the sterilizing chemical through semi-permeable membranes that it contacts. On the other hand, it is recognized that if “cold spots” are too cold, that is, if there is too much of a temperature difference (delta) between the load or portions of the load and the surrounding sterilization environment (e.g., the temperature of sterilization chamber 102), the presence of the “cold spots” may prevent distribution and penetration of VHP over the entire load. Thus, it is recognized that a balanced temperature differential between the temperature of the sterilization environment (e.g., sterilization chamber 102) and the temperature of “cold spots” on items to be sterilized (e.g., products 105) is required, such that VHP is drawn to condense at “cold spots,” but not to the detriment of diffusion over the load as a while.
Referring now to the figures, FIG. 1 depicts in schematic form an exemplary sterilization system 100. Sterilization system 100 includes a sterilization chamber 102, surrounded by a temperature control jacket 104. Sterilization chamber 102 has an interior cavity, including an upper interior 101 and a lower interior 103. Sterilization chamber 102 is configured to house one or more products 105 for sterilization. An inlet conduit 134, fluidly connected to sterilization chamber 102, is configured to allow various fluids to enter sterilization chamber 102 via a distribution manifold 107 in sterilization chamber 102. A second inlet conduit 135 is also fluidly connected to sterilization chamber 102, also to allow fluids to enter sterilization chamber 102 via an inlet 109. A blower 106 is fluidly connected to sterilization chamber 102 via a blower exit conduit 108. A blower circulation conduit 118 fluidly connects blower 106 to move fluids from blower exit conduit 108 either towards an exhaust 116, or back towards sterilization chamber 102 via inlet conduit 134. An exhaust valve 120 is located between blower circulation conduit 118 and exhaust 116, and selectively closes or opens a connection between blower circulation conduit 118 and exhaust 116. A recirculation valve 119 is located between blower circulation conduit and inlet conduit 134, and selectively closes or opens a connection between blower circulation conduit 118 and inlet conduit 134. A vacuum pump 110 is also fluidly connected to sterilization chamber 102, via a vacuum conduit 112 and a catalytic converter 115. A vacuum valve 113 is located on vacuum conduit 112, and selectively allows, partially allows, or blocks flow from sterilization chamber 102 through catalytic converter 115 and vacuum pump 110. A vacuum exhaust conduit 114 fluidly connects vacuum pump 110 to exhaust 116.
Several fluid supplies are also fluidly connected to sterilization chamber 102 via inlet conduit 134 or inlet conduit 135. An air supply 117 is configured to supply air to sterilization chamber 102 via inlet conduit 134. An air valve 124 is coupled to the fluid connection between air supply 117 and inlet conduit 134, and selectively allows, partially allows, or blocks flow of air from air supply 117 to sterilization chamber 102 via inlet conduit 134. Further, a VHP injector 132, fluidly connected to inlet conduit 134, is configured to inject VHP to sterilization chamber 102 via inlet conduit 134. A VHP injector valve 128 is coupled to the fluid connection between VHP injector 132 and inlet conduit 134, and selectively allows, partially allows, or blocks flow of VHP from VHP injector 132 to sterilization chamber 102 via inlet conduit 134. Additionally, a dry air supply 130 fluidly connected to inlet conduit 135 is configured to supply dry air to sterilization chamber 102 via inlet conduit 135. A dry air supply valve 126 is coupled to the fluid connection between dry air supply 130 and inlet conduit 135, and is configured to selectively allow, partially allow, or block flow of dry air from dry air supply 130 to sterilization chamber 102 via inlet conduit 134. A controller 140 is connected to one or more other components of sterilization system 100, such as sterilization chamber 102, temperature control jacket 104, blower 106, VHP injector 132, air supply 117, dry air supply 130, and/or any other components of sterilization system 100.
Sterilization system 100 may be configured to run sterilization cycles within sterilization chamber 102 at a variety of temperatures and pressures, and for a variety of time durations and/or time intervals. In some embodiments, the temperature(s), pressure(s), and time interval(s) at which sterilization system 100 may run sterilization cycles may be selectively and individually modified and customized. Sterilization system 100 may be configured to control the environment in the interior of sterilization chamber 102, including temperature, pressure, humidity, atmosphere, intake of fluids via, e.g., inlet conduit 134, exit of fluids via one or more of temperature or pressure controls, and/or via e.g., blower exit conduit 108 and/or vacuum conduit 112. Further, sterilization system 100 may include any suitable number and location of sensors configured to sense, e.g., temperature, pressure, flow, chemical concentration, or other parameters throughout sterilization system 100, including in sterilization chamber 102, temperature control jacket 104, blower 106, vacuum pump 110, and/or any of conduits 108, 112, 114, 118, and 134. Such sensors may be configured to transmit sensed data to, e.g., controller 140 and/or a human-machine interface.
Sterilization chamber 102 may be a sealable chamber defining an interior, including upper interior 101 and lower interior 103. Sterilization chamber 102 may be openable into an open configuration, such that one or more items, e.g., products 105, may be placed inside as a part of a load for sterilization, and may be removed subsequent to sterilization. In some embodiments, sterilization chamber 102 may have an operating orientation, e.g., such that upper interior 101 is located above lower interior 103, and such that matter may fall (e.g., under the forces of gravity) from the vicinity of upper interior 101 towards lower interior 103. Sterilization chamber 102 may have one or more delivery apparatus to which one or more of inlet conduit 134 and inlet conduit 135 may be connected. As depicted in FIG. 1, for example, distribution manifold 107 is one such delivery apparatus. Distribution manifold 107 may be configured to disperse gas, vapor, or liquid into sterilization chamber 102 in a given configuration, such as a stream or an even spray across upper interior 101 of sterilization chamber 102. Inlet 109 is another such delivery apparatus. Inlet 109 may also be configured to disperse gas, vapor, or liquid into sterilization chamber 102 in a given configuration, such as a stream, or an even spray across upper interior 101. In some embodiments, distribution manifold 107 may be configured to disperse gas, vapor, or liquid into sterilization chamber 102 in one configuration, such as an even spray, and inlet 109 may be configured to disperse gas or vapor into sterilization chamber 102 in a different configuration, such as in a stream. In some embodiments, there may be no inlet 109, and both inlet conduits 134 and 135 may be connected to distribution manifold 107.
Temperature control jacket 104 may be any material surrounding sterilization chamber 102, that is configured or effective to afford temperature control to the environment inside sterilization chamber 102. In some embodiments, for example, temperature control jacket 104 may be a water jacket surrounding sterilization chamber 102. In such embodiments, a temperature and/or a flow of water or other liquid through temperature control jacket 104 may be controlled by, e.g. controller 140.
Products 105 may be any item or items suitable for sterilization using sterilization system 100. In some embodiments, products 105 may be medical products in primary packaging, secondary packaging, or both. In some embodiments, products 105 may be medical products having moving parts or parts otherwise sensitive to deep vacuum environments, such as environments having pressure of less than about 100 millibars. Products 105, therefore, may be, e.g., containers filled with a volume of formulated drug substance, such as, e.g., vials or PFS. In further embodiments, products 105 may be or include medical products sensitive to high temperatures, e.g., above 30° C. Such medical products may include, for example, formulated drug substances or other compositions that may be sensitive to high temperatures, such as proteins (e.g., antibodies or enzymes), nucleic acids, blood, blood components, vaccines, allergenics, gene therapy medicaments, tissues, other biologics, etc. For example, products 105 may be packaged PFS containing a formulated drug substance that includes an antibody.
Blower 106 may be, for example, a blower having the capacity to forcibly draw vapor and gas from lower interior 103 of sterilization chamber 102 through blower exit conduit 108, and to reintroduce said vapor and gas back to upper interior 101 of sterilization chamber 102 via inlet conduit 134 (or, alternatively, to draw such vapor and gas through exhaust valve 120 and catalytic converter 121, to exhaust 116). Blower 106 may be any device or mechanism configured or effective to perform this function. For example, blower 106 may have an impeller and rotating blades, or rotating vanes configured to draw vapor and gas from lower interior 103 out of blower exit conduit 108, through blower circulation conduit 118, and back to upper interior 101 of sterilization chamber 102 via inlet conduit 134. In some embodiments, blower 106 may be external to sterilization chamber 102, as shown in FIG. 1. In other embodiments, blower 106 may be disposed within sterilization chamber 102. In some embodiments, blower 106 may be configured to draw vapor and gas from lower interior 103 of sterilization chamber 102 and reintroduce said vapor and gas back to upper interior 101 with sufficient force to create a flow of vapor and gas from upper interior 101 to lower interior 103 of sterilization chamber 102. This flow may be termed a “turbulent flow.” In some embodiments, the force with which blower 106 may operate may be adjustable (via, for example, controller 140), such that a more turbulent (e.g., more forceful), or less turbulent, flow of vapor and gas within sterilization chamber 102 may be generated. In some embodiments, blower 106 may be configured to generate a stronger force to draw vapor and gas than, e.g., vacuum pump 110.
Vacuum pump 110 may be a vacuum pump having the capacity to draw gas from the interior (e.g., lower interior 103) of sterilization chamber 102, via vacuum conduit 112 and catalytic converter 115, and towards exhaust 116, thereby creating a vacuum within sterilization chamber 102 and/or a closed system containing sterilization chamber 102 and, e.g., blower 106. In some embodiments, vacuum pump 110 may have an impeller, rotating blades, or vanes configured to draw vapor and gas towards exhaust 116. Vacuum pump 110 may be fluidly connected to exhaust 116 via, e.g., vacuum exhaust conduit 114. In some embodiments, exhausts from vacuum pump 110 and blower 106 may be separated instead of being combined into one.
In some embodiments, vacuum-type functions may also or alternately be performed by, e.g., blower 106, which may selectively circulate vapor and gas out of and into sterilization chamber 102 or out of sterilization chamber 102, through exhaust valve 120, and towards exhaust 116. Exhaust valve 120 may be selectively opened or closed so as to permit or prevent flow of gas or vapor from blower circulation conduit 118 towards exhaust 116 or towards inlet conduit 134 for reintroduction into sterilization chamber 102. Exhaust valve 120 may be manually controlled, or may be controlled by, e.g., controller 140.
Sterilization system 100 may include several supplies of air and/or vapor from which fluid may be introduced into sterilization chamber 102 via inlet conduit 134 or inlet conduit 135. Air supply 117, for example, may be any supply of air (e.g., room air, or compressed dry air) or other fluid external from the rest of sterilization system 100. In some embodiments, air supply 117 may be a supply of “room air” surrounding sterilization system 100, which may have gone through an indoor filtration system. In some embodiments, air supply 117 may include more water vapor than “room air.” In some embodiments, air supply 117 may be a supply of filtered outdoor air. Air valve 124, coupled to the fluid connection between air supply 117 and inlet conduit 134, may be configured to selectively allow, partially allow, or block flow of air from air supply 117 to sterilization chamber 102 via inlet conduit 134, thus controlling the intake of air into closed portions of sterilization system 100. Air valve 124 may be manually controllable and/or controllable by, e.g., controller 140.
Dry air supply 130 may be a supply of air having a relatively low humidity, such that it may be used to dry the interior of, e.g., sterilization chamber 102 and/or one or more of conduits 108, 112, 114, 118, and 134. In some embodiments, for example, air in dry air supply 130 may include a dew point of, e.g., −10 degrees Celsius or less, −40 degrees Celsius or less, or anywhere between −10 degrees Celsius and −40 degrees Celsius. In some embodiments, dry air supply 130 may be a supply of hygienic dry air, such as air that has been sterilized or otherwise filtered to at least 0.2 microns. In some embodiments, dry air supply 130 may be a sealed supply of air. In some embodiments, dry air supply 130 may be a supply of compressed air. Dry air supply valve 126, coupled to the fluid connection between dry air supply 130 and inlet conduit 135, may be configured to selectively allow, partially allow, or block flow of dry air from dry air supply 130 to sterilization chamber 102 via inlet conduit 135. Dry air supply valve 126 may be manually controllable and/or may be controllable by, e.g., controller 140. In some embodiments, dry air supply 130 may be connected to inlet conduit 134 instead of inlet conduit 135. In further embodiments, air supply 117 may supply any of the types of air that dry air supply 130 includes.
VHP injector 132 may include a supply of VHP, or VHP and vaporized water, and may be configured to inject VHP or a combination of VHP and vaporized water into sterilization chamber 102 via, e.g., inlet conduit 134. VHP injector 132 may be configured to inject vapor into sterilization chamber 102 at an adjustable concentration. VHP injector valve 128 may be coupled to the fluid connection between VHP injector 132 and inlet conduit 134, and may be configured to selectively allow or block flow of VHP from VHP injector 132 to sterilization chamber 102 via inlet conduit 134. VHP injector valve 128 may be manually controllable and/or may be controllable by, e.g., controller 140. Dry air supply valve 126 and VHP injector valve 128 may also be used in concert to allow a desired combination of dry air and vaporized VHP/water into sterilization chamber 102, via inlet conduit 134.
Catalytic converter 115 and catalytic converter 121 may be, for example, any catalytic converters known in the art suitable for converting toxic gaseous or vaporized fluids circulated within sterilization system 100, e.g., during a sterilization cycle, to less toxic gases or vapors. For example, catalytic converters 115, 121 may be configured to convert VHP injected into sterilization system 100 by VHP injector 132 into water vapor, oxygen, or other non-toxic fluids.
Some or all aspects of sterilization system 100 may be controllable by, e.g., controller 140. Controller 140 may be, for example, an analog or digital controller configured to alter aspects of the environment of sterilization chamber 102 such as an internal temperature or pressure of sterilization chamber 102 and/or one or more of blower 106, vacuum pump 110, air supply 117, dry air supply 130, VHP injector 132, exhaust 116, one or more of valves 113, 119, 120, 124, 126, and 128, one or more of catalytic converters 115, 121, one or more of conduits 108, 112, 114, 116, 118, and 134, and any and/or other aspects of sterilization system 100. In some embodiments, sterilization system 100 may be controllable by multiple controllers 140. In other embodiments, sterilization system may only have one controller 140. In some embodiments, controller 140 may be a digital controller, such as a programmable logic controller.
In some embodiments, controller 140 may be pre-programmed to execute one or more sterilization cycles using sterilization system 100. In some embodiments, sterilization system 100 may be controllable by a controller having one or more human machine interface (“HMI”) elements, which may be configured to allow a user to input or alter desired parameters for a sterilization cycle, which may be executable by a controller on or operably coupled to sterilization system 100. Thus, in some embodiments, HMI elements may be used to program a customized sterilization cycle for execution by sterilization system 100. For example, in some embodiments, sterilization system 100 may be controllable by a controller connected to, e.g., a computer, tablet, or handheld device having a display. Such a display may include, for example, options to select or alter a desired temperature, pressure, time, amount of VHP intake, etc., for one or more steps of a sterilization cycle.
FIGS. 2 and 3A-3C depict flow diagrams of phases and steps in methods for sterilization according to the present disclosure. As will be recognized by one of ordinary skill in the art, some phases and/or steps in FIGS. 2 and 3A-3C may be omitted, combined, and/or performed out of order while remaining consistent with the present disclosure. In some embodiments, the phases and steps in FIGS. 2 and 3A-3C may be performed using, e.g., sterilization system 100 or a variation of sterilization system 100. It will be recognized that the customizable and controllable aspects of sterilization system 100 may be used in order to carry out phases and steps depicted in FIGS. 2 and 3A-3C. For example, in some embodiments, controller 140 may be employed to direct, adjust, or modify a series of sterilization steps, setpoints, and phases performable by sterilization system 100. Additionally, although the phases and steps described in FIGS. 2 and 3A-3C are recited in relation to sterilization system 100, one of ordinary skill in the art will understand that these phases and steps may be performed by another sterilization system, or another system having the capacity to carry out the steps.
FIG. 2 depicts a flow diagram of a series of steps in a method 200 for sterilization according to the present disclosure in a sterilization system, such as sterilization system 100. According to step 202, a leak test may be performed on sterilization system 100. According to step 204, sterilization system 100 may be preconditioned. According to step 206, a sterilization phase may be performed. According to step 208, a first aeration phase may be performed. According to step 208, a second aeration phase may be performed.
Prior to performance of the steps of method 200, a sterilization load, such as products 105, may be placed within a sterilization chamber, such as sterilization chamber 102, of a sterilization system, such as sterilization system 100. The closed-system sterilization environment—including sterilization chamber 102, blower exit conduit 108, blower 106, blower circulation conduit 118, inlet conduit 134, and any elements connecting these components—may then be sealed.
According to step 202, a leak test may be performed on the closed-system sterilization environment. The leak test may include, for example, creating a vacuum through the closed system. The vacuum may be created by, e.g., expelling gas and vapor from the closed system using vacuum pump 110. During the leak test, blower 106 may be in operation, so as to circulate any remaining air through the closed system and create a homogenous environment. The leak test may be performed in this manner in part to verify that a suitable vacuum may be held within the closed system. Additionally, inclusion of, and circulation of air through, the entirety of the closed system in the leak test may assist in increasing the heat transfer coefficient between the environment within the closed system and the load to be sterilized, which may assist in equalizing the temperature between the environment within the closed system and the load to be sterilized prior to sterilization.
According to step 204, the sterilization system (e.g., sterilization system 100) may be preconditioned. Preconditioning may include, for example, increasing the temperature of the closed system to temperatures intended to be maintained during a sterilization phase (e.g., between about 25° C. and about 50° C.). In some embodiments, preconditioning may be performed for longer than is performed in standard chemical sterilization procedures, which may allow more time for any temperature difference between the environment in the closed system (including, e.g., the environment of sterilization chamber 102) and the load to be sterilized to decrease. In some embodiments, preconditioning may be performed for between about 15 minutes and about two hours, such as between about 20 minutes and about 1.5 hours, between about 25 minutes and about 1 hour, between about 30 minutes and about 1 hour, between about 30 minutes and about 45 minutes, between about 45 minutes and about 1 hour, such as about 30 minutes, about 40 minutes, about 45 minutes, or about 1 hour. Preconditioning according to step 204 also may include operating at pressures which are at or near atmospheric pressure, e.g., between about 400 millibars and about 700 millibars, between about 500 millibars and about 700 millibars, between about 500 millibars and about 600 millibars, between about 800 millibars and about 1000 millibars, or between about 900 millibars and about 1100 millibars. Operation of the preconditioning step at or near atmospheric pressure may promote convective heat transfer from the chamber environment to the load, assisting in minimizing the difference in temperature between the chamber environment and the load. Additionally, blower 106 may be operated during preconditioning according to step 204, which may contribute to a higher heat transfer coefficient, and thus potentially faster equalization of temperature between the closed system, including the environment of sterilization chamber 102, and the load to be sterilized. Equalization of temperature between the closed system and the load to be sterilized may allow for warming of “cold spots,” or locations on or in the load having a cooler temperature than the majority of the load and/or the surrounding environment. For example, liquid contents of PFS may absorb heat more slowly than their non-liquid packaging, thus acting as “cold spots” within a load containing the PFS. Reduction of such cold spots by equalizing the temperature throughout the closed system and the load to be sterilized may advantageously allow for even diffusion of a vaporized sterilizing chemical (e.g., VHP) through sterilization chamber 102, across the load to be sterilized, and/or diffusion through permeable membranes and barriers in the load to be sterilized. Maintaining some temperature difference between the closed system and the “cold spots” may be desirable, however, to promote preferential surface adsorption and condensation of VHP and water vapor onto the load to be sterilized.
As is discussed elsewhere herein, it is also contemplated that, in some embodiments, maintaining “cold spots” via keeping a temperature differential between the load to be sterilized and the surrounding closed system may also have advantages; for example, controlled condensation of vaporized sterilizing chemical (e.g., VHP) on “cold spots” of a load to be sterilized may concentrate the sterilizing chemical on the load and lead to more efficient diffusion of the chemical into the load, thus decreasing the overall amount of sterilizing chemical needed in the sterilization chamber 102 to achieve effective sterilization. In such embodiments, preconditioning according to step 204 may be performed for a shorter amount of time and/or in a shallow vacuum created by, e.g., vacuum pump 110, in order to allow for or maintain “cold spots” within the load to be sterilized.
According to step 206, a sterilization phase may be performed. The sterilization phase may include, for example, initiating circulation of fluid through the sterilization system, achieving a vacuum level, injecting vaporized chemical into the sterilization chamber, maintaining a post-injection hold, injecting gas into the sterilization chamber to transition to a shallower vacuum, and maintaining a post-transition hold. The sterilization phase according to step 206 may be repeated multiple times. A sterilization phase according to step 206 is depicted in further detail in FIG. 3A.
According to step 208, a first aeration phase may be performed. The first aeration phase may include, for example, achieving a vacuum level, holding the vacuum level, breaking the vacuum level, and aerating and exhausting the system. The first aeration phase may be performed multiple times. A first aeration phase according to step 208 is depicted in further detail in FIG. 3B.
According to step 210, a second aeration phase may be performed. The second aeration phase may include, for example, achieving a vacuum level, holding the vacuum level, and breaking the vacuum level. The second aeration phase may be performed multiple times. A second aeration phase according to step 210 is depicted in further detail in FIG. 3C.
Both steps 208 and 210 may be performed multiple times. Additionally, while in some embodiments, step 208 may be performed before step 210, in alternative embodiments, step 210 may be performed before step 208.
FIG. 3A is a flow diagram of a sterilization phase 300 that may be performed as step 206 of sterilization method 200. Prior to sterilization phase 300, a sterilization load (e.g., products 105) may be introduced into sterilization chamber 102. According to step 302, a vacuum level may be achieved. According to step 304, vaporized chemical may be injected into the sterilization chamber. According to step 306, a post-injection hold may be maintained. According to step 308, gas may be injected into the sterilization chamber to transition to a shallower vacuum. According to step 310, a post-injection hold may be maintained.
As a part of sterilization phase 300, a turbulent flow may be initiated and maintained in sterilization system 100.
According to step 302, a vacuum level may be achieved within sterilization chamber 102 of sterilization system 100. The vacuum level may be, for example, between about 400 millibars and about 700 millibars, such as between about 450 millibars and about 650 millibars, or between about 450 millibars and about 550 millibars. For example, the vacuum may be about 450 millibars, about 500 millibars, about 550 millibars, or about 600 millibars. This vacuum may promote a higher concentration of sterilizing chemical on the sterilization load, extending the amount of time at which the closed system is kept at a deeper vacuum increases exposure of the sterilization load to the sterilizing chemical.
According to step 304, vaporized chemical may be injected into the sterilization chamber. In some embodiments, the vaporized chemical may include VHP. In some embodiments, the vaporized sterilization chemical may be a vaporized aqueous hydrogen peroxide solution, having a concentration of, for example, between about 5% and about 75% hydrogen peroxide by weight. In some embodiments, the vaporized chemical may be a vaporized aqueous hydrogen peroxide solution having a concentration of, for example, between about 10% and about 65% hydrogen peroxide by weight, between about 15% and about 60% hydrogen peroxide by weight, between about 30% and about 60% hydrogen peroxide by weight, between about 30% and about 60% hydrogen peroxide by weight, or between about 45% and about 60% hydrogen peroxide by weight. In some embodiments, the vaporized chemical may be a vaporized aqueous hydrogen peroxide having a concentration of about 35% hydrogen peroxide (and 65% water) by weight. In further embodiments, the vaporized chemical may be a vaporized aqueous hydrogen peroxide having a concentration of about 59% hydrogen peroxide (and 41% water) by weight.
In some embodiments, an injected supply of VHP may be, for example, between about 50 g and about 700 g of aqueous VHP. For example, the injected supply of VHP may be between about 50 g and about 600 g, between about 100 g and about 600 g, between about 300 g and about 550 g, or between about 450 g and about 550 g. For example, the injected supply of VHP may be about 100 g, about 200 g, about 300 g, about 400 g, about 450 g, about 475 g, about 500 g, about 525 g, about 550 g, about 600 g, or about 650 g. In some embodiments, an injected supply of VHP may be quantified based on the volume or amount of load to be sterilized inside sterilization chamber 102. For example, if a number of drug products, such as pre-filled syringes, are to be sterilized in sterilization chamber 102, an injected supply of VHP may be between about 0.01 and about 0.15 grams of VHP per unit of the drug product inside sterilization chamber 102, such as between about 0.01 and about 0.10 grams of VHP, such as about 0.015 grams, 0.02 grams, 0.025 grams, 0.03 grams, 0.04 grams, 0.05 grams, 0.06 grams, 0.07 grams, 0.08 grams, 0.09 grams, 0.1 grams, or 0.11 grams per drug product. In other embodiments, an injected supply of VHP may be quantified based on the volume of the sterilization environment, such as the interior of sterilization chamber 102. For example, an injected supply of VHP may be between about 0.2 and 3.0 grams per cubic foot of volume in a sterilization chamber. For example, an injected supply of VHP may be between about 0.2 and about 2.0 grams per cubic foot, such as about 0.25 grams, about 0.50 grams, about 0.75 grams, about 1.0 gram, about 1.2 grams, about 1.4 grams, about 1.5 grams, about 1.6 grams, about 1.8 grams, or about 2.0 grams per cubic foot.
In some embodiments, step 210 may also include injecting dry air from, e.g., dry air supply 130, into the sterilization system, so as to create a desired balance between concentrations of vaporized chemical and water vapor, at different pressures, inside the chamber.
According to step 306, a post-injection hold may be maintained. During the post-injection hold, turbulent flow is maintained through the closed system including sterilization chamber 102 and blower 106. No fluids are added or removed from the closed system in which the turbulent flow is maintained. The time for which a post-injection hold is maintained (or the “post-injection hold time”) may be selected so as to allow the vaporized sterilization chemical adequate time to contact the load to be sterilized. In some embodiments, the post-injection hold time may be between about 2 minutes and about 20 minutes. In some embodiments, the post-injection hold time may be at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, the post-injection hold time may be between about 5 minutes and about 20 minutes, between about 8 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, or between about 10 minutes and about 15 minutes. In such a manner, the need for adding excess VHP into the system to ensure its contact with the sterilization load may be avoided.
According to step 308, gas may be injected into the sterilization chamber to transition to a shallower vacuum (i.e., a higher pressure) in the sterilization chamber. The gas may be any suitable gas that can break or lessen the vacuum in sterilization chamber 102. In some embodiments, the gas may be a dry gas, such as a gas containing nitrogen (e.g., commercially available supplies of only nitrogen or primarily nitrogen), or air having a dew point of, for example, −10° C. or colder. In some embodiments, gas may be injected from dry air supply 130. The gas may be injected in a volume to achieve a pressure between about 500 millibars and about 1100 millibars, such as between about 550 millibars and about 1000 millibars, between about 600 millibars and about 1000 millibars, between about 700 millibars and about 700 millibars and about 900 millibars, or between about 750 millibars and about 850 millibars. For example, the second post-injection pressure may be about 700 millibars, about 750 millibars, about 800 millibars, about 850 millibars, or about 900 millibars.
According to step 310, a post-transition hold may be maintained. During the post-transition hold, the pressure achieved during step 308 may be maintained for, for example, at least about 5 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, the second post-injection pressure may be maintained for between about 5 minutes and about 20 minutes, between about 8 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, or between about 10 minutes and about 15 minutes.
The steps of sterilization phase 300 may be repeated, for example, between 1 and 10 times, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. This may aid in ensuring full sterilization of the sterilization load within sterilization chamber 102. In some embodiments, the number of times that sterilization phase 300 may be repeated may be inversely proportional to the time that the post-injection hold is maintained in each repetition. For example, if the time that the post-injection hold is maintained is short (e.g., 10 minutes), then steps 210 through 216 may be repeated a greater number of times. In some embodiments, the post-injection hold is maintained for a longer period of time (e.g., 15-20 minutes), to increase the time during which the sterilization load is exposed to the sterilizing chemical in each repetition of sterilization phase 300. In further embodiments, the number of times that sterilization phase 300 may be repeated may depend on a total desired amount of VHP for the sterilization process. In some embodiments, for example, injection of a total amount of at least 200 g of VHP may be desired. For example, in some embodiments, injection of a total amount of at least 250 g may be desired. In some embodiments, injection of a total amount of between about 200 g and about 700 g of VHP may be desired.
FIG. 3B is a flow diagram of a first aeration phase 320 that may be performed as step 208 of sterilization method 200, after performing one or more repetitions of sterilization phase according to step 206. According to step 322, a vacuum level may be achieved. According to step 324, the vacuum level may be held. According to step 326, the vacuum level may be broken. According to step 328, the sterilization system (e.g., sterilization system 100) may be aerated and exhausted.
According to step 322, a vacuum level may be achieved in sterilization chamber 102, while also injecting dry gas into sterilization chamber 102 near upper interior 101 of sterilization chamber 102, such as via distribution manifold 107 or inlet 109. The dry gas may include, for example, oxygen and/or nitrogen. The dry gas may have a dew point of, for example, −10° C. or lower. The dry gas may be injected from, e.g., dry air supply 130. While dry gas is being injected into sterilization chamber 102, a vacuum may be pulled by, e.g., vacuum pump 110 via vacuum conduit 112, catalytic converter 115, and vacuum exhaust conduit 114. The vacuum may be pulled at a greater rate than the rate of injection of dry gas, such that a vacuum level is gradually achieved. The vacuum level may be, for example, between about 500 millibars and about 850 millibars, such as between about 500 millibars and about 800 millibars, between about 550 millibars and about 750 millibars, or between about 600 millibars and about 700 millibars. For example, the vacuum level may be 500 millibars, 550 millibars, 600 millibars, 650 millibars, or 700 millibars. Injection of the dry gas near upper interior 101 of sterilization chamber 102 while achieving a desired vacuum level reduces condensation of VHP and water vapor at upper interior 101 of the chamber, and promotes the movement of denser molecules in sterilization chamber towards the lower interior (e.g., lower interior 103) of sterilization chamber 102, and to some extent out of sterilization system 100 through vacuum exhaust conduit 114.
According to step 324, injection of dry gas may be stopped and the vacuum level may be held for, e.g., between about 1 minute and about 20 minutes, such as between about 2 min and about 20 min, between about 5 min and about 20 min, between about 5 min and about 15 min, or between about 5 min and about 10 min. For example, the vacuum level may be maintained for about 2, 5, 8, 10, or 15 minutes. Holding the vacuum level may continue to promote settling of denser molecules (e.g., sterilization chemical molecules) down towards the lower interior 103 of sterilization chamber 102, and away from the sterilization load.
According to step 326, the vacuum level may be broken by the addition of more dry gas near upper interior 101 of sterilization chamber 102, via, for example, distribution manifold 107 or inlet 109. A volume of dry gas sufficient to achieve a higher pressure may be added. The higher pressure may be, for example, between 50 and 200 millibars higher than the vacuum level achieved in step 322. The vacuum level may be, for example, between about 550 millibars and about 1000 millibars, such as between about 550 millibars and about 850 millibars, between about 600 millibars and about 700 millibars, or between about 650 millibars and about 750 millibars. For example, the vacuum level may be about 550 millibars, 600 millibars, 650 millibars, 700 millibars, 750 millibars, or 800 millibars. The addition of more dry gas may continue to force sterilization chemicals to settle to the lower interior 101 of sterilization chamber 102, thus moving them away from the sterilization load and positioning them for removal via vacuum conduit 112 or blower exit conduit 108.
According to step 328, the sterilization system (e.g., sterilization system 100) may be aerated and exhausted. During this step, blower 106 may be turned on while recirculation valve 119 is closed and exhaust valve 120 is opened, such that blower 106 pulls fluid from within sterilization chamber 102 and expels it through exhaust 116 via catalytic converter 121. Because blower exit conduit 108 is connected to sterilization chamber 102 at lower interior 103 of sterilization chamber 102, denser fluids that have settled to lower interior 103 (such as sterilizing chemicals) may be removed by this step. Air (e.g., from air supply 117) may be concurrently allowed to vent into sterilization chamber 102, such that the pressure in sterilization chamber 102 returns to, or near, atmospheric pressure.
First aeration phase 320 may be repeated, for example, between 1 and 35 times, such as 2, 5, 10, 15, 17, 19, 22, 25, 27, 29, 30, 32, or 35 times. Repetition of first aeration phase 320 may ensure that the majority of sterilization chemical (e.g., VHP) is removed from sterilization system 100.
FIG. 3C is a flow diagram of a second aeration phase 340 that may be performed as step 210 of sterilization method 200. According to step 342, a vacuum level may be achieved. According to step 344, a vacuum level may be held. According to step 346, the vacuum level may be broken.
According to step 342, a vacuum level may be achieved in sterilization chamber 102. Like with the first aeration phase, the vacuum level achieved in this phase may be, for example, between about 500 millibars and about 850 millibars, such as between about 500 millibars and about 800 millibars, between about 550 millibars and about 750 millibars, or between about 600 millibars and about 700 millibars. For example, the vacuum level may be 500 millibars, 550 millibars, 600 millibars, 650 millibars, or 700 millibars. Achieving a vacuum level may promote removing of moisture from sterilization chamber 102 and thus the sterilization load. Thus, the sterilization load may be dried.
According to step 344, the vacuum level may be held for, e.g., between about 1 minute and about 20 minutes, such as between about 2 min and about 20 min, between about 5 min and about 20 min, between about 5 min and about 15 min, or between about 5 min and about 10 min. For example, the vacuum level may be maintained for about 2, 5, 8, 10, or 15 minutes. Holding the vacuum level may continue to promote removal of moisture from sterilization chamber 102, and thus the sterilization load. Thus, the sterilization load may be further dried. In some embodiments, step 344 may be omitted.
According to step 346, the vacuum level in sterilization chamber 102 may be broken, or raised to a higher pressure, by the addition of dry gas from, e.g., dry air supply 130.
Second aeration phase 340 may be repeated, for example, between 1 and 50 times, such as 2, 5, 10, 15, 20, 25, 30, 35, 38, 40, 42, 45, 47, 49, or 50 times. Repetition of second aeration phase 340 may ensure drying of sterilization chamber 102 and the sterilization load.
As has been previously described, second aeration phase 340 may be performed either before or after first aeration phase 320. First aeration phase 320 may ensure, for example, that the concentration of sterilizing chemical (e.g., VHP) in sterilization chamber 102 is relatively low, and second aeration phase 340 may ensure that the sterilization load is dried, and may also remove residual sterilizing chemical remaining in sterilization chamber 102 after first aeration phase 320. In cases where second aeration phase 340 is performed after first aeration phase 320, first aeration phase may ensure that the concentration of sterilization chemical (e.g., VHP) in sterilization chamber 102 is relatively low so that when sterilization chamber 102 and the sterilization load are dried in second aeration phase 340, there is little remaining need to remove residual sterilization chemical from the sterilization system 100.
FIGS. 4A-4C depict, in schematic form, sterilization system 100, and in particular, which parts of sterilization system 100 may be active, open, or on (as opposed to inactive, closed, or off) during phases 300, 320, and 340. For clarity, controller 140 and thermal jacket 104 are not pictured.
FIG. 4A depicts, in schematic form, the various parts of sterilization system 100 in various stages of activity or inactivity during sterilization phase 300. As is shown, during sterilization phase 300, blower exit conduit 108, blower circulation conduit 118, blower 106, and recirculation valve 119 remain open, on, or active throughout sterilization phase 300. Air supply 117, air supply valve 124, exhaust valve 120, and catalytic converter 121 remain closed, off, or inactive throughout sterilization phase 300. The remaining components are sometimes open, on, or active during sterilization phase 300. The following table indicates when these components are open, on or active:
TABLE 1
Vacuum valve 113;
vacuum conduit 112;
Dry air supply
catalytic converter 115;
VHP injector
130;
vacuum pump 110;
132;
dry air supply
vacuum exhaust conduit
VHP injector
valve 126;
Components
114; exhaust 116
valve 128
inlet 109
Steps
Achieving vacuum
On/open/active
level (step 302)
Injecting vaporized
On/open/active
chemical (step
304)
Maintaining post-
injection hold (step
306)
Transitioning to
On/open/active
shallower vacuum
(step 308)
Maintaining post-
transition hold
(step 310)
FIG. 4B depicts, in schematic form, the various parts of sterilization system 100 during first aeration phase 320. As is shown, during first aeration phase 320, VHP injector 132, VHP injector valve 128, and recirculation valve 119 remain off or closed. The remaining components are sometimes open, on, or active during first aeration phase 320, as indicated in the following table:
TABLE 2
air supply 117;
air valve 124;
inlet 134;
Vacuum
distribution
conduit 112;
manifold 107;
vacuum valve 113;
blower 106;
catalytic
Dry air
blower exit
converter 115;
supply 130;
conduit 108;
vacuum
dry air
exhaust valve 120;
pump 110;
supply valve
catalytic
vacuum exhaust
126;
Exhaust
Components
converter 121
conduit 114
inlet 109
116
Steps
Achieving
On/open/active
On/open/
On/open/
vacuum level
active
active
(step 322)
Holding the
vacuum level
(step 324)
Breaking the
On/open/
vacuum level
active
(step 326)
Aerating and
On/open/active
On/open/
exhausting
active
the system
(step 328)
FIG. 4C depicts, in schematic form, the various parts of sterilization system 100 during second aeration phase 340. As is shown, during second aeration phase 340, air supply 117, air supply valve 124, VHP injector, VHP injector valve 128, exhaust valve 120, and catalytic converter 121 remain closed. Blower exit conduit 108, blower 108, blower circulation conduit 118, recirculation valve 119, inlet 134, and distribution manifold 107 remain open during aeration phase 340. The remaining components are sometimes open, on, or active during aeration phase 340. The following table indicates when these components are open, on or active:
TABLE 3
Vacuum conduit 112;
vacuum valve 113;
catalytic converter 115;
vacuum pump 110;
Dry air supply 130;
vacuum exhaust conduit 114;
dry air supply valve 126;
Components
exhaust 116
inlet 109
Steps
Achieving vacuum level
On/open/active
On/open/active
(step 342)
Holding the vacuum
level (step 344)
Breaking the vacuum
On/open/active
level (step 346)
In some embodiments, any or all of the above-described steps and phases may be executed automatically by sterilization system 100 as directed by, e.g., controller 140, which may be programmed or otherwise configured in advance by e.g., a user. The methods of sterilization disclosed herein may be qualified as “limited overkill” sterilization methods, in that they may ensure sterilization of a load of, e.g., PFS while minimizing impact of the sterilization method on the product.
The above description is illustrative, and is not intended to be restrictive. One of ordinary skill in the art may make numerous modification and/or changes without departing from the general scope of the invention. For example, and as has been described, the above-described embodiments (and/or aspects thereof) may be used in combination with each other. Additionally, portions of the above-described embodiments may be removed without departing from the scope of the invention. In addition, modifications may be made to adapt a particular situation or material to the teachings of the various embodiments without departing from their scope. Many other embodiments will also be apparent to those of skill in the art upon reviewing the above description.
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16702909
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:regn
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Regeneron Pharmaceuticals
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Apr 28th, 2020 12:00AM
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Jun 13th, 2017 12:00AM
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https://www.uspto.gov?id=US10633434-20200428
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Anti-C5 antibodies
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The present invention provides monoclonal antibodies that bind to the complement factor 5 (C5) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to C5 protein. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing C5 activity, thus providing a means of treating or preventing a C5-related disease or disorder in humans. In some embodiments, the invention provides for an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, e.g., a half-life of more than 10 days.
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10633434
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1. An antibody or antigen-binding fragment thereof that binds specifically to C5 comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising amino acid sequences of SEQ ID NOs: 100-102-104-108-110-112.
2. An antibody or antigen-binding fragment thereof that binds specifically to C5, wherein the antibody or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 98/106.
3. The antibody or antigen-binding fragment thereof of claim 2 comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 353.
4. The antibody or antigen-binding fragment thereof of claim 2 comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence of SEQ ID NO: 354.
5. An antibody or antigen-binding fragment thereof that binds specifically to C5 comprising a heavy chain/light chain amino acid sequence pair of SEQ ID NOs: 353/354.
6. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds specifically to C5 according to claim 1 and a pharmaceutically acceptable carrier or diluent.
7. The antibody or antigen-binding fragment thereof of claim 1 which is an antibody.
8. The antibody or antigen-binding fragment thereof of claim 2 which is an antibody.
9. The antibody or antigen-binding fragment thereof of claim 5 which is an antibody.
10. A pharmaceutical composition comprising the antibody of claim 7 and a pharmaceutically acceptable carrier or diluent.
11. A pharmaceutical composition comprising the antibody of claim 8 and a pharmaceutically acceptable carrier or diluent.
12. A pharmaceutical composition comprising the antibody of claim 9 and a pharmaceutically acceptable carrier or diluent.
13. A reusable pen delivery device comprising the pharmaceutical composition of claim 10.
14. A reusable pen delivery device comprising the pharmaceutical composition of claim 11.
15. A reusable pen delivery device comprising the pharmaceutical composition of claim 12.
16. An autoinjector delivery device comprising the pharmaceutical composition of claim 10.
17. An autoinjector delivery device comprising the pharmaceutical composition of claim 11.
18. An autoinjector delivery device comprising the pharmaceutical composition of claim 12.
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18
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/349,705, filed on Jun. 14, 2016; 62/405,561, filed on Oct. 7, 2016; and 62/422,107, filed on Nov. 15, 2016, the disclosures of each herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention is related to antibodies and antigen-binding fragments of antibodies that specifically bind to complement factor C5, and therapeutic and diagnostic methods of using those antibodies.
BACKGROUND OF THE INVENTION
The complement system is a group of plasma proteins that when activated lead to target cell lysis and facilitate phagocytosis through opsonization. Complement is activated through a series of proteolytic steps by three major pathways: the classical pathway, which is typically activated by immune-complexes, the alternative pathway that can be induced by unprotected cell surfaces, and the mannose binding lectin pathway. All three pathways of complement cascade converge on proteolytic cleavage of complement component 5 (C5) protein. Cleavage of complement component 5 (C5) results in the production of fragments C5a and C5b, a process that is critical during the activation of the complement cascade. C5a can generate pleiotropic physiological responses through binding to its receptors (Monk et al 2007, Br. J. Pharmacol. 152: 429-448). C5a is a potent pro-inflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates the oxidative burst, and induces the release of various inflammatory mediators such as histamine or cytokines. C5b mediates the formation of the membrane-attack complex (MAC, or C5b-9) leading to cell lysis in the late phases of the complement dependent cytotoxicity (CDC). Further, in nucleated cells that are resistant to cytolysis by C5b-9, sublytic quantities of C5b-9 can cause cellular activation which results in cell proliferation, generation of pro-inflammatory mediators and production of extracellular matrix.
Monoclonal antibodies to C5 are known in the art and have been described, for example, in US Patent/Publication Nos. 9206251, 9107861, 9079949, 9051365, 8999340, 8883158, 8241628, 7999081, 7432356, 7361339, 7279158, 6534058, 6355245, 6074642, 20160299305, 20160051673, 20160031975, 20150158936, 20140056888, 20130022615, 20120308559, and in WO2015198243, WO2015134894, WO2015120130, EP2563813B1, EP2328616B1, and EP2061810B1.
Fully human antibodies that specifically bind to C5 protein with high affinity and have improved pharmacokinetic properties could be important in the prevention and treatment of various C5-associated diseases (e.g., atypical hemolytic uremic syndrome).
BRIEF SUMMARY OF THE INVENTION
The present invention provides antibodies and antigen-binding fragments thereof that specifically bind complement factor 5 (C5) protein. The antibodies of the present invention are useful, inter alia, for inhibiting or neutralizing the activity of C5 protein. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom or indication of a C5-associated disease or disorder in a subject. In certain embodiments, the antibodies may be administered prophylactically or therapeutically to a subject having or at risk of having a C5-associated disease or disorder. In certain embodiments, the anti-C5 antibodies are fully human antibodies that bind to C5 with high affinity and have improved pharmacokinetic (PK) and pharmacodynamic (PD) properties. Such high-affinity antibodies with improved PK/PD can be used to provide superior efficacy, along with less frequent dosing in a subject with a C5-associated disease or disorder.
The antibodies of the invention can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab′)2 or scFv fragment), and may be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.
In a first aspect, the present invention provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that bind specifically to the C5 protein. In some embodiments, the antibodies are fully human monoclonal antibodies.
Exemplary anti-C5 antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of exemplary anti-C5 antibodies. Table 2 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-C5 antibodies.
The present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 98/114, 122/106, 98/130, 138/106, 146/106, 122/130, 146/114, 146/130, 138/130, 154/162, 170/178, 186/194, 202/210, 218/226, 234/242, 250/258, 266/258, 274/282, 290/298, 306/314, 322/330, and 338/346. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 50/58 (e.g., H4H12161P), 98/106 (e.g., H4H12166P), 138/106 (e.g., H4H12166P5), or 202/210 (e.g., H4H12170P). In certain embodiments, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence listed in Table 1 having no more than five amino acid substitutions, and said LCVR comprising an amino acid sequence listed in Table 1 having no more than two amino acid substitutions. For example, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence of SEQ ID NO: 98 having no more than five amino acid substitutions, and said LCVR comprising an amino acid sequence of SEQ ID NO: 106 having no more than two amino acid substitutions. In another embodiment, the present invention provides anti-C5 antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence of SEQ ID NO: 98 having at least one amino acid substitution, and said LCVR comprising an amino acid sequence of SEQ ID NO: 106 having one amino acid substitution.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 56/64 (e.g., H4H12161P), 104/112 (e.g., H4H12166P), 144/112 (e.g., H4H12166P5), and 208/216 (e.g., H4H12170P).
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, HCDR2 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, and HCDR3 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid. In certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, LCDR2 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, and LCDR3 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid. For example, the present invention provides anti-C5 antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 100 or an amino acid sequence differing from SEQ ID NO: 100 by 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO: 102 or an amino acid sequence differing from SEQ ID NO: 102 by 1 amino acid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 104 or an amino acid sequence differing from SEQ ID NO: 104 by 1 amino acid. In another exemplary embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 108 or an amino acid sequence differing from SEQ ID NO: 108 by 1 amino acid, LCDR2 comprising an amino acid sequence of SEQ ID NO: 110 or an amino acid sequence differing from SEQ ID NO: 110 by 1 amino acid, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 112 or an amino acid sequence differing from SEQ ID NO: 112 by 1 amino acid.
The present invention provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 353, or a substantially similar sequence thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain comprising an amino acid sequence of SEQ ID NO: 354, or a substantially similar sequence thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
In certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 353, or a substantially similar sequence thereof having at least 80%, or at least 90% sequence identity thereto; and a light chain comprising an amino acid sequence of SEQ ID NO: 354, or a substantially similar sequence thereof having at least 80%, or at least 90% sequence identity thereto.
The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-C5 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 52-54-56-60-62-64 (e.g., H4H12161P), 100-102-104-108-110-112 (e.g., H4H12166P), 140-142-144-108-110-112 (e.g., H4H12166P5), and 204-206-208-212-214-216 (e.g., H4H12170P).
In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-C5 antibodies listed in Table 1. For example, the present invention includes antibodies, or antigen-binding fragments thereof, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50/58 (e.g., H4H12161P), 98/106 (e.g., H4H12166P), 138/106 (e.g., H4H12166P5), or 202/210 (e.g., H4H12170P). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
In certain embodiments, the present invention includes an antibody or antigen-binding fragment thereof that binds specifically to C5, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVR comprises: (i) the amino acid sequence of SEQ ID NO: 98, (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 98, (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 98; or (iv) the amino acid sequence of SEQ ID NO: 98 having no more than 5 amino acid substitutions; and the LCVR comprises: (i) the amino acid sequence of SEQ ID NO: 106, (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 106, (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 106; or (iv) the amino acid sequence of SEQ ID NO: 106 having no more than 5 amino acid substitutions.
The present invention includes anti-C5 antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to C5. For example, the present invention includes antibodies and antigen-binding fragment thereof that bind C5 with higher affinity at neutral pH than at acidic pH (i.e., reduced binding at acidic pH).
In certain embodiments, the present invention provides antibodies and antigen-binding fragments that exhibit improved pharmacokinetic and pharmacodynamic properties, for example, the present invention provides anti-C5 antibodies that have extended serum half-life. In certain embodiments, the anti-C5 antibodies of the present invention have serum concentration of more than 10 μg/mL through day 40 in C5-humanized mice. In certain embodiments, the anti-C5 antibodies of the present invention block CP- and AP hemolysis through day 35 upon administration to C5-humanized mice.
The present invention also provides for antibodies and antigen-binding fragments thereof that compete for specific binding to C5 with an antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.
The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to C5 with a reference antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.
The present invention also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR/LCVR amino acid sequence pair has SEQ ID NOs: 98/106.
The present invention also includes anti-C5 antibodies and antigen-binding fragments thereof that bind to one or more amino acid residues comprised in the alpha chain and/or the beta chain of C5. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to one or more amino acids in the alpha chain of C5 and one or more amino acids in the beta chain of C5. In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that bind to one or more amino acids in the alpha and beta chains of C5, wherein the antibodies do not bind to the C5a anaphylatoxin domain. In certain embodiments, the present invention provides anti-C5 antibodies that interact with one or more amino acids contained within human C5 (SEQ ID NO: 359). In certain embodiments, the present invention provides anti-C5 antibodies that interact with one or more amino acids contained within human C5 (SEQ ID NO: 359), wherein the antibodies do not bind to the C5a anaphylatoxin domain of C5. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with an amino acid sequence selected from the group consisting of (a) amino acids 591 to 599 of SEQ ID NO: 359; (b) amino acids 593 to 599 of SEQ ID NO: 359; (c) amino acids 775 to 787 of SEQ ID NO: 359; (d) amino acids 775 to 794 of SEQ ID NO: 359; and (e) amino acids 779 to 787 of SEQ ID NO: 359. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained within SEQ ID NO: 359, for example, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids, at least 10 amino acids, or at least 15 amino acids contained within SEQ ID NO: 361.
In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained within SEQ ID NO: 359, for example, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids contained within SEQ ID NO: 360. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids contained within SEQ ID NOs: 360 and 361. In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that interact with the amino acid sequence of SEQ ID NO: 360 (corresponding to amino acids 591 to 599 of SEQ ID NO: 359) and with the amino acid sequence of SEQ ID NO: 361 (corresponding to amino acids 775 to 794 of SEQ ID NO: 359).
In some embodiments, the antibody or antigen binding fragment thereof may bind specifically to C5 in an agonist manner, i.e., it may enhance or stimulate C5 binding and/or activity; in other embodiments, the antibody may bind specifically to C5 in an antagonist manner, i.e., it may block C5 binding and/or activity.
The present invention also provides isolated antibodies and antigen-binding fragments thereof that block C5 binding to C5 convertase. In some embodiments, the antibody or antigen-binding fragment thereof that blocks C5 binding to C5 convertase may bind to the same epitope on C5 as C5 convertase or may bind to a different epitope on C5 as C5 convertase. In some embodiments, the present invention provides antibodies or antigen-binding fragments thereof that block the binding of C5 to monkey C5 convertase.
In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first binding specificity to a first epitope of C5 protein and a second binding specificity to a second epitope of C5 protein wherein the first and second epitopes are distinct and non-overlapping.
In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to C5a with an IC50 of less than 0.5 nM. In certain embodiments, the antibodies comprise an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 290, 306, 322, and 338. In certain embodiments, the antibodies comprise an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298, 314, 330, and 346.
In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that has one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C., as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM, as measured in a surface plasmon resonance assay; (e) binds to human C5 variant R885H (SEQ ID NO: 356) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (f) binds to human C5 variant R885C (SEQ ID NO: 357) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (CP) hemolysis by more than 95% and with IC50 less than 6 nM, as measured in a CP hemolysis assay; (h) blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70% and with IC50 less than 165 nM, as measured in a AP hemolysis assay; (i) inhibits African green monkey C5-mediated CP hemolysis with IC50 less than 185 nM, as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with IC50 less than 235 nM, as measured in a AP hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM, as measured in a CP hemolysis assay; and (I) inhibits cynomolgus monkey C5-mediated AP hemolysis with IC50 less than 30 nM, as measured in a AP hemolysis assay.
In certain embodiments, the present invention provides an isolated recombinant monoclonal anti-C5 antibody or antigen-binding fragment thereof that has one or more of the following characteristics: (a) comprises a set of six CDRs comprising the amino acid sequences of SEQ ID NOs: 100-102-104-108-110-112; (b) binds to human C5 with a dissociation constant (KD) of less than 0.2 nM at 25° C., as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d) binds to a human C5 variant (R885H) with a KD of less than 0.4 nM at 37° C., as measured in a surface plasmon resonance assay; (e) inhibits classical pathway (CP)-mediated hemolysis of human serum with an IC50 of less than 3 nM; (f) inhibits alternative pathway (AP)-mediated hemolysis of human serum with an IC50 of less than 27 nM; (g) inhibits CP-mediated hemolysis of monkey serum with an IC50 of less than 21 nM; (g) inhibits AP-mediated hemolysis of monkey serum with an IC50 of less than 10 nM; (h) has serum half-life (t1/2) of more than 10 days in C5-humanized mice; (i) has serum concentration of more than 10 μg/mL through day 40 upon administering to C5-humanized mice; (j) blocks CP-mediated hemolysis through day 50 in C5-humanized mice; and (k) binds to one or more amino acids comprised in the alpha chain and/or the beta chain of SEQ ID NO: 359, wherein the antibody does not bind to the C5a anaphylatoxin domain of C5.
In a second aspect, the present invention provides nucleic acid molecules encoding anti-C5 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-C5 antibodies listed in Table 1.
The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-C5 antibodies listed in Table 1.
The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-C5 antibody listed in Table 1.
In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-C5 antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 2. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.
In a third aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof which specifically binds C5 and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition which is a combination of an anti-C5 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-C5 antibody. Exemplary agents that may be advantageously combined with an anti-C5 antibody include, without limitation, other agents that bind and/or inhibit C5 activity (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents which do not directly bind C5 but nonetheless treat or ameliorate at least one symptom or indication of a C5-associated disease or disorder. Additional combination therapies and co-formulations involving the anti-C5 antibodies of the present invention are disclosed elsewhere herein.
In a fourth aspect, the invention provides therapeutic methods for treating a disease or disorder associated with C5 in a subject using an anti-C5 antibody or antigen-binding portion of an antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention to the subject in need thereof. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by inhibition of C5 activity. In certain embodiments, the invention provides methods to prevent, treat or ameliorate at least one symptom of atypical hemolytic uremic syndrome (aHUS), the method comprising administering a therapeutically effective amount of an anti-C5 antibody or antigen-binding fragment thereof of the invention to a subject in need thereof. In some embodiments, the present invention provides methods to ameliorate or reduce the severity of at least one symptom or indication of paroxysmal nocturnal hemoglobinuria (PNH) in a subject by administering an anti-C5 antibody of the invention. In some embodiments, the antibody or antigen-binding fragment thereof may be administered prophylactically or therapeutically to a subject having or at risk of having a C5-associated disease or disorder. In certain embodiments, the antibody or antigen-binding fragment thereof the invention is administered in combination with a second therapeutic agent to the subject in need thereof. The second therapeutic agent may be selected from the group consisting of an anti-inflammatory drug (such as corticosteroids, and non-steroidal anti-inflammatory drugs), a different antibody to C5 protein, a dietary supplement such as anti-oxidants and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with an antibody or antigen-binding fragment thereof of the invention, if such side effect(s) should occur. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof may be administered at a dose of about 0.1 mg/kg of body weight to about 100 mg/kg of body weight of the subject. In certain embodiments, an antibody of the present invention may be administered at one or more doses comprising between 50 mg to 600 mg.
The present invention also includes use of an anti-C5 antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from the blockade of C5 binding and/or activity.
Other embodiments will become apparent from a review of the ensuing detailed description.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows inhibition of C5a levels by anti-C5 antibody H4H12166P in a dose-dependent manner, as determined by ELISA (described in Example 9 herein).
FIG. 2 shows total serum concentration vs. time following a single 15 mg/kg intravenous injection of H4H12166P, H4H12161P, or Comparator 2 to male cynomolgus monkeys (described in Example 10 herein). Concentration-time profiles were plotted through the first post dose below the limit of quantitation (BLQ) result, if applicable, which was imputed as LLOQ/2. Each data point represents Mean (±SD) (n=4 animals/group); concentrations considered to be impacted by ADA were excluded from 1 animal in the H4H12166P group and 1 animal in the H4H12161P group starting at Day 36 and Day 29, respectively. LLOQ=Lower limit of quantification
FIG. 3A and FIG. 3B show percent hemolysis vs. time in ex vivo red blood cell (A) Classical Pathway and (B) Alternative Pathway assays following a single intravenous injection of H4H12166P, H4H12161P or Comparator 2 to male cynomolgus monkeys. % hemolysis, calculated as a ratio of experimental vs. maximal lysis with % background lysis subtracted from both values, is related to the amount of C5 inhibited by the particular anti-C5 antibody present in the serum at a given time point. Each data point represents Mean (±SD).
FIG. 4 shows total serum concentration vs. time profiles of selected anti-C5 antibodies in mice humanized for C5 (described in Example 11 herein). Humanized C5 mice were administered a single 15 mg/kg subcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each data point represents the mean±s.e.m. (n=4-5 each). Antibody concentrations in sera were monitored 1, 10, 20, 30 and 40 days post injection using a sandwich ELISA.
FIG. 5 shows percent hemolysis vs. time in an ex vivo complement classical pathway hemolysis assay of selected anti-C5 antibodies in mice humanized for C5. Humanized C5 mice were administered a single 15 mg/kg subcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each data point represents the mean±s.e.m. (n=4-5 each). The percent hemolysis in serum was monitored at predose, 10, 20, 30, 40 and 50 days post injection. % hemolysis, calculated as a ratio of experimental vs. maximal lysis with % background lysis subtracted from both values, is related to the amount of C5 inhibited by the particular anti-C5 antibody present in the serum at a given time point.
FIG. 6 shows total serum concentration vs. time profiles of selected anti-C5 antibodies in mice humanized for C5 (described in Example 11 herein). Mice were administered a single 15 mg/kg subcutaneous dose of H4H12166P, H4H12161P, Comparator 1 or IgG4P isotype control. Each data point represents the mean±s.e.m. (n=5 each). Antibody levels in sera were monitored 6 hours, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45 and 59 days post injection using a sandwich ELISA.
FIG. 7 is a graph showing optical coherence tomography (OCT) scores in mice treated with isotype control or with anti-C5 antibody M1M17628N at 10 mg/kg or 50 mg/kg (described in Example 14 herein). ****p<0.0001, two-way ANOVA treatment with anti-C5 antibody at 50 mg/kg vs. no treatment or treatment with isotype control.
FIG. 8 shows inhibition of classical pathway hemolysis by anti-C5 antibody M1M17628N in the absence of C3 (A); and in the presence of 80 μg/mL of human C3 (B) (described in Example 14 herein).
FIG. 9 shows cell cluster count in C5 humanized mice treated with isotype control or with anti-human C5 antibody H4H12170P at 10 mg/kg or 50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group
FIG. 10 is a graph showing OCT scores in C5 humanized mice treated with isotype control or with anti-human C5 antibody H4H12170P at 10 mg/kg or 50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group
FIG. 11 is a graph showing OCT scores in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg/kg or 10 mg/kg, or Comparator 2 at 10 mg/kg. n=6-12 eyes for each group (described in Example 15 herein).
FIG. 12 shows cell cluster count in C5 humanized mice treated with isotype control, anti-human C5 antibody H4H12166P at 3 mg/kg or 10 mg/kg, or Comparator 2 at 10 mg/kg. n=6-12 eyes for each group (described in Example 15 herein).
FIG. 13 is a survival curve of NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein).
FIG. 14A and FIG. 14B show levels of (A) urinary albumin and (B) urinary albumin normalized to urinary creatinine in NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein).
FIG. 15 shows levels of blood urea nitrogen in NZBWF1 mice treated with isotype control or with anti-C5 antibodies M1M17628N or M1M17627N (described in Example 17 herein).
FIG. 16 is a graph showing inhibition of antibody-dependent cytotoxicity of astrocytes by anti-C5 antibodies H4H12166P, H4H12170P, Comparator 1 and Comparator 2, as described in Example 18.
DETAILED DESCRIPTION
Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
Definitions
The term “C05”, also called “complement component 5” or “complement factor 5” refers to the serum protein of the complement cascade. The C5 protein is a 1676 amino acid protein comprising two chains, alpha and beta. The protein represents the convergence point for three complement activation pathways: classical pathway, alternative pathway and the mannose binding lectin pathway. The amino acid sequence of full-length C5 protein is exemplified by the amino acid sequence provided in GenBank as accession number NP_001726.2 (SEQ ID NO: 355). The term “C5” includes recombinant C5 protein or a fragment thereof. The term also encompasses C5 protein or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence such as ROR1. For example, the term includes sequences exemplified by the sequence shown in SEQ ID NO: 356 or 357, comprising a histidine tag at the C-terminal, coupled to amino acid residues 19-1676 of full-length C5 protein. The term also includes protein variants that comprise a histidine tag at the C-terminal, coupled to amino acid residues 19-1676 of full-length C5 protein with a R885H change or a R885C change.
The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CH1, CH2 and CH3).
Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (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. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).
CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.
The fully human anti-C5 monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
The present invention also includes fully human anti-C5 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-C5 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
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 mAbs 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), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.
The term “recombinant”, as used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.
The term “specifically binds,” or “binds specifically to”, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10−8 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, which bind specifically to C5. Moreover, multi-specific antibodies that bind to one domain in C5 and one or more additional antigens or a bi-specific that binds to two different regions of C5 are nonetheless considered antibodies that “specifically bind”, as used herein.
The term “high affinity” antibody refers to those mAbs having a binding affinity to C5, expressed as KD, of at least 10−8 M; preferably 10−9 M; more preferably 10−10M, even more preferably 10−11 M, even more preferably 10−12 M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.
By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from C5, with a rate constant of 1×10−3 s−1 or less, preferably 1×10−4 s−1 or less, as determined by surface plasmon resonance, e.g., BIACORE™.
The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms “antigen-binding fragment” of an antibody, or “antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to C5 protein.
In specific embodiments, antibody or antibody fragments of the invention may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), a second anti-C5 antibody, or any other therapeutic moiety useful for treating a C5-associated disease or disorder.
An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds C5, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than C5.
A “blocking antibody” or a “neutralizing antibody”, as used herein (or an “antibody that neutralizes C5 activity” or “antagonist antibody”), is intended to refer to an antibody whose binding to C5 results in inhibition of at least one biological activity of C5. For example, an antibody of the invention may prevent or block complement-mediated hemolysis by classical pathway or alternative pathway.
The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
The term “cross-competes”, as used herein, means an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice-versa. In certain embodiments, the first antibody and second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different, but overlapping epitopes such that binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Cross-competition between two antibodies may be expressed as the binding of the second antibody that is less than the background signal due to self-self binding (wherein first and second antibodies is the same antibody). Cross-competition between 2 antibodies may be expressed, for example, as % binding of the second antibody that is less than the baseline self-self background binding (wherein first and second antibodies is the same antibody).
The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.
By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
As used herein, the term “subject” refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and/or treatment of a C5-associated disease or disorder such as atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria (PNH). The term includes human subjects who have or are at risk of having such a disease or disorder.
As used herein, the terms “treat”, “treating”, or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of a C5-associated disease or disorder due to the administration of a therapeutic agent such as an antibody of the present invention to a subject in need thereof. The terms include inhibition of progression of disease or of worsening of a symptom/indication. The terms also include positive prognosis of disease, i.e., the subject may be free of disease or may have reduced disease upon administration of a therapeutic agent such as an antibody of the present invention. The therapeutic agent may be administered at a therapeutic dose to the subject.
The terms “prevent”, “preventing” or “prevention” refer to inhibition of manifestation of a C5-associated disease or disorder or any symptoms or indications of such a disease or disorder upon administration of an antibody of the present invention.
Antigen-Binding Fragments of Antibodies
Unless specifically indicated otherwise, the term “antibody,” as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “full antibody molecules”) as well as antigen-binding fragments thereof. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms “antigen-binding fragment” of an antibody, or “antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to C5 protein. An antibody fragment may include a Fab fragment, a F(ab′)2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multi-specific antigen-binding molecule. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL—CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific). A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.
Preparation of Human Antibodies
Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to C5 protein.
An immunogen comprising any one of the following can be used to generate antibodies to C5 protein. In certain embodiments, the antibodies of the invention are obtained from mice immunized with a full length, native C5 protein (See, for example, GenBank accession number NP_001726.2) (SEQ ID NO: 355), or with DNA encoding the protein or fragment thereof. Alternatively, the protein or a fragment thereof may be produced using standard biochemical techniques and modified and used as immunogen. In certain embodiments of the invention, the immunogen is a fragment of C5 protein that ranges from about amino acid residues 19-1676 of SEQ ID NO: 355.
In some embodiments, the immunogen may be a recombinant C5 protein or fragment thereof expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.
Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to C5 are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.
Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.
Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
Bioequivalents
The anti-C5 antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind C5 protein. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody or antibody fragment of the invention.
Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.
In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.
Bioequivalent variants of the antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.
Anti-C5 Antibodies Comprising Fc Variants
According to certain embodiments of the present invention, anti-C5 antibodies are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes anti-C5 antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A) modification.
For example, the present invention includes anti-C5 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 2571 and 3111 (e.g., P2571 and Q3111); 2571 and 434H (e.g., P2571 and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.
The present invention also includes anti-C5 antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Patent Application Publication 2014/0243504, the disclosure of which is hereby incorporated by reference in its entirety).
Biological Characteristics of the Antibodies
In general, the antibodies of the present invention function by binding to C5 protein and preventing its cleavage to C5a and C5b. For example, the present invention includes antibodies and antigen-binding fragments of antibodies that bind C5 protein (e.g., at 25° C. or at 37° C.) with a KD of less than 9 nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind C5 with a KD of less than about 9 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments thereof that bind human C5 protein with a dissociative half-life (t %2) of greater than about 2 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind C5 protein with a t % of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, greater than about 200 minutes, or greater than about 250 minutes, as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in Example 3 herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments thereof that bind human C5 protein with a dissociative half-life (t %) of greater than about 1.5 minutes as measured by surface plasmon resonance at 37° C., e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind C5 protein with a t % of greater than about 2 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 25 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 150 minutes, or greater than about 200 minutes, as measured by surface plasmon resonance at 37° C., e.g., using an assay format as defined in Example 3 herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments of antibodies that bind monkey C5 protein (e.g., at 25° C. or at 37° C.) with a KD of less than 120 nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind monkey C5 with a KD of less than about 120 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, or less than 250 pM, as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments of antibodies that bind modified human C5 protein with R885H change (exemplified by SEQ ID NO: 356) with a KD of less than 70 nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. C5 variants have shown poor response to anti-C5 antibodies previously disclosed in the art (e.g., Nishimura et al 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, the antibodies or antigen-binding fragments thereof bind the modified human C5 with a KD of less than about 65 nM, less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 3 nM, or less than 2 nM, as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments of antibodies that bind modified human C5 protein with R885C change (exemplified by SEQ ID NO: 357) with a KD of less than 160 nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. C5 variants have shown poor response to anti-C5 antibodies previously disclosed in the art (e.g., Nishimura et al 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, the antibodies or antigen-binding fragments thereof bind the modified human C5 with a KD of less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than 2 nM, as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments of antibodies that inhibit complement dependent cytotoxicity (CDC) with IC50 less than 10 nM as measured by a luminescence assay, e.g., using the assay format as defined in Example 6 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof inhibit CDC with IC50 less than about 5 nM, less than about 3.5 nM, or less than about 2 nM, as measured by a B-cell luminescence assay, e.g., using the assay format as defined in Example 6 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block human C5-mediated classical pathway (CP) hemolysis by more than 94% and with an IC50 less than 6 nM, as measured by a CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block CP hemolysis with IC50 less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, or less than about 2 nM, as measured by CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block human C5-mediated alternative pathway (AP) hemolysis by more than 70% and with an IC50 less than 165 nM, as measured by a AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block AP hemolysis with IC50 less than about 160 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less than about 20 nM, as measured by AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block African green monkey C5-mediated classical pathway (CP) hemolysis by more than 40% and with an IC50 less than 185 nM, as measured by a CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block CP hemolysis with IC50 less than about 180 nM, less than about 150 nM, less than about 100 nM, less than about 75 nM, or less than about 50 nM, as measured by CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block African green monkey C5-mediated alternative pathway (AP) hemolysis with an IC50 less than 235 nM, as measured by a AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block AP hemolysis with IC50 less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, or less than about 20 nM, as measured by AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block more than 90% of cynomolgus monkey C5-mediated classical pathway (CP) hemolysis with an IC50 less than 145 nM, as measured by a CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block CP hemolysis with IC50 less than about 140 nM, less than about 120 nM, less than about 100 nM, less than about 75 nM, or less than about 50 nM, as measured by CP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that block cynomolgus monkey C5-mediated alternative pathway (AP) hemolysis with an IC50 less than 30 nM, as measured by an AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof block AP hemolysis with IC50 less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than about 2 nM, as measured by AP hemolysis assay, e.g., using the assay format as defined in Example 8 herein, or a substantially similar assay.
The present invention also includes antibodies and antigen-binding fragments that show improved pharmacokinetic (PK) and pharmacodynamic (PD) properties as compared to anti-C5 antibodies in the art. The anti-C5 antibodies of the present invention show less susceptibility to target-mediated clearance upon administration, as shown in Examples 9 and 10 herein. In certain embodiments, the present invention includes anti-C5 antibodies and antigen-binding fragments thereof that show serum concentrations for extended periods, e.g., more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 50 days, more than 55 days, or more than 60 days, as described in Examples 9 and 10 herein. In certain embodiments, the anti-C5 antibodies of the present invention show an extended serum half-life of more than 10 days, as compared to anti-C5 antibodies in the art.
In certain embodiments, the present invention provides anti-C5 antibodies and antigen-binding fragments thereof that have high affinity for human C5 (e.g., KD less than 0.3 nM) and a lower clearance (e.g., extended serum half-life, improved pharmacodynamic activity over more days than previously known anti-C5 antibodies). Such antibodies of the present invention may be advantageously used with less frequent dosing in a subject with a C5-associated disease or disorder.
In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that binds specifically to C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C., as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d) has serum concentration of more than 10 μg/mL through day 70 upon administration to cynomolgus monkey; (e) blocks CP- and AP hemolysis through day 35 upon administration to cynomolgus monkey, as measured in an ex vivo hemolysis assay; (f) has serum half-life of more than 10 days in cynomolgus monkey; (g) has serum concentration of more than 10 μg/mL through day 40 upon administration to C5-humanized mice; (h) blocks CP hemolysis through day 30 upon administration to C5-humanized mice, as measured in an ex vivo hemolysis assay; and (i) has serum half-life of more than 10 days in C5-humanized mice.
In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that binds specifically to C5 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C., as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM, as measured in a surface plasmon resonance assay; (e) binds to human C5 variant R885H (SEQ ID NO: 356) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (f) binds to human C5 variant R885C (SEQ ID NO: 357) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (CP) hemolysis by more than 95% and with IC50 less than 6 nM, as measured in a CP hemolysis assay; (h) blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70% and with IC50 less than 165 nM, as measured in a AP hemolysis assay; (i) inhibits African green monkey C5-mediated CP hemolysis with IC50 less than 185 nM, as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with IC50 less than 235 nM, as measured in a AP hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM, as measured in a CP hemolysis assay; and (I) inhibits cynomolgus monkey C5-mediated AP hemolysis with IC50 less than 30 nM, as measured in a AP hemolysis assay.
The antibodies of the present invention may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present invention will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein.
Epitope Mapping and Related Technologies
The present invention includes anti-C5 antibodies which interact with one or more amino acids found within one or more regions of the C5 protein molecule including, the alpha polypeptide and beta polypeptide. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the C5 protein molecule (e.g. a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within either or both of the aforementioned domains of the protein molecule (e.g. a conformational epitope).
Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the invention into groups of antibodies binding different epitopes.
In certain embodiments, the anti-C5 antibodies or antigen-binding fragments thereof bind an epitope within any one or more of the regions exemplified in C5 protein, either in natural form, as exemplified in SEQ ID NO: 355, or recombinantly produced, or to a fragment thereof. In some embodiments, the antibodies of the invention bind to a region comprising one or more amino acids selected from the group consisting of amino acid residues 19-1676 of human C5 protein.
In certain embodiments, the antibodies of the invention, interact with at least one amino acid sequence selected from the group consisting of amino acid residues ranging from about position 19 to about position 750; or amino acid residues ranging from about position 751 to about position 1676 of SEQ ID NO: 355.
In certain embodiments, the present invention includes anti-C5 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the alpha and/or the beta chain of C5 (SEQ ID NO: 359). The epitope(s) may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within alpha chain and/or beta chain of C5. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within C5. As shown in Example 11 herein, the epitope of C5 with which the exemplary antibody of the invention H4H12166P interacts is defined by: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) which corresponds to amino acids 591 to 599 comprised in the beta chain of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775 to 794 comprised in the alpha chain of SEQ ID NO: 359. Accordingly, the present invention includes anti-C5 antibodies that interact with one or more amino acids contained within the region consisting of (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360), which corresponds to amino acids 591 to 599 of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQ ID NO: 361), which corresponds to amino acids 775 to 794 of SEQ ID NO: 359.
The present invention includes anti-C5 antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in Table 1. Likewise, the present invention also includes anti-C5 antibodies that compete for binding to C5 protein or a fragment thereof with any of the specific exemplary antibodies listed in Table 1. For example, the present invention includes anti-C5 antibodies that cross-compete for binding to C5 protein with one or more antibodies listed in Table 1.
One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-C5 antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-C5 antibody of the invention, the reference antibody is allowed to bind to a C5 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the C5 protein molecule is assessed. If the test antibody is able to bind to C5 following saturation binding with the reference anti-C5 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-C5 antibody. On the other hand, if the test antibody is not able to bind to the C5 protein following saturation binding with the reference anti-C5 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-C5 antibody of the invention.
To determine if an antibody competes for binding with a reference anti-C5 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a C5 protein under saturating conditions followed by assessment of binding of the test antibody to the C5 molecule. In a second orientation, the test antibody is allowed to bind to a C5 molecule under saturating conditions followed by assessment of binding of the reference antibody to the C5 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the C5 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to C5. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.
Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
Immunoconjugates
The invention encompasses a human anti-C5 monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), to treat a C5-associated disease or disorder (e.g., atypical hemolytic uremic syndrome). As used herein, the term “immunoconjugate” refers to an antibody which is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein or a therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second different antibody to C5 protein. The type of therapeutic moiety that may be conjugated to the anti-C5 antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see for example, WO 05/103081.
Multi-Specific Antibodies
The antibodies of the present invention may be mono-specific, bi-specific, or multi-specific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.
Any of the multi-specific antigen-binding molecules of the invention, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of ordinary skill in the art.
In some embodiments, C5-specific antibodies are generated in a bi-specific format (a “bi-specific”) in which variable regions binding to distinct domains of C5 protein are linked together to confer dual-domain specificity within a single binding molecule. Appropriately designed bi-specifics may enhance overall C5-protein inhibitory efficacy through increasing both specificity and binding avidity. Variable regions with specificity for individual domains, (e.g., segments of the N-terminal domain), or that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to bind simultaneously to the separate epitopes, or to different regions within one domain. In one example for a bi-specific, heavy chain variable regions (VH) from a binder with specificity for one domain are recombined with light chain variable regions (VL) from a series of binders with specificity for a second domain to identify non-cognate VL partners that can be paired with an original VH without disrupting the original specificity for that VH. In this way, a single VL segment (e.g., VL1) can be combined with two different VH domains (e.g., VH1 and VH2) to generate a bi-specific comprised of two binding “arms” (VH1-VL1 and VH2-VL1). Use of a single VL segment reduces the complexity of the system and thereby simplifies and increases efficiency in cloning, expression, and purification processes used to generate the bi-specific (See, for example, U.S. Ser. No. 13/022,759 and US2010/0331527).
Alternatively, antibodies that bind more than one domains and a second target, such as, but not limited to, for example, a second different anti-C5 antibody, may be prepared in a bi-specific format using techniques described herein, or other techniques known to those skilled in the art. Antibody variable regions binding to distinct regions may be linked together with variable regions that bind to relevant sites on, for example, the extracellular domain of C5, to confer dual-antigen specificity within a single binding molecule. Appropriately designed bi-specifics of this nature serve a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for outside the extracellular domain and are paired on a structural scaffold that allows each variable region to bind to the separate antigens.
An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.
Other exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).
Therapeutic Administration and Formulations
The invention provides therapeutic compositions comprising the anti-C5 antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
The dose of antibody may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When an antibody of the present invention is used for treating a disease or disorder in an adult patient, or for preventing such a disease, it is advantageous to administer the antibody of the present invention normally at a single dose of about 0.1 to about 100 mg/kg body weight, more preferably about 5 to about 80, about 10 to about 70, or about 20 to about 50 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).
The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M., et al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995, each incorporated herein in its entirety.
In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.
The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.
Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 300 mg and in about 10 to about 300 mg for the other dosage forms.
Therapeutic Uses of the Antibodies
The antibodies of the present invention are useful for the treatment, and/or prevention of a disease or disorder or condition associated with C5 and/or for ameliorating at least one symptom associated with such disease, disorder or condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the invention may be administered at a therapeutic dose to a patient with a disease or disorder or condition associated with C5.
In certain embodiments, the antibodies of the present invention are useful in treating or preventing a symptom or indication of atypical hemolytic uremic syndrome (aHUS). Symptoms and indications of aHUS include, but are not limited to, platelet activation, hemolysis, systemic thrombotic microangiopathy (formation of blood clots in small blood vessels throughout the body) leading to stroke, heart attack, kidney failure and/or death, end-stage renal disease, permanent renal damage, abdominal pain, confusion, edema, fatigue, nausea/vomiting, diarrhea, and microangiopathic anemia.
In certain embodiments, the antibodies of the present invention are useful in treating or preventing a symptom or indication of paroxysmal nocturnal hemoglobinuria (PNH). Symptoms and indications of PNH include, but are not limited to, destruction of red blood cells, thrombosis (including deep vein thrombosis, pulmonary embolism), intravascular hemolytic anemia, red discoloration of urine, symptoms of anemia such as tiredness, shortness of breath, and palpitations, abdominal pain and difficulty swallowing.
In certain embodiments, the antibodies of the present invention are useful for treating or preventing at least one symptom or indication of a C5-associated disease or disorder selected from the group consisting of neurological disorders, renal disorders, multiple sclerosis, stroke, Guillain Barre Syndrome, traumatic brain injury, Parkinson's disease, disorders of inappropriate or undesirable complement activation, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 therapy, inflammatory disorders, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal injury including burns or frostbite, post-ischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membraneproliferative glomerulonephritis, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disorders and autoimmune diseases, diabetic nephropathy, Alport's syndrome, progressive kidney failure, proteinuric kidney diseases, renal ischemia-reperfusion injury, lupus nephritis, glomerulopathy, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, membrano-proliferative nephritis, hemolytic anemia, neuromyelitis optica, renal transplant, inherited CD59 deficiency, psoriasis, and myasthenia gravis. In certain other embodiments, the antibodies of the present invention are useful for treating or preventing at least one symptom or indication of a C5-associated disease or disorder selected from the group consisting of lung disease and disorders such as dyspnea, hemoptysis, ARDS, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolisms and infarcts, pneumonia, fibrogenic dust diseases, injury due to inert dusts and minerals (e.g., silicon, coal dust, beryllium, and asbestos), pulmonary fibrosis, organic dust diseases, chemical injury (due to irritant gasses and chemicals, e.g., chlorine, phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia, and hydrochloric acid), smoke injury, thermal injury (e.g., burn, freeze), asthma, allergy, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's Syndrome, pulmonary vasculitis, hereditary angioedema, and immune complex-associated inflammation.
In certain embodiments, the antibodies of the invention are useful to treat subjects suffering from an ocular disease such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, ocular angiogenesis (ocular neovascularization affecting choroidal, corneal or retinal tissue), geographic atrophy (GA), uveitis and neuromyelitis optica. The antibodies of the present invention may be used to treat or to ameliorate at least one symptom or indication of dry AMD or wet AMD. In some embodiments, the antibodies of the invention are useful in preventing or slowing rate of loss of vision. In one embodiment, the antibodies of the present invention are useful in reducing drusen in the eye of a subject with dry AMD. In one embodiment, the antibodies of the present invention are useful in preventing or reducing/slowing loss of vision in a subject with AMD.
One or more antibodies of the present invention may be administered to relieve or prevent or decrease the severity of one or more of the symptoms or conditions/indications of the ocular disease or disorder. The antibodies may be used to ameliorate or reduce the severity of at least one symptom including, but not limited to loss of vision, visual distortion, difficulty adapting to low light levels, crooked central vision, increase in haziness of central/overall vision, presence of drusen (tiny accumulations of extracellular material that build up on the retina), pigmentary changes, distorted vision in the form of metamorphopsia, in which a grid of straight lines appears wavy and parts of the grid may appear blank, exudative changes (hemorrhages in the eye, hard exudates, subretinal/sub-RPE/intraretinal fluid), slow recovery of visual function after exposure to bright light (photostress test), incipient and geographic atrophy, visual acuity drastically decreasing (two levels or more), e.g., 20/20 to 20/80, preferential hyperacuity perimetry changes (for wet AMD), blurred vision, gradual loss of central vision (for those with non-exudative macular degeneration, rapid onset of vision loss (often caused by leakage and bleeding of abnormal blood vessels in subjects with exudative macular degeneration, central scotomas (shadows or missing areas of vision), trouble discerning colors, specifically dark ones from dark ones and light ones from light ones, loss in contrast sensitivity, straight lines appear curved in an Amsler grid.
It is also contemplated herein to use one or more antibodies of the present invention prophylactically to subjects at risk for developing macular degeneration such as subjects over the age of 50, subjects with a family history of macular degeneration, smokers, and subjects with obesity, high cholesterol, cardiovascular disease, or unhealthy diet.
In a further embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition or medicament for treating patients suffering from a disease or disorder associated with C5. In another embodiment of the invention, the present antibodies are used as adjunct therapy with any other agent or any other therapy known to those skilled in the art useful for treating or ameliorating a disease or disorder associated with C5.
Combination Therapies
Combination therapies may include an anti-C5 antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention, or with a biologically active fragment of an antibody of the invention. The antibodies of the present invention may be combined synergistically with one or more drugs or therapy used to treat a disease or disorder associated with C5. In some embodiments, the antibodies of the invention may be combined with a second therapeutic agent to ameliorate one or more symptoms of said disease.
Depending upon the C5-associated disease or disorder, the antibodies of the present invention may be used in combination with one or more additional therapeutic agents including, but not limited to, an anti-coagulant (e.g., warfarin, aspirin, heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors such as argatroban, lepirudin, bivalirudin, or dabigatran) an anti-inflammatory drug (e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), an antihypertensive (e.g., an angiotensin-converting enzyme inhibitor), an immunosuppressive agent (e.g., vincristine, cyclosporine A, or methotrexate), a fibrinolytic agent (e.g., ancrod, E-aminocaproic acid, antiplasmin-a1, prostacyclin, and defibrotide), a lipid-lowering agent such as an inhibitor of hydroxymethylglutaryl CoA reductase, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an anti-seizure agent (e.g., magnesium sulfate), a C3 inhibitor, or an anti-thrombotic agent.
In certain embodiments, the second therapeutic agent is another antibody to C5 protein. It is contemplated herein to use a combination (“cocktail”) of antibodies with broad neutralization or inhibitory activity against C5. In some embodiments, non-competing antibodies may be combined and administered to a subject in need thereof. In some embodiments, the antibodies comprising the combination bind to distinct non-overlapping epitopes on the protein. The antibodies comprising the combination may block the C5 binding to C5 convertase and/or may prevent/inhibit cleavage of C5 into C5a and C5b. In certain embodiments, the second antibody may possess longer half-life in human serum.
As used herein, the term “in combination with” means that additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti-C5 antibody of the present invention. The term “in combination with” also includes sequential or concomitant administration of an anti-C5 antibody and a second therapeutic agent.
The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-C5 antibody of the present invention. For example, a first component may be deemed to be administered “prior to” a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-C5 antibody of the present invention. For example, a first component may be deemed to be administered “after” a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti-C5 antibody of the present invention. “Concurrent” administration, for purposes of the present invention, includes, e.g., administration of an anti-C5 antibody and an additional therapeutically active component to a subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-C5 antibody and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-C5 antibody may be administered intravenously, and the additional therapeutically active component may be administered orally). In any event, administering the components in a single dosage from, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered “concurrent administration,” for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-C5 antibody “prior to”, “concurrent with,” or “after” (as those terms are defined herein above) administration of an additional therapeutically active component is considered administration of an anti-C5 antibody “in combination with” an additional therapeutically active component.
The present invention includes pharmaceutical compositions in which an anti-C5 antibody of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.
Administration Regimens
According to certain embodiments, a single dose of an anti-C5 antibody of the invention (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of an anti-C5 antibody (or a pharmaceutical composition comprising a combination of an anti-C5 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an anti-C5 antibody of the invention. As used herein, “sequentially administering” means that each dose of anti-C5 antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an anti-C5 antibody, followed by one or more secondary doses of the anti-C5 antibody, and optionally followed by one or more tertiary doses of the anti-C5 antibody.
The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the anti-C5 antibody of the invention. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-C5 antibody, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-C5 antibody contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
In certain exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered 1 to 48 hours (e.g., 1, 1%, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more) after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of anti-C5 antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-C5 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
In certain embodiments of the invention, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
Diagnostic Uses of the Antibodies
The anti-C5 antibodies of the present invention may be used to detect and/or measure C5 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present invention in assays to detect a C5-associated-disease or disorder. Exemplary diagnostic assays for C5 may comprise, e.g., contacting a sample, obtained from a patient, with an anti-C5 antibody of the invention, wherein the anti-C5 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate C5 from patient samples. Alternatively, an unlabeled anti-C5 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure C5 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Samples that can be used in C5 diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of either C5 protein, or fragments thereof, under normal or pathological conditions. Generally, levels of C5 protein in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with C5) will be measured to initially establish a baseline, or standard, level of C5. This baseline level of C5 can then be compared against the levels of C5 measured in samples obtained from individuals suspected of having a C5-associated condition, or symptoms associated with such condition.
The antibodies specific for C5 protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.
Selected Embodiments
Selected embodiments of the present disclosure include the following:
In Embodiment 1, the present invention includes an isolated antibody or antigen-binding fragment thereof that binds specifically to complement factor 5 (C05) protein, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within C5 (SEQ ID NO: 359), as determined by hydrogen/deuterium exchange.
In Embodiment 2, the present invention includes the isolated antibody of antigen-binding fragment of Embodiment 1, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within the alpha chain and/or the beta chain of C5, as determined by hydrogen/deuterium exchange.
In Embodiment 3, the present invention includes the isolated antibody of antigen-binding fragment of Embodiments 1 or 2, wherein the antibody or antigen-binding fragment thereof does not interact with an amino acid of the C5a anaphylatoxin region of C5, as determined by hydrogen/deuterium exchange.
In Embodiment 4, the present invention includes the isolated antibody of antigen-binding fragment of any one of Embodiments 1 to 3, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within SEQ ID NO: 360 and/or SEQ ID NO: 361, as determined by hydrogen/deuterium exchange.
In Embodiment 5, the present invention includes the isolated antibody or antigen-binding fragment of any one of Embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of (a) amino acids 591 to 599 of SEQ ID NO: 359; (b) amino acids 593 to 599 of SEQ ID NO: 359; (c) amino acids 775 to 787 of SEQ ID NO: 359; (d) amino acids 775 to 794 of SEQ ID NO: 359; and (e) amino acids 779 to 787 of SEQ ID NO: 359.
In Embodiment 6, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with at least five amino acids contained within an amino acid sequence selected from the group consisting of SEQ ID NOs: 360 and 361.
In Embodiment 7, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 5, wherein the antibody or antigen-binding fragment thereof interacts with the amino acid sequences of SEQ ID NOs: 360 and 361.
In Embodiment 8, the present invention includes an isolated antibody or antigen-binding fragment thereof that binds specifically to complement factor 5 (C5) protein, wherein the antibody or antigen-binding fragment thereof interacts with at least one of the following amino acid residues: N591, M592, A593, T594, G595, M596, D597, S598, W599, W775, E776, V777, H778, L779, V780, P781, R782, R783, K784, Q785, L786, Q787, F788, A789, L790, P791, D792, S793, or L794 of SEQ ID NO: 359.
In Embodiment 9, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 8, wherein the antibody has one or more of the following characteristics: (a) has serum concentration of more than 10 μg/mL through day 70 upon administration to cynomolgus monkey; (b) blocks classical pathway (CP) hemolysis through day 35 upon administration to cynomolgus monkey, as measured in an ex vivo hemolysis assay; (c) blocks alternative pathway (AP) hemolysis through day 35 upon administration to cynomolgus monkey, as measured in an ex vivo hemolysis assay; (d) has serum half-life of more than 10 days in cynomolgus monkey; (e) has serum concentration of more than 10 μg/mL through day 40 upon administration to C5-humanized mice; (f) blocks CP hemolysis through day 30 upon administration to C5-humanized mice, as measured in an ex vivo hemolysis assay; and (g) has serum half-life of more than 10 days in C5-humanized mice.
In Embodiment 10, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 9, wherein the antibody has an additional characteristic selected from the group consisting of: (a) is a fully human monoclonal antibody; (b) binds to human C5 with a dissociation constant (KD) of less than 0.9 nM at 25° C., as measured in a surface plasmon resonance assay; (c) binds to human C5 with a KD of less than 0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d) binds to monkey C5 with a KD of less than 65 nM, as measured in a surface plasmon resonance assay; (e) binds to human 05 variant R885H (SEQ ID NO: 356) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (f) binds to human C5 variant R885C (SEQ ID NO: 357) with a KD of less than 0.5 nM, as measured in a surface plasmon resonance assay; (g) blocks human C5-mediated classical pathway (CP) hemolysis by more than 95% and with IC50 less than 6 nM, as measured in a CP hemolysis assay; (h) blocks human C5-mediated alternative pathway (AP) hemolysis by more than 70% and with IC50 less than 165 nM, as measured in a AP hemolysis assay; (i) inhibits African green monkey C5-mediated CP hemolysis with IC50 less than 185 nM, as measured in a CP hemolysis assay; (j) inhibits African green monkey C5-mediated AP hemolysis with IC50 less than 235 nM, as measured in a AP hemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC50 less than 145 nM, as measured in a CP hemolysis assay; and (I) inhibits cynomolgus monkey C5-mediated AP hemolysis with IC50 less than 30 nM, as measured in a AP hemolysis assay.
In Embodiment 11, the present invention includes the isolated antibody or antigen-binding fragment of any one of Embodiments 1 to 10, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) sequences listed in Table 1.
In Embodiment 12, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 11, comprising: (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292, 308, 324, and 340; (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 126, 142, 150, 158, 174, 190, 206, 222, 238, 254, 270, 278, 294, 310, 326, and 342; (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 128, 144, 152, 160, 176, 192, 208, 224, 240, 256, 272, 280, 296, 312, 328, and 344; (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332, and 348; (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 118, 134, 166, 182, 198, 214, 230, 246, 262, 286, 302, 318, 334, and 350; and (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 120, 136, 168, 184, 200, 216, 232, 248, 264, 288, 304, 320, 336, and 352.
In Embodiment 13, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 12 comprising a HCVR having an amino acid sequence selected from the group consisting of HCVR sequences listed in Table 1.
In Embodiment 14, the present invention includes an isolated antibody or antigen-binding fragment thereof of Embodiment 13 comprising a LCVR having an amino acid sequence selected from the group consisting of LCVR sequences listed in Table 1.
In Embodiment 15, the present invention includes the isolated antibody or antigen-binding fragment of any one of Embodiments 11 to 14 comprising a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 98/114, 122/106, 98/130, 138/106, 146/106, 122/130, 146/114, 146/130, 138/130, 154/162, 170/178, 186/194, 202/210, 218/226, 234/242, 250/258, 266/258, 274/282, 290/298, 306/314, 322/330, and 338/346.
In Embodiment 16, the present invention includes the isolated antibody or antigen-binding fragment thereof of any one of Embodiments 11 to 15 comprising three CDRs contained within a HCVR selected from the group consisting of SEQ ID NOs: 50, 98, 138, and 202; and three CDRs contained within a LCVR selected from the group consisting of SEQ ID NOs: 58, 106, and 210.
In Embodiment 17, the present invention includes the isolated antibody or antigen-binding fragment thereof of Embodiment 16 comprising CDRs selected from the group consisting of: (a) SEQ ID NOs: 52, 54, 56, 60, 62, and 64; (b) SEQ ID NOs: 100, 102, 104, 108, 110, and 112; (c) SEQ ID NOs: 140, 142, 144, 108, 110, and 112; and (d) SEQ ID NOs: 204, 206, 208, 212, 214, and 216.
In Embodiment 18, the present invention includes the isolated antibody or antigen-binding fragment thereof of Embodiment 17 comprising a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50/58, 98/106, 138/106, and 202/210.
In Embodiment 19, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding to C5 with the antibody or antigen-binding fragment thereof of Embodiment 17.
In Embodiment 20, the present invention includes an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof of Embodiment 17.
In Embodiment 21, the present invention includes the antibody or antigen-binding fragment thereof of Embodiment 9 or 10 comprising a heavy chain variable region comprising an amino acid sequence listed in Table 1 having no more than 5 amino acid substitutions.
In Embodiment 22, the present invention includes the antibody or antigen-binding fragment thereof of Embodiment 21, comprising a light chain variable region comprising an amino acid sequence listed in Table 1 having no more than 5 amino acid substitutions.
In Embodiment 23, the present invention includes the antibody or antigen-binding fragment thereof of Embodiment 9 or 10 comprising a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 98.
In Embodiment 24, the present invention includes an antibody or antigen-binding fragment thereof of Embodiment 23 comprising a light chain variable region having at least 90% sequence identity to SEQ ID NO: 106.
In Embodiment 25, the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that blocks C5 cleavage to C5a and C5b comprising three CDRs of a HCVR, wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 122, 138, 146, 154, 170, 186, 202, 218, 234, 250, 266, 274, 290, 306, 322, and 338; and three CDRs of a LCVR, wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 114, 130, 162, 178, 194, 210, 226, 242, 258, 282, 298, 314, 330, and 346.
In Embodiment 26, the present invention includes a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to C5 according to any one of Embodiments 1 to 25 and a pharmaceutically acceptable carrier or diluent.
In Embodiment 27, the present invention includes an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a HCVR of an antibody as set forth in any one of Embodiments 1 to 25.
In Embodiment 28, the present invention includes an isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a LCVR of an antibody as set forth in any one of Embodiments 1 to 25.
In Embodiment 29, the present invention includes a vector comprising the polynucleotide sequence of Embodiment 27 or 28.
In Embodiment 30, the present invention includes a cell expressing the vector of Embodiment 29.
In Embodiment 31, the present invention includes a method of preventing, treating or ameliorating at least one symptom or indication of a disease or disorder associated with C5, the method comprising administering an antibody or antigen-binding fragment of any one of Embodiments 1 to 25 to a subject in need thereof.
In Embodiment 32, the present invention includes the method of Embodiment 31, wherein the disease or disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, geographic atrophy, uveitis, neuromyelitis optica, multiple sclerosis, stroke, Guillain Barre Syndrome, traumatic brain injury, Parkinson's disease, disorders of inappropriate or undesirable complement activation, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 therapy, inflammatory disorders, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, thermal injury including burns or frostbite, post-ischemic reperfusion conditions, myocardial infarction, capillary leak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy, membraneproliferative glomerulonephritis, diabetic nephropathy, Alport's syndrome, progressive kidney failure, proteinuric kidney diseases, renal ischemia-reperfusion injury, lupus nephritis, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infectious disease or sepsis, immune complex disorders and autoimmune diseases, renal disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolisms and infarcts, pneumonia, and myasthenia gravis.
In Embodiment 33, the present invention includes the method of Embodiment 31, wherein the disease or disorder is aHUS.
In Embodiment 34, the present invention includes the method of Embodiment 31, wherein the disease or disorder is PNH.
In Embodiment 35, the present invention includes the method of any one of Embodiments 31-34, wherein the pharmaceutical composition is administered prophylactically or therapeutically to the subject in need thereof.
In Embodiment 36, the present invention includes the method of any one of Embodiments 31 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.
In Embodiment 37, the present invention includes the method of Embodiment 36, wherein the second therapeutic agent is selected from the group consisting of an anti-coagulant, an anti-inflammatory drug, an antihypertensive, an immunosuppressive agent, a lipid-lowering agent, an anti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab, an anti-seizure agent, a C3 inhibitor, a second anti-C5 antibody, and an anti-thrombotic agent.
In Embodiment 38, the present invention includes the method of any one of Embodiments 31 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25° C., and pressure is at or near atmospheric.
Example 1: Generation of Human Antibodies to Complement Factor 5 (C5) Protein
Human antibodies to C5 protein were generated in a VELOCIMMUNE® mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions. The mice were immunized with serum purified human C5 protein (CALBIOCHEM® Cat #20-4888).
The antibody immune response was monitored by a C5-specific immunoassay. When a desired immune response was achieved splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce C5-specific antibodies. The cell lines were used to obtain several anti-C5 chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains); exemplary antibodies generated in this manner were designated as H2M11683N and H2M11686N.
Anti-C5 antibodies were also isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Pat. No. 7,582,298, herein specifically incorporated by reference in its entirety. Using this method, several fully human anti-C5 antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as H4H12159P, H4H12161P, H4H12163P, H4H12164P, H4H12166P, H4H12167P, H4H12168P, H4H12169P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2, H4H12177P2 and H4H12183P2.
The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.
Example 2: Heavy and Light Chain Variable Region Amino Acid and Nucleotide Sequences
Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-C5 antibodies of the invention.
TABLE 1
Amino Acid Sequence Identifiers
Antibody
SEQ ID NOs:
Designation
HCVR
HCDR1
HCDR2
HCDR3
LCVR
LCDR1
LCDR2
LCDR3
H2M11683N
2
4
6
8
10
12
14
16
H2M11686N
18
20
22
24
26
28
30
32
H4H12159P
34
36
38
40
42
44
46
48
H4H12161P
50
52
54
56
58
60
62
64
H4H12163P
66
68
70
72
74
76
78
80
H4H12164P
82
84
86
88
90
92
94
96
H4H12166P
98
100
102
104
106
108
110
112
H4H12166P2
98
100
102
104
114
116
118
120
H4H12166P3
122
124
126
128
106
108
110
112
H4H12166P4
98
100
102
104
130
132
134
136
H4H12166P5
138
140
142
144
106
108
110
112
H4H12166P6
146
148
150
152
106
108
110
112
H4H12166P7
122
124
126
128
130
132
134
136
H4H12166P8
146
148
150
152
114
116
118
120
H4H12166P9
146
148
150
152
130
132
134
136
H4H12166P10
138
140
142
144
130
132
134
136
H4H12167P
154
156
158
160
162
164
166
168
H4H12168P
170
172
174
176
178
180
182
184
H4H12169P
186
188
190
192
194
196
198
200
H4H12170P
202
204
206
208
210
212
214
216
H4H12171P
218
220
222
224
226
228
230
232
H4H12175P
234
236
238
240
242
244
246
248
H4H12176P2
250
252
254
256
258
260
262
264
H4H12177P2
266
268
270
272
258
260
262
264
H4H12183P2
274
276
278
280
282
284
286
288
H2M11682N
290
292
294
296
298
300
302
304
H2M11684N
306
308
310
312
314
316
318
320
H2M11694N
322
324
326
328
330
332
334
336
H2M11695N
338
340
342
344
346
348
350
352
The corresponding nucleic acid sequence identifiers are set forth in Table 2.
TABLE 2
Nucleic Acid Sequence Identifiers
Antibody
SEQ ID NOs:
Designation
HCVR
HCDR1
HCDR2
HCDR3
LCVR
LCDR1
LCDR2
LCDR3
H2M11683N
1
3
5
7
9
11
13
15
H2M11686N
17
19
21
23
25
27
29
31
H4H12159P
33
35
37
39
41
43
45
47
H4H12161P
49
51
53
55
57
59
61
63
H4H12163P
65
67
69
71
73
75
77
79
H4H12164P
81
83
85
87
89
91
93
95
H4H12166P
97
99
101
103
105
107
109
111
H4H12166P2
97
99
101
103
113
115
117
119
H4H12166P3
121
123
125
127
105
107
109
111
H4H12166P4
97
99
101
103
129
131
133
135
H4H12166P5
137
139
141
143
105
107
109
111
H4H12166P6
145
147
149
151
105
107
109
111
H4H12166P7
121
123
125
127
129
131
133
135
H4H12166P8
145
147
149
151
113
115
117
119
H4H12166P9
145
147
149
151
129
131
133
135
H4H12166P10
137
139
141
143
129
131
133
135
H4H12167P
153
155
157
159
161
163
165
167
H4H12168P
169
171
173
175
177
179
181
183
H4H12169P
185
187
189
191
193
195
197
199
H4H12170P
201
203
205
207
209
211
213
215
H4H12171P
217
219
221
223
225
227
229
231
H4H12175P
233
235
237
239
241
243
245
247
H4H12176P2
249
251
253
255
257
259
261
263
H4H12177P2
265
267
269
271
257
259
261
263
H4H12183P2
273
275
277
279
281
283
285
287
H2M11682N
289
291
293
295
297
299
301
303
H2M11684N
305
307
309
311
313
315
317
319
H2M11694N
321
323
325
327
329
331
333
335
H2M11695N
337
339
341
343
345
347
349
351
Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. “H4H,” “H2M,” etc.), followed by a numerical identifier (e.g. “11686,” “12166,” “12183,” etc., as shown in Table 2), followed by a “P,” “P2,” or “N” suffix. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., “H2M11686N,” “H4H12183P2,” “H4H12168P,” etc. The H4H and H2M prefixes on the antibody designations used herein indicate the particular Fc region isotype of the antibody. For example, an “H4H” antibody has a human IgG4 Fc comprising a serine to proline mutation in the hinge region (S108P) to promote dimer stabilization, and an “H2M” antibody has a mouse IgG2 Fc (a or b isotype) (all variable regions are fully human as denoted by the first ‘H’ in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs)—which are indicated by the numerical identifiers shown in Table 2—will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the Fc domain.
In certain embodiments, selected antibodies with a mouse IgG1 Fc were converted to antibodies with human IgG4 Fc. In one embodiment, the IgG4 Fc domain comprises 2 or more amino acid changes as disclosed in US20100331527.
To generate mutated antibodies, various residues in the complementary determining regions (CDRs) of H4H12166P were mutated to histidine to generate 9 mutated antibodies, identified as H4H12166P2 to H4H12166P10. Histidine mutations in the CDRs have been shown to confer pH-dependence of binding to target antigen leading to improved pharmacokinetics (Igawa et al. 2010, Nat. Biotechnol. 28: 1203-1207).
Control Constructs Used in the Following Examples
The following control constructs (anti-C5 antibodies) were included in the experiments disclosed herein, for comparative purposes: “Comparator 1,” a monoclonal antibody against human C5 having VH/VL sequences of antibody “h5G1.1” according to U.S. Pat. No. 6,355,245 (Alexion Pharmaceuticals, Inc.); and “Comparator 2,” a human monoclonal antibody against human C5 having VH/VL sequences of antibody “8109” according to US Patent Application Publication No. 2013/0022615 (Novartis).
Example 3: Antibody Binding to C5 as Determined by Surface Plasmon Resonance
Equilibrium dissociation constants (KD values) for C5 binding to purified anti-C5 antibodies were determined using a real-time surface plasmon resonance biosensor assay on a BIACORE™ T200 instrument. The BIACORE™ sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE Healthcare, # BR-1008-39) to capture anti-C5 antibodies expressed with human Fc constant regions. BIACORE™ binding studies were conducted in HBST running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20). Human C5 was obtained from a commercial source (EMD). Other C5 reagents were expressed with a C-terminal myc-myc-hexahistidine tag (subsequently referred to as C5-mmh). Human C5-mmh reagents were also expressed containing histidine and cysteine point mutations at arginine 885 (subsequently referred to as C5 R885H-mmh and C5 R885C-mmh, respectively). Different concentrations of human C5, human C5 R885H-mmh (SEQ ID No: 356), human C5 R885C-mmh (SEQ ID No: 357) and monkey C5-mmh (SEQ ID No: 358) (ranging from 100 nM to 1.23 nM, 3-fold dilutions) prepared in HBST running buffer were injected over the anti-C5 antibody captured surface at a flow rate of 30 μL/min. Association of all the C5 reagents to each of the captured monoclonal antibodies was monitored for 3 minutes and their dissociation in HBST running buffer was monitored for 8 minutes. All the binding kinetics experiments were performed at either 25° C. or 37° C. Kinetic association (ka) and dissociation (kd) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (t1/2) were calculated from the kinetic rate constants as: KD (M)=kd/ka and t1/2 (min)=ln 2/(60×kd)
Binding kinetic parameters for human C5 binding to anti-C5 antibodies at 25° C. and 37° C. are shown in Tables 3 and 4.
TABLE 3
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human C5
at 25° C.
Amount of
100 nM
Antibody
Human C5
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
231
439
4.17E+05
5.14E−05
1.23E−10
225
H4H12171P
51
64
1.49E+05
8.16E−05
5.49E−10
142
H4H12161P
38
64
2.58E+05
4.37E−05
1.70E−10
264
H4H12176P2
50
96
3.36E+05
6.75E−05
2.01E−10
171
H4H12163P
51
108
6.43E+05
2.08E−04
3.24E−10
55
H4H12167P
52
116
1.09E+06
1.31E−04
1.21E−10
88
H4H12175P
51
100
2.16E+05
1.96E−04
9.10E−10
59
H4H12159P
53
118
9.75E+05
7.13E−05
7.31E−11
162
H4H12164P
52
103
2.92E+05
8.84E−05
3.02E−10
131
H4H12168P
50
113
4.23E+05
4.75E−05
1.12E−10
243
H4H12169P
51
18
2.24E+05
4.40E−04
1.96E−09
26
H4H11686N
200
341
2.20E+05
3.31E−05
1.50E−10
349
H4H12170P
51
119
5.25E+05
6.79E−05
1.29E−10
170
H4H12177P2
47
60
6.56E+04
6.29E−05
9.59E−10
184
H4H12183P2
46
50
1.66E+05
2.70E−05
1.63E−10
427
H4H12166P
53
105
6.42E+05
1.10E−04
1.71E−10
105
H4H12166P2
53
95
8.26E+05
3.61E−04
4.38E−10
32
H4H12166P3
59
124
4.03E+05
4.65E−04
1.15E−09
25
H4H12166P4
49
92
4.46E+05
1.76E−04
3.95E−10
66
H4H12166P5
59
110
2.85E+05
3.28E−04
1.15E−09
35
H4H12166P6
64
131
4.89E+05
1.84E−04
3.75E−10
63
H4H12166P7
50
92
2.74E+05
1.01E−03
3.67E−09
11
H4H12166P8
50
91
4.84E+05
6.86E−04
1.42E−09
17
H4H12166P9
52
100
3.32E+05
2.64E−04
7.94E−10
44
H4H12166P10
49
69
1.57E+05
1.32E−03
8.38E−09
9
Comparator 1
232
250
9.69E+04
1.46E−04
1.51E−09
79
Comparator 2
117
170
2.62E+05
2.39E−04
9.12E−10
48
TABLE 4
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human C5
at 37° C.
Amount of
100 nM
Antibody
Human C5
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
257
492
4.54E+05
2.41E−04
5.32E−10
48
H4H12171P
59
58
1.22E+05
7.62E−04
6.27E−09
15
H4H12161P
40
66
1.16E+05
1.15E−04
9.90E−10
101
H4H12176P2
38
71
1.47E+05
2.34E−04
1.59E−09
49
H4H12163P
65
139
9.11E+05
6.65E−04
7.29E−10
17
H4H12167P
75
153
1.29E+06
3.81E−04
2.95E−10
30
H4H12175P
74
132
2.96E+05
6.37E−04
2.15E−09
18
H4H12159P
70
145
1.04E+06
1.07E−04
1.03E−10
108
H4H12164P
66
140
3.96E+05
1.28E−04
3.23E−10
90
H4H12168P
34
12
2.50E+04
4.64E−04
1.85E−08
25
H4H12169P
59
65
1.15E+05
3.52E−04
3.06E−09
33
H4H11686N
206
406
3.33E+05
1.56E−04
4.69E−10
74
H4H12170P
34
55
2.97E+05
4.15E−04
1.40E−09
28
H4H12177P2
41
37
4.42E+04
5.78E−04
1.31E−08
20
H4H12183P2
29
30
4.30E+04
2.50E−04
5.81E−09
46
H4H12166P
69
127
8.80E+05
2.30E−04
2.62E−10
50
H4H12166P2
68
110
9.50E+05
1.23E−03
1.29E−09
9
H4H12166P3
86
147
6.12E+05
1.27E−03
2.07E−09
9
H4H12166P4
63
108
5.05E+05
4.69E−04
9.30E−10
25
H4H12166P5
76
129
4.40E+05
1.22E−03
2.77E−09
9
H4H12166P6
90
157
5.42E+05
4.74E−04
8.75E−10
24
H4H12166P7
64
105
3.49E+05
2.58E−03
7.39E−09
4
H4H12166P8
65
98
6.75E+05
2.09E−03
3.10E−09
6
H4H12166P9
76
122
3.75E+05
6.39E−04
1.70E−09
18
H4H12166P10
64
82
2.27E+05
3.14E−03
1.38E−08
4
Comparator 1
185
246
1.47E+05
5.30E−04
3.61E−09
22
Comparator 2
119
205
2.85E+05
6.57E−04
2.30E−10
18
Monkey C5-mmh binding to anti-C5 antibodies at 25° C. and 37° C. are shown in Tables 5 and 6.
TABLE 5
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to monkey
C5-mmh at 25° C.
Amount
100 nM
of
monkey
Antibody
C5-mmh
Captured
Bound
ka
kd
KD
t 1/2
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
228
403
3.86E+05
2.47E−04
6.40E−10
47
H4H12171P
51
17
4.60E+04
2.26E−04
4.92E−09
51
H4H12161P
38
45
6.33E+04
2.48E−05
3.92E−10
465
H4H12176P2
50
69
1.82E+05
5.88E−05
3.22E−10
196
H4H12163P
50
98
3.11E+05
7.75E−04
2.49E−09
15
H4H12167P
52
111
4.19E+05
1.32E−04
3.15E−10
88
H4H12175P
51
59
6.42E+04
1.65E−03
2.57E−08
7
H4H12159P
53
116
3.54E+05
4.69E−05
1.33E−10
246
H4H12164P
51
66
1.27E+05
1.53E−03
1.20E−08
8
H4H12168P
50
86
1.73E+05
1.14E−04
6.60E−10
101
H4H12169P
51
22
1.64E+05
4.55E−03
2.78E−08
3
H4H11686N
196
247
1.57E+05
4.89E−04
3.11E−09
24
H4H12170P
51
92
2.62E+05
5.21E−05
1.99E−10
222
H4H12177P2
47
32
4.62E+04
9.92E−04
2.15E−08
12
H4H12183P2
47
23
4.88E+04
4.94E−04
1.01E−08
23
H4H12166P
52
90
2.05E+05
1.06E−03
5.15E−09
11
H4H12166P2
53
71
3.00E+05
3.16E−03
1.05E−08
4
H4H12166P3
59
72
1.68E+05
4.47E−03
2.66E−08
3
H4H12166P4
49
69
2.10E+05
1.78E−03
8.50E−09
6
H4H12166P5
59
56
1.44E+05
3.46E−03
2.40E−08
3
H4H12166P6
64
94
2.39E+05
2.66E−03
1.11E−08
4
H4H12166P7
50
36
1.36E+05
6.33E−03
4.65E−08
2
H4H12166P8
50
47
2.31E+05
4.99E−03
2.16E−08
2
H4H12166P9
52
55
1.70E+05
3.18E−03
1.87E−08
4
H4H12166P10
49
15
9.56E+04
6.16E−03
6.44E−08
2
Comparator 1*
228
11
N/A
N/A
3.11E−07
N/A
N/A = Not Available;
*SS = steady state analysis
TABLE 6
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to monkey
C5-mmh at 37° C.
100 nM
Amount of
monkey
Antibody
C5-mmh
Captured
Bound
ka
kd
KD
t 1/2
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
192
303
5.35E+05
1.15E−03
2.16E−09
10
H4H12171P
59
78
5.56E+05
1.03E−03
1.85E−09
11
H4H12161P
41
53
1.34E+05
7.45E−04
5.56E−09
16
H4H12176P2
36
47
1.35E+05
1.29E−03
9.60E−09
9
H4H12163P
64
129
3.90E+05
1.25E−03
3.20E−09
9
H4H12167P
74
146
5.37E+05
2.89E−04
5.39E−10
40
H4H12175P
74
74
1.77E+05
2.76E−03
1.56E−08
4
H4H12159P
70
137
4.12E+05
5.50E−05
1.33E−10
210
H4H12164P
65
99
1.86E+05
1.17E−03
6.30E−09
10
H4H12168P
34
29
5.33E+04
6.76E−04
1.27E−08
17
H4H12169P
59
64
2.51E+05
3.61E−03
1.43E−08
3
H4H11686N
145
195
2.33E+05
2.07E−03
8.88E−09
6
H4H12170P
34
60
5.21E+05
8.71E−04
1.67E−09
13
H4H12177P2
41
27
1.50E+05
7.17E−03
4.77E−08
2
H4H12183P2
28
13
5.40E+04
6.37E−03
1.18E−07
2
H4H12166P
68
110
2.19E+05
1.87E−03
8.55E−09
6
H4H12166P2
68
83
3.93E+05
2.97E−03
7.56E−09
4
H4H12166P3
85
92
2.23E+05
2.92E−03
1.31E−08
4
H4H12166P4
62
80
2.23E+05
1.83E−03
8.20E−09
6
H4H12166P5
75
70
1.50E+05
3.13E−03
2.09E−08
4
H4H12166P6
90
112
2.53E+05
2.32E−03
9.18E−09
5
H4H12166P7
63
48
1.25E+05
2.41E−03
1.93E−08
5
H4H12166P8
64
53
2.03E+05
2.69E−03
1.33E−08
4
H4H12166P9
75
69
1.81E+05
2.61E−03
1.44E−08
4
H4H12166P10
63
24
6.60E+04
2.79E−03
4.22E−08
4
Comparator 1
132
4
NB
NB
NB
NB
Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodies at 25° C. are shown in Tables 7 and 8, respectively.
TABLE 7
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human C5
R885H-mmh at 25° C.
100 nM
Amount of
Human C5
Antibody
R885H-mmh
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
183
118
5.26E+05
2.46E−04
4.68E−10
47
H4H12171P
119
51
6.59E+05
1.42E−04
2.16E−10
81
H4H12161P
105
199
8.36E+04
8.32E−05
9.96E−10
139
H4H12176P2
170
65
1.78E+05
2.17E−04
1.22E−09
53
H4H12163P
111
214
6.72E+05
4.34E−04
6.46E−10
27
H4H12167P
93
187
6.89E+05
2.98E−04
4.33E−10
39
H4H12175P
104
207
1.81E+05
1.98E−03
1.09E−08
6
H4H12159P
101
177
7.06E+05
1.76E−04
2.50E−10
66
H4H12164P
143
295
1.58E+05
1.87E−04
1.19E−09
62
H4H12168P
138
197
5.29E+04
2.14E−04
4.05E−09
54
H4H12169P
116
173
4.84E+05
7.09E−05
1.47E−10
163
H4H11686N
145
259
2.16E+05
1.06E−04
4.91E−10
109
H4H12170P
244
442
4.09E+05
1.61E−04
3.94E−10
72
H4H12177P2
137
232
1.48E+05
5.92E−04
4.01E−09
20
H4H12183P2
158
99
3.77E+04
4.37E−05
1.16E−09
264
H4H12166P
188
366
5.28E+05
2.12E−04
4.02E−10
54
Comparator 1
87
11
NB
NB
NB
NB
Comparator 2
118
249
1.08E+06
6.53E−04
6.06E−10
18
TABLE 8
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human C5
R885C-mmh at 25° C.
100 nM
Amount of
Human C5
Antibody
R885C-mmh
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
174
116
4.99E+05
2.39E−04
4.79E−10
48
H4H12171P
109
51
3.79E+05
1.39E−04
3.66E−10
83
H4H12161P
103
147
1.30E+05
8.71E−05
6.72E−10
133
H4H12176P2
164
63
1.07E+05
2.18E−04
2.03E−09
53
H4H12163P
110
211
5.04E+05
4.32E−04
8.58E−10
27
H4H12167P
85
163
7.11E+05
2.94E−04
4.13E−10
39
H4H12175P
99
128
8.18E+04
1.55E−02
1.90E−07
1
H4H12159P
93
168
5.86E+05
1.69E−04
2.89E−10
68
H4H12164P
139
249
1.53E+05
1.82E−04
1.19E−09
63
H4H12168P
128
144
6.09E+04
1.99E−04
3.27E−09
58
H4H12169P
108
168
2.78E+05
6.99E−05
2.51E−10
165
H4H11686N
143
253
1.78E+05
9.49E−05
5.34E−10
122
H4H12170P
244
427
3.57E+05
1.60E−04
4.47E−10
72
H4H12177P2
138
177
1.00E+05
1.32E−03
1.32E−08
9
H4H12183P2
158
80
2.99E+04
2.20E−05
7.37E−10
525
H4H12166P
188
356
4.26E+05
2.07E−04
4.87E−10
56
Comparator 1
87
9
NB
NB
NB
NB
Comparator 2
117
241
1.17E+06
6.19E−04
5.30E−10
19
Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodies at 37° C. are shown in Tables 9 and 10, respectively.
TABLE 9
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human C5
R885H-mmh at 37° C.
100 nM
Amount of
Human C5
Antibody
R885H-mmh
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
49
81
5.48E+05
1.47E−03
2.69E−09
8
H4H12171P
59
80
5.92E+05
9.63E−04
1.63E−09
12
H4H12161P
41
54
1.18E+05
9.25E−04
7.84E−09
12
H4H12176P2
45
69
2.57E+05
9.58E−04
3.73E−09
12
H4H12163P
60
85
7.24E+05
2.90E−03
4.00E−09
4
H4H12167P
38
65
8.81E+05
2.57E−03
2.91E−09
5
H4H12175P
25
30
1.37E+05
9.50E−03
6.94E−08
1
H4H12159P
51
82
6.38E+05
9.48E−04
1.49E−09
12
H4H12164P
59
68
1.95E+05
1.06E−03
5.46E−09
11
H4H12168P
34
29
2.43E+04
1.23E−03
5.04E−08
9
H4H12169P
61
79
4.29E+05
7.39E−04
1.72E−09
16
H4H11686N
40
74
4.19E+05
8.00E−04
1.91E−09
14
H4H12170P
36
64
5.59E+05
8.39E−04
1.50E−09
14
H4H12177P2
45
51
2.76E+05
2.76E−03
1.00E−08
4
H4H12183P2
33
36
9.58E+04
7.12E−04
7.43E−09
16
H4H12166P
71
58
6.24E+05
1.31E−03
2.09E−09
9
Comparator 1
41
5
NB
NB
NB
NB
Comparator 2
23
47
8.39E+05
1.05E−03
1.25E−09
11
TABLE 10
Binding Kinetics parameters of anti-C5 monoclonal antibodies binding to human
C5 R885C-mmh at 37° C.
100 nM
Amount of
Human C5
Antibody
R885C-mmh
Captured
Bound
ka
kd
KD
t½
Antibody
(RU)
(RU)
(1/Ms)
(1/s)
(M)
(min)
H4H11683N
48
78
4.38E+05
1.43E−03
3.25E−09
8
H4H12171P
59
78
4.77E+05
9.57E−04
2.01E−09
12
H4H12161P
41
49
1.10E+05
9.01E−04
8.17E−09
13
H4H12176P2
44
55
1.41E+05
1.03E−03
7.32E−09
11
H4H12163P
59
83
5.66E+05
2.81E−03
4.97E−09
4
H4H12167P
38
64
6.84E+05
2.49E−03
3.64E−09
5
H4H12175P
25
4
1.12E+05
1.79E−02
1.59E−07
1
H4H12159P
51
68
5.61E+05
9.75E−04
1.74E−09
12
H4H12164P
59
64
1.77E+05
1.04E−03
5.85E−09
11
H4H12168P
34
21
6.38E+04
5.69E−04
8.90E−09
20
H4H12169P
61
75
3.29E+05
7.37E−04
2.24E−09
16
H4H11686N
39
69
2.84E+05
7.91E−04
2.78E−09
15
H4H12170P
36
61
4.24E+05
8.70E−04
2.05E−09
13
H4H12177P2
43
31
1.07E+05
5.07E−03
4.76E−08
2
H4H12183P2
31
25
5.12E+04
9.97E−04
1.95E−08
12
H4H12166P
72
54
4.91E+05
1.26E−03
2.56E−09
9
Comparator 1
41
2
NB
NB
NB
NB
Comparator 2
23
42
7.34E+05
1.07E−03
1.45E−09
11
At 25° C., all 25 anti-C5 antibodies of the invention bound to human C5 with KD values ranging from 73 pM to 8.4 nM as shown in Table 3. At 37° C., the anti-C5 antibodies of the invention bound to human C5 with KD values ranging from 103 pM to 18.5 nM as shown in Table 4. At 25° C., 25 out of the 25 anti-C5 antibodies of the invention tested bound to monkey C5-mmh with KD values ranging from 133 pM to 64 nM as shown in Table 5. At 37° C., 25 out of the 25 anti-C5 antibodies of the invention tested bound to monkey C5-mmh with KD values ranging from 133 pM to 118 nM as shown in Table 6. At 25° C., 16 out of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885H-mmh with KD values ranging from 147 pM to 10.9 nM as shown in Table 7. At 25° C., 16 out of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885C-mmh with KD values ranging from 251 pM to 190 nM as shown in Table 8. At 37° C., 16 out of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885H-mmh with KD values ranging from 1.49 nM to 69.4 nM as shown in Table 9. At 25° C., 16 out of the 16 anti-C5 antibodies of the invention tested bound to human C5 R885C-mmh with KD values ranging from 1.74 nM to 159 nM as shown in Table 10.
Example 4: Antibody Binding to C5 Through Different pH
Effect of pH on the rate of dissociation of recombinant human C5 bound to purified anti-C5 monoclonal antibodies was determined using a real-time surface plasmon resonance biosensor using a BIACORE™ T200 instrument. The BIACORE™ sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, # BR-1008-39) to capture anti-C5 monoclonal antibodies expressed with human IgG4 Fc. All BIACORE™ binding studies were performed using two running buffers PBS-T, pH7.4 (0.01M Na2HPO4/NaH2PO4, 0.15M NaCl, 0.05% v/v Tween-20, adjusted to pH7.4) and PBS-T, pH6.0 (0.01M Na2HPO4/NaH2PO4, 0.15M NaCl, 0.05% v/v Tween-20, adjusted to pH6.0). Different concentrations of human C5 (EMD, Catalog #204888) or monkey C5.mmh (prepared in PBS-T, pH7.4 running buffer (ranging from 100 nM to 11.11 nM, 3-fold dilutions) were injected over the anti-C5 monoclonal antibody captured surface for 3 minutes at a flow rate of 50 μL/minute and their dissociation in two running buffers PBS-T, pH7.4 and PBS-T, pH6.0 was monitored for 6 minutes. All the binding kinetics experiments were performed at 25° C. and 37° C. Kinetic dissociation constant (kd) were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociative half-lives (t1/2) were calculated from kd as:
t
1
/
2
(
min
)
=
ln
(
2
)
60
*
kd
Half-life ratios for human C5 binding to different anti-C5 monoclonal antibodies at 25° C. and 37° C. in two running buffers PBS-T, pH7.4 and PBS-T, pH6.0 are shown in Tables 11 and 12.
TABLE 11
Half-life ratios of selected anti-C5 antibodies for human C5 at 25° C.
t½ Ratio
mAb Captured
pH7.4/pH6.0
H4H12169P
IC
H4H12176P2
IC
H4H12161P
IC
H4H12159P
≤0.3
H4H12170P
≤0.5
H4H12166P
4.5
H4H12183P2
IC
H4H12167P
0.6
H4H12164P
0.3
H4H12163P
1.2
H4H12175P
0.9
H4H12177P2
≤0.5
H4H12171P
0.6
H4H12168P
1.5
H4H12166P2
9.3
H4H12166P3
7.9
H4H12166P4
7.8
H4H12166P5
8.3
H4H12166P6
7.8
H4H12166P7
35
H4H12166P8
47
H4H12166P9
31
H4H12166P10
33
H4H11683N
2
H4H11686N
2
IC = inconclusive
TABLE 12
Half-life ratios of selected anti-C5 antibodies on human C5 at 37° C.
t½ Ratio
mAb Captured
pH7.4/pH6.0
H4H12169P
IC
H4H12176P2
≤0.4
H4H12161P
≤0.7
H4H12159P
≤0.2
H4H12170P
≤0.2
H4H12166P
3.8
H4H12183P2
IC
H4H12167P
0.2
H4H12164P
≤0.1
H4H12163P
0.8
H4H12175P
0.9
H4H12177P2
1.3
H4H12171P
3.7
H4H12168P
1
H4H12166P2
7.3
H4H12166P3
6.6
H4H12166P4
7.6
H4H12166P5
7.6
H4H12166P6
8.2
H4H12166P7
21
H4H12166P8
36
H4H12166P9
28
H4H12166P10
19
H4H11683N
1.4
H4H11686N
0.8
IC = inconclusive
Half-life ratios for monkey C5 binding to different anti-C5 monoclonal antibodies at 25° C. and 37° C. in two running buffers PBS-T, pH7.4 and PBS-T, pH6.0 are shown in Tables 13 and 14.
TABLE 13
Half-life ratios of selected anti-C5 antibodies on monkey C5 at 25° C.
mAb
t½ Ratio
Captured
pH7.4/pH6.0
H4H12169P
3.4
H4H12176P2
≤9.1
H4H12161P
IC
H4H12159P
1.2
H4H12170P
≤1.7
H4H12166P
18.5
H4H12183P2
5.8
H4H12167P
9.2
H4H12164P
2.9
H4H12163P
9.7
H4H12175P
3.6
H4H12177P2
3.7
H4H12171P
2.1
H4H12168P
3.8
H4H11683N
0.34
H4H11686N
0.37
IC = inconclusive
TABLE 14
Half-life ratios of selected anti-C5 antibodies on monkey C5 at 37° C.
mAb
t½ Ratio
Captured
pH7.4/pH6.0
H4H12169P
2
H4H12176P2
2.8
H4H12161P
10.7
H4H12159P
6.3
H4H12170P
4.7
H4H12166P
7.1
H4H12183P2
2.4
H4H12167P
4.4
H4H12164P
1.1
H4H12163P
3.3
H4H12175P
0.4
H4H12177P2
1.5
H4H12171P
4.7
H4H12168P
4
H4H11683N
0.7
H4H11686N
0.5
IC = inconclusive
As shown in Tables 11-14, selected anti-C5 antibodies showed pH-dependent binding, as seen by the t % ratios.
Example 5: OCTET® Cross-Competition Between Anti-C5 Antibodies
Binding competition between anti-C5 monoclonal antibodies (mAbs) was determined using a real time, label-free bio-layer interferometry assay on an OCTET™ RED384 biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25° C. in 0.01 M HEPES pH7.4, 0.15M NaCl, 0.05% v/v Surfactant Tween-20, 0.1 mg/mL BSA (OCTET™ HBS-P buffer) with the plate shaking at the speed of 1000 rpm. To assess whether 2 antibodies were able to compete with one another for binding to their respective epitopes on a human C5 (hC5 purified from plasma, EMD), around 1.5 nm of anti-human C5 mAb was first captured onto anti-hFc antibody coated OCTET™® biosensor tips (Pall ForteBio Corp., #18-5060) by submerging the tips for 3 minutes into wells containing a 50 μg/mL solution of anti-human C5 mAb (subsequently referred to as mAb1). The antibody captured biosensor tips were then saturated with a blocking H4H isotype control mAb (subsequently referred to as blocking mAb) by dipping into wells containing 200 μg/mL solution of blocking mAb for 4 minutes. The biosensor tips were then subsequently dipped into wells containing a co-complexed solution of 50 nM hC5 and 1 μM of a second anti-human C5 mAb (subsequently referred to as mAb2), that had been preincubated for 2 hours, for 4 minutes. The bio sensor tips were washed in OCTET™ HBS-P buffer in between every step of the experiment. The real-time binding response was monitored during the course of the experiment and the binding response at the end of every step was recorded. The response of human C5 pre-complexed mAb2 binding to mAb1 was corrected for background binding, compared and competitive/non-competitive behavior of different anti-C5 monoclonal antibodies was determined.
Table 15 explicitly defines the relationships of antibodies competing in both directions, independent of the order of binding.
TABLE 15
Cross-competition between pairs of selected anti-C5 antibodies
First mAb (mAb1)
Captured using AHC
mAb2 Antibodies Shown to Compete
Octet Biosensors
with mAb1
H4H12183P2
H4H12167P; H4H12166P; H4H12163P
H4H12167P
H4H12183P2; H4H12166P; H4H12163P
H4H12166P
H4H12183P2; H4H12167P; H4H12163P
H4H12163P
H4H12183P2; H4H12167P; H4H12166P
H4H12159P
H4H12169P; H4H11683N; H4H12170P
H4H12169P
H4H12159P; H4H11683N; H4H12170P
H4H11683N
H4H12159P; H4H12169P; H4H12170P
H4H12170P
H4H12159P; H4H12169P; H4H11683N
H4H12175P
H4H12177P2
H4H12177P2
H4H12175P
H4H12176P2
H4H12164P
H4H12164P
H4H12176P2
H4H12168P
none
H4H12161P
none
H4H11686N
none
Example 6: Inhibition of C5-Mediated Complement-Dependent Cytotoxicity in a B-Cell Bioassay
This Example describes a bioassay to test the role of C5 using an anti-CD20 antibody in the classical complement pathway. Therapeutic anti-CD20 antibodies against the B-cell specific cell-surface antigen CD20, have been shown to lead to CDC of B-cells (Glennie et al. 2007, Mol. Immunol. 44: 3823-3837) and CDC assay using cell lines expressing CD20 has been described previously (Flieger et al. 2000, Cell. Immunol. 204: 55-63). Daudi cells, a human B cell line expressing CD20, complement preserved serum or C5 depleted serum with exogenous C5 variants and an anti-CD20 antibody (antibody comprising VH/VL of “2F2” from U.S. Pat. No. 8,529,902) were used to assess the role of C5 activity in CDC.
For the C5 CDC bioassay, Daudi cells were seeded onto a 96-well assay plates at 10,000 cells/well in either RPMI containing 10% FBS, penicillin/streptomycin, L-glutamine, sodium pyruvate and Non-Essential Amino Acids (RPMI Complete media) or RPMI containing 1% BSA, penicillin/streptomycin and L-glutamine (RPMI/BSA). All assays testing mutated anti-hC5 antibodies, along with testing of the non-mutated antibodies with C5 containing human serum were tested in RPMI Complete media, while assays testing the non-mutated antibodies with African Green monkey serum and human C5 variants were tested in RPMI/BSA media. To measure CDC with human or monkey serum, the anti-CD20 antibody was diluted 1:3 from 100 nM to 2 pM (including a control sample containing no antibody) and incubated with cells for 10 minutes at 25° C. followed by addition of 1.66% serum or 1.66% of C5 depleted serum and 6.6 nM C5 variant proteins. The amount of C5 protein to be added to the C5 depleted serum was based on the reported value of C5 concentration in human serum of 0.37 uM (Rawal et al 2008, J. Biol. Chem. 283: 7853-7863). To test C5 antibody inhibition of CDC, C5 antibodies were diluted 1:3 from 100 nM to 2 pM (including a control sample containing no antibody) and incubated with 1.66% serum or 1.66% of C5 depleted serum and 6.6 nM C5 variant proteins for 30 minutes. Ten minutes prior to addition of antibodies with serum to cells, the anti-CD20 antibody was added to cells at 1 nM, 2 nM, 3 nM, 3.5 nM, 7 nM, 10 nM, or 30 nM. At the conclusion of the incubation with the anti-CD20 antibody, the antibody/serum mixture was added to cells. Cytotoxicity was measured after 3.5 hours of incubation at 37° C. and in 5% CO2, followed by 15 minute incubation at 25° C., and addition of CYTOTOX-GLO™ reagent (Promega PROMEGA™, # G9292). CYTOTOX-GLO™ is a luminescence-based reagent that measures cell killing such that increased luminescence is observed with increased cytotoxicity (measured in relative light units, RLUs). Untreated cells in control wells were lysed by treatment with digitonin immediately after addition of CYTOTOX-GLO™ reagent to determine maximal killing of cells. Plates were read for luminescence by a VICTOR™ X instrument (Perkin Elmer) 15 minutes following the addition of CYTOTOX-GLO™. Where calculated, the percentage of cytotoxicity was calculated with the RLU values by using the following equation:
%
Cytoxicity
=
100
×
(
Experimental
Cell
Lysis
-
Background
Cell
Lysis
)
(
Maximum
Cell
Lysis
-
Background
Cell
Lysis
)
In this equation “background cell lysis” is the luminescence from the cells treated with media and serum alone without any anti-CD20 antibody and the “maximum cell lysis” is the luminescence from the cells treated with digitonin. The results, expressed as % cytotoxicity or RLUs, were analyzed using nonlinear regression (4-parameter logistics) with PRISM™ 5 software (GRAPHPAD™) to obtain EC50 and IC50 values. Inhibition of antibodies was calculated such that 0-100% inhibition is the range of inhibition of the concentration of anti-CD20 antibody used in the assay without inhibitor to 0 nM anti-CD20 antibody.
Results
A total of 25 anti-human C5 antibodies, 16 non-mutated and 9 mutated, were tested for their ability to inhibit C5 in the CDC assay using Daudi cells with an anti-CD20 antibody and either human sera (with normal hC5 or C5 variants) or African green monkey sera. Various residues in the complementary determining regions (CDRs) of H4H12166P were mutated to histidines to generate 9 mutated antibodies, H4H12166P2-H4H12166P10. Histidine mutations in the CDRs have been shown to confer pH-dependence of binding to target antigen leading to improved pharmacokinetics (Igawa et al. 2010, Nat. Biotechnol. 28: 1203-1207).
TABLE 16
Non-mutated anti-hC5 antibody inhibition of CDC with 1.66% serum and anti-CD20
antibody in Daudi Cells
C5 depleted
C5 depleted
Human and
Human and
African
6.6 nM C5
6.6 nM C5
Green
Variant
Variant
Serum
Human
Human
monkey
R885H
R885C
EC50 [M] of anti-
1.0E−09
1.4E−09
2.4E−09
1.9E−09
2.7E−09
CD20 antibody
(with 1.66% Serum)
Constant anti-CD20
1 nM
3 nM
3.5 nM
30 nM
antibody (with
1.66% serum)
IC50 [M]
IC50 [M]
(Max %
(Max %
Antibody
IC50 [M]
Inhibition)*
IC50 [M]
Inhibition)*
H4H11683N
Not Tested
1.2E−09
4.0E−09
1.3E−09
9.0E−10
H4H11686N
Not Tested
1.5E−09
4.4E−09
1.1E−09
4.5E−10
H4H12159P
3.2E−09
Not Tested
3.4E−09
1.4E−09
1.0E−09
H4H12161P
2.4E−09
Not Tested
2.6E−09
1.8E−09
1.0E−09
H4H12163P
3.4E−09
Not Tested
3.7E−09
2.1E−09
1.1E−09
H4H12164P
2.4E−09
Not Tested
5.8E−09
1.8E−09
8.2E−10
H4H12166P
2.6E−09
Not Tested
4.5E−09
1.3E−09
4.6E−10
H4H12167P
2.5E−09
Not Tested
3.5E−09
1.9E−09
1.0E−09
H4H12168P
1.5E−09
Not Tested
2.0E−09
2.3E−09
8.6E−10
H4H12169P
1.7E−09
Not Tested
2.9E−09
1.3E−09
6.7E−10
H4H12170P
2.0E−09
Not Tested
3.7E−09
4.8E−10
4.3E−10
H4H12171P
1.9E−09
Not Tested
3.3E−09
1.6E−09
6.5E−10
H4H12175P
2.2E−09
Not Tested
5.2E−09
4.2E−09
>2.0E−08
(67%)
H4H12176P2
2.7E−09
Not Tested
3.5E−09
2.1E−09
1.3E−09
H4H12177P2
2.2E−09
Not Tested
6.1E−09
2.4E−09
1.6E−09
H4H12183P2
1.7E−09
Not Tested
1.4E−08
1.2E−09
4.5E−10
Comparator 1
2.3E−09
1.8E−09
>9.0E−08
No inhibition
No inhibition
(49%)
Control mAb 1
No Inhibition
No Inhibition
Not Tested
Not Tested
Not Tested
Control mAb 2
Not Tested
Not Tested
No Inhibition
No Inhibition
No Inhibition
*Unless otherwise noted, all inhibition is ~100%
As shown in Tables 16 and 17, all 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by C5 present in 1.66% of human serum. The IC50s of the non-mutated antibodies ranged from 1.2 to 3.4 nM. The IC50s of the mutated antibodies ranged from 3.0 nM to 12 nM. The parental, non-mutated antibody H4H12166P gave complete inhibition with IC50s of 2.6 nM and 2.9 nM.
TABLE 17
Mutated anti-hC5 antibody inhibition of CDC with 1.66%
serum and anti-CD20 antibody in Daudi Cells
C5 depleted
C5 depleted
Human and
Human and
6.6 nM C5
6.6 nM C5
African Green
Variant
Variant
Serum
Human
monkey
R885H
R885C
EC50 [M] of
1.9E−09
2.6E−09
6.3E−09
9.5E−09
anti-CD20
antibody (with
1.66% Serum)
Constant
2 nM
10 nM
7 nM
30 nM
anti-CD20
antibody (with
1.66% serum)
IC50 [M]
IC50
(Max %
Antibody
[M]
Inhibition)*
IC50 [M]
IC50 [M]
H4H12166P
2.9E−09
5.6E−09
1.3E−09
7.6E−10
H4H12166P2
3.7E−09
9.7E−09
1.7E−09
1.2E−09
H4H12166P3
7.8E−09
>3.0E−08 (64%)
2.9E−09
1.7E−09
H4H12166P4
3.5E−09
7.9E−09
1.5E−09
9.6E−10
H4H12166P5
4.9E−09
>3.0E−08 (75%)
2.1E−09
1.4E−09
H4H12166P6
3.0E−09
9.9E−09
1.3E−09
7.9E−10
H4H12166P7
7.3E−09
>6.0E−08 (61%)
4.2E−09
2.3E−09
H4H12166P8
4.1E−09
>2.0E−08 (79%)
2.1E−09
1.2E−09
H4H12166P9
3.9E−09
>1.0E−08 (85%)
1.7E−09
7.7E−10
H4H12166P10
1.2E−08
>1.0E−07 (34%)
7.0E−09
3.5E−09
Comparator 1
2.7E−09
>1.0E−07 (35%)
No
No
Inhibition
Inhibition
Control
No
No Inhibition
No
No
mAb 2
Inhibition
Inhibition
Inhibition
*Unless otherwise noted, all inhibition is ~100%
The sixteen non-mutated anti-hC5 antibodies showed complete inhibition of CDC mediated by African Green monkey C5 with IC50s ranging from 2.0 nM to 14 nM.
Four of the 9 mutated antibodies showed complete inhibition of CDC mediated by African Green monkey C5 with IC50s ranging from 7.1 nM to 9.9 nM. The remaining six mutated antibodies were blockers with IC50s greater than 10 nM, and maximum inhibition (at 100 nM antibody) ranging from 34% to 85%. The parental, non-mutated antibody H4H12166P gave complete inhibition with IC50s of 4.5 nM and 5.6 nM.
To test whether the anti-hC5 antibodies inhibit human C5 variants, R885H and R885C, C5-Depleted Human Serum was tested with 6.6 nM of each C5 variant. All 25 anti-hC5 antibodies showed complete inhibition of CDC mediated by C5 variant R885H, with IC50s of the non-mutated antibodies ranging from 0.48 nM to 4.2 nM, while IC50s of the mutated antibodies ranged from 1.3 nM to 7.0 nM. The parental, non-mutated antibody H4H12166P gave complete inhibition with IC50s of 1.3 nM and 1.3 nM.
Fifteen out of 16 non-mutated anti-hC5 antibodies showed complete inhibition of CDC mediated by C5 variant R885C with IC50s ranging from 0.43 nM to 1.6 nM. One non-mutated antibody showed weak inhibition of CDC with maximum inhibition of 67% (at 100 nM antibody) and an IC50>20 nM. All nine mutated antibodies showed complete inhibition of CDC mediated by C5 variant R885C with IC50s ranging from 0.77 nM to 3.5 nM. The parental, non-mutated antibody H4H12166P gave complete inhibition with an IC50s of 0.46 nM and 0.76 nM.
Anti-CD20 antibody showed CDC of Daudi cells with 1.66% serum with EC50s of 1.0 nM, 1.4 nM, and 1.9 nM for human serum, 2.4 nM and 2.6 nM for African Green monkey serum, 1.9 nM and 6.3 nM for hC5 depleted serum with hC5 variant R885H, and 2.7 nM and 9.5 nM for hC5 depleted serum with hC5 variant R885C. Neither of the irrelevant IgG control antibodies, Control mAb1 and Control mAb2, demonstrated any inhibition of CDC.
Example 7: Inhibition of C5a Activity as Determined by Luciferase Assay
This Example describes an assay to test the activation of C5a through one of its receptors, C5aR1. C5aR1 is a G-protein coupled receptor (GPCR) and can initiate various GPCR coupled signaling pathways (Monk et al. 2007, Br. J. Pharmacol. 152: 429-448). A bioassay was established using HEK293 cells stably transfected with human C5aR1 (Accession No. NP_001727.1) and human Gα16 (Accession No. NP_002059.3) along with a luciferase reporter [NFAT response element (4×)-luciferase]. Gα16 is a relatively promiscuous G protein that can couple to different types of GPCRs leading to PLC-β activation and subsequent elevation of Ca++, which in turn activates NFAT translocation and reporter gene transcription (Kostenis et al. 2005, Trends Pharmacol. Sci. 26: 595-602). The resulting cell line, HEK293/hGα16/hC5aR1/NFAT-luc, was isolated and maintained in 10% DMEM containing 10% FBS, NEAA, pencillin/streptomycin, 500 μg/mL G418, 100 μg/mL hygromycin B, and 7 μg/mL blasticidin.
For the C5a luciferase bioassay, HEK293/hGα16/hC5aR1/NFAT-luc cells were seeded into 96-well assay plates at 20,000 cells/well in OPTI-MEM™ reduced serum medium (Invitrogen, #31985-070) supplemented with 0.5% BSA, penicillin/streptomycin and L-glutamine, and then incubated at 37° C. and 5% CO2 overnight. BSA was used instead of FBS, since serum has been shown to cleave and inactivate hC5a (Klos et al., 2013, Pharmacol. Rev. 65: 500-543). The next morning, hC5a was titrated from 100 nM to 2 pM (including a control sample containing no hC5a) and added to cells to determine the dose response titration curve for the cell line. To test hC5a antibody inhibition of hC5a, 500 pM of hC5a was added to cells. Immediately afterwards, antibodies diluted 1:3 from 100 nM to 2 pM (including a control sample containing no antibody) were added to cells. Cells were incubated for 5.5 hours at 37° C. in the presence of 5% CO2 The luciferase activity was detected after the incubation with ONEGLO™ reagent (PROMEGA™, # E6051). ONEGLO™ is a luminescence-based reagent that measures the amount of luciferase present in cells. In this assay, increased hC5a activation leads to increased luciferase production and luminescence (measured in relative light units, RLUs). Measurement of luminescence was performed using a VICTOR™ X instrument (Perkin Elmer). The results were analyzed using nonlinear regression (4-parameter logistics) with PRISM™ 5 software (GRAPHPAD™) to obtain EC50 and IC50 values. Inhibition of antibodies was calculated such that 0-100% inhibition is the range of inhibition from 500 pM hC5a without inhibitor to 0 nM hC5a.
Four anti-hC5 antibodies were tested for their ability to inhibit hC5a activation of its receptor, hC5aR1, by measuring the extent of inhibition of 500 pM hC5a activation of HEK293/hGα16/hC5aR1/NFAT-luc cells.
TABLE 18
Anti-hC5 antibody inhibition of 500 pM hC5a in HEK293/
Gα16/hC5aR1/NFAT-luc cells
3.9E−10
EC50[M] hC5a
Inhibition of 500 pM hC5a
mAb PID or REGN #
IC50 [NA]
H2aM11682N
4.6E−10
H2aM11684N
3.5E−11
H2aM11694N
1.4E−10
H2aM11695N
4.2E−11
Control mAb
No Inhibition
As shown in Table 18, all four antibodies of the invention, showed complete inhibition of 500 pM hC5a with IC50s ranging from 0.035 nM to 0.46 nM. An Irrelevant IgG control antibody, Control mAb3, did not demonstrate any inhibition of hC5a. hC5a activated HEK293/Gα16/hC5aR1/NFAT-luc cells with an EC50 of 0.39 nM.
Example 8: Hemolysis Bioassay
Classical pathway hemolysis assay (CH) and alternative pathway hemolysis assay (AH) were developed to test the antibody activity.
The CH is a screening assay for the activation of the classical complement pathway, which is sensitive to the decrease, absence, and/or inactivity of any component of the pathway. The CH tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (hemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and hemolysis results. If a complement component is absent, the CH level will be zero; if one or more components of the classical pathway are decreased, the CH will be decreased. (Nilsson et al 1984, J. Immunol. Meth. 72: 49-59). This assay is used for characterization and screening of high-affinity anti-human C5 antibodies.
Methods
(A) Classical Pathway Complement Hemolysis Assay
Desired number of sheep red blood cells (SRBCs) were washed in GVB++ buffer and re suspended at 1×10{circumflex over ( )}9 cells/mL. To sensitize the SRBCs, were mixed with equal volume of the 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for 20 minutes. Sensitized SRBC cells were diluted to 2×10{circumflex over ( )}8 cells/ml in GVB++ prior to using in hemolysis assay. Normal human serum or cynomolgus monkey serum was diluted to 2% or 10% in GVB++ buffer. To test the inhibition of C5 mediated hemolysis activity, test antibodies were pre-incubated for 20 minutes at 4° C., at concentrations ranging from 0.6 nM to 800 nM in 2%-10% normal human or 10% cynomolgus monkey serum or African green monkey serum. Round bottom 96 well plates were used to measure hemolysis activity. A total of 100 ul sensitized sheep RBCs (2×10{circumflex over ( )}8 cells/ml) were plated into 96-well plate followed by addition of 100 ul of respective serum samples that was pre-incubated with the test antibodies. Cells were gently mixed and incubated at 37° C. for 60 minutes. After the incubation time, cells were spun down by centrifugation at 1250×g at 4° C. A total of 100 uL of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on Spectramax microplate reader. The hemolytic activity was calculated at final serum concentration of 1-5% for treatments.
Percent hemolysis was calculated as follows:
%
Hemolysis
=
100
×
(
Experimental
Cell
Lysis
-
Background
Cell
Lysis
)
(
Maximum
Cell
Lysis
-
Background
Cell
Lysis
)
In this equation “background cell lysis” is the OD at A412 nm from the cells incubated in GVB++ buffer only containing no serum. The “maximum cell lysis” is the OD at A412 nm from the cells treated with water. The results, expressed as % hemolysis were analyzed using nonlinear regression (4-parameter logistics) with PRISM™ 5 software (GRAPHPAD™) to obtain IC50 values. Data represented as mean±Standard error of mean.
(B) Alternative Complement Assay
Desired number of rabbit red blood cells (RbRBCs) were washed in GVB-Mg2+/EGTA buffer and re suspended at 2×10{circumflex over ( )}8 cells/ml. Normal human or cynomolgus monkey serum was diluted to 10% in GVB-Mg2+/EGTA buffer. To test the inhibition of C5 mediated hemolysis activity, antibodies at concentrations ranging from 3 nM to 800 nM were pre-incubated for 20 minutes at 4° C. in 5-10% normal human serum or cynomolgus monkey serum. Round bottom 96 well plates were used to measure hemolysis activity. A total of 100 ul RbRBCs (2×10{circumflex over ( )}8 cells/ml) were plated into 96-well plate followed by addition of 100 ul of 10% normal human serum or cynomolgus monkey serum or African green monkey serum that was pre-incubated with the anti-C5 antibodies. Cells were gently mixed and incubated at 37° C. for 60 minutes. After incubation time, the cells were spun down by centrifugation at 1250×g at 4° C. A total of 100 uL of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on Spectramax microplate reader. The hemolytic activity was calculated at final serum concentration of 5% serum.
Percent hemolysis was calculated as follows:
%
Hemolysis
=
100
×
(
Experimental
Cell
Lysis
-
Background
Cell
Lysis
)
(
Maximum
Cell
Lysis
-
Background
Cell
Lysis
)
In this equation “background cell lysis” is the OD at A412 nm from the cells incubated in GVB-Mg/EGTA buffer only containing no serum or without any anti-C5 antibody. The “maximum cell lysis” is the OD at A412 nm from the cells treated with water. Inhibition by anti-C5 antibodies, IC50 values were calculated using nonlinear regression (4-parameter logistics) with PRISM™ 6 software (GRAPHPAD™)
Results
(A) Inhibition of Human C5 Hemolysis
A total of 25 anti-human C5 (hC5) antibodies, 16 non-mutated and 9 mutated, were tested for their ability to inhibit C5 from normal human serum (NHS) in the CH50 assay using sensitized sheep red blood (SRBCs) and AH50 assay using rabbit red blood cells (RRBCs).
TABLE 19
Anti-hC5 antibody inhibition of CP and AP activity in 1%
or 5% normal human serum (NHS)
Human CP
Human AP
Human CP
Human AP
% Max
% Max
mAb PID
IC50 [M]
IC50 [M]
Inhibition
Inhibition
H4H12183P2
5.88E−09
1.60E−07
99.9%
78.9%
H4H12176P2
4.58E−09
1.65E−08
94.1%
69.9%
H4H12168P
3.33E−09
2.85E−08
97.5%
66.2%
H4H11686N
3.09E−09
1.30E−08
97.4%
76.2%
H4H12167P
3.68E−09
1.55E−08
99.9%
64.8%
H4H12161P
2.56E−09
2.55E−08
93.7%
56.1%
H4H12163P
2.72E−09
2.05E−08
96.1%
66.0%
H4H12166P
2.80E−09
2.60E−08
95.0%
70.9%
H4H11683N
2.54E−09
3.40E−08
98.1%
73.2%
H4H12159P
2.50E−09
1.75E−08
97.9%
73.4%
H4H12177P2
2.34E−09
1.70E−08
97.5%
71.0%
H4H12170P
2.39E−09
1.80E−08
98.2%
81.1%
H4H12175P
2.36E−09
2.00E−08
98.0%
80.2%
H4H12171P
2.33E−09
1.55E−08
94.9%
42.0%
H4H12164P
2.10E−09
1.45E−08
95.9%
69.7%
H4H12169P
2.36E−09
2.00E−08
98.3%
44.5%
Isotype control
No Activity
No Activity
No Activity
No Activity
As shown in Table 19, sixteen anti-hC5 antibodies of this invention showed more than 94% inhibition of hemolysis in classical pathway (CP) mediated by C5 present in 1% of human serum. The IC50s of antibodies ranged from 2.1 to 5.9 nM and the percent inhibition ranged from 95%-99%. All 16 anti-C5 antibodies showed more than 60% inhibition (except H4H12169P) in the alternative pathway (AP) hemolysis assay mediated by C5 present in 5% NHS. The IC50s of antibodies ranged from 13 to 160 nM and the percent inhibition activity ranged from 44% to 81%.
TABLE 20
Anti-hC5 antibody inhibition of CP and AP activity in 5%
normal human serum (NHS)
Human
Human
Human CP
Human AP
CP
AP
% Max
% Max
mAb PID
IC50 [M]
IC50 [M]
Inhibition
Inhibition
H4H12166P
1.09E−08
2.09E−08
99.4%
86.9%
H4H12166P2
1.59E−08
4.78E−08
98.2%
81.3%
H4H12166P3
1.34E−08
6.00E−08
95.9%
78.3%
H4H12166P4
1.32E−08
3.17E−08
98.6%
77.0%
H4H12166P5
1.49E−08
6.55E−08
97.1%
77.7%
H4H12166P6
1.03E−08
2.84E−08
98.1%
82.4%
H4H12166P7
2.43E−08
1.56E−07
93.7%
83.2%
H4H12166P8
1.41E−08
7.30E−08
95.7%
73.3%
H4H12166P9
1.16E−08
5.35E−08
93.7%
72.2%
H4H12166P10
4.44E−08
No Activity
74.0%
No Activity
Isotype control
No Activity
No Activity
No Activity
No Activity
As shown in Table 20, all 9 mutated anti-hC5 antibodies showed inhibition of CP and AP hemolysis activity mediated by C5 present in 5% of human serum. In the CP hemolysis assay, the parental, non-mutated antibody H4H12166P showed more than 98% inhibition with IC50 of 10.9 nM. Eight mutated anti-hC5 antibodies showed more than 90% inhibition with IC50 ranging from 10.3 nM to 24.3 nM. Mutant anti-C5 antibody 12166P10 showed partial inhibition of 74%. In the AP hemolysis assay the parental, non-mutated antibody H4H12166P showed more than 85% inhibition with IC50s of 20.9 nM. The mutated anti-hC5 antibodies showed inhibition range from 72-83% and the IC50s antibodies ranged from 28 nM to 0.15 pM.
(B) Inhibition of Monkey C5 Hemolysis
A total of 25 anti-human C5 (hC5) antibodies, 16 non-mutated and 9 mutated, were tested for their ability to inhibit C5 from Cynomolgus monkey and African green monkey in the CH50 assay using sensitized sheep red blood (SRBCs) and AH50 assay using rabbit red blood cells (RRBCs).
TABLE 21
Anti-hC5 antibody inhibition of CP and AP activity in 5% normal
African green monkey (AGM) sera
AGM Serum
AGM Serum
AGM CP
AGM AP
CP
AP
% Max
% Max
mAb PID
IC50 [M]
IC50 [M]
Inhibition
Inhibition
H4H12183P2
No Activity
No Activity
No Activity
No Activity
H4H12176P2
3.04E−08
4.77E−08
91.0%
83.2%
H4H12168P
2.80E−08
2.25E−08
90.8%
88.2%
H4H11686N
4.82E−08
1.63E−07
49.3%
50.4%
H4H12167P
6.95E−08
6.95E−08
90.5%
53.4%
H4H12161P
3.19E−08
4.75E−08
78.9%
35.7%
H4H12163P
6.90E−08
2.16E−07
83.2%
58.5%
H4H12166P
1.30E−07
2.33E−07
81.0%
44.2%
H4H11683N
2.92E−08
4.08E−08
81.1%
88.1%
H4H12159P
2.58E−08
2.70E−08
93.4%
93.5%
H4H12177P2
1.80E−07
1.01E−07
80.6%
8.80%
H4H12170P
2.54E−08
2.69E−08
94.9%
90.9%
H4H12175P
1.18E−07
9.85E−08
84.5%
17.4%
H4H12171P
No Activity
2.33E−08
17.8%
69.70%
H4H12164P
2.47E−07
1.78E−07
85.8%
15.60%
H4H12169P
3.44E−08
9.15E−08
43.3%
45.70%
As shown in Table 21, the anti-hC5 antibodies showed different levels of inhibition of CP or AP hemolysis activity in 5% African green monkey sera. In the CP assay, two of the 16 anti-hC5 antibodies showed no inhibition of the hemolysis activity. Fourteen antibodies showed inhibition ranging from 43-94%, with IC50s ranging from 25 nM to 180 nM. In the AP hemolysis assay, thirteen of the 17 antibodies showed inhibition activity ranging from 17%-93% with IC50s ranging from 22.5 nM to 233 nM.
TABLE 22
Anti-hC5 antibody inhibition of CP and AP activity in 5%
normal Cynomolgus (Cyno) monkey sera
Cyno
Cyno
Cyno CP
Cyno AP
Serum CP
Serum AP
% Max
% Max
mAb PID
IC50 [M]
IC50 [M]
Inhibition
Inhibition
H4H12183P2
1.42E−07
2.96E−08
64.6%
100.0%
H4H12171P
8.70E−09
7.20E−09
92.9%
98.8%
H4H12170P
8.20E−09
7.00E−09
99.0%
99.2%
H4H12159P
7.75E−09
7.05E−09
99.3%
99.6%
H4H12168P
1.03E−08
5.45E−09
99.0%
99.7%
H4H11683N
9.00E−09
7.15E−09
98.8%
98.9%
H4H12176P2
1.47E−08
7.70E−09
97.5%
99.3%
H4H12161P
2.79E−08
7.80E−09
100.0%
98.4%
H4H12169P
1.99E−08
7.95E−09
92.8%
96.30%
H4H11686N
1.41E−08
9.00E−09
94.5%
98.7%
H4H12163P
1.65E−08
1.02E−08
96.4%
98.5%
H4H12167P
2.13E−08
7.60E−09
100.0%
98.30%
H4H12175P
1.09E−08
8.05E−09
96.7%
98.10%
H4H12166P
2.01E−08
8.85E−09
94.2%
98.60%
H4H12177P2
1.71E−08
8.80E−09
94.90%
98.10%
H4H12164P
1.96E−08
9.10E−09
94.7%
98.70%
Isotype control
No Activity
No Activity
No Activity
No Activity
As shown in Table 22, anti-hC5 antibodies (except H4H12183P2, which showed 64% CP inhibition) showed more than 90% inhibition of CP or AP hemolysis assay in 5% of Cynomolgus monkey serum. In CP hemolysis assay, the IC50s of antibodies ranged from 7.15 nM to 142 nM. In AP hemolysis assay, the IC50s of antibodies ranged from 5.4 to 29.6 nM.
(C) Inhibition of Variant Human C5 Hemolysis
Selected anti-C5 antibodies were tested for their ability to inhibit variant human C5 (see Example 3 herein) from C5-depleted human serum in the CH50 assay. In C5-depleted human serum supplemented with exogenous C5 variant R885H, H4H12166P, and Comparator 2 blocked CP hemolysis with IC50 values of 6.0 nM and 4.4 nM, respectively, and IC80 values of 7.6 nM and 5.5 nM, respectively. For variant R885C, H4H12166P and Comparator 2 blocked CP hemolysis in C5-depleted human serum with exogenous C5 variants with an IC50 of 9.3 nM and 6.8 nM, respectively, and IC80 values of 1 nM and 8.2 nM, respectively. As expected, Comparator 1 did not block the hemolytic activity of human C5 variants.
(D) Inhibition of Human C5b-6 Complex
Selected anti-C5 antibodies were tested for their ability to inhibit human C5b-6 complex from C5-depleted human serum in CH50 assay. H4H12166P potently blocked CP hemolysis in C5-depleted human serum supplemented with exogenous huC5b-6 complex with an IC50 of 3.8 nM and IC80 value of 5.8 nM. In contrast, Comparator 1 blocked C5b-6 complex-mediated hemolysis with lower potency, with IC50 values of 5.0 nM and IC80 value of 46 nM respectively. Comparator 1 inhibited only 70% total hemolysis at the highest concentrations tested. Comparator 2 did not block human C5b-6 complex hemolytic activity.
Example 9: Anti-C5 Antibodies Block Generation of C5a in CP Hemolysis Assay
To assess whether anti-C5 antibodies inhibit the generation of C5a, supernatants from the assay for classical pathway (CP) hemolysis were analyzed for C5a levels by ELISA.
C5a, generated as the result of C5 cleavage, is a protein fragment of 74 amino acids. C5a is rapidly metabolized by serum carboxypeptidases to a more stable, less active, 73 amino acid form, C5a des-Arg, by removal of the C-terminal arginine. The quantitation of C5a des-Arg therefore provides a reliable measurement for monitoring the generation of C5a in vivo and in vitro. The MicroVue C5a ELISA kit used here detects C5a des-Arg according to information provided by the manufacturer. Preliminary experiments (data not shown) indicate that the primary 74 amino acid form of C5a is also detected. For the purpose of this Example, both forms will be collectively referred to as “C5a”.
C5a protein levels were determined in supernatants from the CP hemolysis assay using complement-preserved normal human serum (NHS) pre-incubated with H4H12166P or isotype control antibody as described in Example 8. C5a protein levels were measured using the MicroVue C5a ELISA kit according to the manufacturer's instructions. Briefly, samples were diluted and incubated on plates pre-coated with capture antibody (mouse anti-C5a specific for a neo-epitope on human C5a). Human C5a protein provided by the manufacturer was used as a standard for calibration. C5a in the supernatants was detected by HRP-conjugated detection antibody (mouse monoclonal antibody to the C5a region of C5). The chromogenic HRP-substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), was added to detect HRP activity. A solution of IN hydrochloric acid was used to stop the reaction, and the optical density at 450 nm (OD450) was measured on a SPECTRAMAX™ plate reader. Data were analyzed using nonlinear regression (4-parameter logistics) in GRAPHPAD™ PRISM™. C5a concentration was expressed as ng/mL of supernatant.
In the assay using 5% NHS, H4H12166P potently blocked increases in C5a protein levels in a dose-dependent manner with an IC50 of 8.5 nM, while the isotype control antibody had no effect on C5a levels (FIG. 1). Maximal blockade at the highest tested H4H12166P concentration (267 nM) resulted in a ˜10-fold decrease in C5a levels to 3.8 ng/mL (0.3 nM), compared with 34 ng/mL (2.8 nM) observed at the lowest tested H4H12166P concentration (1 nM) in 5% serum. The C5a concentration observed for maximal blockade was close to the baseline C5a level of 2.3 ng/mL (0.2 nM) in untreated 5% NHS.
Example 10: Characterization of Pharmacokinetics and Pharmacodynamics of Anti-C5 Antibodies in Cynomolgus Monkey
This Example describes the characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of selected anti-C5 antibodies conducted in male cynomolgus monkeys. Endogenous C5 levels were determined prior to anti-C5 antibody dosing and used to stratify animal dose groups.
Total circulating C5 levels in cynomolgus monkeys were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations. Average concentrations of C5 protein in monkeys were determined to be 90.85 μg/mL±19.17 μg/mL.
For each anti-C5 antibody, 4 cynomolgus monkeys were each administered a single intravenous (IV) injection at a dose of 15 mg/kg. Blood samples were collected from each animal from pre-dose through 1680 hours (70 days), processed into serum and frozen at −80° C. until analyzed for PK and PD.
Total IG Antibody Level Analysis by ELISA Immunoassay
Total antibody concentrations in monkey serum samples were measured using a non-validated direct ELISA. The ELISA procedure employed a microtiter plate coated with a mouse anti-human IgG1/IgG4 Fc monoclonal antibody. Different anti-C5 antibodies were added to the plate and the anti-C5 antibodies captured on the plate were detected using a biotinylated mouse anti-human IgG4 Fc monoclonal antibody, followed by NeutrAvidin conjugated with Horseradish Peroxidase (NeutrAvidin HRP). A luminol-based substrate specific for peroxidase was then added to achieve a signal intensity that is proportional to the concentration of total captured anti-C5 antibody. The relative light unit (RLU) measurements of the calibration standards and their respective nominal concentrations were fitted using a weighted 4 Parameter Logistic equation to generate a calibration equation that described the concentration of anti-C5 antibodies and response relationship of the assay. The lower limit of quantitation (LLOQ) was 1.56 ng/mL in the assay (2% monkey serum) and 78 ng/mL in neat monkey serum.
Determination of PK Parameters
PK parameters were determined by non-compartmental analysis (NCA) using Phoenix®WinNonlin® software (Version 6.4, Certara, L.P.) and an IV bolus dosing model.
All PK parameters were derived from the respective mean concentration values, including the observed maximum concentration in serum (Cmax), the time of observed peak concentration, tmax, and the estimated half-life observed (T1/2). For each antibody, the area under the concentration versus time curve up to the last measurable concentration (AUClast) and extrapolated from time zero to infinity (AUCinf) were determined using a linear trapezoidal rule with linear interpolation and uniform weighting.
PD Analysis by Ex Vivo Hemolysis Assay
Pharmacodynamics of selected anti-C5 antibodies was analyzed using ex vivo classical pathway and alternative pathway hemolysis assays.
Classical Pathway Hemolysis Assay:
Sheep red blood cells (SRBCs) were washed in GVB++ buffer (Gelatin Veronal Buffer with CaCl2 and MgCl2) (Boston BioProducts) and re-suspended at 1×10{circumflex over ( )}9 cells/mL. To sensitize the SRBCs, a total of 1×10{circumflex over ( )}9/mL of SRBCs were mixed with equal volume of the 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for 20 minutes. Sensitized SRBCs were diluted to 2×10{circumflex over ( )}8 cells/mL in GVB++ buffer prior to using in the hemolysis assay. Blood from cynomolgus monkeys was collected prior to dosing and at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56, 63 and 70 days post dose for PD analysis. Serum was prepared and frozen until further use. On day of the assay, cynomolgus serum from respective time points was diluted to 10% in GVB++ buffer. Round bottom 96 well plates were used to measure hemolysis activity. A total of 100 μl of sensitized SRBCs (2×10{circumflex over ( )}8 cells/mL) were plated into 96-well plate at 37° C. followed by addition of 100 ul of 10% cynomolgus monkey serum from respective time points. SRBCs were gently mixed and incubated at 37° C. for 10 minutes. After incubation time, the cells were centrifuged at 1250×g at 4° C. A total of 100 μL of the supernatant was transferred to a fresh 96 flat bottom plate and read at 412 nm on a Spectramax microplate reader. The hemolytic activity was calculated at final serum concentration of 5%. The percent hemolysis was calculated with the absorbance values by using the following equation:
%
Hemolysis
=
100
×
(
Experimental
Cell
Lysis
-
Background
Cell
Lysis
)
(
Maximum
Cell
Lysis
-
Background
Cell
Lysis
)
In this equation “background cell lysis” is the OD at A412 nm from the SRBCs incubated in GVB++ buffer only containing no serum. The “maximum cell lysis” is the OD at A412 nm from SRBCs treated with water. The results, expressed as % hemolysis were analyzed using nonlinear regression (4-parameter logistics) with PRISM™5 software (GRAPHPAD™) to obtain IC50 values. Data are represented as mean±Standard Error of Mean.
Alternative Pathway Hemolysis Assay:
The desired number of rabbit red blood cells (RbRBCs) were washed in GVB-Mg2+/EGTA buffer and re suspended at 2×10{circumflex over ( )}8 cells/mL. Blood from cynomolgus monkeys was collected prior to dosing and at 5 minutes, 4 and 8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42 and 49 days post dose for PD analysis. Serum was prepared and frozen until further use. Round bottom 96 well plates were used to measure hemolysis activity. A total of 100 μl RbRBCs (2×10{circumflex over ( )}8 cells/mL) were plated into 96-well plate at 37° C. followed by addition of 100 μl of 10% cynomolgus monkey serum from the respective time points listed above. RbRBCs were gently mixed and incubated at 37° C. for 60 minutes. After incubation time, the cells were centrifuged at 1250×g at 4° C. A total of 100 μL of the supernatant was transferred to a fresh 96 flat bottom plate which was read at 412 nm on a Spectramax microplate reader. The hemolytic activity was calculated for final serum concentration of 5% and expressed as percentage of total hemolysis of RBCs by water. The percent of hemolysis was calculated as described above.
Results
Selected anti-C5 antibodies (listed in Table 1) were tested in initial experiments for prolonged pharmacokinetic profiles in cynomolgus monkeys and C5-humanized mice (described in Example 10). H4H12166P and H4H12161P were selected as having high affinity coupled with prolonged PK and used in subsequent experiments herein with Comparator 1 and Comparator 2.
Cynomolgus monkeys were administered a single 15 mg/kg IV bolus dose of H4H12166P, H4H12161P, or Comparator 2. Serum concentrations of total antibody and percent classical pathway (CP) hemolysis activity were determined at 19 time points during a 70-day in-life period. Alternative pathway (AP) hemolysis was determined at 17 time points during a 50-day in-life period. Table 23 summarizes the mean antibody concentrations for all 3 antibodies. Mean total antibody concentrations versus time profiles are shown in FIG. 2. Mean PK parameters are described in Table 24.
TABLE 23
Mean Concentrations of Total IgG in Serum Following a Single
15 mg/kg Intravenous Injection of Selected Anti-C5
Antibodies to Male Cynomolgus Monkeys
Time
(hours
Serum concentration of Ab (μg/mL)
post-
Mean ± SD
dose)
#
H4H12166P
H4H12161P
Comparator 2
0
4
BLQ
BLQ
BLQ
0.083
4
445 ± 30
456 ± 26.2
459 ± 55.2
4
4
328 ± 27
360 ± 28.2
363 ± 43.8
8
4
353 ± 29.9
316 ± 21.5
357 ± 16.1
24
4
282 ± 43.6
276 ± 32.4
248 ± 19.6
48
4
225 ± 15.2
221 ± 21.5
212 ± 19.3
72
4
180 ± 15.0
181 ± 17.2
196 ± 36.2
120
4
194 ± 20.9
162 ± 10.7
179 ± 23.3
168
4
171 ± 29.9
132 ± 17.0
157 ± 18.4
240
4
157 ± 12.7
96.1 ± 17.3
114 ± 13.0
336
4
120 ± 10.2
49.3 ± 18.7
67.9 ± 25.8
432
4
105 ± 13.9
24.6 ± 11.8
42.6 ± 15.9
504
4
92.2 ± 10.6
13.8 ± 9.95
28.6 ± 13.1
672
4
75.1 ± 15.8
6.16 ± 2.40
10.9 ± 6.25
840
3
59.6 ± 4.79
2.44 ± 0.85
4.45 ± 2.63
1008
3
43.3 ± 2.89
1.16 ± 0.52
2.17 ± 1.58
1176
3
30.6 ± 1.42
0.57 ± 0.25
1.29 ± 1.16
1344
3
25.9 ± 3.74
0.315 ± 0.16
0.492 ± 0.49
1512
3
18.2 ± 2.41
0.17 ± 0.08
0.270 ± 0.27
1680
3
11.5 ± 1.51
0.079 ± 0.07
0.123 ± 0.15
Time = Time in hours post single-dose injection;
SD = Standard deviation;
BLQ = Below Limit of Quantitation
Following IV bolus administration, the total IgG concentration-time profiles of H4H12166P, H4H12161P, and Comparator 2 were characterized by an initial brief distribution phase followed by single elimination phases throughout the in-life period. Peak H4H12166P, H4H12161P, and Comparator 2 concentrations were highly comparable, as corresponding Cmax/Dose values between all the antibodies were within 1.1-fold (29.7, 30.4, and 30.6 [(ug/mL)/(mg/kg)], respectively) (Table 24).
TABLE 24
Mean Pharmacokinetic Parameters of Total IgG Concentrations in Serum Following
a Single 15 mg/kg Intravenous Injection of Selected Anti-C5 Antibodies to Male Cynomolgus
Monkeys
H4H12166P
H4H12161P
Comparator 2
15 mg/kg IV (n = 4)
Parameter
Mean
SD
Mean
SD
Mean
SD
Cmax (μg/mL)
445
30.0
456
26.2
459
55.2
Cmax /Dose (μg/mL)/(mg/kg)
29.7
2.00
30.4
1.75
30.6
3.68
C0 (μg/mL)
448
30.5
458
26.6
461
55.6
tmax (hours)
0.083
0
0.083
0
0.083
0
AUClast day · (μg/mL)
5080
1040
2350
357
2810
470
AUClast/Dose
339
69.3
157
23.8
187
31.3
day · (μg/mL)/(mg/kg)
AUCinf day · (μg/mL)
5550
671
2350
356
2810
470
AUCinf/Dose
370
44.7
157
23.7
188
31.4
day · (μg/mL)/(mg/kg)
CL (mL/h/kg)
0.114
0.0143
0.270
0.0411
0.228
0.0425
Vss (mL/kg)
60.4
4.85
44.0
4.34
45.3
3.06
t1/2 (day)
15.6
1.43
5.50
2.45
5.91
1.13
IV = Intravenous;
n = Number of animals;
Cmax = Peak concentration;
C0 = Initial concentration determined by extrapolation;
tmax = Time to Cmax;
AUC = Area under the concentration-time curve;
AUClast = AUC computed from time zero to the time of the last positive concentration;
AUCinf = AUC from time zero extrapolated to infinity;
CL = Total body clearance;
Vss = Volume of distribution at steady state;
t1/2 = Half-life;
SD = Standard Deviation.
Note:
tmax is expressed in nominal hours
Assessment of concentration-time profiles revealed that H4H12166P demonstrated the slowest elimination with terminal antibody concentrations >10 μg/mL through study day 71. Kinetics of H4H12161P and of Comparator 2 were similar; both demonstrated more rapid elimination than H4H12166P, with mAb concentrations >10 μg/mL through day 22 and 29, respectively.
Consequently, dose-normalized exposures (AUClast/Dose) indicated that H4H12166P had the highest exposure at 339 day*(μg/mL)/(mg/kg), while H4H12161P and Comparator 2 had approximately 2-fold lower exposure, 157 and 187 day*(μg/mL)/(mg/kg), respectively, than that of H4H12166P.
Antibody half-life (t1/2) calculated during the elimination phase ranged from 5.5 to 15.6 days across the dose groups and also correlated with exposure, as H4H12166P had the correspondingly highest t1/2 of 15.6 days, while H4H12161P and Comparator 2 had t1/2 values of 5.5 and 5.9 days, respectively.
The pharmacologic effects of the anti-C5 antibodies from cynomolgus monkey serum samples were determined ex vivo by complement classical pathway (CP) hemolysis of sensitized sheep red blood cells (SRBCs) and alternative pathway (AP) hemolysis of rabbit red blood cells (RbRBCs). The inhibition of hemolytic activity was calculated for a final serum concentration of 5% and expressed as percentage of total hemolysis of RBCs by water. Table 25 summarizes ex vivo activity of the 3 antibodies as determined by mean percent hemolysis.
TABLE 25
Ex Vivo Classical and Alternative Pathway Percent Hemolysis Activity of Selected
anti-C5 Antibodies
Time
(hours
Classical Pathway % Hemolysis in
Alternative Pathway % Hemolysis in
post-
cynomolgus serum, 10 min, Mean ± SEM
cynomolgus serum, 60 min, Mean ± SEM
dose)
#
H4H12166P
H4H12161P
Comparator 2
H4H12166P
H4H12161P
Comparator 2
0
4
91.34 ± 7.6
Not tested
84.36 ± 20.28
73.44 ± 17.26
64.90 ± 19.51
55.77 ± 10.82
0.083
4
3.5 ± 1.4
Not tested
6.6 ± 6.5
5.53 ± 1.98
5.90 ± 3.92
3.83 ± 3.93
4
4
2.35 ± 1.06
Not tested
3.16 ± 2.2
7.43 ± 2.54
6.53 ± 2.7
5.30 ± 2.80
8
4
1.55 ± 0.21
Not tested
1.25 ± 0.21
3.53 ± 0.91
4.70 ± 2.77
1.98 ± 0.83
24
4
7.7 ± 6.08
Not tested
4.55 ± 2.05
13.40 ± 2.77
4.68 ± 1.89
4.65 ± 2.35
48
4
2.85 ± 2.19
Not tested
2.6 ± 0.7
5.53 ± 2.40
7.68 ± 5.22
2.28 ± 0.67
72
4
0.9 ± 0.42
Not tested
1.3 ± 0.28
7.95 ± 3.36
5.95 ± 2.23
1.45 ± 0.33
120
4
1.75 ± 0.07
Not tested
1.3 ± 0.14
16.38 ± 6.91
7.60 ± 1.94
1.68 ± 0.22
168
4
1.6 ± 1.13
Not tested
1.4 ± 0.56
21.28 ± 8.24
10.75 ± 2.27
2.15 ± 0.19
240
4
1 ± 0.14
Not tested
3.7 ± 2.83
19.18 ± 10.20
13.53 ± 7.17
14.20 ± 16.73
336
4
2.55 ± 2.05
Not tested
37.85 ± 5.3
21.10 ± 7.55
50.58 ± 12.91
65.60 ± 26.04
432
4
1.35 ± 0.91
Not tested
105.25 ± 3.3
15.20 ± 10.86
59.75 ± 12.65
54.55 ± 19.11
504
4
3.55 ± 2.05
Not tested
107.1 ± 4.38
33.15 ± 8.80
88.55 ± 24.63
85.63 ± 27.48
672
4
2.2 ± 0.56
Not tested
88.9 ± 23.05
75.25 ± 18.30
88.55 ± 8.53
91.58 ± 18.55
840
3
3.075 ± 2.70
Not tested
105.37 ± 53.4
46.65 ± 5.30
92.33 ± 5.16
91.85 ± 2.33
1008
3
15.5 ± 26.6
Not tested
108.85 ± 2.35
58.60 ± 9.48
92.45 ± 6.27
92.30 ± 2.69
1176
3
58.33 ± 39.55
Not tested
113.85 ± 2.62
72.95 ± 5.87
104.90 ± 3.5
101.90 ± 0.42
1344
3
71.55 ± 43.02
Not tested
110.3 ± 1.98
Not tested
Not tested
Not tested
1512
3
91.375 ± 29.7
Not tested
110.6 ± 0.85
Not tested
Not tested
Not tested
1680
3
112.22 ± 4.06
Not tested
112.15 ± 0.5
Not tested
Not tested
Not tested
Time = Time in hours post single-dose injection;
SEM = Standard error of mean;
BLQ = Below Limit of Quantitation;
NC = Not calculated
As shown in Table 25 and FIG. 2, PD effects were measured by complement CP (10 minute incubation) to Day 70. H4H12166P blocked more than 95% of CP hemolytic activity until day 35. Activity returned to pre-study maximum hemolysis levels by day 70. Comparator 2 blocked about 95% of CP hemolytic activity through day 10, and activity rapidly returned to pre-study maximum hemolysis levels by day 18.
PD effects were also measured by complement AP pathway (60 minute incubation) hemolysis assays to day 49. As shown in Table 25 and FIG. 3, H4H12166P blocked 80% of the total AP hemolytic activity until day 18 and activity returned to pre-study maximum hemolysis level at day 50. H4H1216P and Comparator 2 blocked 90% of AP hemolytic activity through day 7, and activity returned to pre-study maximum hemolysis levels by day 21.
Example 11: Characterization of PK/PD of Anti-C5 Antibodies in C5-Humanized Mice
In this set of experiments, the pharmacokinetics and pharmacodynamics of selected anti-C5 antibodies were assessed in mice humanized to express human C5 protein using Velocigene® technology (Valenzuela et a 2003, Nat. Biotechnol. 21: 652-659). Humanized mice were engineered to replace exon 2 through exon 41 of murine C5 gene with exons 2-42 of human C5 gene (disclosed in US Patent Application Publication 2015/0313194, herein incorporated in its entirety).
Total circulating human C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to manufacturer's recommendations.
Determination of Total Drug Level in Serum by ELISA
Circulating anti-C5 antibody concentrations, both C5-bound and -unbound, were determined by total human antibody analysis using ELISA. Briefly, a goat anti-human IgG polyclonal antibody at 1 μg/mL in PBS was immobilized on 96-well plates overnight; plates were washed to remove unbound IgG and then blocked with 5% BSA. Serial dilutions of anti-C5 antibody containing serum samples (6 points) and the reference standards (12 points) of the respective antibodies were transferred to the anti-human IgG coated plates and incubated for one hour. The plate-bound anti-C5 antibodies were then detected using a goat anti-human IgG polyclonal antibody conjugated with horseradish peroxidase. Plates were developed with TMB substrate according to the manufacturer's recommended protocol and signals of optical density (OD) at 450 nm were recorded using a Perkin Elmer VICTOR™ X4 Multimode Plate Reader. Anti-05 antibody concentrations in serum were calculated based on the reference standard calibration curve generated using GRAPHPAD™ PRISM™ software.
Determination of PK Parameters
PK parameters were determined by non-compartmental analysis (NCA) using Phoenix®WinNonlin® software (Version 6.3, Certara, L.P.) and an extravascular dosing model. Using the respective mean concentration values for each antibody, all PK parameters, including estimated half-life observed (t1/2), and area under the concentration versus time curve up to the last measurable concentration (AUClast) were determined using a linear trapezoidal rule with linear interpolation and uniform weighting.
PD Analysis by Hemolysis Assay
Pharmacodynamics of selected anti-C5 antibodies was determined using a classical pathway complement hemolysis assay. Sheep red blood cells (SRBCs) (Sheep blood in Alsevers solution) were washed in GVB++ buffer (Gelatin Veronal Buffer with CaCl2 and MgCl2) (Boston BioProducts) and re suspended at 1×10{circumflex over ( )}9 cells/mL. To sensitize, 1×10{circumflex over ( )}9/mL of SRBCs were mixed with equal volume of the 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for 20 minutes. Sensitized SRBCs were diluted to 2×10{circumflex over ( )}8 cells/mL in GVB++ prior to use in the hemolysis assay. Serum samples from pre-dosed animals or humanized C5 mice dosed with anti-C5 antibodies collected on days 10, 20, 30, 40 and 50 post-dose were diluted to 20% in GVB++ buffer. A total of 100 μl sensitized SRBCs (2×10{circumflex over ( )}8 cells/mL) were plated into 96-well round bottom plates at 37° C. followed by addition of 100 μl of 20% serum that was supplemented with 160-180 μg/mL human complement 3 (huC3) protein. Cells were gently mixed and incubated at 37° C. for 1 hour. After incubation, the cells were centrifuged at 1250×g at 4° C. A total of 100 μL of supernatant was transferred to a fresh 96 flat bottom plate and read at A412 nm on a Spectramax microplate reader. The percent hemolysis was calculated with the absorbance values by using the following equation:
%
Hemolysis
=
100
×
(
Experimental
Cell
Lysis
-
Background
Cell
Lysis
)
(
Maximum
Cell
Lysis
-
Background
Cell
Lysis
)
In this equation “background cell lysis” is the OD at A412 nm from SRBCs incubated in GVB++ buffer only containing no serum. The “maximum cell lysis” is the OD at A412 nm from SRBCs treated with water. The results, expressed as % hemolysis, were analyzed using nonlinear regression (4-parameter logistics) with PRISM™ 6 software (GRAPHPAD™) to obtain IC50 values. Data represented as Mean±(Standard Error of Mean).
Experiment 1
In this experiment, the pharmacokinetics and pharmacodynamics of exemplary antibody H4H12166P were assessed in comparison with Comparator 1 and Comparator 2 in humanized C5 mice. Total circulating human C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to manufacturer's recommendations. Average concentrations of human C5 in the mice were determined to be 39.73 μg/mL±17.82 μg/mL. There was a difference between male (55.4±1.7 μg/ml, n=47) and female (24.7±0.6 μg/ml, n=49) mice.
Prior to antibody dosing, male and female humanized C5 mice were stratified according to human C5 levels that averaged 40 μg/mL. For each anti-C5 antibody, cohorts of twenty-two mice received a single 15 mg/kg dose of H4H12166P, Comparator 1 or Comparator 2 by subcutaneous (s.c.) injection. All mice were bled predose and at one day post-injection for PK analysis. In addition, at 10, 20, 30, 40 and 50 days post injection, groups of 4 or 5 mice from each cohort were euthanized and terminal bleeds were collected for PK and PD analysis. Day 1 serum samples were the mean of the entire cohort of 22 mice. Blood was processed into serum and frozen at −80° C. until analyzed.
Total antibody concentrations were determined at 7 time points and percent hemolysis activity was determined at 6 time points over the 50-day in-life period. Total anti-C5 antibody concentrations are summarized in Table 26. The mean total antibody concentrations versus time profile are shown in FIG. 4. Mean PK parameters are described in Table 27.
TABLE 26
Mean Concentrations of Total IgG in Serum Following a Single 15 mg/kg
Subcutaneous Injection of anti-C5 antibodies in Humanized C5 mice
Serum concentration of Ab (μg/mL)
Time
Mean ± SD
(Day)
#
H4H12166P
Comparator 1
Comparator 2
1
22
178 ± 22.7
229 ± 40.7
164 ± 24.1
10
4
83.7 ± 22.2
102 ± 22.9
44 ± 24.1
20
5
57.1 ± 26.8
29 ± 31.3
11.4 ± 10.3
30
5
38.1 ± 7.6
30.1 ± 34.2
3.6 ± 3.2
40
4
11.9 ± 5.0
0.4 ± 0.4
0.5 ± 0.4
50
4*
9.3 ± 12.2
0.3 ± 0.3
0.3 ± 0.2
Time = Time in hours post single-dose injection;
Day = Day of study;
SD = Standard deviation;
SEM = Standard error of mean;
ND = Not detected;
NS = No sample.
*For Comparator 2, day 50, n = three due to the inability of one sample to be analyzed due to technical issues.
TABLE 27
PK parameters
Parameter
Units
H4H12166P
Comparator 1
Comparator 2
Day 1 mAb
μg/mL
178
229
164
concentration
AUClast
day · μg/mL
2801
2708
1418
t1/2
d
11.3
4.7
7.6
Cmax = Peak concentration;
AUC = Area under the concentration-time curve;
AUClast = AUC computed from time zero to the time of the last positive concentration;
T1/2 = Estimated half-life observed
Mean concentration versus time profiles at day 1 show that the three antibodies, H4H12166P, Comparator 1 and Comparator 2 had comparable serum concentrations of 178, 229 and 164 μg/mL, respectively. Comparator 1 had a similar elimination profile to H4H12166P up to day 30, but at days 40 and 50 exhibited a rapid increase in clearance versus H4H12166P. At day 50, H4H12166P had an average antibody serum concentration of approximately 9 μg/mL, whereas Comparator 1 and Comparator 2 both had a 30-fold lower average antibody serum concentration of 0.3 μg/mL. Comparator 2 exhibited the lowest exposure of the three antibodies tested, with an approximately 2-fold lower AUClast (1408 day pg/mL) as compared to H4H12166P (2801 day pg/mL) and Comparator 1 (2708 day pg/mL).
The pharmacologic effects of anti-C5 antibodies H4H12166P, Comparator 1 and Comparator 2 from humanized C5 mouse serum samples supplemented with human C3 were measured out to day 50 and were determined ex vivo by complement classical pathway (CP) hemolysis of sensitized SRBC. Mean percent hemolysis for each anti-C5 antibody is summarized in Table 28 and the mean percent hemolysis versus time profile is shown in FIG. 5.
TABLE 28
Ex Vivo Classical Pathway Percent Hemolysis
Activity of anti-human C5 antibodies
Classical pathway % hemolysis in 10% mouse
Time
serum, 60 min, Mean ± SEM
(Day)
#
H4H12166P
Comparator 1
Comparator 2
1
22
NS
NS
NS
10
4
12.6 ± 7.79
10.39 ± 2.88
12.06 ± 9.12
20
5
18.8 ± 8.1
21.59 ± 17.53
65.08 ± 52.87
30
5
13.76 ± 10.9
78.98 ± 40.3
91.67 ± 16.74
40
4
41.71 ± 40.7
101.09 ± 4.01
68.99 ± 42.47
50
4*
62.2 ± 56.6
88.99 ± 17.51
105.14 ± 4.07
Time = Time in hours post single-dose injection;
Day = Day of study;
SEM = Standard error of mean;
ND = Not detected;
NS = No sample.
*For Comparator 2, day 50, n = three due to the inability of one sample to be analyzed due to technical issues
H4H12166P, Comparator 1 and Comparator 2 inhibited the terminal complement hemolytic activity that appeared to correlate with antibody exposures. H4H12166P blocked more than 85% of hemolytic activity until day 30 with activity returning to predose baseline levels by day 50. Comparator 1 and Comparator 2 blocked about 80% hemolytic activity until day 20 and day 10, respectively, with activity returning to baseline levels by day 30 for both.
Experiment 2
In this experiment, the pharmacokinetics and pharmacodynamics of anti-C5 antibodies H4H12166P, H4H12161P, Comparator 1, and an isotype control was assessed in humanized C5 mice (mice homozygous for human C5 expression). Total circulating C5 levels were determined using a Human Complement C5 ELISA (Abcam, cat # ab125963), which was performed according to the manufacturer's recommendations. Average concentrations of human C5 in the mice were determined to be 48.98 μg/mL±15.1 μg/mL.
Prior to antibody dosing, humanized male and female C5 mice were stratified according to human C5 levels that averaged 50 μg/mL. For each anti-C5 mAb, cohorts of five mice received a single 15 mg/kg subcutaneous (s.c.) injection of H4H12166P, H4H12161P, Comparator 1 or an isotype control. All mice were bled predose, 6 hours, 1, 2, 3, 4, 7, 10, 13, 21, 30 and 45 days post injection for PK analysis. In addition, on day 59, all mice from each cohort were euthanized and terminal bleeds were collected for PK and PD analysis. Blood was processed into serum and frozen at −80° C. until analyzed.
Total antibody concentrations were determined at 12 time points and percent hemolysis activity was determined at 1 time point during a 59-day in-life period. Total serum antibody concentrations for each anti-C5 antibody are summarized in Table 29. Mean total antibody concentration versus time profiles are shown in FIG. 6. Mean PK parameters are described in Table 30.
TABLE 29
Mean Concentrations of Total IgG in Serum Following
a Single 15 mg/kg Subcutaneous Injection of
selected anti-C5 antibodies in Humanized C5 mice
Serum concentration of Ab (μg/mL)
Mean ± SD
Time
Comparator
Isotype
(Day)
H4H12166P
H4H12161P
1
Control
0
ND
ND
ND
ND
0.25
31.2 ± 4.2
43.5 ± 16.3
59.2 ± 24.1
61.5 ± 29.4
1
149.9 ± 16.1
193.8 ± 24.1
179.0 ± 9.8
218.1 ± 17
2
160.8 ± 20
221 ± 26.5
166.6 ± 22.3
188.8 ± 25.8
3
166.2 ± 12.4
210 ± 31.2
159.2 ± 33.2
177.9 ± 26.2
7
158.6 ± 8.5
162.5 ± 34.8
136.1 ± 38.1
184.9 ± 33.9
10
123.5 ± 28.7
133.2 ± 20.2
107.2 ± 45.7
159.5 ± 28.8
13
93.7 ± 23.6
97.2 ± 24.6
70.6 ± 38
117.2 ± 24.1
21
60.4 ± 14.9
42.4 ± 30.3
29.5 ± 20.6
80.0 ± 17.5
30
37.8 ± 10.8
15.3 ± 19.7
4.2 ± 3.5
42.1 ± 6.7
45
20.7 ± 5.2
3.5 ± 5.2
0.4 ± 0.3
16.5 ± 13.9
59
4.1 ± 1.9
0.6 ± 1.0
0.08 ± 0.04
4.6 ± 4.5
Time = Time in hours post single-dose injection;
Day = Day of study;
SD = Standard deviation;
SEM = Standard error of mean,;
ND = Not detected;
NS = No sample.
TABLE 30
PK parameters
Test Antibody (mean ± SD)
Param-
Comparator
Isotype
eter
Units
H4H12166P
H4H12161P
1
control
Cmax
μg/mL
178 ± 10
225 ± 22
183 ± 18
221 ± 19
AUClast
d · μg/
3490 ± 590
3040 ± 900
2240 ± 780
4080 ±
mL
480
t1/2
D
11 ± 1
5.8 ± 2
4.2 ± 1
9.9 ± 4
Cmax = Peak concentration;
AUC = Area under the concentration-time curve;
AUClast = AUC computed from time zero to the time of the last positive concentration;
T1/2 = Estimated half-life observed.
Mean concentration versus time profiles show that H4H12166P, H4H12161P, Comparator 1 and isotype control reached a maximum serum concentration (Cmax) between days 1 to 3, with comparable Cmax values within 1.3-fold (178, 225, 183 and 221) pg/mL, respectively. H4H12166P and isotype control had similar elimination profiles, with remaining drug levels of approximately 4 μg/mL at day 59. H4H12161P exhibited faster clearance than H4H12166P and isotype control but cleared more slowly than Comparator 1. At day 59, H4H12161P had mean serum drug level of 0.6 μg/mL while Comparator 1 had an almost undetectable drug level of 0.08 μg/mL.
The isotype control, H4H12166P and H4H12161P exhibited comparable exposure (AUClast) values within 1.3-fold (4080, 3490 and 3040 day pg/mL, respectively) whereas Comparator 1 exhibited a 1.6-fold lower exposure (2240 day·μg/mL) compared to H4H12166P.
Example 12: LC-MRM-MS-Based Assay to Determine the Concentration of Total Human C5
In this Example, serum concentrations of total human C5 were determined using a liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) method in a pharmacokinetics/pharmacodynamics study of anti-C5 antibody H4H12166P.
The serum concentrations of total human C5 were determined by measuring the concentration of a 10-amino acid peptide contained in the C5 sequence LQGTLPVEAR (aa 1129-1138 of SEQ ID NO: 359) as a proxy for C5. Theoretically, this method could also detect the C5 split product, C5b. However, due to the instability of free C5b, concentrations C5b in serum are generally low with the majority of C5b being bound to cell surfaces in the form of MAC complexes (Cooper & Muller-Eberhard 1970, J. Exp. Med. 132: 775-93; Hadders et al 2012, Cell Rep. 1: 200-7). Therefore, the processed serum samples analyzed here are likely to contain only negligible amounts of C5b product, if any.
Methods
For the PK/PD study, mice received a single 15 mg/kg dose of H4H12166P by subcutaneous (s.c.) injection. All mice were bled predose and at one day post-injection for PK analysis. In addition, at 10, 20, 30, 40, 50 and 60 days post injection, mice were euthanized and terminal bleeds were collected for PK and PD analysis.
Human C5 was used as a reference standard for calibration; and a human C5 peptide produced with a C-terminal stable isotope-labeled arginine residue was used as the internal standard (LQGTLPVEAR-13C615N4). Reference standard was used at concentrations ranging from 3.9 to 250 μg/mL (1:2 serial dilutions) in serum from in house-generated C5 knock-out mice, in which the mouse C5 gene was deleted (C5−/−). Serum from C5−/− mice was also used as a negative control (blank). Calibration standards, blanks, and study serum samples (10 μL each) were dried and then were denatured in 100 μL of 8M urea/20 mM Tris(2-carboxyethyl)phosphine (TCEP) buffer at 37° C. for 1 hour. Next, 10 μL of 25 nM internal standard was added to all samples. Samples were alkylated with 10 mM of 2-iodoacetamide at room temperature for 30 minutes and were diluted using 50 mM ammonium bicarbonate to a final volume of 500 μL. The samples were then digested by trypsin (1:20 w/w) overnight at 37° C. The tryptic peptide LQGTLPVEAR derived from C5 was detected and quantified by LC-MRM-MS using a Waters Xevo TQ-S with ACQUITY UPLC system. Each processed sample (10 μL) was injected onto a pre-equilibrated ACQUITY UPLC BEH C18 Column. The flow rate was 0.6 mL/min (Mobile Phase A:water:formic acid/100:0.1 [V:V] and Mobile Phase B: acetonitrile:formic acid/100:0.1 [V:V]). Retention time and peak area were determined using Masslynx Analyst Data software (Waters). Concentrations of C5 analyte were calculated from the calibration curve which was constructed by plotting the peak area ratio of C5 reference standard (unlabeled C5 peptide LQGTLPVEAR-12C614N4 generated by tryptic digest of hC5) to internal standard (stable isotope-labeled C5 peptide) versus the nominal concentration of C5 reference standard. Concentrations were calculated using linear regression. The lowest concentration of C5 reference standard (3.9 μg/mL) was within the dynamic range of the assay and was defined as the assay's LLOQ.
Results
Concentrations of total human C5 in serum were evaluated for samples collected and via tail bleed in advance of dosing (predose) and via terminal bleed on days 10, 30 and 35, from the corresponding animals. Total hC5 concentrations following H4H12166P dosing were similar (within ˜1 to 0.9-fold) to predose levels on days 10, 30, and 35 post dosing. The observed minor differences were not statistically significant as assessed by Mann-Whitney test using GRAPHPAD™ PRISM™ software. Analysis of the C5/H4H12166P molar ratio demonstrated that H4H12166P remained in molar excess of C5 through day 35 post dosing (Table 31).
TABLE 31
Summary of PD characteristics of H4H12166P
% CP
C5 (μg/mL)
H4H12166P
Molar ratio
Day Post
Hemolysis
%
Fold
(μg/mL)
terminal
dose
Mean ± SD
Inhibition
Predose
Terminal
Change
mean ± SD
C5:H4H12166P
0
77.2
0
n/d
n/a
n/a
10
5.2
93
34.2
30.0
1.0
78.8
0.3
30
7.4
90
31.9
28.4
0.9
30.4
0.8
35
6.0
92
36.1
32.7
1.0
29.6
0.9
40
37.1
52
n/d
20.9
n/d
50
All animals in group excluded due to MAHA titers >1000
60
73.57
5
n/d
5.9
n/d
aPercentage of inhibition of CP hemolytic activity was calculated from mean % CP hemolysis values on the indicated day post dosing relative to the mean % CP hemolysis value on day 0.
bFold Change = terminal (indicated day post dosing) C5: predose C5.
SD = standard deviation;
MAHA = mouse anti-human antibody;
n/d = not determined
Animals with MAHA-impacted data were completely excluded from calculations (2x day 30, 1x day 35, 2x day 40, and all 4 mice on day 50)
Example 13: Epitope Mapping of H4H12166P Binding to C5 by Hydrogen/Deuterium Exchange
H/D exchange epitope mapping with mass spectrometry was carried out to determine the amino acid residues of hC5 [(amino acids M1-C1676 of SEQ ID No: 359) with which H4H12166P interacts. A general description of the H/D exchange method is set forth in e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.
HDX-MS experiments were performed on an integrated Waters HDX/MS platform, consisting of a Leaptec HDX PAL system for the deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for the sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for the analytical column gradient, and Synapt G2-Si mass spectrometer for peptic peptide mass measurement.
The labeling solution was prepared in 10 mM PBS buffer in D2O at pD 7.0 (equivalent to pH 6.6). For deuterium labeling, 3.8 μL of C5 (6 pmol/μL) or C5 premixed with the antibody in 1:1 molar ratio was incubated with 56.2 μL D2O labeling solution for various time-points (e.g., undeuterated control=0 sec, labeled for 1 min and 20 min). The deuteration was quenched by transferring 50 μL sample to 50 μL pre-chilled quench buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0° C. for two minutes. The quenched sample was then injected into a Waters HDX Manager for online pepsin/protease XIII digestion. The digested peptides were trapped onto an ACQUITY UPLC BEH C18 1.7-μm, 2.1×5 mm VanGuard pre-column at 0° C. and eluted to an analytical column ACQUITY UPLC BEH C18 1.7-μm, 1.0×50 mm for a 9-minute gradient separation of 5%-40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set at cone voltage of 37 V, scan time of 0.5 s, and mass/charge range of 50-1700 Thomson units (Th).
For the identification of the peptides from human C5, LC-MSE data from undeuterated sample were processed and searched against the database including human C5, pepsin, and their randomized sequence via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported to DynamX software and filtered by two criteria: (1) minimum products per amino acid=0.3 and (2) replication file threshold=3. DynamX software then automatically determined deuterium uptake of each peptide based on retention time and high mass accuracy (<10 ppm) across multiple time points with 3 replicates at each time.
Using the online pepsin/protease XIII column coupled with MSE data acquisition, total 189 peptides from human C5 were identified in the absence or presence of the antibody, representing 62% sequence coverage. Five peptides had significantly reduced deuteration uptake (centroid delta values >0.9 daltons with p-values <0.05) when bound to H4H12166P and are illustrated in the Table 32.
TABLE 32
Deuteration of Human C5 peptides upon binding to H4H12166P
1 min Deuteration
20 min Deuteration
C5
C5 + H4H12166P
C5
C5 + H4H12166P
Residues
Centroid H+
Centroid MH+
Δ
Centroid MH+
Centroid MH+
Δ
591-599
1015.38 ± 0.09
1014.44 ± 0.16
−0.93
1015.64 ± 0.04
1014.60 ± 0.08
−1.04
593-599
769.41 ± 0.11
768.33 ± 0.05
−1.08
769.65 ± 0.01
768.30 ± 0.004
−1.35
775-787
1693.81 ± 0.11
1692.85 ± 0.07
−0.96
1694.06 ± 0.04
1692.96 ± 0.02
−1.10
775-794
2439.62 ± 0.29
2438.42 ± 0.20
−1.20
2440.16 ± 0.06
2439.17 ± 0.21
−0.99
779-787
1141.14 ± 0.04
1140.21 ± 0.05
−0.93
1141.23 ± 0.03
1140.21 ± 0.02
−1.02
The recorded peptide mass corresponds to the average value of the centroid MH+ mass from three replicates. These peptides, corresponding to amino acids 591-599 and 775-794, had slower deuteration rate upon binding to H4H12166P. These identified residues also correspond to the residues 591-599 and 775-794 of human C5 as defined by Uniprot entry P01031 (CO5_HUMAN; SEQ ID NO: 359).
Example 14: Effect of Anti-C5 Antibodies on Ocular Inflammation in Experimental Autoimmune Uveitis in Mice
The present study was undertaken to evaluate the role of C5 in experimental autoimmune uveitis (EAU). Both genetic [C5 knockout (KO), C3/C5 double KO mice], and pharmacologic (anti-C5 antibody) experimental approaches were used.
Methods
Adult C57BL/6J mice (n=25, Jackson labs), C5 KO (n=13) and C3/C5 KO (n=8) mice (Regeneron Pharmaceuticals Inc.) were used. EAU was induced by subcutaneous injection of human interphotoreceptor retinoid-binding protein peptide (IRBP, New England Peptide) in complete Freund's adjuvant and intraperitoneal injection of pertussis toxin. Anti-mouse C5 mAb or isotype control mAb was administered through subcutaneous injections every 3 days from day 5 to 28. The anti-mouse C5 antibody used in this study (M1M17628N) comprised a HCVR/LCVR of SEQ ID NOs: 362/363. SPECTRALIS® HRA+OCT (Heidelberg Engineering, Inc.) was used to assess levels of inflammation on days −1, 7, 14, 21 and 28. All animals were euthanized on day 28 for eye and blood collection. Hemolysis assay with/without human C3 was performed to validate complement inhibition. Data were analyzed by ANOVA.
Results
Compared to wild type mice, inflammation occurrence (30-50%) and vitreous cell cluster counts were significantly decreased in C5 KO mice (p<0.01). Optical coherence tomography (OCT) scores in C5 KO mice also significantly reduced 50% at week 3 (p<0.0001). Interestingly, in C3/C5 double KO mice, there were significantly more vitreous cell clusters and higher disease scores on day 28 compared to wild type mice (p<0.05). In animals that received anti-mC5 Ab (50 mg/kg), inflammation incidence and vitreous cell clusters were significantly lower compared to either no treatment or isotype control group on day 21 (p<0.01). At weeks 3 and 4, OCT scores in anti-C5 antibody-treated group were significantly lower compared to no treatment or isotype control (p<0.0001). (FIG. 7) Hemolysis assays with/without human C3 confirmed the inhibition effect of anti-C5 antibody at week 4 (FIG. 8).
Conclusion
Ocular inflammation due to EAU was mitigated by inhibiting C5 activity, either by genetic deletion or pharmacologic inhibition with a specific anti-C5 antibody. C5 depletion delayed EAU occurrence and reduced OCT disease score. These results indicate that C5 is a potential therapeutic target for autoimmune uveitis. Anti-C5 antibody has protective effect on EAU disease in wild type mice. Our findings also suggest that C3 might be beneficial for EAU disease in mice.
Example 15: Effect of Anti-Human C5 Antibodies on Experimental Autoimmune Uveitis
This Example describes the effects of anti-C5 antibodies against human C5 in a mouse model of experimental autoimmune uveitis (EAU). The mice used for this study were humanized to express human C5 protein using Velocigene® technology (Valenzuela et al 2003, Nat. Biotechnol. 21: 652-659). Humanized mice were engineered to replace exon 2 through exon 41 of murine C5 gene with exons 2-42 of human C5 gene (disclosed in US Patent Application Publication 2015/0313194, herein incorporated in its entirety).
Methods
Adult, male mice were immunized subcutaneously in each thigh with 150 μg of human interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20 (GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO: 364) (Avichezer et al 2000, Invest. Ophthalmol. Vis. Sci. 41:127-131) in 0.2 ml emulsion of CFA, supplemented with Mycobacterium tuberculosis strain H37RA to 2.5 mg/ml.
Mice were then inoculated intraperitoneally with 1.0 μg of pertussis toxin (PTX) to facilitate induction of cell-mediated antoimmunity by promoting a Th1 polarization of the immune response (Thurau et al 1997, Clin. Exp. Immunol. 109: 370-376; Silver et al 1999, Invest. Ophthalmol. Vis. Sci. 40: 2898-2905). The animal body weights were monitored twice a week.
Ophthalmic examinations were carried out on day −1, before EAU induction and on days 7, 14, 21 and 28. Mice were anesthetized with ketamine (120 mg/kg, IP) and xylazine (5 mg/kg, IP). Pupils were dilated using a 0.5% ophthalmic solution of Tropicamide, and the fundus of the eye was examined using a contact lens with a fundus camera in a Spectralis Heidelberg retinal angiography platform (HRA)+OCT system (Heidelberg Engineering, Carlsbad, Calif., USA).
A series of 61 lateral optical sections were obtained for each eye using the OCT function on the Spectralis HRA+OCT system (Heidelberg Engineering, Carlsbad, Calif., USA)). The OCT imaging area was centered on the optic disc allowing for equal imaging above and below the optic nerve head. The retinal thickness was measured as the distance between the bottom of the RPE layer to the inner limiting membrane of the eye. Measurements were taken 1500 μm from the optic disc, and the values from 4 different retinal quadrants (e.g., superior, inferior, temporal and nasal) were averaged for a mean retinal thickness of the eye.
The severity of inflammatory cell infiltration into the vitreous also was graded in OCT images, by assessing the average number of inflammatory cell clusters in the vitreous, in four lateral OCT scans that transected the optic nerve, per eye.
An 4-point scale was developed for assessment of disease severity in OCT images (OCT Scores) (Table 33).
TABLE 33
Scoring EAU in vivo using Optical Coherence Tomography (OCT)
Grade
Criteria
0
No Change
0.5 (Trace)
Minor inflammatory cell infiltration in the vitreous, primarily near the optic nerve head (<15
clusters)
1
Minor inflammatory cell infiltration in the vitreous, primarily near the optic nerve head (<25
clusters); minor focal subretinal lesions (grey spots) in the periphery; minor retinal folds in the
periphery; retinal vascular dilation; perivasculitis and vasculitis
2
Moderate inflammatory cell infiltration in the vitreous more diffuse but not in the far periphery
(<50 clusters); retinal layer disruptions; small- to medium-sized granuloma formations with
retinal folds primarily in the periphery; vessel dilation; minor focal choroidal
neovascularization; perivasculitis and vasculitis; minor retinal edema (<10 μm)
3
Moderate to severe diffuse inflammatory cell infiltration in the vitreous (>50 clusters); diffuse
retinal layer disruptions and dilated vessels in the inner nuclear layer; medium- to large-
granuloma formations with retinal folds throughout the retina; severe retinal vascular dilation;
perivasculitis and vasculitis; moderate diffuse choroidal neovascularization; moderate- to
severe-retinal edema (10-40 μm); minor retinal detachments
4
Severe diffuse inflammatory cell infiltration in the vitreous (>70 clusters); diffuse layer
disruptions and dilated vessels in the inner nuclear layer; severe diffuse granuloma formation
with retinal folds; severe diffuse choroidal neovascularization; perivasculitis and vasculitis;
retinal degeneration or severe retinal edema (>20 μm loss or >40 μm gain respectively); large
retinal detachments
Statistical Analysis
Statistical analyses for parametric data (body weight, inflammatory cell clusters in the vitreous, and retinal thickness) were performed by one-way ANOVA test and Tukey's multiple comparison test. For nonparametric data (OCT scores and histology scores) analyses were performed by the Kruskal-Wallis test and Dunn's test with the GRAPHPAD™ PRISM™ version 5.0d software relative to isotype control or no-treatment groups. Data show mean values±SEM. A p-value of less than 0.05 was considered as statistically significant.
Results
In a first study (Study A), mice were treated subcutaneously every 3 days from day 5 with isotype control antibody (50 mg/kg), or 10 mg/kg or 50 mg/kg of H4H12170P. Treatment with 10 mg/kg H4H12170P resulted in a reduction in inflammation and retinal damage (FIG. 9). Mice treated with 10 mg/kg H4H12170P also showed a statistically significant reduction in OCT scores on day 21 and day 28 (FIG. 10).
In a second study (study B), mice were treated subcutaneously every 3 days from day 6 with either isotype control (10 mg/kg), 3 mg/kg or 10 mg/kg of H4H12166P, or with Comparator 2 (see Example 2 herein; “Control Constructs used in the following Examples”). Treatment with H4H12166P either at 3 mg/kg or 10 mg/kg produced a dose-related reduction in OCT scores that was statistically significant on days 14 to 28 (FIG. 11). Treatment with 10 mg/kg H4H12166P in C5 humanized mice starting 6 days following EAU induction resulted in a dose-related reduction in inflammation and retinal damage as determined by OCT obtained on day 14 to 28 (FIG. 12).
For both studies, non-invasive, in-life evaluation by OCT showed a progressive development of inflammation, increased retinal thickness and morphological abnormalities in control animals following immunization with IRBP.
Conclusion
These experiments provide further pharmacological evidence that C5 plays a role in the pathogenesis of autoimmune uveitis. Pharmacologic depletion of human C5 by fully human anti-human C5 antibodies postponed EAU incidence and reduced disease severity, establishing the efficacy of these antibodies in autoimmune uveitis.
Example 16: Effect of Anti-C5 Antibodies on Renal Ischemia-Reperfusion Injury
The present study was carried out to evaluate the role of C5 in renal ischemia-reperfusion injury. Both genetic (using C3 knockout and C5 knockout mice) and pharmacological approaches (using anti-C5 antibodies) were used. Ischemia-reperfusion model was induced by bilateral renal pedicle clamping for 45 min followed by 48 h of reperfusion. Sham laparotomy served as controls. Anti-C5 antibody was administered at 50 mg/kg intravenously as a single dose immediately after ischemia (curative); or subcutaneously as two doses, day −1 and day 1 surgery (preventive). The anti-C5 antibody used for this study was M1M17628N comprising HCVR/LCVR of SEQ ID NO: 362/363. Blood urea nitrogen (BUN) and serum creatinine markers were used to assess levels of disease and protection in the mice.
TABLE 34
Percent change in blood urea nitrogen levels in
mice treated with anti-C5 antibody
(M1M17628N) in preventive and therapeutic modes
RIRI + Veh
RIRI + Iso. Ctl
BUN, % Change Vs.
Day 2
Day 2
RIRI + M1M17628N
−37.19
−34.68
(Prev)
RIRI + M1M17628N
−53.70
−51.85
(Cur)
TABLE 35
Percent change in serum creatinine levels in mice treated with anti-
C5 antibody (M1M17628N) in preventive and therapeutic modes
RIRI + Veh
RIRI + Iso. Ctl
SCr, % Change Vs.
Day 2
Day 2
RIRI + M1M17628N
−53.09
−49.34
(Prev)
RIRI + M1M17628N
−59.40
−56.16
(Cur)
Compared to wild type mice, C3 and C5 knockout mice showed significant functional protection in the RIRI model of acute kidney injury, as evidenced by reduction in the blood urea nitrogen and serum creatinine levels. The anti-C5 antibody showed functional protection in RIRI model in both preventive and therapeutic modes (Tables 34-35).
Example 17: Effect of Anti-C5 Antibodies on Lupus Nephritis
This Example describes the efficacy of anti-C5 antibodies in treating lupus nephritis in a mouse model.
Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. Complement activation in the kidneys of lupus nephritis patients contributes to inflammation and tissue damage. The efficacy of anti-C5 antibodies in treating lupus nephritis was studied in NZBWF1 mice, a spontaneous mouse model of lupus nephritis (Yang et al 1996, PNAS). The mice develop autoimmune disease resembling human SLE, autoantibodies to nuclear antigens and cell membrane proteins, hypergammaglobulinemia, albuminuria, proteinuria, initiate immune complex glomerulonephritis and die of kidney failure and end-stage renal disease at 35 to 50 weeks of age.
For this study, 25-week old NZBWF1 mice were subcutaneously treated with 30 mg/kg of isotype control, or anti-C5 antibodies twice a week for 8 weeks followed by thrice a week for 10 weeks. The anti-mouse C5 antibodies used for this study were M1M17628N and M1M17627N, comprising HCVR/LCVR of SEQ ID NOs: 362/363 and 365/366, respectively.
Treatment with anti-C5 antibodies significantly improved survival rate in mice (FIG. 13). Both antibodies improved albuminuria at 8-14 weeks of treatment (FIG. 14), and blood urea nitrogen levels at 12-16 weeks of treatment (FIG. 15).
Example 18: Effect of Anti-C5 Antibodies Against Astrocyte Cell Death
Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS) that mainly affects the optic nerve and spinal cord. In NMO, anti-aquaporin-4 autoantibodies (AQP4-Ab) cause damage to astrocytes by activating complement-dependent cytotoxicity (CDC). The goals of this study were to evaluate the role of the complement system in NMO progression and the use of an antibody against a complement protein as a potential therapeutic treatment for NMO.
Primary rat cortical astrocytes were obtained from cerebral brain cortex of post-natal rat pups and were cultured with AQP4-Ab (antibody “rAb-53” from US Patent Application Publication 2014/0170140; Bennett et al 2009, Ann. Neurol. 66: 617-629) and complement proteins to demonstrate cell-mediated cytotoxicity. Then the experiments were repeated with addition of an anti-C5 antibody to demonstrate blocking of the astrocyte cell destruction.
To quantify cell death, a CYTOTOX-GLO™ luminescence cytotoxicity assay was performed. The assay used various concentrations of anti-C5 antibody (0.001m/ml, 0.01m/ml, 0.1m/ml, 1m/ml, 10m/ml, 100m/ml, or 1000m/ml) or an isotype control antibody.
In order to determine whether anti-C5 antibody could block the AQP4-Ab induced CDC, astrocytes were plated and the CYTOTOX-GLO™ Cytotoxicity Assay was repeated to find an optimal dose of AQP4-Ab for that plating. The optimal concentration of AQP4-Ab was found to be 50m/mL, and in a following experiment a constant dose of AQP4-Ab (50m/mL) was used, while the dose of anti-C5 antibody was varied. As shown in FIG. 16, a decrease in RLU was seen (on average 300 k to on average 100 k) with increasing amounts of anti-C5 antibody demonstrating that the anti-C5 antibody blocked astrocyte cell death. For both experiments, the RLU did not vary with the isotype control antibody. As shown in FIG. 16, anti-C5 antibodies inhibited AQP4 Ab induced cytotoxicity on primary cortical astrocyte with IC50 of 15-17 nM.
In a subsequent study, anti-AQP4 antibody and anti-C5 antibody will be injected into rat brains to assess the therapeutic efficacy against complement-mediated cytotoxicity of astrocytes in CNS.
Example 19: Endothelial Assay
This Example describes an in vitro glomerular endothelial assay to examine if anti-C5 antibodies block C5b-9 and C3 deposition.
Reproducible methods for evaluating inhibitory effects of drug candidates on complement activation are essential for preclinical development. Due to the complexity of complement activation pathways, an assay should use relevant cells and endpoints to the given therapeutic indication. Here, using an immortalized human glomerular endothelial cell line (HGECs), a complement C3 & C5 deposition model was validated for use evaluating the blocking activity of anti-C3 or C5 mAbs.
Methods
Human primary kidney glomerular endothelial cells (HGEC; Cell Biologics) were plated overnight in complete media into collagen I—coated black clear bottom 96-well plates. The cells were treated with either PBS (control) or activated for 10 mins with 10 uM ADP. After PBS washing, 50% human serum (complement-preserved, C3-depleted, or C5-depleted) was added for 4 hours. Anti-C5 antibodies were added at 1 mg/mL to the serum prior to the treatment. The cells were washed and fixed and probed with anti-C3b antibodies (Thermofisher) and/or anti-C5b-9 antibodies (Abcam), secondary antibodies and counterstained with DAPI. Images were captured on ImagExpress and high content image analysis was used to quantify fluorescent staining for each image and averaged per condition.
Results
C3 and C5b-9 deposition was observed on ADP-activated HGECs exposed to normal human serum but not on non-activated HGECs (C3: 1.5×107±1.0×107; C5: 7.9×106±6.6×106, P<0.05 vs non-ADP-activated HGEC). The deposition of C3 and C5b-9 were significantly reduced on ADP-activated HGEC exposed to C3 or C5 depleted serum (C3: 3.3×105±4.8×104; C5: 1.5×106±6.0×105, P<0.05). Addition of a blocking anti-C5 mAb significantly reduced normal human serum derived C5b-9 deposition onto ADP-activated HGEC, deposition was comparable to C5 depleted sera (C5 mAb: 1.02×106+6.0×105, Control mAb 3.7×106±1.6×106, P<0.05 vs. control mAb).
CONCLUSION
These data demonstrate the utility of an in vitro human glomerular endothelial assay to model complement C3 & C5 deposition. In addition to in-vitro screening, this assay offers potential as a translational model to evaluate anti-complement strategies in renal disease using patient derived serum samples.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
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15621689
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regeneron pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Regeneron Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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