MONOVALENT ANTIBODY FRAGMENTS USEFUL AS THERAPEUTICS

• US 20080063641A1
• Filed: 10/03/2006
• Published: 03/13/2008
• Est. Priority Date: 12/19/2003
• Status: Abandoned Application

• Current Assignee: Arthur Huang, Daniel Yansura, Ralph Schwall
• Original Assignee: Arthur Huang, Daniel Yansura, Ralph Schwall
• Inventors: Yansura, Daniel, Schwall, Ralph, Huang, Arthur
• Application Number:
  US11/538,375
• Publication Number:
  US 20080063641A1
• Time in Patent Office: Days
• US Class Current: 424/138.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/53 Hinge
• 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 2317/622 Single chain antibody (scFv)
• 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 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The invention provides methods and compositions comprising a novel stabilized monovalent antibody fragment.

Claims

1. An antibody fragment comprising a single antigen binding arm and an Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm, wherein the Fc region comprises a complex of a first and a second Fc polypeptide, wherein one but not both of the Fc polypeptides is an N-terminally truncated heavy chain.

2. The antibody fragment of claim 1 wherein the antibody fragment is a glycosylated.

3. The antibody fragment of claim 1 or 2 wherein the antibody fragment has little to no immunosuppressive properties.

4. The antibody fragment of claim 3 wherein said immunosuppressive properties comprise ability to effect T cell depletion.

5. The antibody fragment of any of claims 1-4 which does not possess substantial effector function other than FcRn binding.

6. The antibody fragment of claim 5 wherein said effector function is complement lysis.

7. The antibody fragment of any of claims 1-6 wherein the antibody fragment binds FcRn.

8. The antibody fragment of any of claims 1-7 which does not specifically bind a T cell surface antigen.

9. The antibody fragment of claim 8 wherein said T cell surface antigen is CD3 or CD4.

10. The antibody fragment of claim 9 wherein said T cell surface antigen is CD3.

11. The antibody fragment of any of claims 1-10 which specifically binds a tumor antigen.

12. The antibody fragment of any of claims 1-11 which specifically binds a cell surface receptor that is activated upon receptor dimerization.

13. The antibody fragment of any of claims 1-12 wherein the antibody fragment comprises a first polypeptide comprising a light chain variable domain, a second polypeptide comprising a heavy chain variable domain and said first Fc polypeptide, and a third polypeptide comprising said second Fc polypeptide.

14. The antibody fragment of claim 13 wherein the first polypeptide comprises a non-human light chain variable domain fused to a human light chain constant domain.

15. The antibody fragment of claim 13 wherein the first polypeptide comprises a CDR from a non-human species fused to a humanized or human framework sequence.

16. The antibody fragment of claim 13 wherein the second polypeptide comprises a non-human heavy chain variable domain fused to a human heavy chain constant domain.

17. The antibody fragment of claim 13 wherein the second polypeptide comprises a CDR from a non-human species fused to a humanized or human framework sequence.

18. The antibody fragment of claim 13 wherein the third polypeptide comprises an N-terminally truncated heavy chain which comprises at least a portion of the hinge sequence at the N terminus.

19. The antibody fragment of any of claims 1-18 wherein the two Fc polypeptides are covalently linked.

20. The antibody fragment of any of claims 1-19 wherein the two Fc polypeptides are linked through intermolecular disulfide bonds at the hinge region.

21. The antibody fragment of any of claims 1-20 wherein the antibody fragment when bound to a target molecule inhibits target molecule multimerization.

22. The antibody fragment of any of claims 1-21 wherein the antibody fragment when bound to a target molecule inhibits binding of a cognate binding partner to the target molecule.

23. The antibody fragment of any of claims 1-22 wherein the first Fc polypeptide and the second Fc polypeptide meet at an interface, and the interface of the second Fc polypeptide comprises a protuberance which is positionable in a cavity in the interface of the first Fc polypeptide.

24. The antibody fragment of any of claims 1-23 wherein the second Fc polypeptide has been altered from a template/original polypeptide to encode the protuberance or the first Fc polypeptide has been altered from a template/original polypeptide to encode the cavity, or both.

25. The antibody fragment of any of claims 1-24 wherein the second Fc polypeptide has been altered from a template/original polypeptide to encode the protuberance and the first Fc polypeptide has been altered from a template/original polypeptide to encode the cavity, or both.

26. The antibody fragment of any of claims 1-25 wherein the first Fc polypeptide and the second Fc polypeptide meet at an interface, wherein the interface of the second Fc polypeptide comprises a protuberance which is positionable in a cavity in the interface of the first Fc polypeptide, and wherein the cavity or protuberance, or both, have been introduced into the interface of the first and second Fc polypeptides respectively.

27. The antibody fragment of any of claims 24-26 wherein the protuberance and cavity have been introduced into the interface of the respective Fc polypeptides.

28. The antibody of any of claims 24-27 wherein the protuberance and cavity each comprise a naturally occurring amino acid residue.

29. The antibody fragment of any of claims 24-28 wherein the Fc polypeptide comprising the protuberance is generated by replacing an original residue from the interface of a template/original polypeptide with an import residue having a larger side chain volume than the original residue.

30. The antibody fragment of any of claims 24-29 wherein the Fc polypeptide comprising the protuberance is generated by a method comprising a step wherein nucleic acid encoding an original residue from the interface of said polypeptide is replaced with nucleic acid encoding an import residue having a larger side chain volume than the original.

31. The antibody fragment of claim 29 or 30 wherein the original residue is threonine.

32. The antibody fragment of any of claims 29-31 wherein the import residue is arginine (R).

33. The antibody fragment of any of claims 29-31 wherein the import residue is phenylalanine (F).

34. The antibody fragment of any of claims 29-31 wherein the import residue is tyrosine (Y).

35. The antibody fragment of any of claims 29-31 wherein the import residue is tryptophan (W).

36. The antibody fragment of any of claims 23-28 wherein the Fc polypeptide comprising the cavity is generated by replacing an original residue in the interface of a template/original polypeptide with an import residue having a smaller side chain volume than the original residue.

37. The antibody fragment of any of claims 23-28 and 36 wherein the Fc polypeptide comprising the cavity is generated by a method comprising a step wherein nucleic acid encoding an original residue from the interface of said polypeptide is replaced with nucleic acid encoding an import residue having a smaller side chain volume than the original.

38. The antibody fragment of claim 36 or 37 wherein the original residue is threonine.

39. The antibody fragment of claim 36 or 37 wherein the original residue is leucine.

40. The antibody fragment of claim 36 or 37 wherein the original residue is tyrosine.

41. The antibody fragment of any of claims 36-40 wherein the import residue is not cysteine (C).

42. The antibody fragment of any of claims 36-40 wherein the import residue is alanine (A).

43. The antibody fragment of any of claims 36-40 wherein the import residue is serine (S).

44. The antibody fragment of any of claims 36-40 wherein the import residue is threonine (T).

45. The antibody fragment of any of claims 36-40 wherein the import residue is valine (V).

46. The antibody fragment of any of claims 36-45 wherein the Fc polypeptide comprising the cavity comprises replacement of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine.

47. The antibody fragment of any of claims 36-46 wherein the Fc polypeptide comprising the cavity comprises two or more import residues selected from the group consisting of alanine, serine, threonine and valine.

48. The antibody fragment of any of claims 36-47 wherein the Fc polypeptide comprising the cavity comprises replacement of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine, and wherein said original amino acids are replaced with import residues selected from the group consisting of alanine, serine, threonine and valine.

49. The antibody fragment of any of claims 23-48 wherein the Fc polypeptide comprising the cavity comprises replacement of threonine at position 366 with serine, amino acid numbering according to the EU numbering scheme of Kabat.

50. The antibody fragment of any of claims 23-48 wherein the Fc polypeptide comprising the cavity comprises replacement of leucine at position 368 with alanine, amino acid numbering according to the EU numbering scheme of Kabat.

51. The antibody fragment of any of claims 23-50 wherein the Fc polypeptide comprising the cavity comprises replacement of tyrosine with valine.

52. The antibody fragment of any of claims 23-51 wherein the Fc polypeptide comprising the cavity comprises two or more amino acid replacements selected from the group consisting of T366S, L368A and Y407V.

53. The antibody fragment of any of claims 23-52 wherein the Fc polypeptide comprising the protuberance comprises replacement of threonine at position 366 with tryptophan, amino acid numbering according to the EU numbering scheme of Kabat.

54. The antibody fragment of any of claims 1-53 wherein the first and second Fc polypeptides each comprise an antibody constant domain.

55. The antibody fragment of claim 54 wherein the antibody constant domain is a CH₂ and/or CH3 domain.

56. The antibody fragment of claim 54 or 55 wherein the antibody constant domain is from an IgG.

57. The antibody fragment of claim 56 wherein the IgG is human IgG₁.

58. The antibody fragment of any of claims 1-57 which is monospecific.

59. The antibody fragment of any of claims 1-58 which is a monospecific immunoadhesin.

60. The antibody fragment of any of claims 1-59 which is an antibody-immunoadhesin chimera.

61. A composition comprising a population of immunoglobulins wherein at least 75% of the immunoglobulins is the antibody fragment of any of claims 1-60.

62. A method of preparing the antibody fragment of any of claims 1-60, the comprising the steps of:

(a) culturing a host cell comprising nucleic acid encoding the antibody fragment; and

(b) recovering the antibody fragment from the host cell culture.

63. The method of claim 62, wherein polypeptides comprising the antibody fragment are expressed at ratios that results in a population of immunoglobulins wherein at least 50% of the immunoglobulins are the antibody fragment of any of claims 1-60.

64. The method of claim 63, wherein approximately equimolar amounts of said polypeptides are expressed.

65. The method of claim 64, wherein nucleic acids encoding the polypeptides are operably linked to translational initiation regions (TIRs) of approximately equal strength.

66. The method of any of claims 62-65 wherein said host cell is prokaryotic.

67. The method of claim 66, wherein the host cell is E. coli.

68. The method of claim 67, wherein the E. coli is of a strain deficient in endogenous protease activities.

69. The method of any of claims 62-65, wherein said host cell is eukaryotic.

70. The method of claim 69, wherein the host cell is CHO.

71. The method of any of claims 62-70, where the antibody fragment is recovered from culture medium.

72. The method of any of claims 62-70, wherein the antibody fragment is recovered from cell lysate.

73. A method of preparing the antibody fragment of any of claims 23-60, the comprising the steps of:

(a) culturing a host cell comprising nucleic acid encoding the antibody fragment, wherein the nucleic acid encoding the interface of the second Fc polypeptide has been altered from nucleic acid encoding the original interface of the second Fc polypeptide to encode the protuberance or the nucleic acid encoding the interface of the first Fc polypeptide has been altered from nucleic acid encoding the original interface of the first Fc polypeptide to encode the cavity or both; and

(b) recovering the antibody fragment from the host cell culture.

74. The method of claim 73, wherein the nucleic acid encoding the second Fc polypeptide has been altered from the original nucleic acid to encode the protuberance and the nucleic acid encoding the first polypeptide has been altered from the original nucleic acid to encode the cavity.

75. The method of claim 73 wherein step (a) is preceded by a step wherein nucleic acid encoding an original amino acid residue from the interface of the second Fc polypeptide is replaced with nucleic acid encoding an import amino acid residue having a larger side chain volume than the original amino acid residue, wherein the import residue with the larger side chain volume comprises the protuberance.

76. The method of claim 73, wherein step (a) is preceded by a step wherein nucleic acid encoding an original amino acid residue in the interface of the first Fc polypeptide is replaced with nucleic acid encoding an import amino acid residue having a smaller side chain volume than the original amino acid residue so as to form the cavity.

77. The method of claims 73 wherein step (a) is preceded by a step wherein the nucleic acid encoding the first and second Fc polypeptide is introduced in the host cell.

78. A method of preparing the antibody fragment of any of claims 1-60 comprising the steps of:

(a) preparing polypeptides that form the antibody fragment; and

(b) allowing heteromultimerization to occur;

whereby the antibody fragment is formed.

79. The method of any of claims 62-78 wherein at least 50% of the immunoglobulin polypeptide complexes that are formed are the antibody fragment of any of claims 1-60.

80. The method of any of claims 62-78 wherein at least 50% of the immunoglobulin polypeptide complexes that are formed are heterotrimers.

81. The method of any of claims 62-78 wherein step (b) comprises coupling the first Fc polypeptide and the second Fc polypeptide in vitro.

82. The method of any of claims 62-78 wherein the amino acid sequence of the original interface has been altered so as to generate the protuberance and the cavity in the engineered interface.

83. Isolated nucleic acid encoding the antibody fragment of any of claims 1-60.

84. A composition comprising two or more recombinant nucleic acids which collectively encode the antibody fragment of any of claims 1-60.

85. A host cell comprising the nucleic acid of claim 83 or 84.

86. The host cell of claim 85 wherein the nucleic acid encoding the antigen binding arm is present in a single vector.

87. The host cell of claim 85 wherein the nucleic acid encoding the antigen binding arm is present in separate vectors.

88. The host cell of claim 85 wherein the nucleic acid encoding the antigen binding arm and N-terminally truncated heavy chain is present in a single vector.

89. A method of making the antibody fragment of any of claims 1-60 comprising culturing a host cell comprising the nucleic acid of claim 83 or 84 so that polypeptides are expressed, and recovering the antibody fragment from the cell culture.

90. The method of claim 89 wherein the antibody fragment is recovered from the cell lysate.

91. The method of claim 89 wherein the antibody fragment is recovered from the cell culture medium.

92. The method of claim 89 wherein the host cell is a prokaryotic cell.

93. The method of claim 90 wherein the host cell is E. coli.

94. The method of claim 89 wherein the host cell is mammalian.

95. A composition comprising the antibody fragment of any of claims 1-60 and a carrier.

96. A method of generating an antibody fragment comprising a single antigen binding arm and an Fc region that increases stability of the antibody fragment compared to a Fab molecule comprising said antigen binding arm, said method comprising expressing in a suitable host cell nucleic encoding the antigen binding arm and a first and second Fc polypeptide under conditions the permit formation of the antigen binding arm and dimerization of the first and second Fc polypeptides to form said Fc region, wherein one but not both of the Fc polypeptides is an N-terminally truncated heavy chain.

97. The method of claim 96 wherein said method generates a heterogeneous population of immunoglobulins, and wherein at least 50% of the immunoglobulins comprise a single antigen binding arm and an Fc region that increases stability of the antibody fragment compared to a Fab molecule comprising said antigen binding arm.