CONSTANT CHAIN MODIFIED BISPECIFIC, PENTA- AND HEXAVALENT IG-M ANTIBODIES

• US 20160222132A1
• Filed: 09/04/2014
• Published: 08/04/2016
• Est. Priority Date: 09/05/2013
• Status: Active Grant

• Current Assignee: IGM Biosciences, Inc.
• Original Assignee: IGM Biosciences, Inc.
• Inventors: Yoshimura, Rency, Keyt, Bruce, Zhang, Fen, Presta, Leonard
• Granted Patent: US 10,351,631 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/461 Igs containing Ig-regions, -domains or -residues form different species
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• 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 2317/35 Valency
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/522 CH1 domain
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/528 CH4 domain
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components

Abstract

The present invention concerns binding molecules having a penta- or hexameric ring structure, such as for example, isolated IgM antibodies with five or six bispecific binding units, and methods and means for making and using the same. The invention further concerns multi-specific binding molecules having a penta- or hexameric ring structure, such as, for example, isolated IgM antibodies with five or six bispecific binding units, and methods and means for making and using the same.

Claims

1. A binding molecule having a penta- or hexameric ring structure comprising five or six bispecific binding units, wherein each of said bispecific binding units has the same two binding specificities and comprises a first chain comprising at least a Cμ

4 domain of an IgM heavy chain constant region conjugated to a first binding region to a first binding target, and a second chain comprising at least a Cμ

4 domain of an IgM heavy chain constant region and a second binding region to a second binding target, wherein said first and second binding targets are different, and wherein said first and second chains are assembled to create a bispecific binding unit as a result of an asymmetric interface created between their respective IgM heavy chain constant regions.

2. The binding molecule of claim 1, wherein said bispecific binding units are identical.

3. The binding molecule of claim 2, which further comprises an IgM J chain.

4. The binding molecule of claim 3, which has a pentameric ring structure.

5. The binding molecule of claim 2, which has a hexameric structure.

6. The binding molecule of claim 2, wherein said first and second chains further comprise a Cμ

3 domain of an IgM heavy chain constant region.

7. The binding molecule of claim 2 or claim 6, wherein said first and second chains further comprise a Cμ

2 domain of an IgM heavy chain constant region.

8. The binding molecule of claim 2, wherein said first and second binding targets are selected from peptides, polypeptides, glycoproteins, nucleic acid molecules, and organic and non-organic small molecules.

9. The binding molecule of claim 2, wherein said first and second binding targets are selected from soluble polypeptides, cell surface receptors, ligands, molecular transporters, enzymes and substrates of enzymes.

10. The binding molecule of claim 2, wherein said first and second binding targets bind to two sites on the same soluble target, two sites on the same cell surface receptor target, two different soluble targets, two cell surface receptor targets, one soluble target and one cell surface receptor target, one soluble or cell surface receptor target and one long residence time target, one soluble target and one matrix protein or substrate target, one soluble or receptor target and one molecular transporter target, or two different cell types.

11. The binding molecule of any one of claims 1 to 10, wherein conjugation is by fusion.

12. The binding molecule of claim 11, wherein said first and second binding regions are fused to the N-termini of said first and said second IOW heavy chain constant region, respectively.

13. The binding molecule of claim 12, wherein said first and second binding regions are variable regions of an antibody.

14. The binding molecule of claim 13, wherein said first and second binding targets are two different antigens.

15. The binding molecule of claim 13, wherein said first and second binding targets are different epitopes on the same antigen.

16. The binding molecule of claim 13, wherein said first and second binding regions are two different antibody heavy chain variable regions, binding to said first and said second binding target, respectively.

17. The binding molecule of claim 13, wherein said first and second binding regions are two different antibody heavy chain variable regions, each binding to a different epitope on the same binding target.

18. The binding molecule of claim 16 or claim 17, wherein said antibody heavy chain variable regions are from an IgG, IgA, IgE, or IgM antibody.

19. The binding molecule of claim 18, wherein said antibody heavy chain variable regions are from an IgM antibody.

20. The binding molecule of claim 16 or claim 17, which is a bispecific IgM molecule.

21. The binding molecule of claim 20, further comprising at least one IgM light chain variable region sequence associated with at least one of said two different IgM heavy chain variable regions.

22. The binding molecule of claim 20, further comprising an IgM light chain variable region sequence associated with each of said two different IgM heavy chain variable regions.

23. The binding molecule of any one of claims 1 to 22, wherein at least some of the asymmetric interfaces between the IgM heavy chain constant regions of the two chains of a binding unit are created by a salt bridge formed by pair-wise switches between oppositely charged amino acid residues in at least one of the Cμ

2, Cμ

3 and/or Cμ

4 domains of the two chains of said binding unit.

24. The binding molecule of claim 23, wherein a salt bridge is formed between at least one of the Cμ

2-Cμ

2, Cμ

4-Cν

4, and Cμ

2-Cμ

3-Cμ

4 domains of the two chains of said binding unit.

25. The binding molecule of claim 23, wherein the pair-wise switches are selected from the group consisting of E→

K, K→

E;

R→

E, E→

R;

D→

K, K→

D; and

R→

D, D→

R.

26. The binding molecule of claim 24, comprising at least one pair-wise charged amino acid residue switch in the Cμ

4-Cμ

4 domains.

27. The binding molecule of claim 26, wherein said switch is selected from the group consisting of R328E,D⇄

E339R,K;

R344E,D⇄

S330R,K;

K376E,D⇄

E385R,K;

R427E,D⇄

E339R,K; and

T354E,D⇄

I397R,K.

28. The binding molecule of claim 24, comprising at least one pair-wise charged amino acid switch between the Cμ

2-Cμ

2 domains.

29. The binding molecule of claim 28, wherein said switch is selected from the group consisting of E167R,K⇄

K177E,D and K169E,D⇄

E170R,K.

30. The binding molecule of claim 24, comprising at least one pair-wise charged amino acid residue switch in the Cμ

2-Cμ

3-Cμ

4 domains.

31. The binding molecule of claim 29, wherein said switch is selected from the group consisting of D121K,R⇄

K315D,E;

K150E,D⇄

E385K,R; and

K185D,E⇄

D360K,R.

32. The binding molecule of any one of claims 1 to 22, wherein at least some of the asymmetric interfaces between the IgM heavy chain constant regions of the two chains of a binding unit are created through knobs-into-holes connections.

33. The binding molecule of claim 32, wherein at least one knobs-into-hole connection is created by mutations selected from the group consisting of knobs:

T350→

Y,F,W; and

H395→

Y,F; and

holes;

L352→

G,A,V,I,M,S,T;

H395→

A,V,I,L,M,F,Y;

F393→

W,Y;

I397→

A,V,S,T;

T350→

S,A,V; and

T348→

S.

34. The binding molecule of any one of claims 21 to 33, wherein said light chain variable region sequences are coupled to their matching heavy chain variable region by creating an asymmetric interface between the light and heavy chains.

35. The binding molecule of claim 34, wherein the asymmetric interface is created by CrossMab technique, knobs-into-holes coupling and/or salt bridges coupling.

36. The binding molecule of any one of claims 21 to 35, comprising a common light chain.

37. The binding molecule of any one of claims 1 to 36, conjugated to a toxin.

38. The binding molecule of any one of claims 1 to 36, conjugated to a chemotherapeutic agent.

39. The binding molecule of claim 37 or claim 38, wherein conjugation is by fusion.

40. The binding molecule of claim 37 or claim 38, wherein conjugation is by a chemical linker.

41. The binding molecule of any one of claims 20 to 40, which is chimeric or humanized.

44. A composition comprising at least about 95% of the binding molecule according to any one of claims 1 to 41.

45. A composition comprising at least about 98% of a binding molecule according to any one of claims 1 to 41.

46. A composition comprising at least about 99% of a binding molecule according to any one of claims 1 to 42.

42. A composition comprising at least about 70% of the binding molecule according to any one of 1 to 41.

47. The composition according to any one of claims 42 to 46, which is a pharmaceutical composition.

43. A composition comprising at least about 80% of the binding molecule according to any one of 1 to 41.

48. A multi-specific binding molecule having a penta- or hexameric ring structure comprising five or six monospecific binding units, wherein (i) each of said monospecific binding units comprises two IgM heavy chain constant regions each comprising at least a Cμ

3 and Cμ

4 domain conjugated to a binding region to a binding target, (ii) at least two of said monospecific binding units bind to different binding targets, and (iii) an external asymmetric interface is created between the heavy chain constant regions of the neighboring monospecific binding units that bind to different binding targets.

49. The multi-specific binding molecule of claim 48, wherein at least three of said monospecific binding units bind to different binding targets.

50. The multi-specific binding molecule of claim 48, wherein at least four of said monospecific binding units bind to different binding targets.

51. The multi-specific binding molecule of claim 48, wherein the binding molecule has a pentameric ring structure and all five monospecific binding units bind to different targets.

52. The multi-specific binding molecule of claim 48, wherein the binding molecule has a hexameric ring structure and at least five of said monospecific binding units bind to different targets.

53. The multi-specific binding molecule of claim 52, wherein all six monospecific binding units bind to different targets.

59. The multi-specific binding molecule of claim 52, wherein all six bispecific binding units bind to different targets.

60. The multi-specific binding molecule of claim 48 or claim 54, which further comprises an IgM chain.

61. The multi-specific binding molecule of claim 48 or claim 54, which has a pentameric ring structure.

62. The multi-specific binding molecule of claim 48 or claim 54, which has a hexameric structure.

63. The multi specific binding molecule of claim 48 or claim 54, wherein in at least one of said binding units the IgM heavy chain constant regions further comprise a Cμ

2 domain.

64. The multi-specific binding molecule of claim 63, wherein in all of said binding units the IgM heavy chain constant regions further comprise a Cμ

2 domain.

65. The multi-specific binding molecule of claim 48 or claim 54, wherein in all of said binding units the heavy chain constant regions are identical.

66. The multi-specific binding molecule of claim 48 or claim 54, wherein the binding targets are selected from peptides, polypeptides, glycoproteins, nucleic acid molecules, and organic and non-organic small molecules.

67. The multi-specific binding molecule of claim 48 or claim 54, wherein the binding targets are selected from soluble polypeptides, cell surface receptors, ligands, molecular transporters, enzymes and substrates of enzymes.

68. The multi-specific binding molecule of claim 48 or claim 54, wherein the binding units binding to different targets are selected from the group consisting of binding units binding to sites on the same soluble target;

sites on the same cell surface receptor target;

different soluble targets;

different cell surface receptor targets;

soluble and cell surface receptor targets;

soluble or cell surface receptor and long residence time targets;

soluble and matrix protein or substrate targets;

soluble or receptor and molecular transporter targets, and different cell types.

69. The multi-specific binding molecule of any one of claims 48 to 68, wherein conjugation is by fusion.

70. The multi-specific binding molecule of claim 67, wherein said binding regions are fused to the N-termini of the IgM heavy chain constant regions.

71. The multi-specific binding molecule of claim 70, wherein at least one of said binding regions is a variable region of an antibody.

72. The multi-specific binding molecule of claim 71, wherein all of said binding regions are antibody heavy chain variable regions.

73. The multi-specific binding molecule of claim 72, wherein at least two binding targets are different antigens.

74. The multi-specific binding molecule of claim 72, wherein at least two binding targets are different epitopes on the same antigen.

75. The multi-specific binding molecule of claim 71, wherein the antibody heavy chain variable regions are from an IgG, IgA, IgE, or IgM antibody.

76. The multi-specific binding molecule of claim 75, wherein the antibody heavy chain variable regions are from an IgM antibody.

77. The multi-specific binding molecule of claim 76, which is a multi-specific IgM molecule.

78. The multi-specific binding molecule of claim 77, further comprising at least one IgM light chain variable region sequence associated with an IgM heavy chain variable region in at least one of the binding units.

79. The multi-specific binding molecule of claim 78, further comprising an IgM light chain variable region sequence associated with each of the IgM heavy chain variable regions.

80. The multi-specific binding molecule of any one of claims 48 to 79, wherein said internal asymmetric interface is created by a salt bridge formed by pair-wise switches between oppositely charged amino acid residues in at least one of the Cμ

2, Cμ

3 and/or Cμ

4 domains.

81. The multi-specific binding molecule of claim 80, wherein a salt bridge is formed between at least one of the Cμ

2-Cμ

2, Cμ

4-Cν

4, and Cμ

2-Cμ

3-Cμ

4 domains of the two chains of said binding unit.

82. The multi-specific binding molecule of claim 81, wherein the pair-wise switches are selected from the group consisting of E→

K, K→

E;

R→

E, E→

R;

D→

K, K→

D; and

R→

D, D→

R.

83. The multi-specific binding molecule of claim 80, comprising at least one pair-wise charged amino acid residue switch in the Cμ

4-Cμ

4 domains.

84. The multi-specific binding molecule of claim 83, wherein said switch is selected from the group consisting of R328E,D⇄

E339R,K;

R344E,D⇄

S330R,K;

K376E,D⇄

E385R,K;

R427E,D⇄

E339R,K; and

T354E,D⇄

I397R,K.

85. The multi-specific binding molecule of claim 80, comprising at least one pair-wise charged amino acid switch between the Cμ

2-Cμ

2 domains.

86. The multi-specific binding molecule of claim 85, wherein said switch is selected from the group consisting of E167R,K⇄

K177E,D and K169E,D⇄

E170R,K.

87. The multi-specific binding molecule of claim 80, comprising at least one pair-wise charged amino acid residue switch in the Cμ

2-Cμ

3-Cμ

4 domains.

88. The multi-specific binding molecule of claim 87, wherein said switch is selected from the group consisting of D121K,R⇄

K315D,E;

K150E,D⇄

E385K,R; and

K185D,E⇄

D360K,R.

89. The multi-specific binding molecule of any one of claims 48 to 88, wherein the external asymmetric interface is created by at least one pair-wise charged amino acid residue switch in the Cμ

3-Cμ

3 domains.

90. The multi-specific binding molecule of claim 89, wherein the pair-wise changed amino acid switch is K238⇄

D293 or K268⇄

D294.

91. The multi-specific binding molecule of any one of claims 48 to 79, wherein at least some of the external and/or internal asymmetric interfaces between the IgM heavy chain constant regions are created through knobs-into-holes connections.

92. The multi-specific binding molecule of claim 91, wherein at least one knobs-into-hole connection is created by mutations selected from the group consisting of knobs:

T350→

Y,F,W; and

H395→

Y,F; and

holes;

L352→

G,A,V,I,M,S,T;

H395→

A,V,I,L,M,F,Y;

F393→

W,Y;

I397→

A,V,S,T;

T350→

S,A,V; and

T348→

S.

93. The multi-specific binding molecule of any one of claims 79 to 92, wherein said light chain variable region sequences are coupled to their matching heavy chain variable region by creating an asymmetric interface between the light and heavy chains.

94. The multi-specific binding molecule of claim 93, wherein the asymmetric interface is created by CrossMab technique, knobs-into-holes coupling and/or salt bridges coupling.

95. The multi-specific binding molecule of any one of claims 79 to 94, comprising a common light chain.

96. The multi-specific binding molecule of any one of claims 48 to 95, conjugated to a toxin.

97. The multi-specific binding molecule of any one of claims 48 to 95, conjugated to a chemotherapeutic agent.

98. The multi-specific binding molecule of claim 96 or claim 97, wherein conjugation is by fusion.

99. The multi-specific binding molecule of claim 96 or claim 97, wherein conjugation is by a chemical linker.

100. The multi-specific binding molecule of any one of claims 79 to 99, which is chimeric or humanized.

54. A multi-specific binding molecule having penta- or hexameric ring structure comprising five or six bispecific binding units, wherein (i) each of said bispecific binding units comprises two Ig vi heavy chain constant regions each comprising at least a Cμ

3 and Cμ

4 domain conjugated to a binding region to a binding target, (ii) at least two of said bispecific binding units bind to different binding targets, (iii) an internal asymmetric interface is created between two IgM heavy chain constant regions of each bispecific binding unit, and (iv) an external asymmetric interface is created between the heavy chain constant regions of the neighboring bispecific binding units binding to different targets.

55. The multi-specific binding molecule of claim 54, wherein at least three of said bispecific binding units bind to different binding targets.

56. The multi-specific binding molecule of claim 54, wherein at least four of said bispecific binding units bind to different binding targets.

57. The multi-specific binding molecule of claim 54, wherein the binding molecule has a pentameric ring structure and all five bispecific binding units bind to different targets.

58. The multi-specific binding molecule of claim 54, wherein the binding molecule has a hexameric ring structure and at least five of said bispecific binding units bind to different targets.

101. A composition comprising at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% of the multi-specific binding molecule according to any one of 48 to 100.

102. The composition of claims 101, which is a pharmaceutical composition.