Anti-VEGF antibodies

• US 20070141065A1
• Filed: 10/19/2006
• Published: 06/21/2007
• Est. Priority Date: 08/01/2003
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Liang, Wei-Ching, Wiesmann, Christian, Gerber, Hans-Peter, Fuh, Germaine, Fellouse, Frederic, Sidhu, Sachdev
• Granted Patent: US 7,691,977 B2
• Time in Patent Office: Days
• US Class Current: 424/155.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 16/005 : constructed by phage libraries
• CPC Class Codes: C07K 16/22 : against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 : multispecific
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

Anti-VEGF antibodies and variants thereof, including those having high affinity for binding to VEGF, are disclosed. Also provided are methods of using phage display technology with naive libraries to generate and select the anti-VEGF antibodies with desired binding and other biological activities. Further contemplated are uses of the antibodies in research, diagnostic and therapeutic applications.

Claims

1. An antibody capable of binding to mouse VEGF and human VEGF with Kd values within 10 fold of the other and is capable of inhibiting the binding of VEGF to a VEGF receptor.

2. The antibody according to claim 1, wherein the antibody is capable of binding to a human VEGF G88A mutant with a Kd value that is within 10 fold of the Kd value of unmutated human VEGF.

3. The antibody according to claim 1, wherein the antibody is capable of binding a human VEGF G88A mutant with a Kd value that is 10 nM or less.

4. The antibody according to claim 1, wherein the antibody contacts no more than 80% of the surface area of GSS of human VEGF.

5. The antibody according to claim 1, wherein the receptor is a VEGF receptor 2 (KDR).

6. The antibody according to claim 1, wherein the receptor is a VEGF receptor 1 (Flt-1).

7. The antibody according to claim 1, the antibody is capable of inhibiting the binding of VEGF to VEGF receptor 1 and VEGF receptor 2.

9. The antibody according to claim 1, wherein the antibody binds to mouse and human VEGF with Kd values of 10 nM or less.

10. The antibody according to claim 1, wherein the antibody binds to mouse and human VEGF with Kd values of 2 nM or less.

11. The antibody according to claim 1, wherein the antibody binds to human VEGF with an on-rate (k_on) of 1.0 or more (10 M⁻

1 S<sup>−

1</sup>).

12. The antibody according to claim 1, wherein the antibody binds to the 20s helix of VEGF.

13. The antibody according to claim 1, wherein the antibody binds to the 80s loop of VEGF.

14. The antibody according to claim 1, wherein the antibody binds to the 20s helix and 80s loop of VEGF.

15. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residue residue F17 of human VEGF.

16. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17 and I83 of human VEGF.

17. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, I83 and Q89 of human VEGF.

18. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17 and M18 of human VEGF.

19. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, M18 and I89 of human VEGF.

20. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, Y21 and Y25 of human VEGF.

21. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, Y21, Q22, Y25, D63 and I83 of human VEGF.

22. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, Y21, Y25 and Q89 of human VEGF.

23. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103 and C104 of human VEGF.

24. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising residues F17, Y21, Q22, Y25, D63, I83 and Q89 of human VEGF.

25. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising a combination of any of the residues selected from the group consisting of F17, M18, Y21, Q22, Y25, K48, D63, L66, I83, H86, Q89, 191, C104 and P106 of human VEGF.

26. The antibody according to any one of claims 1-11 wherein the antibody has a functional epitope comprising the residues F17, M18, Y21, Q22, Y25, K48, D63, L66, I83, H86, Q89, I91, C104 and P106 of human VEGF.

27. The antibody according to claim 1, wherein the antibody comprises a CDR-H3 comprising the contiguous amino acid sequence X₁X₂FX₄X₅X₆X₇, wherein

X₁ is Y or F;

X₂ is V or A;

X₄ is F or Y;

X₅ is L or A;

X₆ is P or A; and

X₇is Y or F.

28. The antibody according to claim 27, wherein the CDR-H2 comprises a contiguous amino acid sequence GX₂TPX₅G, wherein

X₂ is a I or V or other hydrophobic amino acid;

and

X₅ is any amino acid residue.

29. The antibody according to claim 28, wherein the CDR-H1 comprises a contigous amino acid sequence X₁X₂X_3zIH, wherein

X₁ is any amino acid residue;

X₂ is Y or F; and

X₃is W or L.

30. The antibody according to claim 1, wherein the antibody comprises the CDR-H3 sequence of any one of the antibodies of FIGS. 7, 24-29 and 34-43.

31. The antibody according to claim 1, wherein the antibody comprises a variable region comprising a sequence of any one of the antibodies of FIGS. 7, 24-29 and 34-43.

34. The antibody of any one of claims 1-15 and 27-30, further comprising the framework regions of the G6 antibody.

37. The antibody according to claim 27-36, wherein the antibody binds human VEGF and mouuse VEGF with Kd values within 10 fold of each other.

38. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.

39. The antibody according to claim 1, wherein the antibody is a bispecific antibody.

40. The antibody according to claim 1, wherein the antibody is a synthetic antibody.

46. The antibody of claim 20 comprising at least one variant CDR having a variant amino acid in at least one solvent accessible and highly diverse amino acid position, wherein the variant amino acid is encoded by a nonrandom codon set, and wherein at least 70% of the amino acids encoded by the nonrandom set are target amino acids for that position in known antibody variable domains.

47. The antibody of claim 46 having a heavy chain variable domain which comprises at least 1, 2 or 3 variant CDRs selected from the group consisting of CDR H1I, H2 and H3.

48. The antibody of claim 47, wherein the heavy chain variable domain comprises a variant CDR H3.

49. The antibody of claim 47, comprising following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):

50. The antibody of claim 24, further comprising following amino acid sequence as its light chain:

51. The antibody of claim 21, having a light chain variable domain which comprises at least 1, 2 or 3 variant CDRs selected from the group consisting of CDR L1, L2 and L3.

52. The antibody of claim 49, further comprising following amino acid sequence as its light chain:

54. Isolated nucleic acid encoding the antibody of any one of claims 1-50.

55. A vector comprising the nucleic acid of claim 54.

60. A method of using the antibody of any of claims 1-52 for treating a disorder associated with pathological angiogenesis in a mammal in need of treatment thereof comprising the step of administering said antibody to the mammal.

61. The method of claim 60, wherein the disorder is cancer.

62. The method of claim 61, wherein the cancer is from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma.

63. The method of claim 62, wherein the treatment further comprises the step of administering a second therapeutic agent simultaneously or sequentially with the antibody.

64. The method of claim 63, wherein the second therapeutic agent is an agent selected from the group consisting of an anti-angiogenic agent, an anti-neoplastic composition, a chemotherapeutic agent and a cytotoxic agent.

65. The method of claim 64, wherein the anti-angiogenic agent is an anti-hVEGF antibody capable of binding to the same VEGF epitope as the antibody A4.6.1.

66. A method of treating a mammal suffering from or at risk of developing an inflammatory or immune disorder comprising the step of treating the mammal with a Fab antibody of any of one of claims 1-51.

67. The method according to claim 66, wherein the inflammatory or immune disorder is rheumatoid arthritis.

68. The method according to claim 66, wherein the Fab antibody is a G6 series antibody.

69. The method according to claim 66, wherein the Fab antibody is a B20 series antibody.

32. An antibody comprising the CDR-H1 sequence, CDR-H2 sequence and CDR-H3 sequence of any one of the antibodies of FIGS. 7, 24-29 and 34-43.

33. An antibody comprising a variable region comprising a sequence described in any one of the antibodies of FIGS. 7, 24-29 and 34-43.

35. An antibody comprising:

(a) a CDR-L1 comprising the contiguous amino acid sequence X₁X₂X₃X₄X₅L, wherein;

X₁ and X₂ are any amino acid;

Either X₃ or X₄ or both X₃ and X₄ are R;

X₅is S or A.

(b) a CDR-L2 comprising a contiguous amino acid sequence X_lX₂X₃, wherein

X₁ is S or A or G; and

X₂ and X₃ is any amino acid residue.

(c) a CDR-L3 comprising a contigous amino acid sequence SX₁X₂X₃PL, wherein

X₁ and X₂ are any amino acid residue; and

X₃ is S or A.

36. The antibody according to claim 35, wherein the antibody comprises the framework regions of the B20-4.1 antibody.

41. A synthetic antibody capable of binding hVEGF with a Kd value of no more than about 2 nM.

42. The antibody of claim 41, wherein the Kd value is no more than about 1 nM.

43. The antibody of claim 42, wherein the Kd value is no more than about 500 pM.

44. The antibody of claim 43, which is capable of binding to a VEGF from a non-human mammal species with a Kd value that is within 10 fold of the Kd value for hVEGF binding.

45. The antibody of claim 44, wherein the VEGF from a non-human mammal is murine VEGF.

53. A method of selecting a hVEGF antibody from a library of synthetic antibodies comprising:

a) generating the library of synthetic antibodies having a designed diversity in at least one of the CDRs;

b) contacting the library with hVEGF to form binders;

c) separating the binders from the nonbinders, and eluting the binders from the target hVEGF and incubating the binders in a solution with decreasing amounts of the target hVEGF in a concentration from about 0.1 nM to 1000 nM; and

d) selecting the binders that can bind to the lowest concentration of the target VEGF and that have an affinity of about 500 pM to 2 nM.

56. A host cell transformed with the vector of claim 56.

57. A process of producing an antibody comprising culturing the host cell of claim 56 so that the nucleic acid is expressed.

58. The process of claim 57 further comprising recovering the antibody from the host cell culture.

59. The process of claim 58 wherein the antibody is recovered from the host cell culture medium.