METHOD FOR MAKING ANTIBODY FC-HETERODIMERIC MOLECULES USING ELECTROSTATIC STEERING EFFECTS

• US 20100286374A1
• Filed: 01/06/2009
• Published: 11/11/2010
• Est. Priority Date: 01/07/2008
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Pentony, Martin, Kannan, Gunasekaran, Yan, Wei, Wittekind, Michael
• Granted Patent: US 8,592,562 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• 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 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/526 CH3 domain

Abstract

The invention relates to methods of making Fc-heterodimeric proteins or polypeptides. The invention also relates to the Fc-heterodimeric proteins or polypeptides themselves, including the individual polypeptide components that comprise the heterodimer. Nucleic acids encoding such polypeptides, expression vectors, and host cells. Moreover, the invention relates to pharmaceutical compositions comprising one of more Fc-heterodimeric proteins or polypeptides.

Claims

1. A method of preparing a heterodimeric protein comprising a first CH3-containing polypeptide and a second CH3-containing polypeptide that meet together to form an interface engineered to promote heterodimer formation, wherein said first CH3-containing polypeptide and said second CH3-containing polypeptide comprise one or more charged amino acids within the interface that are electrostatically unfavorable to homodimer formation but electrostatically favorable to heterodimer formation, the method comprising the steps of:

(a) culturing a host cell comprising a nucleic acid encoding the first CH3-containing polypeptide and a nucleic acid comprising the second CH3-containing polypeptide, wherein the cultured host cell expresses the first and second CH3-containing polypeptides; and

(b) recovering the heterodimeric protein from the host cell culture.

2. The method of claim 1, wherein the heterodimeric protein comprises an Fc region.

3. The method of claim 2, wherein the Fc region comprises an IgG Fc region.

4. The method of claim 3, wherein the IgG Fc region comprises a human IgG Fc region.

5. The method of claim 4, wherein the human IgG region comprises an IgG1 Fc region.

6. The method of claim 4, wherein the IgG region comprises an IgG2 Fc region.

7. The method of claim 4, wherein the IgG region comprises an IgG3 Fc region.

8. The method of claim 4, wherein the IgG region comprises an IgG4 Fc region.

9. The method of claim 2, wherein the Fc region comprises an IgA, IgE, IgD, or IgM Fc region.

10. The method of claim 4, wherein the first CH3-containing polypeptide or the second CH3-containing polypeptide comprises a polypeptide sequence differing from wild-type human IgG such that one or more positive-charged amino acids in a wild-type human IgG sequence are replaced with one or more negative-charged amino acids.

11. The method of claim 10, wherein the positive-charged amino acid is selected from the group consisting of lysine, histidine, and arginine.

12. The method of claim 10, wherein the negative-charged amino acid is selected from the group consisting of aspartic acid and glutamic acid.

13. The method of claim 10, wherein the first CH3-containing polypeptide or the second CH3-containing polypeptide comprises a polypeptide sequence differing from wild-type human IgG such that one or more negative-charged amino acids in a wild-type human IgG sequence is replaced with one or more positive-charged amino acids.

14. The method of claim 13, wherein the positive-charged amino acid is selected from the group consisting of lysine, histidine, and arginine.

15. The method of claim 13, wherein the negative-charged amino acid is selected from the group consisting of aspartic acid and glutamic acid.

16. The method of claim 5, wherein the IgG1 Fc comprises a first CH3-containing polypeptide or second CH3-containing polypeptide having a polypeptide sequence differing from wild-type human IgG1 such that one or more positive-charged amino acids in a wild-type human IgG1 sequence are replaced with one or more negative-charged amino acids.

17. The method of claim 16, wherein the first CH3-containing polypeptide comprises a replacement of a lysine with a negative-charged amino acid.

18. The method of claim 17, wherein the lysine is selected from the group consisting of Lys409, Lys392, Lys439, and Lys370.

19. The method of claim 5, wherein the IgG1 Fc comprises a first CH3-containing polypeptide or second CH3-containing polypeptide having a polypeptide sequence differing from wild-type human IgG1 such that one or more negative-charged amino acids in a wild-type human IgG1 sequence are replaced with one or more positive-charged amino acids.

20. The method of claim 19, wherein the first CH3-containing polypeptide comprises a replacement of an aspartic acid with a positive-charged amino acid.

21. The method of claim 20, wherein the aspartic acid is selected from the group consisting of Asp399 and Asp356.

22. The method of claim 19, wherein the first CH3-containing polypeptide comprises a replacement of a glutamic acid with a positive-charged amino acid.

23. The method of claim 22, wherein the glutamic acid is Glu357.

24. The method of claim 18, wherein the first CH3-containing polypeptide comprises a replacement of Lys409 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

25. The method of claim 18, wherein the first CH3-containing polypeptide comprises a replacement of Lys392 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

26. The method of claim 18, wherein the first CH3-containing polypeptide comprises a replacement of Lys439 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

27. The method of claim 18, wherein the first CH3-containing polypeptide comprises a replacement of Lys370 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

28. The method of claim 20, wherein the first CH3-containing polypeptide comprises a replacement of Asp399 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

29. The method of claim 20, wherein the first CH3-containing polypeptide comprises a replacement of Asp356 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

30. The method of claim 23, wherein the first CH3-containing polypeptide comprises a replacement of Glu357 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

31. The method of claim 1, wherein the first CH3-containing polypeptide is an antibody heavy chain.

32. The method of claim 1, wherein the second CH3-containing polypeptide is an antibody heavy chain.

33. The method of claim 1, wherein the host cell expresses one or more antibody light chains.

34. The method of claim 1, wherein the heterodimeric protein is selected from the group consisting of an antibody, a bispecific antibody, a monospecific monovalent antibody, a bispecific maxibody, a monobody, a peptibody, a bispecific peptibody, a monovalent peptibody, and a receptor fusion protein.

35. A host cell comprising a nucleic acid encoding a first CH3-containing polypeptide and a nucleic acid comprising a second CH3-containing polypeptide, wherein the first CH3-containing polypeptide and the second CH3-containing polypeptide meet together to form an interface engineered to promote heterodimer formation, and wherein said first CH3-containing polypeptide and said second CH3-containing polypeptide comprise one or more charged amino acids within the interface that are electrostatically unfavorable to homodimer formation but electrostatically favorable to heterodimer formation.

36. A composition comprising a nucleic acid encoding a first CH3-containing polypeptide and a nucleic acid comprising a second CH3-containing polypeptide, wherein the first CH3-containing polypeptide and the second CH3-containing polypeptide meet together to form an interface engineered to promote heterodimer formation, and wherein said first CH3-containing polypeptide and said second CH3-containing polypeptide comprise one or more charged amino acids within the interface that are electrostatically unfavorable to homodimer formation but electrostatically favorable to heterodimer formation.

37. A heterodimeric protein comprising a first CH3-containing polypeptide and a second CH3-containing polypeptide that meet together to form an interface engineered to promote heterodimer formation, wherein said first CH3-containing polypeptide and said second CH3-containing polypeptide comprise one or more charged amino acids within the interface that are electrostatically unfavorable to homodimer formation but electrostatically favorable to heterodimer formation.

38. The heterodimeric protein of claim 37, wherein the heterodimeric protein comprises an Fc region.

39. The heterodimeric protein of claim 38, wherein the Fc region comprises an IgG Fc region.

40. The heterodimeric protein of claim 39, wherein the IgG Fc region comprises a human IgG Fc region.

41. The heterodimeric protein of claim 40, wherein the human IgG region comprises an IgG1 Fc region.

42. The heterodimeric protein of claim 40, wherein the IgG region comprises an IgG2 Fc region.

43. The heterodimeric protein of claim 40, wherein the IgG region comprises an IgG3 Fc region.

44. The heterodimeric protein of claim 40, wherein the IgG region comprises an IgG4 Fc region.

45. The heterodimeric protein of claim 38, wherein the Fc region comprises an IgA, IgE, IgD, or IgM Fc region.

46. The heterodimeric protein of claim 40, wherein the first CH3-containing polypeptide or the second CH3-containing polypeptide comprises a polypeptide sequence differing from wild-type human IgG such that one or more positive-charged amino acids in a wild-type human IgG sequence are replaced with one or more negative-charged amino acids.

47. The heterodimeric protein of claim 46, wherein the positive-charged amino acid is selected from the group consisting of lysine, histidine, and arginine.

48. The heterodimeric protein of claim 46, wherein the negative-charged amino acid is selected from the group consisting of aspartic acid and glutamic acid.

49. The heterodimeric protein of claim 46, wherein the first CH3-containing polypeptide or the second CH3-containing polypeptide comprises a polypeptide sequence differing from wild-type human IgG such that one or more negative-charged amino acids in a wild-type human IgG sequence is replaced with one or more positive-charged amino acids.

50. The heterodimeric protein of claim 49, wherein the positive-charged amino acid is selected from the group consisting of lysine, histidine, and arginine.

51. The heterodimeric protein of claim 49, wherein the negative-charged amino acid is selected from the group consisting of aspartic acid and glutamic acid.

52. The heterodimeric protein of claim 41, wherein the IgG1Fc comprises a first CH3-containing polypeptide or second CH3-containing polypeptide having a polypeptide sequence differing from wild-type human IgG1 such that one or more positive-charged amino acids in a wild-type human IgG1 sequence are replaced with one or more negative-charged amino acids.

53. The heterodimeric protein of claim 52, wherein the first CH3-containing polypeptide comprises a replacement of a lysine with a negative-charged amino acid.

54. The heterodimeric protein of claim 53, wherein the lysine is selected from the group consisting of Lys409, Lys392, Lys439, and Lys370.

55. The heterodimeric protein of claim 41, wherein the IgG1Fc comprises a first CH3-containing polypeptide or second CH3-containing polypeptide having a polypeptide sequence differing from wild-type human IgG1 such that one or more negative-charged amino acids in a wild-type human IgG1 sequence are replaced with one or more positive-charged amino acids.

56. The heterodimeric protein of claim 55, wherein the first CH3-containing polypeptide comprises a replacement of an aspartic acid with a positive-charged amino acid.

57. The heterodimeric protein of claim 56, wherein the aspartic acid is selected from the group consisting of Asp399 and Asp356.

58. The heterodimeric protein of claim 55, wherein the first CH3-containing polypeptide comprises a replacement of a glutamic acid with a positive-charged amino acid.

59. The heterodimeric protein of claim 58, wherein the glutamic acid is Glu357.

60. The heterodimeric protein of claim 54, wherein the first CH3-containing polypeptide comprises a replacement of Lys409 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

61. The heterodimeric protein of claim 54, wherein the first CH3-containing polypeptide comprises a replacement of Lys392 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

62. The heterodimeric protein of claim 54, wherein the first CH3-containing polypeptide comprises a replacement of Lys439 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

63. The heterodimeric protein of claim 54, wherein the first CH3-containing polypeptide comprises a replacement of Lys370 with a negative-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Asp399, Asp356, or Glu357 with a positive-charged amino acid.

64. The heterodimeric protein of claim 57, wherein the first CH3-containing polypeptide comprises a replacement of Asp399 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

65. The heterodimeric protein of claim 57, wherein the first CH3-containing polypeptide comprises a replacement of Asp356 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

66. The heterodimeric protein of claim 59, wherein the first CH3-containing polypeptide comprises a replacement of Glu357 with a positive-charged amino acid and the second CH3-containing polypeptide comprises a replacement of Lys409, Lys439, Lys370, or Lys392 with a negative-charged amino acid.

67. The heterodimeric protein of claim 37, wherein the first CH3-containing polypeptide is an antibody heavy chain.

68. The heterodimeric protein of claim 37, wherein the second CH3-containing polypeptide is an antibody heavy chain.

69. The heterodimeric protein of claim 37, wherein the heterodimeric protein further comprises one or more antibody light chains.

70. The heterodimeric protein of claim 37, wherein the heterodimeric protein is selected from the group consisting of an antibody, a bispecific antibody, a monospecific monovalent antibody, a bispecific maxibody, a monobody, a peptibody, a bispecific peptibody, a monovalent peptibody, and a receptor fusion protein.

71. A polypeptide comprising a CH3 region of an antibody, wherein the CH3 region comprises a polypeptide sequence differing from a wild-type CH3 region such that one or more positive-charged amino acids in wild-type CH3 are replaced with one or more negative-charged amino acids, wherein the polypeptide has decreased ability to form homodimers compared to a polypeptide comprising a wild-type CH3 region.

73. The polypeptide of claim 71, further comprising a CH2 region.

75. The polypeptide of claim 73, wherein the polypeptide comprises an antibody heavy chain.

77. An antibody comprising the polypeptide of claim 75.

79. An isolated nucleic acid encoding a polypeptide of any of claims 71-76.

80. An expression vector comprising the isolated nucleic acid of claim 79 operably linked to a promoter.

81. A host cell containing the expression vector of claim 80.

82. A pharmaceutical composition comprising a polypeptide of any of claim 71-76.

72. A polypeptide comprising a CH3 region of an antibody, wherein the CH3 region comprises a polypeptide sequence differing from a wild-type CH3 region such that one or more negative-charged amino acids in wild-type CH3 are replaced with one or more positive-charged amino acids, wherein the polypeptide has decreased ability to form homodimers compared to a polypeptide comprising a wild-type CH3 region.

74. The polypeptide of claim 72, further comprising a CH2 region.

76. The polypeptide of claim 74, wherein the polypeptide comprises an antibody heavy chain.

78. An antibody comprising the polypeptide of claim 76.