Knobs and holes heteromeric polypeptides

• US 7,695,936 B2
• Filed: 09/20/2006
• Issued: 04/13/2010
• Est. Priority Date: 03/01/1995
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Presta, Leonard G., Carter, Paul J., Ridgway, John B.
• Primary Examiner(s): Stucker; Jeffrey
• Assistant Examiner(s): Gucker; Stephen
• Application Number:
  US11/533,709
• Publication Number:
  US 20070014794A1
• Time in Patent Office: 1,301 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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

Abstract

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis.

Claims

1. A method of preparing a heteromultimer comprising a first polypeptide and a second polypeptide each comprising a C_H3 antibody constant domain, wherein said polypeptides meet at an interface, comprising the steps of:

(a) altering the nucleic acid encoding at least one amino acid residue of the first polypeptide selected from the group consisting of amino acid residues 366, 405, and 407, according to the amino acid numbering as shown in FIG. 5, so as to create a protuberance or cavity; and

(b) altering the nucleic acid encoding at least one amino acid residue of the second polypeptide selected from the group consisting of amino acid residues 407 if amino acid residue 366 is altered in the first polypeptide, amino acid residue 394 if amino acid residue 405 is altered in the first polypeptide, and amino acid residue 366 if amino acid residue 407 is altered in the first polypeptide, according to the amino acid numbering as shown in FIG. 5, so as to create a cavity or protuberance positionable in a protuberance or cavity, respectively, in the first polypeptide; and

(c) expressing said nucleic acid in a host cell to obtain the heteromultimer;

(d) recovering the heteromultimer from the host cell culture.

2. The method of claim 1, wherein the first polypeptide comprises a protuberance and the second polypeptide comprises a cavity.

3. The method of claim 1, wherein said altering the nucleic acid to create a protuberance comprises replacing the nucleic acid encoding the original residue with nucleic acid encoding an import residue having a larger side chain volume than the original residue.

4. The method of claim 3 wherein the import residue is arginine (R).

5. The method of claim 3 wherein the import residue is phenylalanine (F).

6. The method of claim 3 wherein the import residue is tyrosine (Y).

7. The method of claim 3 wherein the import residue is tryptophan (W).

8. The method of claim 1, wherein said altering the nucleic acid to create a cavity comprises replacing the nucleic acid encoding an original residue with nucleic acid encoding an import residue having a smaller side chain volume than the original residue.

9. The method of claim 8 wherein the import residue is not cysteine (C).

10. The method of claim 8 wherein the import residue is alanine (A).

11. The method of claim 8 wherein the import residue is serine (S).

12. The method of claim 8 wherein the import residue is threonine (T).

13. The method of claim 8 wherein the import residue is valine (V).

14. The method of claim 1 wherein the antibody constant domain is from an IgG.

15. The method of claim 1 wherein the heteromultimer is a bispecific antibody.

16. The method of claim 1 wherein the heteromultimer is a bispecific immunoadhesin.

17. The method of claim 1 wherein the heteromultimer is an antibody-immunoadhesin chimera.

18. An isolated heteromultimer prepared by the method of claim 1.

27. The method of claim 1, comprising altering amino acid residue 366 of the first polypeptide and amino acid residue 407 of the second polypeptide.

28. The method of claim 27, wherein said altering creates a protuberance in the first polypeptide and a cavity in the second polypeptide.

29. The method of claim 27, wherein said altering creates a cavity in the first polypeptide and a protuberance in the second polypeptide.

30. The method of claim 1, comprising altering amino acid residue 405 of the first polypeptide and amino acid residue 394 of the second polypeptide.

31. The method of claim 30, wherein said altering creates a protuberance in the first polypeptide and a cavity in the second polypeptide.

32. The method of claim 30, wherein said altering creates a cavity in the first polypeptide and a protuberance in the second polypeptide.

33. The method of claim 1, comprising altering amino acid residue 407 of the first polypeptide and amino acid residue 366 of the second polypeptide.

34. The method of claim 33, wherein said altering creates a protuberance in the first polypeptide and a cavity in the second polypeptide.

35. The method of claim 33, wherein the alteration in the said altering creates a cavity in the first polypeptide and a protuberance in the second polypeptide.

19. An isolated heteromultimer comprising a first polypeptide and a second polypeptide each comprising a C_H3 antibody constant domain, wherein

(a) the first polypeptide comprises an alteration of at least one amino acid residue selected from the group consisting of amino acid residues 366, 405 and 407, according to the amino acid numbering as shown in FIG. 5, so as to create a protuberance or cavity, and

(b) the second polypeptide comprises an alteration of at least one amino acid residue selected from the group consisting of 407 if amino acid residue 366 is altered in the first polypeptide, amino acid residue 394 if amino acid residue 405 is altered in the first polypeptide, and amino acid residue 366 if amino acid residue 407 is altered in the first polypeptide, according to the amino acid numbering as shown in FIG. 5, so as to create a protuberance or cavity positionable in a cavity or protuberance, respectively, in the first polypeptide, and

wherein the protuberance or cavity, or both, have been introduced into the interface of the first and second polypeptides respectively.

20. A composition comprising the heteromultimer of claim 19 and a pharmaceutically acceptable carrier.

21. A host cell comprising nucleic acid encoding the heteromultimer of claim 19.

22. The host cell of claim 21 wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in a single vector.

23. The host cell of claim 21 wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in separate vectors.

24. A method of making a heteromultimer comprising culturing the host cell of claim 21 so that the nucleic acid is expressed and recovering the heteromultimer from the cell culture.

25. The method of claim 24 wherein the host cell is a mammalian cell.

26. The method of claim 24 wherein the heteromultimer is recovered from the cell culture media.

36. The heteromultimer of claim 19, wherein the alteration of the second polypeptide creates a protuberance and alteration of the first polypeptide creates a cavity.

37. The heteromultimer of claim 19, wherein the protuberance comprises the replacement of the original amino acid residue with an import residue with a larger side chain volume.

38. The heteromultimer of claim 37, wherein the import residue is arginine (R).

39. The heteromultimer of claim 37, wherein the import residue is phenylalanine (F).

40. The heteromultimer of claim 37, wherein the import residue is tyrosine (Y).

41. The heteromultimer of claim 37, wherein the import residue is tryptophan (W).

42. The heteromultimer of claim 19, wherein the alteration of the second polypeptide creates a cavity and alteration of the first polypeptide creates a protuberance.

43. The heteromultimer of claim 19, wherein the cavity comprises the replacement of the original amino acid residue with an import residue with a smaller side chain volume.

44. The heteromultimer of claim 43, wherein the import residue is alanine (A).

45. The heteromultimer of claim 43, wherein the import residue is serine (S).

46. The heteromultimer of claim 43, wherein the import residue is threonine (T).

47. The heteromultimer of claim 43, wherein the import residue is valine (V).

48. The heteromultimer of claim 19, wherein the first polypeptide comprises an alteration of amino acid residue 366 and wherein the second polypeptide comprises an alteration of amino acid residue 407.

49. The heteromultimer of claim 19, wherein the first polypeptide comprises an alteration of amino acid residue 405 and wherein the second polypeptide comprises an alteration of amino acid residue 394.

50. The heteromultimer of claim 19, wherein the first polypeptide comprises alterations of amino acid residues 366 and 405 and wherein the second polypeptide comprises alterations of amino acid residues 407 and 394.

51. The heteromultimer of claim 19, wherein the first polypeptide comprises alterations of amino acid residues 405 and 407 and wherein the second polypeptide comprises alterations of amino acid residues 394 and 366.

52. The heteromultimer of claim 19, wherein the first polypeptide comprises an alteration of amino acid residue 407 and wherein the second polypeptide comprises an alteration of amino acid residue 366.

53. The heteromultimer of claim 19, wherein the C_H3 domain is from an IgG.

54. The heteromultimer of claim 19, wherein the C_H3 domain is from an IgA.

55. The heteromultimer of claim 19, wherein the C_H3 domain is from an IgD.

56. The heteromultimer of claim 19, wherein the C_H3 domain is from an IgE.

57. The heteromultimer of claim 19, wherein the C_H3 domain is from an IgM.

58. The heteromultimer of claim 19, wherein the first or second polypeptide further comprises a binding domain.

59. The heteromultimer of claim 58, wherein the binding domain is an antigen binding domain.

60. The heteromultimer of claim 59, wherein the antigen binding domain is an antibody variable domain.

61. The heteromultimer of claim 58, wherein the binding domain is a ligand binding domain.

62. The heteromultimer of claim 58, wherein the binding domain is a receptor binding domain.

63. The heteromultimer of claim 58, wherein the binding domain is an enzymatic domain.

64. The heteromultimer of claim 19, which is a multi-specific antibody.

65. The heteromultimer of claim 64, which is a bi-specific antibody.

66. The heteromultimer of claim 64, which is a tri-specific antibody.

67. The heteromultimer of claim 19, which is an immunoadhesin.

68. The heteromultimer of claim 67, which is multi-specific.

69. The heteromultimer of claim 67, which is a bi-specific.

70. The heteromultimer of claim 19, which is a heterodimer.

71. The heteromultimer of claim 19, which is a heterotrimer.

72. The heteromultimer of claim 19, which is a heterotetramer.

73. The heteromultimer of claim 19, which is an antibody-immunoadhesin.