Engineering Fc antibody regions to confer effector function

• US 20060134709A1
• Filed: 11/10/2005
• Published: 06/22/2006
• Est. Priority Date: 11/10/2004
• Status: Active Grant

• Current Assignee: MacroGenics, Inc.
• Original Assignee: MacroGenics, Inc.
• Inventors: Koenig, Scott, Stavenhagen, Jeffery
• Granted Patent: US 7,632,497 B2
• Time in Patent Office: Days
• US Class Current: 435/7.230
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: G01N 33/574 for cancer

Abstract

The present invention relates to molecules having a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region. These modified molecules confer an effector function to a molecule, where the parent molecule does not detectably exhibit this effector function. In particular, the molecules of the invention have an increased effector cell function mediated by a FcγR, such as, but not limited to, ADCC. In one embodiment, the variant Fc region binds FcγRIIIA and/or FcγRIIA with a greater affinity, relative to a comparable molecule comprising the wild-type Fc region. The molecules of the invention have particular utility in treatment, prevention or management of a disease or disorder, such as cancer, in a sub-population of patients, wherein the target antigen is expressed at low levels in the target cell population, in particular, in patients refractory to treatment with an existing therapeutic antibody due to the low level of target antigen expression on the cancer or associated cells.

Claims

1. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable effector function activity in cells positive for said antigen, wherein said parent antibody does not exhibit detectable effector function activity in said cells using said in vitro assay.

4. The modified antibody of claim 1 or 2, wherein said antigen is a cancer antigen.

5. The modified antibody of claim 1 or 2, wherein said antigen is expressed on the surface of a cell.

6. The modified antibody of claim 4, wherein said cancer antigen MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15, beta-catenin, MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, human papillomavirus-E7, MUC-1, CD20 or CD32B.

7. The modified antibody of claim 1 or 2, wherein said at least one amino acid modification comprises substitution at position 370 with glutamic acid, at position 396 with leucine and at position 270 with glutamic acid;

at position 419 with histidine, at position 396 with leucine and at position 270 with glutamic acid;

at position 240 with alanine, at position 396 with leucine and at position 270 with glutamic acid;

at position 240 with alanine, at position 396 with leucine and at position 270 with glutamic acid;

at position 255 with leucine, at position 396 with leucine and position 270 with glutamic acid;

at position 255 with leucine, at position 396 with leucine, at position 270 with glutamic acid and at position 292 glycine;

at position 255 with leucine, at position 396 with leucine, at position 270 with glutamic acid and at position 300 leucine;

at position 243 with leucine, at position 270 with glutamic acid, at position 392 with asparagine and at position 396 with leucine;

or at position 243 with leucine, at position 255 with leucine, at position 270 with glutamic acid and at position 396 with leucine.

8. The modified antibody of claim 1 or 2, wherein said amino acid modification comprises at least one amino acid modification in the CH2 domain.

9. The modified antibody of claim 8, wherein said amino acid modification in the CH2 domain comprises substitution at position 240, 243, 247, 255, 270, 292, or 300 with another amino acid at that position.

10. The modified antibody of claim 1 or 2, wherein said amino acid modification comprises at least one amino acid modification in the CH3 domain.

11. The modified antibody of claim 10, wherein said amino acid modification in the CH3 domain comprises substitution at position 370, 392, 396, 419, or 421 with another amino acid at that position.

12. The modified antibody of claim 1 or 2, wherein said amino acid modification comprises at least one amino acid modification in the CH2 domain and at least one amino acid modification in the CH3 domain of the Fc region

13. The modified antibody of claim 1 or 2, comprising at least one amino acid modification in the hinge region of the human IgG heavy chain.

14. The modified antibody of claim 12, comprising at least one amino acid modification in the hinge region of the human IgG heavy chain.

15. The modified antibody of claim 1 or 2 which variant IgG Fc region specifically binds Fcγ

RIIIA with a greater affinity than said parent antibody binds Fcγ

RIIIA.

16. The modified antibody of claim 1 or 2 which variant IgG Fc region specifically binds Fcγ

RIIA with a greater affinity than said parent antibody binds Fcγ

RIIA.

17. The modified antibody of claim 1 or 2 which variant IgG Fc region specifically binds Fcγ

RIIB with a lower affinity than said parent antibody binds Fcγ

RIIB.

18. The modified antibody of claim 15 which variant IgG Fc region specifically binds Fcγ

RIIB with a lower affinity than said parent antibody binds Fcγ

RIIB.

19. The modified antibody of claim 16 which variant IgG Fc region specifically binds Fcγ

RIIB with a lower affinity than said parent antibody binds Fcγ

RIIB.

20. The modified antibody of claim 1 or 2 which detectably binds cells positive for said antigen, which antigen is expressed at a density of 200 to 1,000 molecules/cell on said cells.

21. The modified antibody of claim 1 or 3 wherein said effector function is ADCC.

22. The modified antibody of claim 1 or 3 wherein said effector function is phagocytosis, opsonization, cell binding, rosetting, complement dependent cell mediated cytotoxicity (CDC), or antibody dependent cell-mediated cell cytotoxicity (ADCC).

23. The modified antibody of claim 1 or 3 wherein said in vitro assay is performed at an effector cell:

target cell ratio of 10;

1, 30;

1, 50;

1, 75;

1 or 100;

1.

24. The modified antibody of claim 1 or 2 which is a monoclonal antibody.

25. The modified antibody of claim 1 or 2 which is a chimeric, human or humanized antibody.

26. The modified antibody of claim 25 which is chimeric 2B6 antibody.

27. The modified antibody of claim 4, wherein said parent antibody is HERCEPTIN®

, IC14, PANOREX™

, IMC-225, VITAXIN™

, Campath 1H/LDP-03, LYMPHOCIDE™

, ZEVLIN™

or rituximab.

28. The modified antibody of claim 22, wherein said parent antibody is HERCEPTIN®

, IC14, PANOREX™

, IMC-225, VITAXIN™

, Campath 1H/LDP-03, LYMPHOCIDE™

, or ZEVLIN™

or rituximab.

29. The modified antibody of claim 1 or 2, wherein the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region.

30. The modified antibody of claim 1 or 2 in which the antigen is associated with an infectious disease.

31. The modified antibody of claim 30, in which the antigen is a viral, bacterial or fungal antigen.

32. The modified antibody of claim 1 or 2, in which the parent antibody has immunomodulatory activity.

33. A method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of the modified antibody of claim 1 or 2, which modified antibody binds said cancer antigen.

34. The method of claim 33, wherein said patient is refractory to treatment with said parent antibody.

35. The method of claim 34, wherein said cancer antigen is MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15, beta-catenin, MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, human papillomavirus-E7, MUC-1, CD20 or CD32B.

36. The method of claim 33, wherein said cancer antigen is a colon, breast, ovarian, prostate, cervical, pancreatic carcinoma, non-Hodgkins lymphoma or chronic lymphocytic leukemia antigen.

37. The method of claim 33 further comprising the administration of one or more additional cancer therapies.

38. The method of claim 33, wherein said patient is human.

39. The method of claim 33, which modified antibody is a monoclonal antibody.

40. The method of claim 33, wherein said parent antibody is HERCEPTIN®

, IC14, PANOREX™

, IMC-225, VITAXIN™

, Campath 1H/LDP-03, LYMPHOCIDE™

, ZEVLIN™

or rituximab.

41. The method of claim 33, wherein the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region.

42. A pharmaceutical composition comprising the modified antibody of claims 1 or 2 and a pharmaceutically acceptable carrier.

43. A nucleic acid comprising a nucleotide sequence encoding a heavy or light chain of the modified antibody of claim 1 or 2.

44. A vector comprising the nucleic acid of claim 43.

45. The vector of claim 44 which is an expression vector.

46. A host cell comprising the nucleic acid of claim 45.

47. A host cell comprising a first nucleotide sequence encoding a heavy chain of a modified antibody and a second nucleotide sequence encoding a light chain of a modified antibody, said modified antibody being the modified antibody of claim 1 or 2.

48. A method for recombinantly producing a modified antibody, said method comprising:

a. culturing in a medium the host cell of claim 46 under conditions suitable for the expression of the modified antibody; and

b. recovery of said modified antibody from said medium.

2. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody is therapeutically effective in a patient refractory to treatment with said parent antibody.

3. The modified antibody of claim 2, wherein said modified antibody exhibits, in an in vitro assay, detectable effector function activity in cells derived from said patient, which cells are positive for said antigen, wherein said parent antibody does not exhibit detectable function activity in said cells using said in vitro assay.

49. A method for improving a therapeutic antibody that specifically binds to an antigen and that does not exhibit, in an in vitro assay, detectable effector function activity, said method comprising

a. introducing at least one amino acid modification in the Fc region of said therapeutic antibody to generate a modified therapeutic antibody;

and

b. determining whether said modified therapeutic antibody exhibits detectable effector function activity using said in vitro assay.

50. The method of claim 49, wherein said modified antibody bind to cells expressing said antigen at a density of 200 to 1,000 molecules/cell.

51. The method of claim 49 wherein said target molecule is Fcγ

RIIA or Fcγ

RIIIA.

52. The method of claim 49 wherein said target molecule is said antigen.

53. The method of claim 49, wherein said antigen is a cancer antigen.

54. The method of claim 49, wherein said modified therapeutic antibody has an increase in at least one effector function.

56. A method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of the modified antibody of claim 54, which modified antibody binds said cancer antigen.

55. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable cell killing in cells positive for said antigen, wherein said parent antibody does not exhibit detectable cell killing in said cells using said in vitro assay.

57. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable effector function activity in cells positive for said antigen, wherein said parent antibody does not exhibit detectable effector function activity in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 75;

1.

58. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable effector function activity in cells positive for said antigen, wherein said parent antibody does not exhibit detectable effector function activity in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 30;

1.

59. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable effector function activity in cells positive for said antigen, wherein said parent antibody does not exhibit detectable effector function activity in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 10;

1.

60. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable cell killing in cells positive for said antigen, wherein said parent antibody does not exhibit detectable cell killing in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 75;

1.

61. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable cell killing in cells positive for said antigen, wherein said parent antibody does not exhibit detectable cell killing in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 30;

1.

62. A modified antibody that binds an antigen, said modified antibody comprising a variant human IgG Fc region, wherein said variant human IgG Fc region comprises at least one amino acid modification relative to the human IgG Fc region of a parent antibody that binds said antigen, such that said modified antibody exhibits, in an in vitro assay, detectable cell killing in cells positive for said antigen, wherein said parent antibody does not exhibit detectable cell killing in said cells using said in vitro assay, which in vitro assay is performed at an effector cell:

target cell ratio of 10;

1.