阅读说明：本技术 双特异性抗原结合分子和使用方法 (Bispecific antigen binding molecules and methods of use ) 是由 D·埃勒曼 T·T·云蒂拉 T·N·隆巴纳 D·斯拉格 C·施皮斯 于 2019-02-08 设计创作，主要内容包括：本发明提供具有对第一靶抗原(例如T细胞抗原,诸如CD3)特异性的单价臂和对第二靶抗原(例如肿瘤抗原,诸如HER2)特异性的二价臂的双特异性抗原结合分子。双特异性抗原结合分子在治疗病症,诸如癌症(例如HER2阳性癌症)中是有用的。本发明还特征在于生成双特异性抗原结合分子的方法,使用双特异性抗原结合分子治疗病症的方法,和包括双特异性抗原结合分子的组合物。(The present invention provides bispecific antigen binding molecules having a monovalent arm specific for a first target antigen (e.g., a T cell antigen such as CD3) and a bivalent arm specific for a second target antigen (e.g., a tumor antigen such as HER 2). Bispecific antigen binding molecules are useful in treating disorders, such as cancer (e.g., HER2 positive cancer). The invention also features methods of making the bispecific antigen binding molecules, methods of treating disorders using the bispecific antigen binding molecules, and compositions including the bispecific antigen binding molecules.)

1. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a first antigen binding moiety, wherein the C-terminus of the first antigen binding moiety is fused to the N-terminus of a first Fc subunit;

(b) the bivalent arm comprises a second antigen binding moiety and a third antigen binding moiety, wherein the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety and the C-terminus of the second antigen binding moiety is fused to the N-terminus of a second Fc subunit; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain,

wherein the first antigen binding moiety is capable of specifically binding to CD3, and the second antigen binding moiety and the third antigen binding moiety are each capable of specifically binding to HER 2.

2. The bispecific antigen binding molecule of claim 1, wherein the second and third antigen binding modules bind to domain IV of HER 2.

3. The bispecific antigen binding molecule of claim 1 or 2, wherein the second antigen binding moiety and the third antigen binding moiety each have a monovalent binding affinity (K) _D) Is 10nM to 100 nM.

4. The bispecific antigen binding molecule of claim 3, wherein the monovalent K of each of the second antigen binding moiety and the third antigen binding moiety_DIs 20nM to 50 nM.

5. The bispecific antigen binding molecule of any one of claims 1-4, wherein the monovalent off-rate of the second antigen binding moiety and/or the third antigen binding moiety is 10^-3Second to 10^-1In seconds.

6. The bispecific antigen binding molecule of claim 5, wherein the monovalent off-rate of each of the second antigen binding moiety and the third antigen binding moiety is 10^-2Second to 30^-2In seconds.

7. The bispecific antigen binding molecule of any one of claims 1-6, wherein the monovalent K of the first antigen binding moiety_DIs 10nM to 100 nM.

8. The bispecific antigen binding molecule of claim 7, wherein the monovalent K of the first antigen binding moiety_DIs 20nM to 80 nM.

9. The bispecific antigen binding molecule of claim 8, wherein the monovalent K of the first antigen binding moiety_DIs 40nM to 60 nM.

10. The bispecific antigen binding molecule of any one of claims 1-9, wherein the monovalent K of the second antigen binding moiety_DK of the second antigen binding module in Fab format measured using surface plasmon resonance _DAnd wherein the third antigen binding moiety has a monovalent K_DK of the third antigen binding moiety in Fab format measured using surface plasmon resonance_D。

11. The bispecific antigen binding molecule of any one of claims 1-10, wherein the first antigen binding moiety is a heavy chain comprising a Variable (VH)_A) Domains and variable light chains (VL)_A) Fab molecules (Fab) of the region_A)。

12. The bispecific antigen binding molecule of any one of claims 1-11, wherein the second antigen binding moiety is a heavy chain comprising a Variable (VH)_B1) Domains and variable light chains (VL)_B1) Fab molecules (Fab) of the region_B1) And/or the third antigen binding moiety is a variable heavy chain (VH)_B2) Domains and variable light chains (VL)_B2) Fab molecules (Fab) of the region_B2)。

13. The bispecific antigen binding molecule of claim 12, wherein the first antigen binding moiety comprises a VH_ARegion and VL_AFab of the region_AThe second antigen binding module is a second antigen binding module comprising VH_B1Region and VL_B1Fab of the region_B1And the third antigen binding moiety comprises VH_B2Region and VL_B2Fab of the region_B2。

14. The bispecific antigen binding molecule of any one of claims 12-13, wherein (a) the VH_B1And the VH_B2Share at least 95% sequence identity; (b) the VL_B1And the VL_B2Share at least 95% sequence identity; or (c) the VH_B1And the VH_B2Share at least 95% sequence identity and the VL _B1And the VL_B2Share at least 95% sequence identity.

15. The bispecific antigen binding molecule of any one of claims 12-14, wherein the VH_B1The region and/or the VH_B2The region comprises an amino acid substitution at one, two, three, or all four residues of N54, D98, F100, and/or Y102 according to the numbering system of Kabat.

16. The bispecific antigen binding molecule of claim 15, wherein the VH_B1The region and/or the VH_B2The region comprises an amino acid substitution at one, two, three, four, or all five of the following residues N54E, D98A, D98T, F100A, and/or Y102V according to the Kabat numbering system.

17. The bispecific antigen binding molecule of any one of claims 12-15, wherein the VL_B1A region and/or the VL_B2The region comprises an amino acid substitution at one, two, or all three residues of N30, Y55, and/or H91 according to the numbering system of Kabat.

18. The bispecific antigen binding molecule of claim 17, wherein the VL_B1A region and/or the VL_B2The region comprises amino acid substitutions at one, two, or all three of the following residues N30S, Y55E, and/or H91A.

19. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 20.

20. The bispecific antigen binding molecule of any one of claims 12-19, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24.

21. The bispecific antigen binding molecule of claim 20, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 24.

22. The bispecific antigen binding molecule of any one of claims 12-21, wherein the VL_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 22, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

23. The bispecific antigen binding molecule of any one of claims 12-22, wherein the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27.

24. The bispecific antigen binding molecule of claim 23, wherein the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27.

25. The bispecific antigen binding molecule of any one of claims 12-24, wherein the VH_B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 20;

and the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

26. The bispecific antigen binding molecule of any one of claims 12-25, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27.

27. The bispecific antigen binding molecule of claim 26, wherein the VH_B1The region comprises the amino acids of SEQ ID NO 24, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27.

28. The bispecific antigen binding molecule of any one of claims 12-27, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 20.

29. The bispecific antigen binding molecule of any one of claims 12-28, wherein the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24.

30. The bispecific antigen binding molecule of claim 29, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 24.

31. The bispecific antigen binding molecule of any one of claims 12-30, wherein the VL_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 22, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

32. The bispecific antigen binding molecule of any one of claims 12-31, wherein the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27.

33. The bispecific antigen binding molecule of claim 32, wherein the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27.

34. The bispecific antigen binding molecule of any one of claims 12-33, wherein the VH_B2Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 20;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

35. The bispecific antigen binding molecule of any one of claims 12-34, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27.

36. The bispecific antigen binding molecule of claim 35, wherein the VH_B2The region comprises the amino acids of SEQ ID NO 24, and the VL_B2District bagContains the amino acid sequence of SEQ ID NO. 27.

37. The bispecific antigen binding molecule of any one of claims 12-36, wherein the VL_B1A region and/or the VL_B2A region comprises one or more susceptibility repair residues.

38. The bispecific antigen binding molecule of claim 37, wherein the one or more susceptibility repair residues comprise the amino acid substitution N30S.

39. The bispecific antigen binding molecule of any one of claims 12-38, wherein the VH_B1The region and/or the VH_B2A region comprises one or more susceptibility repair residues.

40. The bispecific antigen binding molecule of claim 39, wherein the one or more susceptibility repair residues comprise one or more amino acid substitutions selected from the group consisting of N54E, D98A, and D98T.

41. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 32.

42. The bispecific antigen binding molecule of any one of claims 12-18 or 41, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ id No. 33.

43. The bispecific antigen binding molecule of claim 42, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 33.

44. Claim 12-18 or 41-43, wherein the VL is_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 22, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 23.

45. The bispecific antigen binding molecule of any one of claims 12-18 or 41-44, wherein the VL is_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 25.

46. The bispecific antigen binding molecule of claim 45, wherein the VL is _B1The region comprises the amino acid sequence of SEQ ID NO 25.

47. The bispecific antigen binding molecule of any one of claims 12-18 or 41-46, wherein the VH_B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 32;

and the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 23.

48. The bispecific antigen binding molecule of any one of claims 12-18 or 41-47, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to seq id No. 33; and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25.

49. The bispecific antigen binding molecule of claim 48, wherein the VH_B1The region comprises the amino acids of SEQ ID NO 33, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25.

50. The bispecific antigen binding molecule of any one of claims 12-18 or 41-49, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 32.

51. The bispecific antigen binding molecule of any one of claims 12-18 or 41-50, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33.

52. The bispecific antigen binding molecule of claim 51, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 33.

53. The bispecific antigen binding molecule of any one of claims 12-18 or 41-52, wherein the VL is_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 22, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 23.

54. The bispecific antigen binding molecule of any one of claims 12-18 or 41-53, wherein the VL is_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 25.

55. The bispecific antigen binding molecule of claim 54, wherein the VL is_B2The region comprises the amino acid sequence of SEQ ID NO 25.

56. The bispecific antigen binding molecule of any one of claims 12-18 or 41-55, wherein the VH_B2Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 32;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 23.

57. The bispecific antigen binding molecule of any one of claims 12-18 or 41-56, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25.

58. The bispecific antigen binding molecule of claim 57, wherein the VH_B2The region comprises the amino acids of SEQ ID NO 33, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25.

59. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37.

60. The bispecific of any one of claims 12-18 or 59 A sex antigen binding molecule, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ id No. 41.

61. The bispecific antigen binding molecule of claim 60, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41.

62. The bispecific antigen binding molecule of any one of claims 12-18 or 59-61, wherein the VL is_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

63. The bispecific antigen binding molecule of any one of claims 12-18 or 59-62, wherein the VL is_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 48.

64. The bispecific antigen binding molecule of claim 63, wherein the VL is_B1The region comprises the amino acid sequence of SEQ ID NO 48.

65. The bispecific antigen binding molecule of any one of claims 12-18 or 59-64, wherein the VH_B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37;

And the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

66. The bispecific antigen binding molecule of any one of claims 12-18 or 59-65, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

67. The bispecific antigen binding molecule of claim 66, wherein the VH_B1The region comprises the amino acids of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48.

68. The bispecific antigen binding molecule of any one of claims 12-18 or 59-67, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37.

69. The bispecific antigen binding molecule of any one of claims 12-18 or 59-68, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 41.

70. The bispecific antigen binding molecule of claim 69, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41.

71. The bispecific antigen binding molecule of any one of claims 12-18 or 59-70, wherein the VL is_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

72. The bispecific antigen binding molecule of any one of claims 12-18 or 59-71, wherein the VL is_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 48.

73. The bispecific antigen binding molecule of claim 72, wherein the VL is_B2The region comprises the amino acid sequence of SEQ ID NO 48.

74. The bispecific antigen binding molecule of any one of claims 12-17 or 59-73, wherein the VH_B2Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

75. The bispecific antigen binding molecule of any one of claims 12-18 or 59-74, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 41, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

76. The bispecific antigen binding molecule of claim 75, wherein the VH_B2The region comprises the amino acids of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

77. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises the followingOne, two, or all three of the HVRs of (a):

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43.

78. The bispecific antigen binding molecule of any one of claims 12-18 or 77, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ id No. 44.

79. The bispecific antigen binding molecule of claim 78, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44.

80. The bispecific antigen binding molecule of any one of claims 12-18 or 77-79, wherein the VL is _B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

81. The bispecific antigen binding molecule of any one of claims 12-18 or 77-80, wherein the VL region of the second antigen binding module comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

82. The bispecific antigen binding molecule of claim 81, wherein the VL is_B1The region comprises the amino acid sequence of SEQ ID NO 48.

83. The bispecific antigen binding molecule of any one of claims 12-18 or 77-82, wherein the VH_B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43;

and the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

84. The bispecific antigen binding molecule of any one of claims 12-18 or 77-83, wherein the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

85. The bispecific antigen binding molecule of claim 84, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48.

86. The bispecific antigen binding molecule of any one of claims 12-18 or 77-85, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43.

87. The bispecific antigen binding molecule of any one of claims 12-18 or 77-86, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 44.

88. The bispecific antigen binding molecule of claim 87, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44.

89. The bispecific antigen binding molecule of any one of claims 12-18 or 77-88, wherein the VL_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

90. The bispecific antigen binding molecule of any one of claims 12-18 or 77-89, wherein the VL is_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 48.

91. The bispecific antigen binding molecule of claim 90, wherein the VL is_B2The region comprises the amino acid sequence of SEQ ID NO 48.

92. The bispecific antigen binding molecule of any one of claims 12-18 or 77-91, wherein the VH_B2Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

93. The bispecific antigen binding molecule of any one of claims 12-18 or 77-92, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 44, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

94. The bispecific antigen binding molecule of claim 93, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

95. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37.

96. The bispecific antigen binding molecule of any one of claims 12-18 or 95, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ id No. 41.

97. The bispecific antigen binding molecule of claim 96, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41.

98. The bispecific antigen binding molecule of any one of claims 12-18 or 95-97, wherein the VL_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

99. The bispecific antigen binding molecule of any one of claims 12-18 or 95-98, wherein the VL _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49.

100. The method of99, wherein the VL is_B1The region comprises the amino acid sequence of SEQ ID NO 49.

101. The bispecific antigen binding molecule of any one of claims 12-18 or 95-100, wherein the VH_B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37;

and the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

102. The bispecific antigen binding molecule of any one of claims 12-18 or 95-101, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

103. The bispecific antigen binding molecule of claim 102, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49.

104. The bispecific antigen binding molecule of any one of claims 12-18 or 95-103, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37.

105. The bispecific antigen binding molecule of any one of claims 12-18 or 95-104, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41.

106. The bispecific antigen binding molecule of claim 105, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41.

107. The bispecific antigen binding molecule of any one of claims 12-18 or 95-106, wherein the VL_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

108. The bispecific antigen binding molecule of any one of claims 12-18 or 95-107, wherein the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

109. The bispecific antigen binding molecule of claim 108, wherein the VL _B2The region comprises the amino acid sequence of SEQ ID NO 49.

110. The bispecific antigen binding molecule of any one of claims 12-18 or 95-109, wherein the VH_B2Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

111. The bispecific antigen binding molecule of any one of claims 12-18 or 95-110, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

112. The bispecific antigen binding molecule of claim 111, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

113. The bispecific antigen binding molecule of any one of claims 12-18, wherein the VH_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43.

114. The bispecific antigen binding molecule of any one of claims 12-18 or 113, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 44.

115. The bispecific antigen binding molecule of claim 114, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44.

116. The bispecific antigen binding molecule of any one of claims 12-18 or 113-115, wherein the VL_B1The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

117. The bispecific antigen binding molecule of any one of claims 12-18 or 113-116, wherein the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

118. The bispecific antigen binding molecule of claim 117, wherein the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49.

119. The bispecific antigen binding molecule of any one of claims 12-18 or 113-118, wherein the VH _B1Comprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43;

and the VL_B1Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

120. The bispecific antigen binding molecule of any one of claims 12-18 or 113-119, wherein the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

121. The bispecific antigen binding molecule of claim 120, wherein the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49.

122. The bispecific antigen binding molecule of any one of claims 12-18 or 113-121, wherein the VH_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43.

123. The bispecific antigen binding molecule of any one of claims 12-18 or 113-122, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44.

124. The bispecific antigen binding molecule of claim 123, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44.

125. The bispecific antigen binding molecule of any one of claims 12-18 or 113-124, wherein the VL_B2The region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

126. The bispecific antigen binding molecule of any one of claims 12-18 or 113-125, wherein the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

127. The bispecific antigen binding molecule of claim 126, wherein the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

128. The bispecific antigen binding molecule of any one of claims 12-18 or 113-127, wherein the VH_B2ComprisesThe following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43;

and the VL_B2Comprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 26.

129. The bispecific antigen binding molecule of any one of claims 12-18 or 113-128, wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

130. The bispecific antigen binding molecule of claim 129, wherein the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

131. The bispecific antigen binding molecule of any one of claims 11-130, wherein the VH_AThe region comprises one, two, or all three of the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, or

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3.

132. The bispecific antigen binding molecule of any one of claims 11-131, wherein the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 7.

133. The method of132, wherein the VH is_AThe region comprises the amino acid sequence of SEQ ID NO 7.

134. The bispecific antigen binding molecule of any one of claims 11-133, wherein the VL_AThe region comprises one, two, or all three of the following HVRs:

(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, or

(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6.

135. The bispecific antigen binding molecule of any one of claims 11-134, wherein the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8.

136. The bispecific antigen binding molecule of claim 135, wherein the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8.

137. The bispecific antigen binding molecule of any one of claims 11-136, wherein the VH_AComprising the following HVRs:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3;

and the VL_AComprising the following HVRs:

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6.

138. The bispecific antigen binding molecule of any one of claims 11-137, wherein the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7And the VL is_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8.

139. The bispecific antigen binding molecule of claim 138, wherein the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8.

140. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 20,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 22,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

141. The bispecific antigen binding molecule of claim 140, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27.

142. The bispecific antigen binding molecule of claim 141, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 24, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 24, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27.

143. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprises the followingThe HVR of (1):

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 19,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 32,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 23; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

144. The bispecific antigen binding molecule of claim 143, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, andthe VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25.

145. The bispecific antigen binding molecule of claim 144, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25.

146. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1C terminal of (2)Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

147. The bispecific antigen binding molecule of claim 146, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

148. The bispecific antigen binding molecule of claim 147, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

149. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

150. The bispecific antigen binding molecule of claim 149, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48.

151. The bispecific antigen binding molecule of claim 150, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

152. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 37,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

153. The bispecific antigen binding molecule of claim 152, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1Zone comprises and49 has an amino acid sequence of at least 95% sequence identity; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

154. The bispecific antigen binding molecule of claim 153, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

155. A bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein:

(a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO 6;

(b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab _B1Is fused to the N-terminus of the second Fc subunitWherein the Fab_B1And the Fab_B2Each comprising the following HVRs:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11,

(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36,

(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 43,

(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38,

(v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and

(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26; and is

(c) The first Fc subunit associates with the second Fc subunit to form an Fc domain.

156. The bispecific antigen binding molecule of claim 155, wherein:

(a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8;

(b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49; and is

(c) The Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49.

157. The bispecific antigen binding molecule of claim 156, wherein:

(a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8;

(b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and is

(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

158. The bispecific antigen binding molecule of any one of claims 1-157, wherein the Fc domain is an IgG Fc domain.

159. The bispecific antigen binding molecule of claim 158, wherein the IgG Fc domain is IgG₁Or IgG₄An Fc domain.

160. The bispecific antigen binding molecule of any one of claims 1-159, wherein the Fc domain is a human Fc domain.

161. The bispecific antigen binding molecule of any one of claims 1-160, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.

162. The bispecific antigen binding molecule of claim 161, wherein the one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function is at N297.

163. The bispecific antigen binding molecule of claim 162, wherein the first Fc subunit and the second Fc subunit each comprise the amino acid substitution N297G.

164. The bispecific antigen binding molecule of any one of claims 161-163, wherein the Fc receptor is an fey receptor.

165. The bispecific antigen binding molecule of any one of claims 161-164, wherein the effector function is antibody dependent cell-mediated cytotoxicity (ADCC).

166. The bispecific antigen binding molecule of any one of claims 1-165, wherein the Fc domain comprises a modification configured to facilitate association of the first Fc subunit with the second Fc subunit.

167. The bispecific antigen binding molecule of claim 166, wherein an amino acid residue in the CH3 domain of the second Fc subunit is substituted with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the second Fc subunit that is placeable in a cavity within the CH3 domain of the first Fc subunit, and an amino acid residue in the CH3 domain of the first Fc subunit is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the first Fc subunit within which the protuberance within the CH3 domain of the second Fc subunit is placeable.

168. The bispecific antigen binding molecule of claim 167, wherein the CH3 domain of the second Fc subunit comprises an amino acid substitution at T366 and the CH3 domain of the first Fc subunit comprises an amino acid substitution at one, two, or all three of T366, L368, and/or Y407.

169. The bispecific antigen binding molecule of claim 168, wherein the CH3 domain of the second Fc subunit comprises the amino acid substitution T366W and the CH3 domain of the first Fc subunit comprises one, two, or all three amino acid substitutions in T366S, L368A, and/or Y407V.

170. The bispecific antigen binding molecule of any one of claims 1-169, wherein the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety via a peptide linker.

171. The bispecific antigen binding molecule of claim 170, wherein the peptide linker is 5 to 20 amino acids in length.

172. The bispecific antigen binding molecule of claim 171, wherein the peptide linker comprises the amino acid sequence of SEQ ID No. 50.

173. The bispecific antigen binding molecule of claim 172, wherein the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 53.

174. The bispecific antigen binding molecule of claim 173, wherein the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs 57,61,81,83,85, and 87.

175. The bispecific antigen-binding molecule of claim 174, wherein the antigen-binding molecule comprises the amino acid sequence of any one of SEQ ID NOs 57,61,81,83,85, and 87.

176. The bispecific antigen binding molecule of claim 175, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 51.

177. The bispecific antigen binding molecule of claim 176, wherein the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 56.

178. The bispecific antigen-binding molecule of claim 177, wherein the antigen-binding molecule comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs 58,62,82,84,86, and 88.

179. The bispecific antigen-binding molecule of claim 178, wherein the antigen-binding molecule comprises the amino acid sequence of any one of SEQ ID NOs 58,62,82,84,86, and 88.

180. An isolated nucleic acid encoding the bispecific antigen binding molecule of any one of claims 1-179.

181. A vector comprising the isolated nucleic acid of claim 180.

182. A host cell comprising the isolated nucleic acid of claim 180 or the vector of claim 181.

183. The host cell of claim 182, wherein the host cell is a mammalian cell.

184. The host cell of claim 183, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

185. The host cell of claim 184, wherein the host cell is a prokaryotic cell.

186. The host cell of claim 185, wherein the prokaryotic cell is an e.

187. A method of producing the bispecific antigen binding molecule of any one of claims 1-179, the method comprising culturing the host cell of any one of claims 182-186 in a culture medium.

188. The method of claim 187, wherein the method further comprises recovering the bispecific antigen binding molecule from the host cell or the culture medium.

189. An isolated set of nucleic acids encoding the bispecific antigen binding molecule of any one of claims 1-179.

190. A set of vectors, wherein each vector in the set comprises an isolated nucleic acid of the set of isolated nucleic acids of claim 189.

191. A set of host cells, wherein each host cell in the set comprises the isolated nucleic acids of the isolated nucleic acid set of claim 189 or the vectors of the vector set of claim 190.

192. The set of host cells of claim 191, wherein the host cells comprise mammalian cells.

193. The host cell kit of claim 192, wherein the mammalian cells are CHO cells.

194. The set of host cells of claim 191, wherein the host cells comprise prokaryotic cells.

195. The set of host cells of claim 194, wherein the prokaryotic cells are e.

196. A method of producing the bispecific antigen binding molecule of any one of claims 1-179, the method comprising culturing the host cell kit of any one of claims 191-195 in a culture medium.

197. The method of claim 196, wherein the method further comprises recovering the bispecific antigen binding molecule from the host cell kit or the culture medium.

198. An immunoconjugate comprising the bispecific antigen binding molecule of any one of claims 1-179 and a cytotoxic agent.

199. A composition comprising the bispecific antigen binding molecule of any one of claims 1-179.

200. The composition of claim 199, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

201. The composition of claim 199 or 200, wherein the composition is a pharmaceutical composition.

202. The composition of any one of claims 199-201, wherein the composition further comprises a PD-1 axis binding antagonist or an additional therapeutic agent.

203. The bispecific antigen binding molecule of any one of claims 1-179 for use as a medicament.

204. The bispecific antigen-binding molecule of any one of claims 1-179, for use in treating or delaying progression of a HER2 positive cancer in a subject in need thereof.

205. The bispecific antigen-binding molecule of any one of claims 1-179 for use in enhancing immune function in a subject having a HER2 positive cancer.

206. The bispecific antigen binding molecule of claim 205, wherein the HER2 positive cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, renal cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, endometrial cancer, and osteosarcoma.

207. The bispecific antigen binding molecule of any one of claims 204-206, wherein the HER2 positive cancer is characterized by tumor cells expressing HER2 at an average copy number per cell of 200,000 or more copies.

208. Use of the bispecific antigen binding molecule of any one of claims 1-179 in the manufacture of a medicament for the treatment of HER2 positive cancer or delaying progression of HER2 positive cancer.

209. Use of the bispecific antigen binding molecule of any one of claims 1-179 in the manufacture of a medicament for enhancing immune function in a subject having a HER2 positive cancer.

210. The use of claim 208 or 209, wherein the HER2 positive cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, renal cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, endometrial cancer, and osteosarcoma.

211. The use of any one of claims 208-210, wherein the HER2 positive cancer is characterized by tumor cells expressing HER2 at an average copy number per cell of 200,000 or more copies.

212. A method of treating or delaying progression of a HER2 positive cancer or a HER2 positive cancer in a subject in need thereof, the method comprising administering to the subject the bispecific antigen binding molecule of any one of claims 1-179.

213. A method of enhancing immune function in a subject having a HER2 positive cancer, the method comprising administering to the subject the bispecific antigen binding molecule of any one of claims 1-179.

214. The method of claim 212 or 213, wherein the HER2 positive cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, renal cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, endometrial cancer, and osteosarcoma.

215. The method of any one of claims 212-214, wherein the HER2 positive cancer is characterized by tumor cells expressing HER2 at an average copy number per cell of 200,000 or more copies.

216. A method of treating or delaying progression of a HER2 positive cancer in a subject in need thereof, the method comprising:

(a) determining HER2 expression on a tumor cell, wherein the tumor cell expresses HER2 at an average copy number per cell of 200,000 copies or more; and are

(b) Administering to the subject the bispecific antigen binding molecule of any one of claims 1-179.

217. A method of enhancing immune function in a subject having a HER2 positive cancer, the method comprising:

(a) determining HER2 expression on a tumor cell, wherein the tumor cell expresses HER2 at an average copy number per cell of 200,000 copies or more; and are

(b) Administering to the subject the bispecific antigen binding molecule of any one of claims 1-179.

218. The method of claim 216 or 217, wherein the HER2 positive cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, renal cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, endometrial cancer, and osteosarcoma.

219. The method of any one of claims 212-218, wherein the bispecific antigen binding molecule is administered to the subject at a dose of about 0.01mg/kg to about 10 mg/kg.

220. The method of claim 219, wherein the bispecific antigen binding molecule is administered to the subject at a dose of about 0.1mg/kg to about 10 mg/kg.

221. The method of claim 220, wherein the bispecific antigen binding molecule is administered to the subject at a dose of about 1 mg/kg.

222. The method of any one of claims 212-221, further comprising administering to the subject a PD-1 axis binding antagonist and/or an additional therapeutic agent.

223. The method of claim 222, wherein the PD-1 axis binding antagonist or additional therapeutic agent is administered before or after the bispecific antigen binding molecule is administered.

224. The method of claim 222, wherein the PD-1 axis binding antagonist or additional therapeutic agent is administered concurrently with the bispecific antigen binding molecule.

225. The method of any one of claims 212-224, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

226. The method of claim 225, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

227. The method of claim 226, wherein the PD-L1 binding antagonist is selected from the group consisting of MPDL3280A (atelizumab), yw243.55.s70, MDX-1105, MEDI4736 (dulvacizumab), and MSB0010718C (avizumab).

228. The method of claim 225, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

229. The method of claim 228, wherein the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680(AMP-514), PDR001 (sibatuzumab), REGN2810 (cimiraprizumab), and BGB-108.

230. The method of claim 225, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

231. The method of claim 230, wherein the PD-L2 binding antagonist is an antibody or an immunoadhesin.

232. The method of any one of claims 212-231, wherein the bispecific antigen binding molecule is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

233. The method of claim 232, wherein the bispecific antigen binding molecule is administered subcutaneously.

234. The method of claim 232, wherein the bispecific antigen binding molecule is administered intravenously.

235. The method of any one of claims 212-234, wherein the subject is a human.

236. A kit, comprising:

(a) the composition of any one of claims 199-202; and

(b) a package insert comprising instructions for administering the composition to a subject to treat or delay progression of a HER2 positive cancer.

237. The kit of claim 236, wherein the HER2 positive cancer is characterized by tumor cells expressing HER2 at an average copy number per cell of 200,000 or more copies.

Technical Field

The present invention relates generally to bispecific antigen binding molecules, compositions thereof, and methods for treating diseases, such as cancer.

Background

Manipulation of cell-to-cell contact between specific cell types in a patient represents a promising approach for the treatment of various disease conditions. For example, bispecific antigen binding molecules (e.g., bispecific antibodies) with two arms, each specific for a different target antigen, are under development for their ability to bring immune cells into contact with target cells. Such bispecific antibodies have shown promise in a variety of disorders, such as cancer, where potent immune-mediated killing of tumor target cells has been observed in clinical trials. To confer tumor specificity, the tumor targeting arm of bispecific antibodies has been designed to target antigens that are overexpressed on tumor cells.

Existing bispecific antibodies may have several limitations, including short half-life and toxicity to healthy tissues. Bispecific antibodies overexpressed by tumor cells dependent on the target antigen often kill healthy, non-tumor cells expressing normal levels of the antigen. Such a targeted, off-tumor effect limits the therapeutic index of bispecific antibody therapy by constraining the maximum dose that a subject is tolerated. As such, there is an unmet need in the art for the development of bispecific antigen binding molecules (e.g., bispecific antibodies) with enhanced selectivity for target cells or tissues.

Summary of The Invention

The present invention relates to bispecific antigen binding molecules having a monovalent arm and a bivalent arm (e.g., T cell dependent bispecific (TDB) antibodies having a monovalent arm and a bivalent arm).

In one aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a first antigen binding moiety, wherein the C-terminus of the first antigen binding moiety is fused to the N-terminus of a first Fc subunit; (b) the bivalent arm comprises a second antigen binding moiety and a third antigen binding moiety, wherein the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety and the C-terminus of the second antigen binding moiety is fused to the N-terminus of a second Fc subunit; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain, wherein the first antigen binding moiety is capable of specifically binding to a first target antigen, and the second antigen binding moiety and the third antigen binding moiety are each capable of specifically binding to a second target cell antigen. In some embodiments, the first target antigen is an activating T cell antigen, such as CD3, and/or the second target cell antigen is a tumor antigen (e.g., HER 2). In some embodiments, the tumor antigen is expressed on (a) tumor cells in the subject and (b) at least one type of non-tumor cells in the subject.

In some embodiments, the ratio of the non-tumor cell to the copy number of tumor antigen on the tumor cell is 1:10 to 1:1,000 (e.g., 1:100 to 1: 200). In some embodiments, the tumor antigen copy number is 10 on non-tumor cells²To 10⁵And 10 on tumor cells³To 10⁷。

In some embodiments, the tumor antigen copy on the tumor cell (e.g., HER2 positive tumor cell)The number (e.g. average tumor antigen copy number, e.g. HER2 copy number, e.g. average HER2 copy number) is greater than 10⁵Per cell (e.g. 10)⁵To 10⁷Per cell, or 10⁵To 10⁶Per cell). In some embodiments, the tumor antigen copy number (e.g., an average tumor antigen copy number, such as a HER2 copy number, such as an average HER2 copy number) is greater than or equal to 200,000 per tumor cell (e.g., a HER2 positive tumor cell). In some embodiments, the tumor antigen copy number (e.g., average tumor antigen copy number, e.g., HER2 copy number, e.g., average HER2 copy number) is no more than 200,000 per non-tumor cell (e.g., non-cancerous cell, e.g., healthy cell).

In some embodiments, the monovalent binding affinity (K) of the second antigen binding moiety and/or the third antigen binding moiety_D) Is 10nM to 100nM (e.g., 20nM to 90nM,30nM to 80nM,40nM to 60nM, e.g., 25nM to 55 nM). In one embodiment, the monovalent binding affinity (K) of the second antigen binding moiety and/or the third antigen binding moiety _D) Is 20nM to 50 nM. In one embodiment, the monovalent binding affinities (K) of the second antigen binding moiety and the third antigen binding moiety_D) Is 20nM to 50 nM.

In some embodiments, the monovalent K of the second antigen binding moiety_DUsing surface plasmon resonance (e.g. usingSurface plasmon resonance) measurement of the K of this second antigen binding moiety in Fab format_DAnd wherein the third antigen binding moiety has a monovalent K_DUsing surface plasmon resonance (e.g. using

Surface plasmon resonance) measurement of the K of this third antigen binding module in Fab format_D。

In some embodiments, the monovalent off rate of the second antigen binding moiety and/or the third antigen binding moiety is 10^-3Second to 10^-1Second (e.g. 10)^-2Second to 30^-2In seconds).In some embodiments, the monovalent K of the first antigen binding moiety_DIs 10nM to 100nM (e.g., 20nM to 90nM,20nM to 80nM,30nM to 70nM, or 40nM to 60 nM).

In any of the preceding embodiments, the first antigen binding moiety may be a heavy chain comprising a Variable (VH)_A) Domains and variable light chains (VL)_A) Fab molecules (Fab) of the region_A) (ii) a The second antigen binding moiety is a heavy chain comprising a Variable (VH)_B1) Domains and variable light chains (VL)_B1) Fab molecules (Fab) of the region_B1) (ii) a And/or the third antigen binding moiety is a variable heavy chain (VH) _B2) Domains and variable light chains (VL)_B2) Fab molecules (Fab) of the region_B2). Thus, in some embodiments, the first antigen-binding moiety is a VH-comprising moiety_ARegion and VL_AFab of the region_AThe second antigen binding module is a second antigen binding module comprising VH_B1Region and VL_B1Fab of the region_B1And the third antigen binding moiety is a VH-containing moiety_B2Region and VL_B2Fab of the region_B2。

In some embodiments, the VH_B1And the VH_B2Share at least 95% sequence identity. Additionally or alternatively, the VL_B1And the VL_B2Share at least 95% sequence identity. Additionally or alternatively, the VH_B1And the VH_B2Share at least 95% sequence identity and/or the VL_B1And the VL_B2Share at least 95% sequence identity.

In some embodiments, the VH_B1The region and/or the VH_B2The region comprises an amino acid substitution at one, two, three, or all four residues of N54, D98, F100, and/or Y102 according to the numbering system of Kabat. For example, the VH_B1The region and/or the VH_B2A region may be characterized by amino acid substitutions at one, two, three, four, or all five of the following residues N54E, D98A, D98T, F100A, and/or Y102V according to the numbering system of Kabat.

In some embodiments, the VL_B1A region and/or the VL_B2The region comprises an amino acid substitution at one, two, or all three residues of N30, Y55, and/or H91 according to the numbering system of Kabat. For example, the VL _B1Zone and/or theVL_B2A region may be characterized by amino acid substitutions at one, two, or all three of the following residues N30S, Y55E, and/or H91A.

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:12, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 17 (e.g., at least 96% sequence identity to SEQ ID No. 17, at least 97% sequence identity to SEQ ID No. 17, at least 98% sequence identity to SEQ ID No. 17, at least 99% sequence identity to SEQ ID No. 17, or 100% sequence identity to SEQ ID No. 17). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16. In some embodiments, the VL _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, theVH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 17 (e.g., at least 96% sequence identity to SEQ ID No. 17, at least 97% sequence identity to SEQ ID No. 17, at least 98% sequence identity to SEQ ID No. 17, at least 99% sequence identity to SEQ ID No. 17, or 100% sequence identity to SEQ ID No. 17); and the VL _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:12, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 17 (e.g., at least 96% sequence identity to SEQ ID NO. 17, at least 97% sequence identity to SEQ ID NO. 17, at least 98% sequence identity to SEQ ID NO. 17, at least 99% sequence identity to SEQ ID NO. 17, or 100% sequence identity to SEQ ID NO. 17). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 17. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, up to SEQ ID NO. 18Less than 99% sequence identity, or 100% sequence identity to SEQ ID NO: 18). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 17 (e.g., at least 96% sequence identity to SEQ ID No. 17, at least 97% sequence identity to SEQ ID No. 17, at least 98% sequence identity to SEQ ID No. 17, at least 99% sequence identity to SEQ ID No. 17, or 100% sequence identity to SEQ ID No. 17); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 17; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution at H91. For example, in some embodiments, the H91 residue is replaced with an amino acid having a non-polar side chain. In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution H91A. In some embodiments, the VL_B1A region and/or the VL _B2The region comprises an amino acid substitution at Y55. For example, in some embodiments, the Y55 residue is replaced with an amino acid having an acidic side chain. In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution Y55E. At one endIn some embodiments, the VH_B1The region and/or the VH_B2The region comprises amino acid substitutions at F100 and/or Y102. For example, in some embodiments, the F100 residue and/or the Y102 residue are replaced with an amino acid having a non-polar side chain. In some embodiments, the VH_B1The region and/or the VH_B2The region comprises amino acid substitutions F100A and/or Y102V.

In some embodiments, the VL_B1A region and/or the VL_B2A region comprises one or more susceptibility repair residues, for example one or more susceptibility repair residues comprising the amino acid substitution N30S. Additionally or alternatively, the VH_B1The region and/or the VH_B2A region may be characterized by one or more susceptibility repair residues, for example, one or more susceptibility repair residues comprising one or more amino acid substitutions selected from the group consisting of N54E, D98A, and D98T.

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 24. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 27 (e.g., at least 96% sequence identity to SEQ ID NO. 27, at least 97% sequence identity to SEQ ID NO. 27, to SEQ ID NO. 27)At least 98% sequence identity, at least 99% sequence identity to SEQ ID NO:27, or 100% sequence identity to SEQ ID NO: 27). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5H 91A-1Fab-IgG TDB).

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 24; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5H 91A-1 Fab-IgGTDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In some casesIn embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 24 (e.g., at least 96% sequence identity to SEQ ID NO. 24, at least 97% sequence identity to SEQ ID NO. 24, at least 98% sequence identity to SEQ ID NO. 24, at least 99% sequence identity to SEQ ID NO. 24, or 100% sequence identity to SEQ ID NO. 24). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 24. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5H 91A-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24); and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID No. 24; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5H 91A-1 Fab-IgGTDB).

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33 (e.g., at least 96% sequence identity to SEQ ID NO. 33, at least 97% sequence identity to SEQ ID NO. 33, at least 98% sequence identity to SEQ ID NO. 33, at least 99% sequence identity to SEQ ID NO. 33, or 100% sequence identity to SEQ ID NO. 33). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO 33. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 25And (4) columns. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5d98a.f100a.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL _B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 33 (e.g., at least 96% sequence identity to SEQ ID No. 33, at least 97% sequence identity to SEQ ID No. 33, at least 98% sequence identity to SEQ ID No. 33, at least 99% sequence identity to SEQ ID No. 33, or 100% sequence identity to SEQ ID No. 33); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 33; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5d98a.f100a.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33 (e.g., at least 96% sequence identity to SEQ ID NO. 33, up to SEQ ID NO. 33Less than 97% sequence identity, at least 98% sequence identity to SEQ ID NO:33, at least 99% sequence identity to SEQ ID NO:33, or 100% sequence identity to SEQ ID NO: 33). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 33. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). In some embodiments, the VL _B2The region comprises the amino acid sequence of SEQ ID NO 25. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5D 98a.f100a.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 33 (e.g., at least 96% sequence identity to SEQ ID No. 33, at least 97% sequence identity to SEQ ID No. 33, at least 98% sequence identity to SEQ ID No. 33, at least 99% sequence identity to SEQ ID No. 33, or 100% sequence identity to SEQ ID No. 33); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence to SEQ ID NO: 25) Identity, at least 98% sequence identity to SEQ ID NO. 25, at least 99% sequence identity to SEQ ID NO. 25, or 100% sequence identity to SEQ ID NO. 25). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 33; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5d98a.f100a.y102v-1Fab-IgG TDB).

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a. n54e.d98t-1Fab-IgG TDB).

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL _B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a. n54e.d98t-1Fab-IgG TDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 41 (e.g., at least 96% sequence identity to SEQ ID NO. 41, at least 97% sequence identity to SEQ ID NO. 41, at least 98% sequence identity to SEQ ID NO. 41, at least 99% sequence identity to SEQ ID NO. 41, or 100% sequence identity to SEQ ID NO. 41Unity). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48 (e.g., at least 96% sequence identity to SEQ ID No. 48, at least 97% sequence identity to SEQ ID No. 48, at least 98% sequence identity to SEQ ID No. 48, at least 99% sequence identity to SEQ ID No. 48, or 100% sequence identity to SEQ ID No. 48). In some embodiments, the VL _B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D5 y55e.h91a. n54e.d98t-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a. n54e.d98t-1Fab-IgG TDB).

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a.n54e.d98t.y102v-1 Fab-IgGTDB).

In some embodiments, the VH_B1Comprising the HVR (a) comprising the amino acid sequence of SEQ ID NO 11(iii) HVR-H1 of (a), (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48 (e.g., at least 96% sequence identity to SEQ ID No. 48, at least 97% sequence identity to SEQ ID No. 48, at least 98% sequence identity to SEQ ID No. 48, at least 99% sequence identity to SEQ ID No. 48, or 100% sequence identity to SEQ ID No. 48). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D5 y55e.h91a. n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL _B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B2The region comprises SEQ ID NO:48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VL _B1The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t-1 Fab-IgGTDB).

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the sameVL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t-1Fab-IgG TDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 38 and (b) comprising S HVR-L2 of the amino acid sequence of EQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49 (e.g., at least 96% sequence identity to SEQ ID No. 49, at least 97% sequence identity to SEQ ID No. 49, at least 98% sequence identity to SEQ ID No. 49, at least 99% sequence identity to SEQ ID No. 49, or 100% sequence identity to SEQ ID No. 49). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D5 n30s.y55e.h91a.n54e.d98t-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen bindingMolecules (e.g., 4D5N30S.Y55E.H91A.N54E.D98T-1Fab-IgG TDB).

In some embodiments, the bispecific antigen binding molecule is characterized as a VH_B1A region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 38 and (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO 29The HVR-L2 of column (a), and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments In (VL)_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49 (e.g., at least 96% sequence identity to SEQ ID No. 49, at least 97% sequence identity to SEQ ID No. 49, at least 98% sequence identity to SEQ ID No. 49, at least 99% sequence identity to SEQ ID No. 49, or 100% sequence identity to SEQ ID No. 49). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D5 n30s.y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some casesIn an embodiment, the bispecific antigen binding molecule of any preceding embodiment is characterized as VH_AA region comprising one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 7 (e.g., at least 96% sequence identity to SEQ ID No. 7, at least 97% sequence identity to SEQ ID No. 7, at least 98% sequence identity to SEQ ID No. 7, at least 99% sequence identity to SEQ ID No. 7, or 100% sequence identity to SEQ ID No. 7). For example, in some embodiments, the VH _AThe region comprises the amino acid sequence of SEQ ID NO 7. In some embodiments, the VL_AThe region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8 (e.g., at least 96% sequence identity to SEQ ID No. 8, at least 97% sequence identity to SEQ ID No. 8, at least 98% sequence identity to SEQ ID No. 8, at least 99% sequence identity to SEQ ID No. 8, or 100% sequence identity to SEQ ID No. 8). For example, in some embodiments, the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3 bispecific antigen binding molecule (e.g., an anti-CD 3/HER 21 Fab-IgG TDB, e.g., an anti-CD 3/HER 21 Fab-IgG TDB with a 40G5c CD3 binding domain).

In some embodiments, the VH_AAn HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL _AComprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6. At one endIn some embodiments, the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 7 (e.g., at least 96% sequence identity to SEQ ID No. 7, at least 97% sequence identity to SEQ ID No. 7, at least 98% sequence identity to SEQ ID No. 7, at least 99% sequence identity to SEQ ID No. 7, or 100% sequence identity to SEQ ID No. 7); and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8 (e.g., at least 96% sequence identity to SEQ ID No. 8, at least 97% sequence identity to SEQ ID No. 8, at least 98% sequence identity to SEQ ID No. 8, at least 99% sequence identity to SEQ ID No. 8, or 100% sequence identity to SEQ ID No. 8). In some embodiments, the VH_AThe region comprises the amino acid sequence of SEQ ID NO. 7; and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3 bispecific antigen binding molecule (e.g., an anti-CD 3/HER 21 Fab-IgG TDB, e.g., an anti-CD 3/HER 21 Fab-IgG TDB with a 40G5c CD3 binding domain).

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14, (v) HVR-H3578(vii) HVR-L2 comprising the amino acid sequence of SEQ ID No. 15, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID No. 16; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab _AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 17, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 18; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 17, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 18. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 17, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 18.

In another aspect, the invention provides a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab _AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:20, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL _AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 24, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 24, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27.

In yet another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab _AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:32, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab _B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ id No. 25; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25.

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab _B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:37, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the sequence VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab _B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48. For example, in some embodiments, (a) the VH _AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., an anti-CD 3/HER21Fab-IgG TDB, e.g., an anti-CD 3/HER21Fab-IgGTDB with one 40G5 CD3 binding domain and two 4D5 y55e.h91a.n54e.d98t.y102v HER2 binding domains).

In yet another aspect, the invention provides a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab _B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11 and (ii) an HVR comprising(ii) HVR-H2 of the amino acid sequence of SEQ ID No. 36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID No. 37, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID No. 38, (v) HVR-L2 comprising the amino acid sequence of SEQ ID No. 29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID No. 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., an anti-CD 3/HER21Fab-IgG TDB, e.g., an anti-CD 3/HER21Fab-IgGTDB with one 40G5 CD3 binding domain and two 4D5 n30s. y55e.h91a. n54e.d98t HER2 binding domains).

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab _B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and is(c) The VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., an anti-CD 3/HER21Fab-IgG TDB, e.g., an anti-CD 3/HER21Fab-IgG TDB having one 40G5 CD3 binding domain and two 4D5 n30s.y55e.h91a.n54e.d98t.y102v HER2 binding domains).

In some embodiments of any of the preceding aspects, the Fc domain is an IgG Fc domain (e.g., IgG₁Or IgG₄Fc domain). The Fc domain may be a human Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. For example, in some embodiments, the one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function are at one or more positions selected from the group of L234, L235, and P329 (e.g., wherein the first Fc subunit and the second Fc subunit each comprise the amino acid substitutions L234A, L235A, and P329G). In some embodiments, the one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function is at N297 (e.g., N297G). In some embodiments, the Fc receptor is an fey receptor. In some embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments of any of the preceding aspects, the Fc domain comprises a modification configured to facilitate association of the first Fc subunit with the second Fc subunit. In some embodiments, an amino acid residue in the CH3 domain of the second Fc subunit is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the second Fc subunit that can be placed in the cavity within the CH3 domain of the first Fc subunit, and an amino acid residue in the CH3 domain of the first Fc subunit is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the first Fc subunit within which the protuberance within the CH3 domain of the second Fc subunit can be placed. In some embodiments, the CH3 domain of the second Fc subunit comprises the amino acid substitution T366, and the CH3 domain of the first Fc subunit comprises the amino acid substitution at one, two, or all three of T366, L368, and/or Y407. In some embodiments, the CH3 domain of the second Fc subunit comprises the amino acid substitution T366W, and the CH3 domain of the first Fc subunit comprises one, two, or all three amino acid substitutions T366S, L368A, and/or Y407V.

In some embodiments of any preceding aspect, the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety via a peptide linker. The peptide linker may be 5-20 amino acids in length (e.g., 5-10,10-15, or 15-20, e.g., 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, or 20 amino acids in length).

In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO 50. In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 55 (e.g., at least 96% sequence identity to SEQ ID No. 55, at least 97% sequence identity to SEQ ID No. 55, at least 98% sequence identity to SEQ ID No. 55, at least 99% sequence identity to SEQ ID No. 55, or 100% sequence identity to SEQ ID No. 55). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 59 (e.g., at least 96% sequence identity to SEQ ID No. 59, at least 97% sequence identity to SEQ ID No. 59, at least 98% sequence identity to SEQ ID No. 59, at least 99% sequence identity to SEQ ID No. 59, or 100% sequence identity to SEQ ID No. 59). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:63 (e.g., at least 96% sequence identity to SEQ ID NO:63, at least 97% sequence identity to SEQ ID NO:63, at least 98% sequence identity to SEQ ID NO:63, at least 99% sequence identity to SEQ ID NO:63, or 100% sequence identity to SEQ ID NO: 63). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:83 (e.g., at least 96% sequence identity to SEQ ID NO:83, at least 97% sequence identity to SEQ ID NO:83, at least 98% sequence identity to SEQ ID NO:83, at least 99% sequence identity to SEQ ID NO:83, or 100% sequence identity to SEQ ID NO: 83). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 85 (e.g., at least 96% sequence identity to SEQ ID No. 85, at least 97% sequence identity to SEQ ID No. 85, at least 98% sequence identity to SEQ ID No. 85, at least 99% sequence identity to SEQ ID No. 85, or 100% sequence identity to SEQ ID No. 85). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 87 (e.g., at least 96% sequence identity to SEQ ID No. 87, at least 97% sequence identity to SEQ ID No. 87, at least 98% sequence identity to SEQ ID No. 87, at least 99% sequence identity to SEQ ID No. 87, or 100% sequence identity to SEQ ID No. 87). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 89 (e.g., at least 96% sequence identity to SEQ ID No. 89, at least 97% sequence identity to SEQ ID No. 89, at least 98% sequence identity to SEQ ID No. 89, at least 99% sequence identity to SEQ ID No. 89, or 100% sequence identity to SEQ ID No. 89).

In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO 51. In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 56 (e.g., at least 96% sequence identity to SEQ ID No. 56, at least 97% sequence identity to SEQ ID No. 56, at least 98% sequence identity to SEQ ID No. 56, at least 99% sequence identity to SEQ ID No. 56, or 100% sequence identity to SEQ ID No. 56). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:60 (e.g., at least 96% sequence identity to SEQ ID NO:60, at least 97% sequence identity to SEQ ID NO:60, at least 98% sequence identity to SEQ ID NO:60, at least 99% sequence identity to SEQ ID NO:60, or 100% sequence identity to SEQ ID NO: 60). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 64 (e.g., at least 96% sequence identity to SEQ ID No. 64, at least 97% sequence identity to SEQ ID No. 64, at least 98% sequence identity to SEQ ID No. 64, at least 99% sequence identity to SEQ ID No. 64, or 100% sequence identity to SEQ ID No. 64). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:84 (e.g., at least 96% sequence identity to SEQ ID NO:84, at least 97% sequence identity to SEQ ID NO:84, at least 98% sequence identity to SEQ ID NO:84, at least 99% sequence identity to SEQ ID NO:84, or 100% sequence identity to SEQ ID NO: 84). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 86 (e.g., at least 96% sequence identity to SEQ ID No. 86, at least 97% sequence identity to SEQ ID No. 86, at least 98% sequence identity to SEQ ID No. 86, at least 99% sequence identity to SEQ ID No. 86, or 100% sequence identity to SEQ ID No. 86). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 88 (e.g., at least 96% sequence identity to SEQ ID No. 88, at least 97% sequence identity to SEQ ID No. 88, at least 98% sequence identity to SEQ ID No. 88, at least 99% sequence identity to SEQ ID No. 88, or 100% sequence identity to SEQ ID No. 88). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90 (e.g., at least 96% sequence identity to SEQ ID NO:90, at least 97% sequence identity to SEQ ID NO:90, at least 98% sequence identity to SEQ ID NO:90, at least 99% sequence identity to SEQ ID NO:90, or 100% sequence identity to SEQ ID NO: 90).

In another aspect, the invention features an isolated nucleic acid encoding the bispecific antigen binding molecule of any of the preceding aspects.

In another aspect, the invention provides a vector comprising an isolated nucleic acid encoding the bispecific antigen binding molecule of any preceding aspect.

In another aspect, the invention provides a host cell (e.g., an isolated host cell) comprising a vector comprising an isolated nucleic acid encoding the bispecific antigen binding molecule of any preceding aspect. In some embodiments, the host cell is a mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell). In other embodiments, the host cell is a prokaryotic cell (e.g., an E.coli cell).

In another aspect, the invention features a method of making a bispecific antigen binding molecule of any of the preceding aspects. In some embodiments, the method comprises culturing any of the host cells described above (e.g., a host cell comprising a vector comprising an isolated nucleic acid encoding the bispecific antigen binding molecule of any preceding aspect) in a culture medium. In some embodiments, the method further comprises recovering the bispecific antigen binding molecule from the host cell or the culture medium.

In another aspect, the invention features an isolated set of nucleic acids encoding a bispecific antigen binding molecule of any preceding aspect (e.g., a set comprising two, three, four, or more isolated nucleic acids encoding a bispecific antigen binding molecule of any preceding aspect). In some embodiments, the kit of isolated nucleic acids comprises a first isolated nucleic acid and a second isolated nucleic acid, wherein the first isolated nucleic acid encodes one or more amino acid sequences of the first arm of the bispecific antigen binding molecule and the second isolated nucleic acid encodes one or more amino acid sequences of the second arm of the bispecific antigen binding molecule.

In another aspect, the invention provides a set of vectors, wherein each vector in the set comprises an isolated nucleic acid of the isolated set of nucleic acids, wherein the isolated set of nucleic acids encodes a bispecific antigen binding molecule of any preceding aspect (e.g., a set comprising two, three, four, or more isolated nucleic acids encoding a bispecific antigen binding molecule of any preceding aspect).

In another aspect, the invention provides a host cell set (e.g., an isolated host cell set). In some embodiments, each host cell in the set comprises an isolated nucleic acid of an isolated set of nucleic acids, wherein the isolated set of nucleic acids encodes a bispecific antigen binding molecule of any preceding aspect (e.g., a set comprising two, three, four, or more isolated nucleic acids encoding a bispecific antigen binding molecule of any preceding aspect). In some embodiments, each host cell in the set comprises a vector comprising an isolated nucleic acid of an isolated nucleic acid set, wherein the isolated nucleic acid set encodes a bispecific antigen binding molecule of any preceding aspect (e.g., a set comprising two, three, four, or more isolated nucleic acids encoding a bispecific antigen binding molecule of any preceding aspect). In some embodiments, the host cell kit comprises mammalian cells (e.g., CHO cells). In some embodiments, the host cell set comprises prokaryotic cells (e.g., e.

In another aspect, the invention provides a method of producing the bispecific antigen binding molecule of any preceding aspect, wherein the method comprises culturing the host cell kit of the preceding aspect in a culture medium. In some embodiments, the method further comprises recovering the bispecific antigen binding molecule from the host cell kit or the culture medium.

In another aspect, the invention features an immunoconjugate comprising the bispecific antigen binding molecule of any preceding aspect and a cytotoxic agent.

In yet another aspect, the invention provides a composition comprising the bispecific antigen binding molecule of any preceding aspect. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. For example, in some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a PD-1 axis binding antagonist or an additional therapeutic agent.

In another aspect, the invention features a bispecific antigen binding molecule of any one of the preceding aspects, for use as a medicament. For example, in some embodiments, the bispecific antigen binding molecules described herein are for use in treating or delaying progression of a cell proliferative disorder (e.g., cancer) or an autoimmune disorder in a subject in need thereof. In some embodiments, the bispecific antigen binding molecule of any preceding aspect is for use in enhancing immune function in a subject having a cell proliferative disorder (e.g., a cancer, e.g., a HER2 positive cancer) or an autoimmune disorder.

In another aspect, the invention features the use of a bispecific antigen binding molecule of any preceding aspect in the manufacture of a medicament for treating or delaying the progression of a disorder. In another aspect, the invention features use of a bispecific antigen binding molecule of any preceding aspect in the manufacture of a medicament for enhancing immune function in a subject having a disorder. In some embodiments, the disorder is a cell proliferative disorder (e.g., a cancer, such as HER2 positive cancer) or an autoimmune disorder.

In yet another aspect, the invention features a method of treating or delaying progression of a disorder in a subject in need thereof, the method comprising administering to the subject a bispecific antigen binding molecule of any preceding aspect. In another aspect, the invention provides a method of enhancing immune function in a subject having a disorder, the method comprising administering to the subject the bispecific antigen binding molecule of any preceding aspect. In some embodiments, the disorder is a cell proliferative disorder (e.g., a cancer, such as HER2 positive cancer) or an autoimmune disorder.

In another aspect, the invention features a method of treating or delaying progression of a disorder in a subject in need thereof, wherein the method comprises (a) determining the expression of HER2 on a tumor cell, wherein the tumor cell expresses HER2 at an average copy number of 200,000 or more copies per cell; and (b) administering to the subject the bispecific antigen binding molecule of any preceding aspect. In some embodiments, the disorder is a cell proliferative disorder (e.g., a cancer, such as HER2 positive cancer) or an autoimmune disorder.

In another aspect, the invention provides a method of enhancing immune function in a subject having a disorder, wherein the method comprises (a) determining the expression of HER2 on a tumor cell, wherein the tumor cell expresses HER2 at an average copy number of 200,000 or more copies per cell; and (b) administering to the subject the bispecific antigen binding molecule of any preceding aspect. In some embodiments, the disorder is a cell proliferative disorder (e.g., a cancer, such as HER2 positive cancer) or an autoimmune disorder.

In some embodiments of any of the preceding aspects, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, renal cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, endometrial cancer, and osteosarcoma. In some embodiments, the cancer is a HER2 positive cancer (e.g., HER2 positive breast cancer, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive non-small cell lung cancer, HER2 positive renal cancer, HER2 positive bladder cancer, HER2 positive pancreatic cancer, HER2 positive prostate cancer, HER2 positive liver cancer, HER2 positive head and neck cancer, HER2 positive melanoma, HER2 positive ovarian cancer, HER2 positive mesothelioma, HER2 positive glioblastoma, HER2 positive endometrial cancer, or HER2 positive osteosarcoma).

In some embodiments, the HER 2-positive cancer (e.g., the HER 2-positive breast cancer, the HER 2-positive gastric cancer, the HER 2-positive colorectal cancer, the HER 2-positive non-small cell lung cancer, the HER 2-positive renal cancer, the HER 2-positive bladder cancer, the HER 2-positive pancreatic cancer, the HER 2-positive prostate cancer, the HER 2-positive liver cancer, the HER 2-positive head and neck cancer, the HER 2-positive melanoma, the HER 2-positive ovarian cancer, the HER 2-positive mesothelioma, the HER 2-positive glioblastoma, the HER 2-positive endometrial cancer, or the HER 2-positive osteosarcoma) is characterized by at least 200,000 copies per cell (e.g., at least 250,000 copies per cell of HER2, at least 300,000 copies per cell of HER2, at least 400,000 copies per cell of HER2, at least 500,000 copies per cell of HER2, at least 600,000 copies per cell, at least 700 copies per cell, at least 874000 copies per cell of HER2 copies per cell, at least 750 copies per 36800, 2 copy 36000 copies per cell, at least 900,000 HER2 copies per cell, at least 1,000,000 HER2 copies per cell, at least 1,200,000 HER2 copies per cell, at least 1,500,000 HER2 copies per cell, at least 2,000,000 HER2 copies per cell, at least 2,500,000 HER2 copies per cell, at least 3,000,000 HER2 copies per cell, or more, e.g. 200,000 to 3,000,000 HER2 copies per cell, 250,000 to 2,500,000 HER2 copies per cell, 300,000 to 2,000,000 HER2 copies per cell, 400,000 to 1,500,000 HER2 copies per cell, or 500,000 to 1,000,000 HER2 copies per cell, e.g. 200,000 to 1,000,000 HER2 copies per cell (e.g. 200,000 to 250,000,000 HER2 copies per cell, or 500,000,000 to 1,000,000,000,000 HER2 copies per cell, e.g. 200,000 to 1,000,000,000,000 HER2 copies per cell (e.g. 200,000 to 250,000,000 HER 46000 copies per cell, 300,000 to 500,000 HER 46000 copies per cell, 300,000 HER 46000 HER 468 copies per cell, 300,465,465 copies per cell, or 750,46000 HER 465,46000 HER 465,000 HER 465,465,465 copies per cell, or 300,000 HER 465 copies per cell, e.g. 5,465,465,000 HER 465, tumor cells expressing HER2 have a copy number (e.g., average copy number) of 1,500,000 to 2,000,000 copies of HER2 per cell, 2,000,000 to 2,500,000 copies of HER2 per cell, or 2,500,000 to 3,000,000 copies of HER2 per cell.

In some embodiments of any preceding aspect, the bispecific antigen binding molecule is administered to the subject at a dose of about 0.01mg/kg to about 10mg/kg (e.g., about 0.1mg/kg to about 10mg/kg, e.g., about 1 mg/kg).

In some embodiments, a PD-1 axis binding antagonist and/or an additional therapeutic agent is administered to the subject. In some embodiments, the PD-1 axis binding antagonist or additional therapeutic agent is administered before or after administration of the bispecific antigen binding molecule. In some embodiments, the PD-1 axis binding antagonist or additional therapeutic agent is administered concurrently with the bispecific antigen binding molecule. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist (e.g., atelizumab (MPDL3280A), yw243.55.s70, MDX-1105, MEDI4736 (kovacizumab), and MSB0010718C (avizumab)), a PD-1 binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidumab), MEDI-0680(AMP-514), PDR001 (sibatuzumab), REGN2810 (cimiralizumab), and BGB-108), and a PD-L2 binding antagonist (e.g., an antibody or immunoadhesin).

In some embodiments, the bispecific antigen binding molecule is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. For example, in some embodiments, the bispecific antigen binding molecule is administered subcutaneously. In other embodiments, the bispecific antigen binding molecule is administered intravenously.

In some embodiments of any of the preceding aspects, the subject is a human.

In another aspect, the invention features a kit comprising (a) a composition comprising the bispecific antigen binding molecule of any preceding aspect; and (b) a package insert comprising instructions for administering the composition to a subject to treat or delay progression of a disorder (e.g., a cell proliferative disorder (e.g., cancer) or an autoimmune disorder). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

Brief Description of Drawings

Figure 1 is an immunoblot showing the relative expression of HER2 protein of SKBR3 cells and MCF7 cells.

Fig. 2A-2F are graphs showing dose response curves for relative killing of various monovalent HER2 tdb (igg tdb) molecules against SKBR3 cells (open squares) and MCF7 cells (solid dots). Data are presented as mean ± standard deviation. Fig. 2A is a graph showing the dose response curve for target cell killing by wild-type 4D5 IgG TDB antibody (trastuzumab). Figure 2B is a graph showing the dose response curve for target cell killing of the 4D5 antibody variant y55e. Figure 2C is a graph showing the dose response curve for target cell killing of the 4D5 antibody variant y55e.d98a.f100a.y102v-IgG TDB. Figure 2D is a graph showing the dose response curve for target cell killing of the 4D5 antibody variant, d98a.f100a.y102v-IgG TDB. FIG. 2E is a graph showing the dose response curve for target cell killing of the 4D5 antibody variant H91A-IgG TDB. FIG. 2F is a graph showing the dose response curve for target cell killing of the 4D5 antibody variant Y100A-IgG TDB.

FIG. 3 is a schematic representation of representative trivalent antibodies in the 1Fab-IgG TDB format. The antibody is characterized by a bivalent arm with two anti-HER 2 binding modules and a monovalent arm with one anti-CD 3 binding module.

Fig. 4A is a graph showing a dose response curve quantifying binding of various 1Fab-IgG TDB antibodies to SKBR3 relative to a wild-type 4D5IgG TDB antibody, as quantified by flow cytometry. The filled lower-pointing triangle represents the wild-type 4D5IgG TDB antibody (trastuzumab); the filled squares represent the 4D5 antibody variant H91A-IgG TDB; the solid upper triangle represents the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; open lower-pointing triangles represent the 4D5 antibody variant Y102V-IgG TDB; while the open diamonds represent the 4D5 antibody variant y55e.y102v-IgG TDB.

Figure 4B is a graph showing a dose response curve quantifying binding of various 1Fab-IgG TDB antibodies to MCF7 relative to a wild-type 4D5IgG TDB antibody, as quantified by flow cytometry. Solid lower-pointing triangles represent wild-type 4D5IgG TDB antibody (trastuzumab); the filled squares represent the 4D5 antibody variant H91A-IgG TDB; the solid upper triangle represents the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; the open lower triangle represents the 4D5 antibody variant Y102V-IgGTDB; while the open diamonds represent the 4D5 antibody variant y55e.y102v-IgG TDB.

Figure 5A is a graph showing induction of apoptosis in SKBR3 cells by various 1Fab-IgG TDB antibodies, as quantified by caspase 3/7 activity over time, relative to wild-type 4D5 IgG TDB antibody. The solid dots represent wild-type 4D5 IgG TDB antibody (trastuzumab); open squares represent the 4D5 antibody variant H91A-IgG TDB; the filled triangles represent the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; open triangles represent the 4D5 antibody variant Y102V-IgGTDB; while the open diamonds represent the 4D5 antibody variant y55e.y102v-IgG TDB. Data are presented as mean ± standard deviation.

Figure 5B is a graph showing the induction of apoptosis in MCF7 cells by various 1Fab-IgG TDB antibodies relative to wild-type 4D5 IgG TDB antibody, as quantified by caspase 3/7 activity over time. The solid dots represent wild-type 4D5 IgG TDB antibody (trastuzumab); open squares represent the 4D5 antibody variant H91A-IgG TDB; the filled triangles represent the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; open triangles represent the 4D5 antibody variant Y102V-IgG TDB; while the open diamonds represent the 4D5 antibody variant y55e.y102v-IgG TDB. Data are presented as mean ± standard deviation.

FIG. 6A is a graph showing cytotoxicity in SKBR3 cells in response to incubation with 50ng/mL of various 1Fab-IgG TDB antibodies, relative to wild-type 4D5 IgG TDB antibody, e.g., by Measured by an assay. The first column (on the left) represents wild-type 4D5 TDB antibody (trastuzumab); the second bar represents the 4D5 antibody variant H91A-IgG TDB; the third bar represents the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; the fourth bar represents the 4D5 antibody variant Y102V-IgG TDB; while the fifth bar (on the right) represents the 4D5 antibody variant y55e.y102v-IgG TDB. Data are presented as mean ± standard deviation.

FIG. 6B is a graph showing cytotoxicity in MCF7 cells in response to incubation with 50ng/mL of various 1Fab-IgG TDB antibodies, relative to wild-type 4D5 IgG TDB antibody, as by

Measured by an assay. The first column (on the left) represents wild-type 4D5 TDB antibody (trastuzumab); the second bar represents the 4D5 antibody variant H91A-IgG TDB; the third bar represents the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; the fourth bar represents the 4D5 antibody variant Y102V-IgG TDB; while the fifth bar (on the right) represents the 4D5 antibody variant y55e.y102v-IgG TDB. Data are presented as mean ± standard deviation.

FIG. 6C is a graph showing cytotoxicity in MCF7 cells in response to incubation with 50 μ g/mL of various 1Fab-IgG TDB antibodies, relative to wild-type 4D5 IgG TDB antibody, as byMeasured by an assay. The first column (on the left) represents wild-type 4D5 TDB antibody (trastuzumab); the second bar represents the 4D5 antibody variant H91A-IgG TDB; the third bar represents the 4D5 antibody variant D98a.f100a.y102v-IgG TDB; the fourth bar represents the 4D5 antibody variant Y102V-IgG TDB; while the fifth bar (on the right) represents the 4D5 antibody variant y55e.y102v-IgG TDB. As mean ± standard deviation table Showing the data.

Figure 7A is a graph showing dose response curves quantifying binding of various 1Fab-IgG TDB antibodies to SKBR3 cells relative to wild-type 4D5 IgG TDB antibody (trastuzumab; filled triangles), as quantified by flow cytometry. Open triangles represent the 4D5 antibody variant y55e.h91a-1Fab-IgG TDB; squares represent the 4D5 antibody variant Y100Aa-1Fab-IgG TDB; while the circles represent the 4D5 antibody variant h91a. n30a-1Fab-IgG TDB.

Figure 7B is a graph showing dose response curves quantifying binding of various 1Fab-IgG TDB antibodies to MCF7 cells relative to wild-type 4D5 IgG TDB antibody (trastuzumab; filled triangles), as quantified by flow cytometry. Open triangles represent the 4D5 antibody variant y55e.h91a-1Fab-IgG TDB; squares represent the 4D5 antibody variant Y100Aa-1 Fab-IgGTDB; while the circles represent the 4D5 antibody variant h91a. n30a-1Fab-IgG TDB.

FIG. 8 is a graph showing dose response curves quantifying cytotoxicity of various 1Fab-IgG TDB antibodies against SKBR3 cells, e.g., byMeasured by an assay. Open squares represent the 4D5 antibody variant H91A-1Fab-IgG TDB; the filled squares represent the 4D5 antibody variant Y100Aa-1Fab-IgG TDB; while the circles represent the 4D5 antibody variant h91a. n30a-1Fab-IgG TDB. Data are presented as mean ± standard deviation.

FIG. 9 is a graph showing a dose response curve comparing the cytotoxicity of 4D5 IgG TDB antibody relative to 4D 5H 91A-1Fab-IgG TDB antibody. Cytotoxicity induced by the 4D5 IgG TDB antibody in SKBR3 cells and MCF7 cells is represented by filled circles and filled squares, respectively. Cytotoxicity induced by the 4D 5H 91A-1Fab-IgG TDB antibody in SKBR3 cells and MCF7 cells is represented by open triangles and open squares, respectively. Data are presented as mean ± standard deviation.

FIG. 10 is a graph showing the results of RNA-seq analysis of ErbB2 RNA expression in 90 breast cancer cell lines. Cell lines were classified as low ErbB2 expressing, medium ErbB2 expressing, and high ErbB2 expressing cell lines.

FIG. 11A is a graph showing cytotoxicity of 4D5 IgG TDB antibody at a concentration of 50ng/mL against low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN ErbB 7), and high ErbB2 expressing cell lines (MDA-MB-361, SKBR3, 474-M1, SK-OV-3, and BT 4). Data are presented as mean ± standard deviation.

FIG. 11B is a graph showing cytotoxicity of 4D5 IgG TDB antibody at a concentration of 50 μ g/mL against low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN7), and high ErbB2 expressing cell lines (MDA-MB-361, SKBR3, BT474-M1, SK-OV-3, and KPL 4). Data are presented as mean ± standard deviation.

FIG. 11C is a graph showing that 4D5 TDB antibody at a concentration of 50ng/mL was raised against human CD8 cultured with low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN ErbB 7), and high ErbB2 expressing cell lines (MDA-MB-361, SKBR3, 474-M1, SK-OV-3, and KPL4)⁺Effect of activation of T cells. T cell activation was measured by dual expression of CD69 and CD 45. Data are presented as mean ± standard deviation.

Figure 11D is an immunoblot showing HER2 protein expression for each of the cell lines presented in figures 11A-11C.

FIG. 12A is a graph showing cytotoxicity of 4D 5H 91A-1Fab-IgG TDB antibody at a concentration of 50ng/mL against low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN7), and high ErbB2 expressing cell lines (MDA-MB-361, SKBR3, BT474-M1, SK-OV-3, and KPL 4). Data are presented as mean ± standard deviation.

FIG. 12B is a graph showing cytotoxicity of 4D 5H 91A-1Fab-IgG TDB1Fab-IgG TDB antibody at a concentration of 50 μ g/mL against low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN7), and high Erb2 expressing cell lines (MDA-MB-361, BR3, BT 474-SKM 1, SK-OV-3, and KPL 4). Data are presented as mean ± standard deviation.

FIG. 12C is a graph showing that 4D5H91A-1Fab-IgG TDB antibody at a concentration of 50ng/mL, raised against human CD8 cultured with low ErbB2 expressing cell lines (MDA-MB-436, PC3, MCF7/neo-cl3, MDA-MB-231, LS1034, and HT55), medium ErbB2 expressing cell lines (MDA-MB-453, MDA-MB-175-VII, JIMT-1, and MKN7), and high Erb2 expressing cell lines (MDA-MB-361, SKBR3, BT-M1, SK-OV-3, and KPL4)⁺Effect of activation of T cells. T cell activation was measured by dual expression of CD69 and CD 45. Data are presented as mean ± standard deviation.

Figure 12D is an immunoblot showing HER2 protein expression for each of the cell lines presented in figures 12A-12C.

FIG. 13 is a graph showing dose response curves quantifying cytotoxicity of 4D5H91A-IgG TDB antibody (squares) and 4D5H91A-1Fab-IgG TDB antibody (circles) against HBL-100 cells.

Figure 14A is a graph showing a dose response curve quantifying cytotoxicity of 4D5H91A-IgG TDB antibody at various concentrations over time on HBL-100 cells. Filled circles represent dose 5,000ng/mL4D 5H91A-IgG TDB, open squares represent dose 500ng/mL 4D5H91A-IgG TDB, filled upper triangles represent dose 50ng/mL 4D5H91A-IgG TDB, open lower triangles represent dose 5ng/mL 4D5H91A-IgG TDB, open diamonds represent dose 0.5ng/mL 4D5H91A-IgG TDB, and filled diamonds represent untreated controls.

FIG. 14B is a graph showing a dose response curve quantifying cytotoxicity of 4D5H91A-1Fab-IgG TDB antibody against HBL-100 cells over time at various concentrations. Filled circles represent dose 5,000ng/mL 4D5H91A-1 Fab-IgGTDB, open squares represent dose 500ng/mL 4D5H91A-1Fab-IgG TDB, filled upper triangles represent dose 50ng/mL 4D5H91A-1Fab-IgG TDB, open lower triangles represent dose 5ng/mL 4D5H91A-1Fab-IgG TDB, open diamonds represent dose 0.5ng/mL 4D5H91A-1Fab-IgG TDB, and filled diamonds represent untreated controls.

Figure 15A is a set of graphs showing the killing effect of 4D5H 91A-IgG TDB and 4D5H91A-1Fab-IgG TDB on cell lines expressing increasing amounts of HER2 from left to right as shown by the immunoblots and bar graphs below.

Figure 15B is a series of photomicrographs showing the results of IHC and FISH detection assays relative to HER2 expression.

FIG. 15C is a graph showing killing of the cells characterized in FIG. 15B by 4D5H91A-1Fab-IgG TDB.

Figure 16 is an immunoblot showing HER2 protein expression in MCF7 cells, HT55 cells, and HER2 expanded KPL4 cells.

FIG. 17 is a trellis diagram showing KPL4 tumor volumes over the course of treatment with various doses of 4D5H91A-1Fab-IgG TDB antibody or wild type 4D5 TDB antibody in mice supplemented with human PBMCs. Mice with established KPL4 tumors received a single intravenous dose of the indicated dose on day 0. Each panel in the grid depicts a dose group, as indicated by the panel header. Bold solid black lines represent the fitted tumor volume for each dose group. The dashed bold line represents the fitted tumor volume of the control group receiving histidine buffer vehicle. Dashed lines with open circles represent individual animals. Solid lines with solid dots represent animals removed from the study. Marking tumor volume by 500mm with short horizontal gray line ³。

FIG. 18 is a trellis diagram showing HT55 tumor volumes in mice supplemented with human PBMCs following treatment with various doses of 4D5H91A-1Fab-IgG TDB antibody or wild type 4D5 IgG TDB antibody. Mice with established HT55 tumors received a single intravenous dose on day 0. Each panel in the grid depicts a dose group, as indicated by the panel header. Bold solid black lines represent the fitted tumor volume for each dose group. The dashed bold line represents the fitted tumor volume of the control group receiving histidine buffer vehicle. Dashed lines with open circles represent individual animals. Solid lines with solid dots represent animals removed from the study. Marking tumor volume by 500mm with short horizontal gray line³。

Figure 19 is a grid graph showing KPL4 tumor volumes over the course of treatment with various doses of 4D5d98a. f100a. y102v-1Fab-IgG TDB antibody or wild-type 4D5 IgG TDB antibody in mice supplemented with human PBMCs. Mice with established KPL4 tumors received a single intravenous dose of the indicated dose on day 0. Each small drawing in the gridOne dose group is plotted, as indicated by the panel header. Bold solid black lines represent the fitted tumor volume for each dose group. The dashed bold line represents the fitted tumor volume of the control group receiving histidine buffer vehicle. Dashed lines with open circles represent individual animals. Solid lines with solid dots represent animals removed from the study. Marking tumor volume with short horizontal red line of 500mm ³。

Figure 20 is a graph showing dose response curves quantifying the cytotoxicity of 4D 5H 91A-1Fab-IgG TDB antibody (filled circles) and 4D5d98a.f100a.y102v-1Fab-IgG TDB antibody (open squares) on SKBR3 cells. Data are presented as mean ± standard deviation.

Figure 21A is a graph showing killing of the high HER2 expressing cell line (n-20) by 4D 5H 91A-1Fab-IgG TDB antibody. The high HER2 expressing cell line was characterized by high expression of ErbB2 RNA. Data are presented as mean ± standard deviation.

Figure 21B is a graph showing killing of high HER2 expressing cell line (n-20) by 4D5D98a.f100a.y102v-1Fab-IgG TDB antibody. The high HER2 expressing cell line was characterized by high expression of ErbB2 RNA. Data are presented as mean ± standard deviation.

FIG. 22 is a graph showing the relative killing potency of 4D5D98A. F100.Y102V-1Fab-IgG TDB antibody versus 4D 5H 91A-1Fab-IgG TDB antibody on high HER2 expressing cell lines. The data is derived from fig. 20A and 20B.

Figure 23A is a graph showing killing of HER2 expressing cell line (n-12) by the 4D 5H 91A-1Fab-IgG TDB antibody. Killing of SKBR3 cells was provided as a positive control. The HER 2-expressing cell line is characterized by expression in ErbB2 RNA. Data are presented as mean ± standard deviation.

Figure 23B is a graph showing killing of HER2 expressing cell line (n-12) by 4D 5D 98a.f100a.y102v-1Fab-IgG TDB antibody. Killing of SKBR3 cells was provided as a positive control. The HER 2-expressing cell line was characterized by expression in ErbB2 RNA. Data are presented as mean ± standard deviation.

FIG. 24 is a graph showing the relative killing potency of 4D 5D 98A. F100.Y102V-1Fab-IgG TDB antibody against HER2 expressing cell lines in the pair of 4D 5H 91A-1Fab-IgG TDB antibodies. Data is derived from fig. 22A and 22B.

Figure 25A is a graph showing killing of low HER2 expressing cell line (n-12) by 4D 5H 91A-1Fab-IgG TDB antibody. Killing of SKBR3 cells was provided as a positive control. The low HER2 expressing cell line was characterized by low expression of ErbB2 RNA. Data are presented as mean ± standard deviation.

Figure 25B is a graph showing killing of low HER2 expressing cell line (n-12) by 4D 5D 98a.f100a.y102v-1Fab-IgG TDB antibody. Killing of SKBR3 cells was provided as a positive control. The low HER2 expressing cell line was characterized by low expression of ErbB2 RNA. Data are presented as mean ± standard deviation.

FIG. 26 is a graph showing the relative killing potency of 4D 5D 98A. F100.Y102V-1Fab-IgG TDB antibody versus 4D 5H 91A-1Fab-IgG TDB antibody against low HER2 expressing cell lines. Data is derived from fig. 24A and 24B.

FIG. 27A is a schematic showing the position and sequence of two peptide linkers tested in FIG. 27B. Each peptide linker fuses the C-terminus of the constant heavy Chain (CH) region of the distal Fab to the N-terminus of the variable heavy chain (VH) region of the proximal Fab. In FIG. 27B, DKTHTGGGGSGG (SEQ ID NO:52) is represented by open squares, and DKKHT (SEQ ID NO:50) is represented by filled circles.

Figure 27B is a graph showing a dose response curve quantifying the binding of 4D5H91A-1Fab-IgG TDB antibody with DKTHTGGGGSGG linker (open squares) versus 4D5H91A-1Fab-IgG TDB antibody with DKTHT linker (filled circles) to MCF7 cells. Data are presented as mean ± standard deviation.

Figure 27C is a graph showing a dose response curve quantifying killing of SKBR3 cells by 4D5H91A-1Fab-IgG TDB antibody with an DKTHTGGGGSGG linker (open squares) versus 4D5H91A-1Fab-IgG TDB antibody with a DKTHT linker (closed circles). Data are presented as mean ± standard deviation.

FIG. 28A is a series of immunoblots showing pAKT against HER2-CD 31 Fab-IgG versus SKBR3 cells over time^S473And the effect of expression of pHER 3.

Figure 28B is a graph showing a dose response curve quantifying the viability of SKBR3 cells in response to treatment with anti-HER 2-CD 31 Fab-IgG, anti-HER 2-CD3IgG TDB, and trastuzumab.

FIG. 29A is a sensorgram showing the binding kinetics of wild-type 4D5Fab to directly immobilized human HER2 (extracellular domain; Novus biologicals), as usedSurface plasmon resonance measurement. Wild type 4D5Fab samples ranged in concentration from 0.27 nM to 200nM, 3-fold diluted.

FIG. 29B is a sensorgram showing the binding kinetics of 4D5Y55E.Y102V-Fab to directly immobilized human HER2 (extracellular domain; Novus biologicals), e.g., using

Surface plasmon resonance measurement. The 4d5y55e.y102v-Fab sample concentration ranges from 0.27 nM to 200nM, 3-fold dilution.

FIG. 29C is a sensorgram showing the binding kinetics of 4D5D98A.F100A.Y102V-Fab to directly immobilized human HER2 (extracellular domain; Novus Biologicals), e.g., usingSurface plasmon resonance measurement. The 4d5d98a.f100a.y102v-Fab sample concentration ranges from 0.27 nM to 200nM, 3-fold dilution.

FIG. 29D is a sensorgram showing the binding kinetics of 4D 5H 91A-Fab to directly immobilized human HER2 (extracellular domain; Novus biologicals), e.g., usingSurface plasmon resonance measurement. The 4D 5H 91A-Fab sample concentration ranged from 0.27 nM to 200nM, 3-fold dilution.

FIG. 30A is a sensorgram showing the binding kinetics of 4D 5N 30S-Fab to directly immobilized human HER2 (extracellular domain; Novus biologicals), as used Surface plasmon resonance measurement. 4D5N 30S-Fab likeThe product concentration range is 0.27nM to 200nM, 3-fold dilution.

FIG. 30B is a sensorgram showing the binding kinetics of 4D5N54E.D98T-Fab to directly immobilized human HER2 (extracellular domain; Novus biologicals), as used

Surface plasmon resonance measurement. The 4D5N54E.D98T-Fab sample concentration ranges from 0.27nM to 200nM, 3-fold dilution.

FIG. 30C is a sensorgram showing the binding kinetics of 4D5N30S.N54E.D98T-Fab to directly immobilized human HER2 (extracellular domain; Novus Biologicals), e.g. using

Surface plasmon resonance measurement. The sample concentration range of 4D5N30S.N54E.D98T-Fab was 0.27nM to 200nM, 3-fold dilution.

FIG. 31A is a sensorgram showing the binding kinetics of 4D5N30S.Y55E.N54E.D98T.Y102V-Fab to directly immobilized human HER2 (extracellular domain; Novus Biologicals), e.g., using

Surface plasmon resonance measurement. The concentration of 4D5N30S.Y55E.N54E.D98T.Y102V-Fab samples ranged from 0.27nM to 200nM, 3-fold dilution.

FIG. 31B is a sensorgram showing binding kinetics of 4D5N30S.N54E.D98T.F100A.Y102V-Fab to directly immobilized human HER2 (extracellular domain; Novus Biologicals), e.g., usingSurface plasmon resonance measurement. The sample concentration range of 4d5N30S.N54E.D98T.F100A.Y102V-Fab was 0.27nM to 200nM, 3-fold dilution.

FIG. 31C is a sensorgram showing binding kinetics of 4D5 N30S.H91A.N54E.D98T-Fab to directly immobilized human HER2 (extracellular domain; Novus Biologicals), e.g., usingSurface plasmon resonance measurementIn (1). The concentration of 4D5 N30S.H91A.N54E.D98T-Fab samples ranged from 0.27nM to 200nM, 3-fold dilution.

FIGS. 32A-32K are a series of sensorgrams showing the binding kinetics of the 4D5 IgG TDB antibody to human HER2, e.g., usingSurface plasmon resonance measurement. FIG. 32A is a sensorgram showing binding kinetics of wild-type 4D5 IgG TDB; FIG. 32B is a sensorgram showing the binding kinetics of 4D 5H 91A-IgG TDB; FIG. 32C is a sensorgram showing binding kinetics of 4D5Y55E.H91A-IgG TDB; FIG. 32D is a sensorgram showing binding kinetics of 4D5 Y55E.D98A.F100A.Y102V-IgG TDB; FIG. 32E is a sensorgram showing the binding kinetics of 4D 5D 98A.F100A.Y102V-IgG TDB; figure 32F is a sensorgram showing binding kinetics of 4D5 h91a.d98a.f100a.y102v-IgG TDB; FIG. 32G is a sensorgram showing the binding kinetics of 4D5 Y55E.H91A.D98A.F100A.Y102V-IgG TDB; FIG. 32H is a sensorgram showing the binding kinetics of 4D5 Y55E.Y102V-IgG TDB; FIG. 32I is a sensorgram showing the binding kinetics of 4D5Y 102V-IgG TDB; FIG. 32J is a sensorgram showing the binding kinetics of 4D5 H91A. Y102V-IgG TDB; and FIG. 32K is a sensorgram showing the binding kinetics of 4D5 Y55E.H91A.Y102V-IgG TDB.

FIG. 33A is a graph showing quantification of 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgG TDB (batch 1) antibody (open squares); 4d5y55e.h91a.n54e.d98t-1Fab-IgG TDB antibody (solid squares); 4d5y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB (batch 2) antibody (open bottom triangle); or 4D5H91A-1Fab-IgG TDB antibody (triangles on solid sides) on SKBR3 cells killing dose response curve.

FIG. 33B is a graph showing quantification of 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgG TDB (batch 1) antibody (open squares); 4D5y55e.h91a.n54e.d98t-1Fab-IgG TDB antibody (filled squares); 4d5y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB (batch 2) antibody (open bottom triangle); or 4D5H91A-1Fab-IgG TDB antibody (triangles on solid side) dose response curve to MCF7 cell killing.

Figure 34A is a graph showing a dose response curve quantifying binding of various 1Fab-IgG TDB variants to SKBR8 cells, as measured by flow cytometry. Open squares represent the 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgGTDB (batch 1) antibody; solid squares represent 4D5Y55E.H91A.N54E.D98T-1Fab-IgG TDB antibody; the open lower triangle represents the 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgG TDB (batch 2) antibody; while the triangles on the solid face represent the 4D5H91A-1Fab-IgG TDB antibody.

Figure 34B is a graph showing a dose response curve quantifying binding of various 1Fab-IgG TDB variants to MCF7 cells, as measured by flow cytometry. Open squares represent the 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgGTDB (batch 1) antibody; solid squares represent 4D5 Y55E.H91A.N54E.D98T-1Fab-IgG TDB antibody; the open lower triangle represents the 4D5Y55E.H91A.N54E.D98T.Y102V-1Fab-IgG TDB (batch 2) antibody; while the triangles on the solid face represent the 4D 5H 91A-1Fab-IgG TDB antibody.

FIG. 35A is a schematic showing the binding and activity of anti-HER 2-CD3 IgG-TDB with monovalent high affinity binding.

FIG. 35B is a schematic showing the binding and activity of anti-HER 2-CD3 IgG-TDB with monovalent low affinity binding.

FIG. 35C is a schematic representation showing the appropriate affinity (monovalent K) due to affinity of anti-HER 2-CD 31 Fab-IgG TDB_D20-50nM) results in high binding to HER2 overexpressing cells.

Figure 36 provides crystal structures of HER2 extracellular domain (ECD) and highlights the region to which different HER2 antibodies bind.

FIG. 37 is a graph showing quantification of 38E4v 14D 5-H91A 1Fab-IgG TDB on SKBR3 target cells (open circles); 40G5c4D5-H91A 1Fab-IgG TDB on SKBR3 target cells (filled circles); 38E4v 14D 5-H91A 1Fab-IgG TDB on MCF7 target cells (open squares); and dose response curves for killing of MCF7 target cells (filled squares) by 40G5c4D5-H91A 1Fab-IgG TDB.

Figure 38A is a graph showing the levels of HER 2-independent T cell activation induced by 4D 5H 91A1Fab-IgG TDB and 4D5 IgG TDB-as tested in the presence and absence of HER2 expressing cells.

Figure 38B is a graph showing the levels of HER 2-independent T cell activation induced by 4D 5H 91A1Fab-IgG TDB and 4D5 IgG TDB, as tested in the presence and absence of bivalent anti-CD 3 OKT 3.

FIG. 38C is a series of graphs showing blood markers of inflammation (C-reactive protein; CRP), T-cell activation (lymphocyte margination), and liver damage (alanine and aspartate aminotransferases; ALT and AST) as measured 2 and 8 days after H91A1Fab-IgG TDB or vehicle control administration to cynomolgus monkeys.

FIG. 38D is a graph and summary table showing PK parameters measured by ELISA from cynomolgus monkeys dosed with H91A1Fab-IgG TDB at 20mg/kg and 3 mg/kg.

Figure 38E is a graph showing H91A1Fab-IgG TDB cynomolgus monkey serum levels at 7 and 14 days post-dose and submitted to healthy donor PBMC and SKBR3 cells for 24 hours using the indicated dilutions. Parallel experiments (control) were performed using dilutions of fresh 4D5-H91A 1 Fab-IgGTDB.

Figure 39 is a graph showing the effect of various amino acid substitutions on binding affinity of 4D5 anti-HER 2 Fab variants.

Detailed Description

I. Definition of

As used herein, the term "about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the art. References herein to "about" a value or parameter include (and describe) embodiments that relate to that value or parameter itself.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K)_D) To express. Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

As used herein, the term "specifically binds," or "specific for … …"Refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has an equilibrium dissociation constant (K.sub.m) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1nM _D). In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but need not be exclusive binding.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antigen binding moiety" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules characterized by an antigen binding moiety include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab' ₂scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies (minibodies), scFv-Fc, affibodies (affibodies), nanobodies (nanobodies), and VH and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules with identified binding partners. "affinity matured" antibody is referred to asAn antibody having one or more alterations in one or more hypervariable regions (HVRs), such alterations resulting in an improved affinity of the antibody for an antigen compared to a parent antibody not possessing such alterations.

As used herein, the term "monovalent," e.g., in the context of a monovalent arm of a bispecific antigen binding molecule, refers to a molecule or portion thereof (e.g., a portion of an antigen binding molecule, e.g., one of the two arms of a bispecific antigen binding molecule) having a single antigen binding moiety. In this way, a monovalent molecule or a portion thereof is capable of specifically binding exactly one antigen. "monovalent binding affinity" or "monovalent K" of one of the two antigen binding modules of the bivalent arm of a bispecific antibody (e.g. one of the HER2 antigen binding modules of 1Fab-IgG TDB)_D"refers to the binding affinity in a monovalent form, i.e., as a monovalent arm of a bispecific antibody capable of specifically binding two different antigens or as an antigen-binding moiety of a Fab molecule.

As used herein, the term "bivalent" refers to a molecule or portion thereof (e.g., a portion of an antigen binding molecule, e.g., one of the two arms of a bispecific antigen binding molecule) having exactly two antigen binding moieties, each of which is capable of specifically binding one antigen, e.g., in the context of a bivalent arm of a bispecific antigen binding molecule. Thus, the bivalent molecule or part thereof is capable of specifically binding two antigens or two different epitopes on the same antigen (e.g. two HER2 antigens expressed on the surface of a single tumor cell).

As used herein, the term "cluster of differentiation 3" or "CD 3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including, for example, CD3, CD3 γ, CD3 α, and CD3 β chains, unless otherwise specified. The term encompasses "full length," unprocessed CD3 (e.g., unprocessed or unmodified CD3 or CD3 γ), as well as any form of CD3 that is processed from a cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3 protein (NCBI RefSeq No. np — 000724) 207 amino acids in length and human CD3 γ protein (NCBI RefSeq No. np — 000064) 182 amino acids in length.

The terms "anti-CD 3 antibody" and "antibody that binds to CD 3" refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting CD 3. In one embodiment, the extent to which the anti-CD 3 antibody binds to an unrelated, non-CD 3 protein is less than about 10% of the binding of the antibody to CD3 as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds CD3 has a molecular weight of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D). In certain embodiments, the anti-CD 3 antibody binds to a CD3 epitope that is conserved among CD3 from different species.

The terms "Fc region" or "Fc domain" are used interchangeably herein to refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The term encompasses truncated Fc regions such as those with C-terminal truncations (e.g., Δ GK truncations, e.g., as described in Hu, et., biotechnol.prog.2017,33: 786-. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of immunological Interest,5th ed. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain, and is capable of stable self-association. In one embodiment, the subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of the variable domains consist of 4 FR domains FR1, FR2, FR3, and FR 4. Thus, the HVR and FR sequences in VH (or VL) generally occur in the order FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.227:381 (1991); Marks et al, J.mol.biol.222:581 (1991)). Also useful for the preparation of human Monoclonal Antibodies are those described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol.147(1):86-95 (1991). See also van Dijk and van deWinkel, curr, opin, pharmacol, 5:368-74 (2001). Human antibodies can be made by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomic), that has been modified to produce human antibodies in response to antigenic challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XeNOMOUS ^TMA technique). See also, e.g., Li et al, Proc. Natl. Ascad. Sci. USA,103:3557-3562(2006), for human antibodies generated via human B-cell hybridoma technology.

A "human consensus framework" is a framework representing the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, a subset of Sequences is a subset as in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3. In one embodiment, for VL, the subgroup is as Kabat et al, supra, subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which contains antigen-contacting residues ("antigen contacts"). Typically, antibodies comprise 6 HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops present at amino acid residues 26-32(L1),50-52(L2),91-96(L3),26-32(H1),53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34(L1),50-56(L2),89-97(L3),31-35b (H1),50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts, present at amino acid residues 27c-36(L1),46-55(L2),89-96(L3),30-35b (H1),47-58(H2), and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)); and (d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2),47-56(L2),48-56(L2),49-56(L2),26-35(H1),26-35b (H1),49-65(H2),93-102(H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). (see, e.g., Kindt et al, Kuby Immunology,6^thed., W.H.Freeman Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, VH from an antibody binding to an antigen orVL domains libraries of complementary VL or VH domains are screened to isolate antibodies that bind a particular antigen. See, e.g., Portolano et al, J.Immunol.150: 880-; clarkson et al, Nature352: 624-.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromager.b 848:79-87 (2007).

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document, where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a relative to, with, or against a certain% amino acid sequence identity of a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the term "susceptibility repair" (and any grammatical evolution thereof) refers to an amino acid substitution configured to enhance the chemical stability (e.g., a reduced deamidation and/or isomerization ratio) of an antibody, for example, by replacing an amino acid residue that is prone to deamidation or isomerization with a relatively inert amino acid residue or by replacing an amino acid flanking the residue that is prone to deamidation or isomerization with a relatively inert amino acid residue. Examples of repair that reduces susceptibility to deamidation include the replacement of asparagine with serine or glutamic acid (e.g., N30S or N54E). Examples of repair that reduces the susceptibility to isomerization include the replacement of aspartic acid with alanine or threonine (e.g., D98A or D98T).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-CD 3 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.

The term "pharmaceutical formulation" refers to a preparation that is in a form such as to allow the biological activity of the active ingredient contained therein to be effective, and free of additional ingredients that have unacceptable toxicity to a subject that will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "administering" refers to a method of administering a dose of a compound (e.g., an anti-CD 3 antibody of the invention or a nucleic acid encoding an anti-CD 3 antibody of the invention) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CD 3 antibody of the invention) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctival, intravesically, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion bathing target cells directly, by catheter, by lavage, in cream, or in lipid compositions. The method of administration may vary depending on a variety of factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be for the purpose of prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, antibodies of the invention are used to delay the development of or slow the progression of disease.

As used herein, "delaying the progression of a disorder or disease" means delaying, impeding, slowing, delaying, stabilizing, and/or delaying the development of a disease or disorder (e.g., a cell proliferative disorder, such as cancer). This delay can be of varying lengths of time depending on the history of the disease and/or the subject being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may essentially encompass prevention, as the individual does not develop disease. For example, the occurrence of advanced cancer, such as metastasis, may be delayed

"reduce" or "inhibit" refers to the ability to cause an overall decrease of, for example, 20% or more, 50% or more, or 75%, 85%, 90%, 95%, or more. In certain embodiments, decreasing or inhibiting may refer to effector functions of an antibody mediated by the Fc region of the antibody, such effector functions specifically including Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Cytotoxicity (ADCC), and Antibody Dependent Cellular Phagocytosis (ADCP).

A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

"disorder" is any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. "early cancer" or "early tumor" refers to a cancer that is non-invasive or metastatic, or is classified as a stage 0, stage I, or stage II cancer. Examples of cancer include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Acute Myeloid Leukemia (AML), Chronic Lymphoid Leukemia (CLL), Marginal Zone Lymphoma (MZL), Small Lymphocytic Leukemia (SLL), Lymphoplasmacytic Lymphoma (LL), Waldenstrom's Macroglobulinemia (WM), Central Nervous System Lymphoma (CNSL), Burkitt's Lymphoma (BL), B-cell prolymphocytic leukemia (BL), splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma/leukemia, splenic diffuse red myeloid small B cell lymphoma, hairy cell leukemia variants, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicular central lymphoma, large B cell lymphoma enriched in T cells/tissue cells, primary DLBCL of the central nervous system, primary DLBCL of the skin (legged form), Epstein Barr Virus (EBV) positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymus) large B cell lymphoma, intravascular large B cell lymphoma, anaplastic Lymphoma Kinase (ALK) positive large B-cell lymphoma, plasmacytic lymphoma, large B-cell lymphoma caused in HHV 8-associated multicenter Castleman (Castleman) disease, primary effusion lymphoma, non-classifiable large B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and burkitt lymphoma, or non-classifiable large B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and classical hodgkin lymphoma)). In some embodiments, the cancer is a HER2 positive cancer (e.g., HER2 positive breast cancer or HER2 positive gastric cancer).

The term "HER 2 positive" cancer comprises cancer cells having higher than normal HER2 levels. Examples of HER2 positive cancers include HER2 positive breast cancer and HER2 positive gastric cancer. Optionally, the HER2 positive cancer has an Immunohistochemistry (IHC) score of 2+ or 3+ and/or an In Situ Hybridization (ISH) amplification ratio of ≧ 2.0.

As used herein, the term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

As used herein, the term "tumor antigen" is understood to mean those antigens that are presented on tumor cells. These antigens can be presented on the cell surface in the extracellular portion, which is often combined with the transmembrane and cytoplasmic portions of the molecule. These antigens can sometimes be presented only by tumor cells and never by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may represent tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. More common are tumor antigens presented by tumor cells and normal cells, and they are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells or accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue.

An "effective amount" of a compound (e.g., a bispecific antigen binding molecule of the invention) or a composition thereof (e.g., a pharmaceutical composition) is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as measurable improvement or prevention of a particular disorder (e.g., a cell proliferative disorder, e.g., cancer). An effective amount herein may vary with factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount where the therapeutically beneficial effect exceeds any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dosage of another drug needed to treat the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumors, an effective amount of the drug is in reducing the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibit tumor growth to some extent; and/or may have an effect in alleviating to some extent one or more symptoms associated with the condition. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical setting, an effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without combination with another drug, compound, or pharmaceutical composition. As such, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or achieved in combination with one or more other agents.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis, one result being restoration or enhancement of T cell function. As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists and PD-L1 binding antagonists as well as molecules that interfere with the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusion).

As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and/or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a particular aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules. In one embodiment, the PD-1 binding antagonist reduces negative signaling through PD-1 or PD-L1 mediated by or through cell surface proteins expressed on T lymphocytes and other cells, thereby rendering dysfunctional T cells less dysfunctional. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab) described herein. In another particular aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab) described herein. In another specific aspect, the PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another particular aspect, the PD-1 binding antagonist is MEDI-0680 (AMP-514). In another specific aspect, the PD-1 binding antagonist is PDR001 (spartalizumab). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cemiplimab). In another particular aspect, the PD-1 binding antagonist is BGB-108. In another particular aspect, the PD-1 binding antagonist is AMP-224 described herein.

As used herein, a "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies and antigen-binding fragments thereof that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules. In one embodiment, the PD-L1 binding antagonist reduces negative signaling through PD-L1 or PD-1 mediated by or through cell surface proteins expressed on T lymphocytes and other cells, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is atelizumab (CAS registry No.: 1422185-06-5), also known as MPDL3280A, described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 (dulafumab) described herein. In yet another specific aspect, the anti-PD-L1 antibody is MSB0010718C (avizumab) described herein.

The terms "programmed death ligand 1" and "PD-L1" refer herein to native sequence PD-L1 polypeptide, polypeptide variants (i.e., PD-L1 polypeptide variants), and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The PD-L1 polypeptides described herein may be isolated from a variety of sources, such as from a human tissue type or from another source, or prepared by recombinant or synthetic methods.

A "native sequence PD-L1 polypeptide" comprises a polypeptide having the same amino acid sequence as a corresponding PD-L1 polypeptide derived from nature.

By "PD-L1 polypeptide variant" or variations thereof is meant a PD-L1 polypeptide, typically an active PD-L1 polypeptide, as defined herein, having at least about 80% amino acid sequence identity to any native sequence PD-L1 polypeptide sequence as disclosed herein. For example, such PD-L1 polypeptide variants include PD-L1 polypeptides in which one or more amino acid residues are added or deleted at the N-or C-terminus of the native amino acid sequence. Typically, a PD-L1 polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a native sequence PD-L1 polypeptide sequence as disclosed herein. Typically, a variant PD-L1 polypeptide is at least about 10 amino acids in length, alternatively at least about 20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,281,282,283,284,285,286,287,288, or 289 amino acids in length, or more. Optionally, the PD-L1 variant polypeptide will have no more than one conservative amino acid substitution as compared to the native PD-L1 polypeptide sequence, or no more than 2,3,4,5,6,7,8,9, or 10 conservative amino acid substitutions as compared to the native PD-L1 polypeptide sequence.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a particular aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 binding antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that mediate signaling via PD-L2, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

Composition II

In one aspect, the invention is based, in part, on bispecific antigen binding molecules (e.g., bispecific antibodies). In certain embodiments, the bispecific antigen binding molecule has a monovalent arm capable of specifically binding a first antigen (e.g., a T cell antigen, such as CD3) and a bivalent arm capable of specifically binding two additional antigens (e.g., a tumor antigen, such as two HER2 antigens). For example, the bivalent arm can comprise two antigen binding modules, each capable of specifically binding a target antigen (e.g., HER2) to increase the avidity of the bispecific antigen binding molecule for cells expressing high levels of the target antigen. Bispecific antigen binding molecules of the invention are useful, for example, for treating or delaying progression of a cell proliferative disorder (e.g., cancer) or an autoimmune disorder, or for enhancing immune function in a subject having such a disorder.

The 1Fab-IgG TDB of the invention selectively kills tumor cells over-expressing the targeted tumor antigen with high potency, while sparing cells expressing a small amount of the targeted tumor antigen, such as cells of normal or healthy human tissue. Selectivity is based on the avidity of two low affinity anti-tumor antigen Fab arms for high target density on cells overexpressing tumor antigens. The increased selectivity for tumor antigen-overexpressing cells mitigates the on-target adverse effects of TDB. For example, using HER2 as a targeted tumor antigen, anti-HER 2-IgG TDB with monovalent high affinity binding to HER2 binds both to HER2 overexpressing cells and to cells expressing low levels of HER 2.The therapeutic index of this TDB is based on the higher activity on HER2 overexpressing cells due to high HER2 density. At high TDB doses, high affinity IgG1 HER2-TDB was able to affect cells expressing low levels of HER2 and thus had the risk of targeting off-tumor autoimmunity on normal tissues (fig. 35A). Engineering HER2 binding arms to have lower affinity resulted in lower binding and TDB activity in both HER2 overexpressing cells and cells expressing low levels of HER2, but did not improve selectivity (fig. 35B). At a suitable affinity (monovalent K) _D20-50nM) results in high binding to HER2 overexpressing cells due to avidity of the anti-HER 21 Fab-IgG TDB. The affinity effect relies on a high HER2 density, so the 1Fab-IgG TDB does not bind to cells expressing low levels of HER2, such as cells of normal or healthy human tissue. Since cellular binding is associated with the ability of TDB to recruit T cell activity, anti-HER 21 Fab-IgG TDB selectively kills only cells overexpressing HER2 and reduces the risk of inducing off-target tumor autoimmunity on normal tissues (fig. 35C).

Exemplary bispecific antigen binding molecules

In one aspect, the invention provides isolated bispecific antigen binding molecules (e.g., bispecific antibodies) having a monovalent arm and a bivalent arm. For example, in one aspect, the invention provides a bispecific antigen binding molecule having a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a first antigen binding moiety, and (b) the bivalent arm comprises a second antigen binding moiety and a third antigen binding moiety. In some embodiments, the C-terminus of the first antigen binding moiety is fused to the N-terminus of the first Fc subunit, the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety, and the C-terminus of the second antigen binding moiety is fused to the N-terminus of the second Fc subunit. In some embodiments, the first Fc subunit is associated with the second Fc subunit to form an Fc domain. In some embodiments, the first antigen binding moiety is capable of specifically binding to a first target cell antigen, and the second antigen binding moiety and the third antigen binding moiety are each capable of specifically binding to a second target cell antigen (e.g., the same epitope on the second target cell antigen or at different epitopes).

The first antigen binding moiety may be a heavy chain comprising a Variable (VH)_A) Domains and variable light chains (VL)_A) Fab molecules (Fab) of the region_A) (ii) a The second antigen binding moiety may be a heavy chain comprising a Variable (VH)_B1) Domains and variable light chains (VL)_B1) Fab molecules (Fab) of the region_B1) (ii) a And/or the third antigen binding moiety may be a variable heavy chain (VH) comprising_B2) Domains and variable light chains (VL)_B2) Fab molecules (Fab) of the region_B2). Thus, in some cases, the first antigen-binding moiety may be a VH-containing moiety_ARegion and VL_AFab of the region_AThe second antigen binding module may be a second antigen binding module comprising VH_B1Region and VL_B1Fab of the region_B1And the third antigen binding moiety may be a VH comprising_B2Region and VL_B2Fab of the region_B2. The VH_B1And the VH_B2Can share at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). Additionally or alternatively, the VL_B1And the VL_B2Can share at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

A1 Fab-IgG molecule refers to a bispecific antigen binding molecule having a first antigen binding moiety, a second antigen binding moiety, and a third antigen binding moiety, wherein the first antigen binding moiety is a VH-containing_ARegion and VL_AFab of the region_AThe second antigen binding module is a second antigen binding module comprising VH _B1Region and VL_B1Fab of the region_B1And the third antigen binding moiety comprises VH_B2Region and VL_B2Fab of the region_B2Wherein the VH_B1And the VH_B2Share at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), and the VL_B1And the VL_B2Share at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). The 1Fab-IgG molecule has a monovalent arm comprising the first antigen binding moiety that binds a first target antigen and a divalent arm comprising the second and third antigen binding moieties, wherein each of the second and third antigen binding molecules specifically binds a second target cell antigen. In thatIn some embodiments, the 1Fab-IgG molecule is a 1Fab-IgG T-cell dependent bispecific molecule (1Fab-IgG TDB), wherein the monovalent arm specifically binds to an antigen on the surface of a T cell and each antigen binding moiety of the bivalent arm specifically binds to an antigen on the surface of a second target cell (e.g., a tumor cell). As with TDB (IgG TDB) having the standard bivalent IgG format, 1Fab-IgG TDB recruits cytotoxic T cells to kill cells expressing the targeted antigen.

For example, an anti-CD 3/HER 21 Fab-IgG TDB has a monovalent arm that specifically binds to CD3 (e.g., a CD3 molecule on the surface of a T cell) and a bivalent arm that has two antigen binding moieties that each specifically binds to HER2 (e.g., a HER2 molecule on the surface of a tumor cell). The two antigen binding modules on the bivalent arm may bind to the same epitope, or each antigen binding module on the bivalent arm may bind to a different epitope on the same antigen. In one embodiment, the bivalent arm binds the same epitope as 4D 5. In one embodiment, the bivalent arm binds domain IV of HER 2. In one embodiment, the antigen binding moiety specific for CD3 on the monovalent arm is a 40G5c Fab and the two antigen binding moieties specific for HER2 on the bivalent arm are each variants of a 4D5 Fab. Specific embodiments are exemplified herein.

The bispecific antigen binding molecules of the present invention can provide increased sensitivity to cells preferentially expressing high densities of antigens (e.g., tumor antigens), which in many cases reduces damage to healthy tissue (e.g., by activating T cells to engage tumor cells rather than healthy cells expressing low levels of tumor antigens). Thus, in some embodiments, a tumor antigen is expressed on (a) tumor cells in a subject and (b) at least one type of non-tumor cells in a subject (e.g., at a lower density than its expression on tumor cells). In some embodiments, the ratio of the non-tumor cell to the number of copies of tumor antigen on the tumor cell is 1:2 to 1:1,000,000 (e.g., 1:3 to 1:500,000,1:4 to 1:100,000,1:5 to 1:50,000,1:6 to 1:40,000,1:7 to 1:20,000,1:8 to 1:10,000,1:9 to 1:5,000,1:10 to 1:1,000,1:20 to 1:500,1:50 to 1:400, or 1:100 to 1:200, e.g., 1:2 to 1:10,1:10 to 1:20,1:20 to 1:50,1:50 to 1:100,1:100 to 1:200,1:200 to 1:300,1:300 to 1:400,1:400 to 1:500,1:500 to 1:600,1:600 to 1:100,1:100 to 1:100,1: 200 to 1:200,1: 300 to 1:400,1: 500 to 1:500,1: 800 to 1,000,1,000 to 1,000,1: 10,000,1:1,000 to 1,200,800,000, 1:50,000 to 1:100,000,1:100,000 to 1:500,000, or 1:500,000 to 1:1,000,000, for example about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1,000, about 1:10,000, about 1:100,000, or about 1:1,000,000.

In some embodiments, the tumor antigen copy number on the non-tumor cell is 10¹To 10⁶(e.g., 10)¹To 10⁶,10¹To 10⁵,10¹To 10⁴,10¹To 10³,10¹To 10²,10²To 10⁶,10²To 10⁵,10²To 10⁴,10²To 10³,10³To 10⁶,10³To 10⁵,10³To 10⁴,10⁴To 10⁶,10⁴To 10⁵Or 10⁵To 10⁶E.g. about 10¹,10²,10³,10⁴,10⁵Or 10⁶). In some embodiments, the tumor antigen copy number on the tumor cell is 10²To 10⁷(e.g., 10)²To 10⁷,10²To 10⁶,10²To 10⁵,10²To 10⁴,10²To 10³,10³To 10⁷,10³To 10⁶,10³To 10⁵,10³To 10⁴,10⁴To 10⁷,10⁴To 10⁶,10⁴To 10⁵,10⁵To 10⁷,10⁵To 10⁶Or 10⁶To 10⁷E.g. about 10²,10³,10⁴,10⁵,10⁶Or 10⁷)。

In some embodiments, the tumor antigen copy number is non10 on tumor cells¹To 10⁶And 10 on tumor cells²To 10⁸(e.g., 10 on non-tumor cells)²To 10⁵And 10 on tumor cells³To 10⁷10 on non-tumor cells²To 10⁵And 10 on tumor cells⁴To 10⁷10 on non-tumor cells³To 10⁵And 10 on tumor cells⁵To 10⁷10 on non-tumor cells³To 10⁴And 10 on tumor cells⁵To 10⁶Or 10 on non-tumor cells⁴To 10⁵And 10 on tumor cells⁶To 10⁷)。

In some embodiments, the tumor antigen copy number (e.g., average tumor antigen copy number, e.g., HER2 copy number, e.g., average HER2 copy number) is 10 on a non-tumor cell¹To 2.0x 10⁵And at least 2.0x 10 on tumor cells ⁵。

Tumor cells bound by the antigen binding molecules of the invention may be treated with at least 200,000 per cell (e.g., at least 250,000 HER2 copies per cell, at least 300,000 HER2 copies per cell, at least 400,000 HER2 copies per cell, at least 500,000 HER2 copies per cell, at least 600,000 HER2 copies per cell, at least 700,000 HER2 copies per cell, at least 750,000 HER2 copies per cell, at least 800,000 HER2 copies per cell, at least 900,000 HER2 copies per cell, at least 1,000,000 HER2 copies per cell, at least 1,200,000 HER2 copies per cell, at least 1,500,000 HER2 copies per cell, at least 2,000,000 HER2 copies per cell, at least 2,500,000 HER2 copies per cell, at least 3,000,000,000 HER2 copies per cell, or more, e.g., 200,000,000,000,000,000,000 to 500,638 copies per cell, at least 500,000,000 HER 638 copies per cell, at least 500,000,000,000,000 HER 638 copies per cell, at least 500,000,000,000,000,000,000,000,000,000 copies per cell, or 500,000 to 1,000,000 HER2 copies per cell, e.g., 200,000 to 1,000,000 HER2 copies per cell (e.g., 200,000 to 250,000 HER2 copies per cell, 250,000 to 300,000 HER2 copies per cell, 300,000 to 400,000 HER2 copies per cell, 400,000 to 500,000 HER2 copies per cell, 500,000 to 750,000 HER2 copies per cell, or 750,000 to 1,000,000 HER2 copies per cell) or 1,000,000 to 3,000,000 HER2 copies per cell (e.g., 1,000,000 to 1,500,000 HER2 copies per cell, 1,500,000 to 2,000,000,000 HER2 copies per cell, 2,000,000 to 2,500,000 HER2 copies per cell, or 2,500,000 to 3,000,000 HER2 copies per cell, e.g., average number of HER 36 2 copies per cell. Thus, upon binding of the antigen binding molecules of the invention, T cells can preferentially kill (e.g., selectively kill) HER2 positive tumor cells having any of the above HER2 expression characteristics, relative to non-tumor cells having little or no HER2 expression (e.g., less than 200,000 copies of HER2 per cell, e.g., 0 to 200,000 copies of HER2 per cell, 0 to 150,000 copies of HER2 per cell, 0 to 100,000 copies of HER2 per cell, 0 to 50,000 copies of HER2 per cell, 0 to 20,000 copies of HER2 per cell, 0 to 10,000 copies of HER2 per cell, 0 to 5,000 copies of HER2 per cell, or 0 to 1,000 copies of HER2 per cell).

Tumor antigen copy number (e.g., average tumor antigen copy number, such as HER2 copy number, such as average HER2 copy number) can be quantified using any suitable means known in the art or described herein. For example, HER2 copy number can be quantified using fluorescence quantification by flow cytometry (e.g., using beads of known equivalent soluble fluorescent dye (MESF) molecules).

In some embodiments, the monovalent binding affinity (K) of the second antigen binding moiety and/or the third antigen binding moiety_D) Is 10nM to 100nM (e.g., 20nM to 90nM,30nM to 80nM,40nM to 60nM, e.g., 25nM to 55 nM). In one embodiment, the monovalent binding affinity (K) of the second antigen binding moiety and/or the third antigen binding moiety_D) Is 20nM to 50 nM. In one embodiment, the monovalent binding affinities (K) of the second antigen binding moiety and the third antigen binding moiety_D) Is 20nM to 50 nM.

In some embodiments, the monovalent off rate of the second antigen binding moiety and/or the third antigen binding moiety is 10^-3Second to 10^-1Second (e.g. 10)^-2Second to 30^-2In seconds).

In some embodiments, the first target antigen is an activating T cell antigen, such as CD 3. A variety of T cell antigen binding modules are known in the art and are suitable for use as part of the present invention. For example, in certain embodiments of the invention, a suitable first antigen-binding moiety is antibody clone 40G5c, or a fragment and/or variant thereof, for example as described in U.S. publication No.2015/0166661, which is incorporated herein by reference in its entirety. In particular embodiments, the first antigen binding moiety is an anti-CD 3 Fab (e.g., 40G5c) comprising HVR-H1 of SEQ ID NO:1, HVR-H2 of SEQ ID NO:2, HVR-H3 of SEQ ID NO:3, HVR-L1 of SEQ ID NO:4, HVR-L2 of SEQ ID NO:5, and HVR-L3 of SEQ ID NO: 6. In some embodiments, the first antigen binding moiety is an anti-CD 3 Fab (e.g., 40G5c) comprising the VH of SEQ ID NO:7 and the VL of SEQ ID NO: 8. The amino acid sequence of 40G5c is also provided in table 1.

TABLE 1 heavy (VH) and light (VL) chain amino acid sequences of SEQ ID NOs corresponding to the hypervariable region (HVR) and variable regions of exemplary antibody clones

In some embodiments, the first antigen-binding moiety binds to a human CD3 polypeptide or a cynomolgus monkey CD3 polypeptide. In some embodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3 polypeptide or a cyno CD3 polypeptide, respectively. In some embodiments, the human CD3 polypeptide or the cynomolgus monkey CD3 polypeptide is a human CD3 gamma polypeptide or a cynomolgus monkey CD3 gamma polypeptide, respectively. Additional antigen binding moieties that bind CD3 (e.g., Fab molecules) are known in the art and described, for example, in U.S. publication No.2015/0166661, and may be adapted for use as part of the present invention.

In some embodiments, the monovalent K of the first antigen binding moiety_DAt 250nK of M or less_DBinding to the human CD3 polypeptide. In some embodiments, the anti-CD 3 antibody has a K of 100nM or less_DBinding to the human CD3 polypeptide. In some embodiments, the anti-CD 3 antibody has a K of 15nM or less_DBinding to the human CD3 polypeptide. In some embodiments, the anti-CD 3 antibody has a K of 10nM or less_DBinding to the human CD3 polypeptide. In some embodiments, the anti-CD 3 antibody has a K of 5nM or less _DBinding to the human CD3 polypeptide. In some embodiments, the monovalent K of the first antigen binding moiety_DIs 10nM to 100nM (e.g., 20nM to 90nM,20nM to 80nM,30nM to 70nM, or 40nM to 60 nM).

In some embodiments, the second target cell antigen is a tumor antigen.

The tumor antigen may be HER 2. One suitable second and/or third antigen-binding moiety that binds HER2 is antibody 4D5, or a fragment and/or variant thereof. The amino acid sequence of 4D5, and substitution variants thereof, are shown in table 1 and described, for example, in U.S. publication No.2015/0166661, which is incorporated herein in its entirety by reference. In one embodiment, the second and/or third antigen binding moiety is conjugated to antibody 4D5 (a humanized version of which is referred to as trastuzumab: (a)Genentech, Inc., South San Francisco, CA), Molina, et al, Cancer Research 2001,61(12): 4744-. In one embodiment, the second and the third antigen binding moiety bind the same epitope as antibody 4D 5.

In one embodiment, the second and/or third antigen binding moiety is conjugated to antibody 2C4 (a humanized version of which is designated pertuzumab: (a)Genentech, inc., South San Francisco, CA), Franklin et al, Cancer Cell 2004,5: 317-. In one embodiment, the second and the third antigen binding moiety bind the same epitope as antibody 2C 4.

In one embodiment, the second and/or third antigen binding moiety binds to the same epitope as antibody 7C2 (U.S. patent No.9,518,118). In one embodiment, the second and the third antigen binding moiety bind the same epitope as antibody 7C 2.

Figure 36 provides the crystal structure of HER2 ECD and highlights the regions to which the different HER2 antibodies bind. 4D5 binds to an epitope in the region of the protein closest to the cell membrane in domain IV of HER 2. 2C4 binds to an epitope in domain II of HER2 that is 50 angstroms from the region to which hu4D5 binds. 7C2 binds to an epitope in domain I of HER2 that is 100 angstroms away from the region of HER2 to which hu4D5 binds.

In one embodiment, the second and/or third antigen binding module binds to domain IV of HER 2. In one embodiment, the second and the third antigen binding module bind domain IV of HER 2. In one embodiment, the second and/or third antigen binding module binds to domain II of HER 2. In one embodiment, the second and the third antigen binding module bind domain II of HER 2.

In one embodiment, the second and/or third antigen binding module binds to domain I of HER 2. In one embodiment, the second and the third antigen binding module bind domain I of HER 2.

In one embodiment, the second binding moiety binds to domain IV of HER2 and the third antigen binding moiety binds to domain I of HER 2.

In one embodiment, the second binding moiety binds to domain IV of HER2 and the third antigen binding moiety binds to domain II of HER 2.

In one embodiment, the second binding moiety binds to domain II of HER2 and the third antigen binding moiety binds to domain I of HER 2.

In some embodiments, the bispecific antigen binding molecule of any of the preceding embodiments is characterized by a VH comprising one, two, or all three of the following HVRs_AThe region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3. In some embodiments, the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 7 (e.g., at least 96% sequence identity to SEQ ID NO. 7)One, at least 97% sequence identity to SEQ ID NO. 7, at least 98% sequence identity to SEQ ID NO. 7, at least 99% sequence identity to SEQ ID NO. 7, or 100% sequence identity to SEQ ID NO. 7). For example, in some embodiments, the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7. In some embodiments, the VL _AThe region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8 (e.g., at least 96% sequence identity to SEQ ID No. 8, at least 97% sequence identity to SEQ ID No. 8, at least 98% sequence identity to SEQ ID No. 8, at least 99% sequence identity to SEQ ID No. 8, or 100% sequence identity to SEQ ID No. 8). For example, in some embodiments, the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3 bispecific antigen binding molecule (e.g., an anti-CD 3/HER 21 Fab-IgG TDB, e.g., an anti-CD 3/HER 21 Fab-IgG TDB with a 40G5c CD3 binding domain).

In some embodiments, the VH_AAn HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL_AComprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the VH _AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 7 (e.g., at least 96% sequence identity to SEQ ID No. 7, at least 97% sequence identity to SEQ ID No. 7, at least 98% sequence identity to SEQ ID No. 7, at least 99% sequence identity to SEQ ID No. 7, or 100% sequence identity to SEQ ID No. 7); and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 8 (e.g., at least 96% to SEQ ID NO. 8Sequence identity, at least 97% sequence identity to SEQ ID NO. 8, at least 98% sequence identity to SEQ ID NO. 8, at least 99% sequence identity to SEQ ID NO. 8, or 100% sequence identity to SEQ ID NO. 8). In some embodiments, the VH_AThe region comprises the amino acid sequence of SEQ ID NO. 7; and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3 bispecific antigen binding molecule (e.g., an anti-CD 3/HER 21 Fab-IgG TDB, e.g., an anti-CD 3/HER 21 Fab-IgG TDB with a 40G5c CD3 binding domain).

In some embodiments, the VH_B1The region and/or the VH_B2The region comprises an amino acid substitution at one, two, three, or all four residues of N54, D98, F100, and/or Y102 according to the numbering system of Kabat. For example, the VH _B1The region and/or the VH_B2A region may be characterized by amino acid substitutions at one, two, three, four, or all five of the following residues N54E, D98A, D98T, F100A, and/or Y102V according to the numbering system of Kabat.

In some embodiments, the VL_B1A region and/or the VL_B2The region comprises an amino acid substitution at one, two, or all three residues of N30, Y55, and/or H91 according to the numbering system of Kabat. For example, the VL_B1A region and/or the VL_B2A region may be characterized by amino acid substitutions at one, two, or all three of the following residues N30S, Y55E, and/or H91A.

In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs_B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 17 (e.g., at least 96% sequence identity to SEQ ID No. 17, at least 97% sequence identity to SEQ ID No. 17, at least 98% sequence identity to SEQ ID No. 17, at least 99% sequence identity to SEQ ID No. 17, or 100% sequence identity to SEQ ID No. 17). For example, in In some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 17 (e.g., at least 96% sequence identity to SEQ ID No. 17, at least 97% sequence identity to SEQ ID No. 17, at least 98% sequence identity to SEQ ID No. 17, at least 99% sequence identity to SEQ ID No. 17, or 100% sequence identity to SEQ ID No. 17); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:12, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 17 (e.g., at least 96% sequence identity to SEQ ID NO. 17, at least 97% sequence identity to SEQ ID NO. 17, at least 98% sequence identity to SEQ ID NO. 17, at least 99% sequence identity to SEQ ID NO. 17, or 100% sequence identity to SEQ ID NO. 17). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 17. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 18 (e.g., at least 96% sequence identity to SEQ ID No. 18, at least 97% sequence identity to SEQ ID No. 18, at least 98% sequence identity to SEQ ID No. 18, at least 99% sequence identity to SEQ ID No. 18, or 100% sequence identity to SEQ ID No. 18). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 18.

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 17 (e.g., at least 96% sequence identity to SEQ ID NO. 17, at least 97% sequence identity to SEQ ID NO. 17At least 98% sequence identity to SEQ ID NO. 17, at least 99% sequence identity to SEQ ID NO. 17, or 100% sequence identity to SEQ ID NO. 17); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 18 (e.g., at least 96% sequence identity to SEQ ID NO. 18, at least 97% sequence identity to SEQ ID NO. 18, at least 98% sequence identity to SEQ ID NO. 18, at least 99% sequence identity to SEQ ID NO. 18, or 100% sequence identity to SEQ ID NO. 18). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 17; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO. 18.

In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution at H91. For example, in some embodiments, the H91 residue is replaced with an amino acid having a non-polar side chain. In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution H91A. In some embodiments, the VL _B1A region and/or the VL_B2The region comprises an amino acid substitution at Y55. For example, in some embodiments, the Y55 residue is replaced with an amino acid having an acidic side chain. In some embodiments, the VL_B1A region and/or the VL_B2The region comprises the amino acid substitution Y55E. In some embodiments, the VH_B1The region and/or the VH_B2The region comprises amino acid substitutions at F100 and/or Y102. For example, in some embodiments, the F100 residue and/or the Y102 residue is replaced with an amino acid having a non-polar side chain. In some embodiments, the VH_B1The region and/or the VH_B2The region comprises amino acid substitutions F100A and/or Y102V.

In some embodiments of the bispecific antigen binding module, the VL_B1A region and/or the VL_B2A region comprises one or more susceptibility repair residues, for example one or more susceptibility repair residues comprising the amino acid substitution N30S. Additionally or alternatively, the VH_B1The region and/or the VH_B2A region may be characterized by one or more susceptibility repair residues, e.g., one comprising one or more amino acid substitutions selected from the group consisting of N54E, D98A, and D98TOne or more susceptible repair residues.

The bispecific antigen binding molecule may be characterized by a mutation at residue H91 of the light chain of the second and/or third antigen binding moiety (e.g. H91A). For example, in some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs _B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 20. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 24. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). For example, in some embodiments, the VL _B1The region comprises the amino acid sequence of SEQ ID NO 27.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26HVR-L3 of amino acid sequence. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 24; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 27.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 24 (e.g., at least 96% sequence identity to SEQ ID NO. 24, at least 97% sequence identity to SEQ ID NO. 24, at least 98% sequence identity to SEQ ID NO. 24, at least 99% sequence identity to SEQ ID NO. 24, or 100% sequence identity to SEQ ID NO. 24). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 24. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 27 (e.g., at least 96% sequence identity to SEQ ID NO. 27, at least 97% sequence identity to SEQ ID NO. 27, and SEQ ID NO. 27) At least 98% sequence identity to SEQ ID NO. 27, at least 99% sequence identity to SEQ ID NO. 27, or 100% sequence identity to SEQ ID NO. 27). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27.

In some cases, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 24 (e.g., at least 96% sequence identity to SEQ ID No. 24, at least 97% sequence identity to SEQ ID No. 24, at least 98% sequence identity to SEQ ID No. 24, at least 99% sequence identity to SEQ ID No. 24, or 100% sequence identity to SEQ ID No. 24); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27 (e.g., at least 96% sequence identity to SEQ ID No. 27, at least 97% sequence identity to SEQ ID No. 27, at least 98% sequence identity to SEQ ID No. 27, at least 99% sequence identity to SEQ ID No. 27, or 100% sequence identity to SEQ ID No. 27). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 24; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4D 5H 91A-1Fab-IgG TDB).

In other cases, the bispecific antigen binding molecule may be characterized by mutations at residues D98, F100, and Y102 of the heavy chain of the second and/or third antigen binding moiety (e.g., D98A, F100A, and Y102V). In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs_B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) amino acid sequence comprising SEQ ID NO. 19HVR-H2 of column (a), or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33 (e.g., at least 96% sequence identity to SEQ ID NO. 33, at least 97% sequence identity to SEQ ID NO. 33, at least 98% sequence identity to SEQ ID NO. 33, at least 99% sequence identity to SEQ ID NO. 33, or 100% sequence identity to SEQ ID NO. 33). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO 33. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 33 (e.g., at least 96% sequence identity to SEQ ID No. 33, at least 97% sequence identity to SEQ ID No. 33, at least 98% sequence identity to SEQ ID No. 33, at least 99% sequence identity to SEQ ID No. 33, or 100% sequence identity to SEQ ID No. 33); and the VL_B1The region comprises at least 95 of SEQ ID NO 25% sequence identity (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 33; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 33 (e.g., at least 96% sequence identity to SEQ ID NO. 33, at least 97% sequence identity to SEQ ID NO. 33, at least 98% sequence identity to SEQ ID NO. 33, at least 99% sequence identity to SEQ ID NO. 33, or 100% sequence identity to SEQ ID NO. 33). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 33. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25.

In some embodiments, the VH_B2Comprising the HVRs of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) ammonia comprising SEQ ID NO:32The HVR-H3 of amino acid sequence; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 33 (e.g., at least 96% sequence identity to SEQ ID No. 33, at least 97% sequence identity to SEQ ID No. 33, at least 98% sequence identity to SEQ ID No. 33, at least 99% sequence identity to SEQ ID No. 33, or 100% sequence identity to SEQ ID No. 33); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25 (e.g., at least 96% sequence identity to SEQ ID NO:25, at least 97% sequence identity to SEQ ID NO:25, at least 98% sequence identity to SEQ ID NO:25, at least 99% sequence identity to SEQ ID NO:25, or 100% sequence identity to SEQ ID NO: 25). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 33; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5d98a.f100a.y102v-1Fab-IgG TDB).

In other embodiments, the bispecific antigen binding molecule may be characterized by mutations at residues Y55 and H91 of the light chain and at residues N54 and D98 of the heavy chain of the second and/or third antigen binding moiety (e.g., Y55E, H91A, N54E, and D98T). In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs _B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or to SEQ ID NO:41100% sequence identity). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VL _B1The region comprises the amino acid sequence of SEQ ID NO 48.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region containing SE48, Q ID NO.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48 (e.g., at least 96% sequence identity to SEQ ID No. 48, at least 97% sequence identity to SEQ ID No. 48, at least 98% sequence identity to SEQ ID No. 48, at least 99% sequence identity to SEQ ID No. 48, or 100% sequence identity to SEQ ID No. 48). In some embodiments, the VL _B2The region comprises the amino acid sequence of SEQ ID NO 48.

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, to SE)At least 97% sequence identity to SEQ ID NO 41, at least 98% sequence identity to SEQ ID NO 41, at least 99% sequence identity to SEQ ID NO 41, or 100% sequence identity to SEQ ID NO 41); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a. n54e.d98t-1Fab-IgG TDB).

In other embodiments, the bispecific antigen binding molecule may be characterized by mutations at residues Y55 and H91 of the light chain and at residues N54, D98, and Y102 of the heavy chain of the second and/or third antigen binding moiety (e.g., Y55E, H91A, N54E, D98T, and Y102). In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs_B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 43. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some casesIn embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48 (e.g., at least 96% sequence identity to SEQ ID No. 48, at least 97% sequence identity to SEQ ID No. 48, at least 98% sequence identity to SEQ ID No. 48, at least 99% sequence identity to SEQ ID No. 48, or 100% sequence identity to SEQ ID No. 48). For example, in some embodiments, the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least 98% sequence identity to SEQ ID NO:48, at least 99% sequence identity to SEQ ID NO:48, or 100% sequence identity to SEQ ID NO: 48). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH_B2The region comprises SEQ ID An amino acid sequence having at least 95% sequence identity to SEQ ID NO. 44 (e.g., at least 96% sequence identity to SEQ ID NO. 44, at least 97% sequence identity to SEQ ID NO. 44, at least 98% sequence identity to SEQ ID NO. 44, at least 99% sequence identity to SEQ ID NO. 44, or 100% sequence identity to SEQ ID NO. 44). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48 (e.g., at least 96% sequence identity to SEQ ID No. 48, at least 97% sequence identity to SEQ ID No. 48, at least 98% sequence identity to SEQ ID No. 48, at least 99% sequence identity to SEQ ID No. 48, or 100% sequence identity to SEQ ID No. 48). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

In some embodiments, the VH _B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 (e.g., at least 96% sequence identity to SEQ ID NO:48, at least 97% sequence identity to SEQ ID NO:48, at least one amino acid sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NOAt least 98% sequence identity to SEQ ID NO 48, at least 99% sequence identity to SEQ ID NO 48, or 100% sequence identity to SEQ ID NO 48). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In other embodiments, the bispecific antigen binding molecule may be characterized by mutations at residues N30, Y55, and H91 of the light chain and at residues N54 and D98 of the heavy chain of the second and/or third antigen binding moiety (e.g., N30S, Y55E, H91A, N54E, and D98T). In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs_B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 37. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodimentsThe VL of_B1The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B1Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH _B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41 (e.g., at least 96% sequence identity to SEQ ID NO:41, at least 97% sequence identity to SEQ ID NO:41, at least 98% sequence identity to SEQ ID NO:41, at least 99% sequence identity to SEQ ID NO:41, or 100% sequence identity to SEQ ID NO: 41). In some embodiments, the VH _B2The region comprises the amino acid sequence of SEQ ID NO 41. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49 (e.g., at least 96% sequence identity to SEQ ID No. 49, at least 97% sequence identity to SEQ ID No. 49, at least 98% sequence identity to SEQ ID No. 49, at least 99% sequence identity to SEQ ID No. 49, or 100% sequence identity to SEQ ID No. 49). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 41 (e.g., at least 96% sequence identity to SEQ ID No. 41, at least 97% sequence identity to SEQ ID No. 41, at least 98% sequence identity to SEQ ID No. 41, at least 99% sequence identity to SEQ ID No. 41, or 100% sequence identity to SEQ ID No. 41); and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 41; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen bindingMolecules (e.g., 4D5N30S.Y55E.H91A.N54E.D98T-1Fab-IgG TDB).

In other embodiments, the bispecific antigen binding molecule may be characterized by mutations at residues N30, Y55, and H91 of the light chain and at residues N54, D98, and Y102 of the heavy chain of the second and/or third antigen binding moiety (e.g., N30S, Y55E, H91A, N54E, D98T, and Y102V). In some embodiments, the bispecific antigen binding molecule is characterized by a VH comprising one, two, or all three of the following HVRs _B1Region (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 43. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). For example, in some embodiments, the VH_B1The region comprises the amino acid sequence of SEQ ID NO. 44. In some embodiments, the VL_B1The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VL _B1The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B1An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B1Comprising the following HVRs (d) comprisingHVR-L1 of the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). For example, in some embodiments, the VH _B1The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B2The region comprises one, two, or all three of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, or (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 (e.g., at least 96% sequence identity to SEQ ID NO:44, at least 97% sequence identity to SEQ ID NO:44, at least 98% sequence identity to SEQ ID NO:44, at least 99% sequence identity to SEQ ID NO:44, or 100% sequence identity to SEQ ID NO: 44). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44. In some embodiments, the VL_B2The region comprises one, two, or all three of (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, or (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL_B2The region comprises an amino group having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO:49, at least 97% sequence identity to SEQ ID NO:49, at least 98% sequence identity to SEQ ID NO:49, at least 99% sequence identity to SEQ ID NO:49, or 100% sequence identity to SEQ ID NO: 49). In some embodiments, the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49.

In some embodiments, the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 44 (e.g., at least 96% sequence identity to SEQ ID No. 44, at least 97% sequence identity to SEQ ID No. 44, at least 98% sequence identity to SEQ ID No. 44, at least 99% sequence identity to SEQ ID No. 44, or 100% sequence identity to SEQ ID No. 44); and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 49 (e.g., at least 96% sequence identity to SEQ ID NO. 49, at least 97% sequence identity to SEQ ID NO. 49, at least 98% sequence identity to SEQ ID NO. 49, at least 99% sequence identity to SEQ ID NO. 49, or 100% sequence identity to SEQ ID NO. 49). In some embodiments, the VH_B2The region comprises the amino acid sequence of SEQ ID NO. 44; and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-HER 2 bispecific antigen binding molecule (e.g., 4d5n30s.y55e.h91a.n54e.d98t.y102v-1Fab-IgG TDB).

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; and the VL_B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH _B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 30 or a sequence of SEQ ID NO. 30 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 31 or a sequence of SEQ ID No. 31.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 35 or a sequence of SEQ ID NO. 35 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 25 or the sequence of SEQ ID No. 25.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 37; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR comprising the amino acid sequence of SEQ ID NO:23HVR-L3 of sequence. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO:39 or a sequence of SEQ ID NO:39 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 40 or the sequence of SEQ ID No. 40.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL _B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 41 or a sequence of SEQ ID NO. 41 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 42 or a sequence of SEQ ID No. 42.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1And/or the VH _B2Comprising at least 95% sequence identity (e.g., at least 96%, at least 97%, at least) to SEQ ID NO. 4198%, at least 99% sequence identity) or the sequence of SEQ ID NO:41 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 27 or a sequence of SEQ ID No. 27.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and the VL_B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 44 or a sequence of SEQ ID NO. 44 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 45 or a sequence of SEQ ID No. 45.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 20; and the VL_B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 24 or a sequence of SEQ ID NO. 24 and the VL_B1And/or the VL_B2Comprising at least 95% sequence identity to SEQ ID NO 40(e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) or the sequence of SEQ ID NO: 40.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; and the VL _B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 41 or a sequence of SEQ ID NO. 41 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 25 or the sequence of SEQ ID No. 25.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO. 34; and the VL_B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH _B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 46 or a sequence of SEQ ID NO. 46 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 40 or the sequence of SEQ ID No. 40.

In some embodimentsThe VH of_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL_B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 47 or the sequence of SEQ ID NO. 47 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 40 or the sequence of SEQ ID No. 40.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 30 or a sequence of SEQ ID NO. 30 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 25 or the sequence of SEQ ID No. 25.

In some embodiments, the VH_B1And/or the VH_B2Comprising the HVRs of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) HVR comprising SHVR-H3 of the amino acid sequence of EQ ID NO: 20; and the VL _B1And/or the VL_B2HVR-L1 comprising the amino acid sequence of SEQ ID NO:53, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 24 or a sequence of SEQ ID NO. 24 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 54 or the sequence of SEQ ID No. 54.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VH_B1And/or the VH _B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 24 or a sequence of SEQ ID NO. 24 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 48 or the sequence of SEQ ID No. 48.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 21, (e) HVR-L1 comprising the amino acid sequence of SEQ ID NO 22, and (f) HVR-L3 comprising the amino acid sequence of SEQ id no: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 33 or the sequence of SEQ ID NO. 33 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 25 or the sequence of SEQ ID No. 25.

In some embodiments, the VH_B1And/or the VH_B2An HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and the VL_B1And/or the VL_B2Comprising the HVRs of (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH_B1And/or the VH_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID NO. 33 or the sequence of SEQ ID NO. 33 and the VL_B1And/or the VL_B2Comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% sequence identity) to SEQ ID No. 31 or a sequence of SEQ ID No. 31.

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR comprising the amino acid sequence of SEQ ID NO:6 -L3; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:12, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:13, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:14, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 17, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 18; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 17, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 18. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 17, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 18; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 17, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 18.

In another aspect, the invention provides a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab _B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:20, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 24, and the VL _B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 27. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 24And the VL is_B1The region comprises the amino acid sequence of SEQ ID NO 27; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 24, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 27.

In yet another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab _B1And the Fab_B2Each comprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:19, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:32, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 33, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 25. For example, in some embodiments, (a) the VH _AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 25; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 33, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 25.

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:37, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab _AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises at least 95% sequence with SEQ ID NO 8An identical amino acid sequence; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO. 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48.

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab _AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2Each comprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 43, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 21, (v) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 29(vii) HVR-L2, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab_AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL _AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 48. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO 48; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 48. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., an anti-CD 3/HER21Fab-IgG TDB, e.g., an anti-CD 3/HER21Fab-IgGTDB with one 40G5 CD3 binding domain and two 4D5 y55e.h91a.n54e.d98t.y102v HER2 binding domains).

In yet another aspect, the invention provides a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AComprising the HVRs of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:37, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab _AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Region of which the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 41, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigenThe binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., anti-CD 3/HER21Fab-IgG TDB, e.g., anti-CD 3/HER21Fab-IgGTDB with one 40G5cCD3 binding domain and two 4D5 n30s.y55e.h91a.n54e.d98t HER2 binding domains).

In another aspect, the invention features a bispecific antigen binding molecule comprising a monovalent arm and a bivalent arm, wherein (a) the monovalent arm comprises a Fab_AWherein the Fab_AIs fused to the N-terminus of the first Fc subunit, and wherein the Fab_AHVRs comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the bivalent arm comprises a Fab_B1And Fab_B2Wherein the Fab_B2C-terminal of (4) and the Fab_B1Is fused to the N-terminus of (A), and the Fab_B1Is fused to the N-terminus of the second Fc subunit, wherein the Fab_B1And the Fab_B2HVRs each comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:43, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and (c) the first Fc subunit associates with the second Fc subunit to form an Fc domain. In some embodiments, (a) the Fab _AComprising VH_ARegion and VL_ARegion of which the VH_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, and the VL_AThe region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 8; (b) the Fab_B1Comprising VH_B1Region and VL_B1Region of which the VH_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B1The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49; and (c) the Fab_B2Comprising VH_B2Region and VL_B2Zone wherein the VH_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 44, and the VL_B2The region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 49. For example, in some embodiments, (a) the VH_AThe region comprises the amino acid sequence of SEQ ID NO 7, and the VL_AThe region comprises the amino acid sequence of SEQ ID NO 8; (b) the VH_B1The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B1The region comprises the amino acid sequence of SEQ ID NO. 49; and (c) the VH_B2The region comprises the amino acid sequence of SEQ ID NO 44, and the VL_B2The region comprises the amino acid sequence of SEQ ID NO 49. For example, in some embodiments, the bispecific antigen binding molecule is an anti-CD 3/HER2 bispecific antigen binding molecule (e.g., an anti-CD 3/HER21Fab-IgG TDB, e.g., an anti-CD 3/HER21Fab-IgG TDB having one 40G5 CD3 binding domain and two 4D5 n30s.y55e.h91a.n54e.d98t.y102v HER2 binding domains).

Peptide linker fusing second and third antigen binding moieties

In some embodiments, the bispecific antigen binding molecule of the invention is characterized by a structure wherein the C-terminus of the third antigen binding moiety is fused to the N-terminus of the second antigen binding moiety via a peptide linker. The peptide linker may be 5-20 amino acids in length (e.g., 5-10,10-15, or 15-20, e.g., 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, or 20 amino acids in length). In some embodiments, the peptide linker is the native amino acid sequence of the variable heavy chain hinge region (e.g., DKKHT; SEQ ID NO: 50). Alternatively, in some embodiments, the peptide linker comprises G₄SG₂Linker (SEQ ID NO: 51). In some embodiments, the peptide linker comprises G₄SG₂A linker and the hinge region (e.g., SEQ ID NO: 52).

With and without G₄SG₂The amino acid sequences of exemplary heavy chain polypeptides of the linker comprising the second and third antigen-binding module heavy chains are provided in table 2.

TABLE 2 AND constitution with and without G₄SG₂Polypeptide of the Fc domain of an exemplary 1Fab-IgG TDB molecule of a linker, a second antigen binding moiety heavy chain region, and a third antigen binding moiety heavy chain regionCorresponding SEQ ID NO. The sequences shown below include a hinge sequence as part of the linker peptide that fuses the second and third antigen binding module heavy chains.

In particular, the bispecific antigen binding molecule of the invention may be characterized as a peptide linker comprising the amino acid sequence of SEQ ID NO. 50. In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 55 (e.g., at least 96% sequence identity to SEQ ID No. 55, at least 97% sequence identity to SEQ ID No. 55, at least 98% sequence identity to SEQ ID No. 55, at least 99% sequence identity to SEQ ID No. 55, or 100% sequence identity to SEQ ID No. 55). In other instances, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 59 (e.g., at least 96% sequence identity to SEQ ID No. 59, at least 97% sequence identity to SEQ ID No. 59, at least 98% sequence identity to SEQ ID No. 59, at least 99% sequence identity to SEQ ID No. 59, or 100% sequence identity to SEQ ID No. 59). Alternatively, the antigen binding molecule may comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO:63 (e.g., at least 96% sequence identity to SEQ ID NO:63, at least 97% sequence identity to SEQ ID NO:63, at least 98% sequence identity to SEQ ID NO:63, at least 99% sequence identity to SEQ ID NO:63, or 100% sequence identity to SEQ ID NO: 63). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:83 (e.g., at least 96% sequence identity to SEQ ID NO:83, at least 97% sequence identity to SEQ ID NO:83, at least 98% sequence identity to SEQ ID NO:83, at least 99% sequence identity to SEQ ID NO:83, or 100% sequence identity to SEQ ID NO: 83). In other instances, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 85 (e.g., at least 96% sequence identity to SEQ ID No. 85, at least 97% sequence identity to SEQ ID No. 85, at least 98% sequence identity to SEQ ID No. 85, at least 99% sequence identity to SEQ ID No. 85, or 100% sequence identity to SEQ ID No. 85). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 87 (e.g., at least 96% sequence identity to SEQ ID No. 87, at least 97% sequence identity to SEQ ID No. 87, at least 98% sequence identity to SEQ ID No. 87, at least 99% sequence identity to SEQ ID No. 87, or 100% sequence identity to SEQ ID No. 87). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 89 (e.g., at least 96% sequence identity to SEQ ID No. 89, at least 97% sequence identity to SEQ ID No. 89, at least 98% sequence identity to SEQ ID No. 89, at least 99% sequence identity to SEQ ID No. 89, or 100% sequence identity to SEQ ID No. 89).

In other cases, the peptide linker comprises the amino acid sequence of SEQ ID NO 51. The antigen binding molecule can comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO:56 (e.g., at least 96% sequence identity to SEQ ID NO:56, at least 97% sequence identity to SEQ ID NO:56, at least 98% sequence identity to SEQ ID NO:56, at least 99% sequence identity to SEQ ID NO:56, or 100% sequence identity to SEQ ID NO: 56). In some embodiments, the antigen binding molecule can comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO:60 (e.g., at least 96% sequence identity to SEQ ID NO:60, at least 97% sequence identity to SEQ ID NO:60, at least 98% sequence identity to SEQ ID NO:60, at least 99% sequence identity to SEQ ID NO:60, or 100% sequence identity to SEQ ID NO: 60). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 64 (e.g., at least 96% sequence identity to SEQ ID No. 64, at least 97% sequence identity to SEQ ID No. 64, at least 98% sequence identity to SEQ ID No. 64, at least 99% sequence identity to SEQ ID No. 64, or 100% sequence identity to SEQ ID No. 64). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 84 (e.g., at least 96% sequence identity to SEQ ID No. 84, at least 97% sequence identity to SEQ ID No. 84, at least 98% sequence identity to SEQ ID No. 84, at least 99% sequence identity to SEQ ID No. 84, or 100% sequence identity to SEQ ID No. 84). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 86 (e.g., at least 96% sequence identity to SEQ ID No. 86, at least 97% sequence identity to SEQ ID No. 86, at least 98% sequence identity to SEQ ID No. 86, at least 99% sequence identity to SEQ ID No. 86, or 100% sequence identity to SEQ ID No. 86). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 88 (e.g., at least 96% sequence identity to SEQ ID No. 88, at least 97% sequence identity to SEQ ID No. 88, at least 98% sequence identity to SEQ ID No. 88, at least 99% sequence identity to SEQ ID No. 88, or 100% sequence identity to SEQ ID No. 88). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90 (e.g., at least 96% sequence identity to SEQ ID NO:90, at least 97% sequence identity to SEQ ID NO:90, at least 98% sequence identity to SEQ ID NO:90, at least 99% sequence identity to SEQ ID NO:90, or 100% sequence identity to SEQ ID NO: 90).

Fc domain

The bispecific antigen binding molecules of the invention may be characterized as Fc domains. The Fc domain can be an IgG Fc domain (e.g., IgG)₁Or IgG₄Fc domain). For example, the Fc domain may be a human Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. For example, in some embodiments, one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function are at one or more positions selected from the group of L234, L235, and P329 (e.g., wherein the first position isThe Fc subunit and the second Fc subunit each comprise the amino acid substitutions L234A, L235A, and P329G). The Fc receptor may be, for example, an Fc γ receptor. As such, the bispecific antigen binding molecules of the invention can be configured to reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

In some cases, the Fc domain comprises a modification configured to facilitate association of the first Fc subunit with the second Fc subunit. The "node-in-pocket" engineering of bispecific antibodies can be utilized to generate a first arm containing a node, which can bind in a pocket of a second arm, and a second arm containing a pocket. In one embodiment, the segments of the multispecific antibodies of the present invention may be monovalent arms (e.g., anti-CD 3 arms). Alternatively, the segments of the multispecific antibodies of the present invention may be bivalent arms (e.g., anti-tumor arms). In one embodiment, the pocket of a multispecific antibody of the invention may be a monovalent arm (e.g., an anti-CD 3 arm). Alternatively, the cavity of a multispecific antibody of the invention may be a bivalent arm (e.g. an anti-tumor arm). Bispecific antibodies can also be engineered using immunoglobulin swapping (also known as Fab domain swapping or CrossMab format) techniques (see, e.g., WO 2009/080253; Schaeferet al, proc.natl.acad.sci.usa,108:11187-11192 (2011)). Bispecific antibodies can also be generated by engineering electrostatically manipulated effects to generate antibody Fc heterodimer molecules (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); or by using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)).

Amino acid residues in the CH3 domain of the second Fc subunit can be replaced with amino acid residues having a larger side chain volume, thereby generating protuberances (e.g., knobs) within the CH3 domain of the second Fc subunit that can be placed in cavities (e.g., holes) within the CH3 domain of the first Fc subunit, and amino acid residues in the CH3 domain of the first Fc subunit can be replaced with amino acid residues having a smaller side chain volume, thereby generating cavities (e.g., holes) within the CH3 domain of the first Fc subunit within which protuberances (e.g., knobs) within the CH3 domain of the second Fc subunit can be placed. In some embodiments, the CH3 domain of the second Fc subunit comprises an amino acid substitution at T366, and the CH3 domain of the first Fc subunit comprises an amino acid substitution at one, two, or all three of T366, L368, and/or Y407. In some embodiments, the CH3 domain of the second Fc subunit comprises the amino acid substitution T366W, and the CH3 domain of the first Fc subunit comprises one, two, or all three amino acid substitutions T366S, L368A, and/or Y407V.

It is expressly contemplated that such bispecific antigen binding molecules or other antibodies described herein for use in any of the instances recited herein can have any of the features described in sections 1-6 below, singly or in combination.

1. Affinity of antibody

In some cases, a bispecific antigen-binding molecule has an equilibrium dissociation constant (K.sub.m) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM_D) (e.g., 10)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one case, K_DIs measured by a radiolabeled antigen binding assay (RIA). In one instance, RIA is performed with Fab versions of the antibody of interest and its antigen. For example, by using the minimum concentration of (in the presence of a titration series of unlabeled antigen)¹²⁵I) The Fab is equilibrated with labeled antigen and then the solution binding affinity of the Fab for the antigen is measured by capturing the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865-881 (1999)). To establish the assay conditions, the

Multi-well plates (Thermo Scientific) were coated with 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) in 50mM sodium carbonate (pH 9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Antigen mixing with serial dilutions of Fab of interest (e.g. consistent with evaluation of anti-VEGF antibodies, Fab-12, anti-VEGF antibodies, Presta et al, Cancer Res.57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation may continue for longer periods of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, will The mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and treated with 0.1% polysorbate 20 in PBS

The plate was washed 8 times. After drying the plates, 150. mu.l/well scintillation fluid (MICROSCINT-20) was added^TM(ii) a Packard), then in TOPCOUNT^TMPlates were counted on a gamma counter (Packard) for 10 minutes. The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays.

According to another aspect, K_DIs to use

Surface plasmon resonance assay. For example, the immobilized antigen CM5 chip was used at about 10 Response Units (RU) at 25 deg.C

Or(Inc., Piscataway, NJ) to perform the assay. In one case, carboxymethylated dextran biosensor chips (CM5,inc.). The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, polysorbate 20 (TWEEN-20) was injected at 25 ℃ at a flow rate of about 25. mu.l/min at 0.05% ^TM) Two-fold serial dilutions of Fab (0.78nM to 500nM) in surfactant PBS (PBST). Simple one-to-one use(Langmuir) binding model: (Evaluation Software version 3.2) calculation of the Association Rate (k) by Simultaneous fitting of Association and dissociation sensorgrams_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) At a ratio of k_off/k_onAnd (4) calculating. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1s^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO^TMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation 295 nM; emission 340nM,16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ in the presence of increasing concentrations of antigen.

2. Antibody fragments

In certain instances, a bispecific antigen binding molecule provided herein is a bispecific antibody comprising one or more antibody fragments. Antibody fragments include, but are not limited to, Fab ', Fab ' -SH, F (ab ')₂for reviews of certain antibody fragments, see Hudson et al, Nat. Med.9:129-134(2003) for reviews of scFv fragments, see, e.g., Pluckthon, compiled by The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, (Springer-Verlag, New York), pp.269-315 (1994), see also WO 93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458, for Fab and F (ab') ₂See U.S. Pat. No.5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, nat. Med.9:129-134 (2003); and Hollinger et al, Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993). Tri-and tetrabodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain instances, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516).

Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain instances, the bispecific antigen binding molecules provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain instances, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some cases, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329 (1988); queen et al, Proc.Natl Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods36: 25-34(2005) (specificity determining region (SDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (described as "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36: 61-68(2005) and Klimka et al, Br.J. cancer,83:252-260(2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al, J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies of the light or heavy chain variable regions (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al, J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

4. Human antibodies

In certain instances, the bispecific antigen binding molecules provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk and van de Winkel, curr. opin. pharmacol.5:368-74(2001) and Lonberg, curr. opin. immunol.20:450-459 (2008).

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application publication No. US 2007/0061900, which describes

A technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, proc.natl.acad.sci.usa,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlens, Histology and Histopathology,20(3):927-937(2005) and Vollmers and Brandlens, Methods and dressings in Experimental and clinical pharmacy, 27(3):185-91 (2005).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

Bispecific antigen binding molecules of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (ed. O' Brien et al, Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature 348: 552-554; clackson et al, Nature 352:624-628 (1991); marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology 248:161-175(Lo eds., Human Press, Totowa, NJ, 2003); sidhu et al, j.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5):1073-1093 (2004); fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472 (2004); and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In some phage display methods, a repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann.Rev.Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g. from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens without any immunization, as described by Griffiths et al, EMBO J,12:725-734 (1993). Finally, non-rearranged V gene segments can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No.5,750,373, and U.S. Pat. Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

6. Antigen binding molecule variants

In certain instances, amino acid sequence variants of the bispecific antigen binding molecules of the invention are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

In certain embodiments, the bispecific antigen binding molecules of the invention comprise one or more modifications in the VH/VL region and/or the CH1/CL region to drive the correct heavy/light chain pairing. In some embodiments, the bispecific antigen binding molecules of the invention comprise one or more modifications in the Fc region to drive heterodimerization of the two arms of the bispecific antigen binding molecule. Such modifications in the VH/VL region, CH1/CL region, and/or FC region are described in International patent publication No. WO 2016/172485, which is incorporated herein by reference in its entirety.

A. Substitution, insertion, and deletion variants

In some cases, antigen binding module variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 3 under the heading of "preferred substitutions". More substantial variations are provided in table 3 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

TABLE 3 exemplary and preferred amino acid substitutions

Initial residue	Exemplary substitutions	Preferred alternatives
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp,Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic Cys, Ser, Thr, Asn, Gln;

(3) acidic Asp, Glu;

(4) basic His, Lys, Arg;

(5) Residues affecting chain orientation Gly, Pro; or

(6) Aromatic, Trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity and/or decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes may be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207:179-196(2008)), and/or antigen-contacting residues, where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ, (2001)). In some cases of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain instances, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. For example, such changes may be outside of antigen-contacting residues in HVRs. In certain instances of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1,2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

B. Glycosylation variants

In certain instances, the antigen binding molecules of the invention can be altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites of the antibodies of the invention can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case where the antigen binding molecule comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some cases, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one instance, antigen binding molecule variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g., complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. publication Nos. US 2003/0157108 and US 2004/0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al, Arch. biochem. Biophys.249:533-545 (1986); U.S. patent application No. US 2003/0157108A 1; and WO2004/056312A1, Adams et al, particularly in example 11), and knock-out cell lines, such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech. Bioeng.87:614 (2004); Kanda, Y.et al, Biotechnol. Bioeng.,94(4):680-688 (2006); and WO 2003/085107).

Further provided are antigen binding molecule variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Pat. Nos. 6,602,684; and US 2005/0123546. Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Fc region variants

In certain instances, one or more amino acid modifications can be introduced into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain instances, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu.Rev.Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.et al, Proc. Natl Acad. Sci.USA 83:7059-7063(1986)) and Hellstrom, I.et al, Proc. Natl Acad. Sci.USA82:1499-1502 (1985); U.S. Pat. No.5,821,337 (see Bruggemann, M.et., J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry) ^TMNon-radioactive cytotoxicity assay (CellTechnology, inctail View, CA; and CYTOTOXNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in animal models such as disclosed in Clynes et al, proc.natl acad.sci.usa 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO2005/100402 for C1q and C3C binding ELISAs. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg et al, Blood 101:1045-1052 (2003); and Cragg et al, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays may also be performed using methods known in the art (see, e.g., Petkova et al, Int' l. immunol.18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those having substitutions of one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. nos. 7,332,581 and 8,219,149).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and shelds et al, j.biol. chem.9(2):6591-6604 (2001)).

In certain instances, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298,333, and/or 334 (EU numbering of residues) of the Fc region.

In some cases, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164:4178-4184 (2000).

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al) (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

Also seen in Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

D. Cysteine engineered antibody variants

In certain instances, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular instances, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In some cases, cysteine may be substituted for any one or more of V205(Kabat numbering) of the light chain; a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

E. Antibody derivatives

In certain instances, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under specified conditions, and the like.

In another aspect, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one case, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

F. Immunoconjugates

The invention also provides immunoconjugates comprising a bispecific antigen binding molecule conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope.

In one instance, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020,5,416,064 and european patent EP 0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342 (1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, CurrentMed. chem.13:477-523 (2006); Jeffrey et al, Bioorganic & Med. chem. Letters16:358-362 (2006); Torgov et al, bioconj. chem.16:717-721 (2005); Nagy et al, Proc. Natl.Acad.Sci.USA 97:829-834 (2000); Dubowchik et al, Bioorg. Med.chem. Letters 12:1529-1532 (2002); King et al, J.Med. chem.chem.45: 4336-4343 (2002); and U.S. Pat. No.6,630,579); methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another instance, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, the enzymatically active toxins include, but are not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, α -fumagillin (sarcin), aleurites fordii (aleurites fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, phytolacca americana (phytolacca americana) protein (PAPI, PAPII and PAP-S), Momordica charantia (momordia) inhibitor, curculin (curcin), crotin (crotin), saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitomycin (strictin), tricin (strictoxin), trichothecin (phytotoxin), trichothecin (triomycin), and enomycin (enomycin).

In another instance, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹,I¹³¹,I¹²⁵,Y⁹⁰,Re¹⁸⁶,Re¹⁸⁸,Sm¹⁵³,Bi²¹²,P³²,Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res 52:127-131 (1992); U.S. Pat. No.5,208,020).

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

Recombinant methods and compositions

Recombinant methods and compositions can be used to produce bispecific antigen binding molecules of the invention, e.g., as described in U.S. Pat. No.4,816,567 and U.S. publication No.2013/0078249, which are incorporated herein by reference in their entirety. In one embodiment, isolated nucleic acids (e.g., polynucleotides) encoding the bispecific antigen binding molecules described herein are provided. Such nucleic acids can encode an amino acid sequence comprising a VL of a bispecific antigen binding molecule and/or an amino acid sequence comprising a VH of a bispecific antigen binding molecule (e.g., the light and/or heavy chains of either arm of a bispecific antigen binding molecule). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided.

The polynucleotides encoding the bispecific antigen binding molecules of the invention may be expressed as a single polynucleotide molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate via, for example, disulfide bonds or other means to form a functional bispecific antigen binding molecule. For example, the light chain portion of the antigen binding module may be encoded by a separate polynucleotide from the portion of the bispecific antigen binding molecule comprising the antigen binding module heavy chain portion, the Fc domain subunit, and optionally the other antigen binding module. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antigen binding module. In another example, the portion of the bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally one or more antigen binding moieties may be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the other of the two Fc domain subunits and optionally the antigen binding moiety. When co-expressed, the Fc domain subunits associate to form an Fc domain.

In certain embodiments, the isolated polynucleotides of the invention encode a fragment of a bispecific antigen binding molecule comprising a first and a second antigen binding moiety and an Fc domain consisting of two subunits. In one embodiment, the isolated polynucleotide of the invention encodes a subunit of the first antigen binding moiety and the Fc domain. In another embodiment, the isolated polynucleotide of the invention encodes a subunit of the heavy chain and Fc domain of the second antigen binding moiety. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab heavy chain shares a C-terminal peptide bond with the Fc domain subunit. In yet another embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the third antigen binding moiety, the heavy chain of the second antigen binding moiety, and a subunit of the Fc domain. In some embodiments, the light chains of the second and third antigen binding modules are co-expressed with the heavy chain region and associate to form a Fab domain.

In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising at least one VL of a bispecific antigen-binding molecule and an amino acid sequence comprising at least one VH of a bispecific antigen-binding molecule of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of a bispecific antigen-binding molecule and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of a bispecific antigen-binding molecule. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell. In one embodiment, a method of producing a bispecific antigen binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a bispecific antigen binding molecule as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture broth).

For recombinant production of bispecific antigen binding molecules, polynucleotides encoding bispecific antigen binding molecules (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the bispecific antigen binding molecule).

Suitable host cells for cloning or expressing the bispecific antigen binding molecule encoding vector include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ,2003), pp.245-254, which describe expression of antibody fragments in E.coli). After expression, the antibody can be isolated from the bacterial cell mass paste in a soluble fraction and can be further purified.

Pharmaceutical formulations

Therapeutic formulations of bispecific antigen binding molecules of the present invention are prepared for storage by mixing the bispecific antigen binding molecule of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. General information on formulations is found, for example, in Gilman et al (eds.) the pharmacological Bases of Therapeutics,8th Ed., Pergamon Press, 1990; gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Co., Pennsylvania, 1990; avis et al (eds.) Pharmaceutical Dosage Forms, Parmental medical Dekker, New York, 1993; lieberman et al (eds.) Pharmaceutical DosageForms, Tablets Dekker, New York, 1990; lieberman et al, (eds.), pharmaceutical dosage Forms: Disperse Systems Dekker, New York, 1990; and Walters (ed.) Dermatological and Transdermal Formulations (Drugs and the pharmaceutical sciences), Vol 119, Marcel Dekker, 2002.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl chloride)Benzalkonium chloride; (ii) hexanediamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN^TM,PLURONICS^TMOr polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs will depend, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma-ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(from lactic acid)-injectable microspheres of glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Methods of treatment

Any bispecific antigen binding molecule of the invention (e.g., a bispecific antigen binding molecule having a monovalent arm capable of specifically binding a first antigen and a divalent arm capable of specifically binding two second antigens) or a composition thereof can be used in any of the methods of treatment described herein.

In one aspect, bispecific antigen binding molecules are provided for use as a medicament. For example, in some embodiments, the bispecific antigen binding molecules described herein are for use in treating or delaying the progression of a cell proliferative disorder (e.g., cancer) or an autoimmune disorder in a subject in need thereof. In some embodiments, the bispecific antigen binding molecule of any preceding aspect is for use in enhancing immune function in a subject having a cell proliferative disorder (e.g., cancer) or an autoimmune disorder. In certain embodiments, the invention provides bispecific antigen binding molecules for use in a method of enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder, comprising administering to the individual an effective amount of the bispecific antigen binding molecule to activate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand (increase) effector cell populations, reduce target cell populations (e.g., cell populations expressing a second target cell antigen recognized by the bispecific antigen binding molecules of the invention), and/or kill target cells (e.g., target tumor cells).

The invention features the use of a bispecific antigen binding molecule of the invention in the manufacture of a medicament for treating or delaying the progression of a disorder, such as a cell proliferative disorder (e.g., cancer) or an autoimmune disorder. In yet another embodiment, the medicament is for use in a method of treating a cell proliferative disorder or an autoimmune disorder, comprising administering to an individual having a cell proliferative disorder or an autoimmune disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, e.g., as described below. In yet another embodiment, the medicament is for activating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expanding (increasing) effector cell populations, decreasing target cell populations (e.g., cell populations expressing a second target cell antigen recognized by a bispecific antigen binding molecule of the invention), and/or killing target cells (e.g., target tumor cells).

In yet another aspect, the invention provides a method of treating or delaying the progression of a disorder in a subject in need thereof, the method comprising administering to the subject a bispecific antigen binding molecule of any preceding aspect. In one embodiment, the method comprises administering to a subject having such a cell proliferative disorder or autoimmune disorder an effective amount of a bispecific antigen binding molecule (e.g., a bispecific antigen binding molecule having a monovalent arm against CD3 and a divalent arm against a tumor cell antigen). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In another aspect, the invention provides a method of enhancing immune function in a subject having a disorder, the method comprising administering to the subject a bispecific antigen binding molecule of any preceding aspect (e.g., a bispecific antigen binding molecule having a monovalent arm against CD3 and a divalent arm against a tumor cell antigen). In some embodiments, the disorder is a cell proliferative disorder (e.g., cancer) or an autoimmune disorder. In certain embodiments, the method comprises administering an effective amount of the bispecific antigen binding molecule to activate effector cells (e.g., T cells, such as CD8+ and/or CD4+ T cells), expand (increase) effector cell populations, decrease target cell populations (e.g., cell populations expressing a second target cell antigen recognized by the bispecific antigen binding molecule of the invention), and/or kill target cells (e.g., target tumor cells).

In some embodiments, the bispecific antigen binding molecules, compositions thereof, and methods of use provided herein are used to treat cancer, such as breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, non-hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Acute Myeloid Leukemia (AML), Chronic Lymphoid Leukemia (CLL), Marginal Zone Lymphoma (MZL), Small Lymphocytic Leukemia (SLL), Lymphoplasmacytic Lymphoma (LL), Waldenstrom's Macroglobulinemia (WM), central Nervous System Lymphoma (CNSL), Burkitt's Lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma/leukemia, splenic diffuse red myeloid small B-cell lymphoma, hairy cell leukemia variants, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicular central lymphoma, large B-cell lymphoma enriched in T-cells/tissue cells, primary DLBCL of the central nervous system, primary DLBCL (legged), Epstein-Barr virus (EBV) positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, Anaplastic Lymphoma Kinase (ALK) positive large B-cell lymphoma, plasmacytic lymphoma, large B-cell lymphoma caused in HHV 8-associated multicenter Castleman (Castleman) disease, primary effusion lymphoma, non-classifiable large B-cell lymphoma having an intermediate characteristic between diffuse large B-cell lymphoma and burkitt lymphoma, or non-classifiable large B-cell lymphoma having an intermediate characteristic between diffuse large B-cell lymphoma and classical hodgkin lymphoma. In some embodiments, the bispecific antigen binding molecules provided herein, compositions thereof, and methods of use are for treating HER2 positive cancer (e.g., HER2 positive breast cancer or HER2 positive gastric cancer).

The invention further provides combination therapies, including parallel or as a combined regimenAnd one or more additional therapeutic agents (e.g., one, two, three, four, five, or more additional therapeutic agents). In one embodiment, the bispecific antigen binding molecule is co-administered with one or more biological modifiers selected from a BCL-2 inhibitor (e.g., GDC-0199/ABT-199), lenalidomide

PI3K inhibitors (such as idelalisib)) Agonists against activating co-stimulatory molecules such as CD40, CD226, CD28, OX40 (e.g., Agonox), GITR, CD137 (also known as TNFRSF9,4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127, such as agonistic antibodies, antagonists against inhibitory co-stimulatory molecules such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIIT, MICA/B, GITR (e.g., TRX518), or arginase, such as antagonistic antibodies, ipilimumab (also known as MDX-010, MDX-101, or ILX-101) tremellimumab (also known as ticilimumab or CP-675,206, urelimumab (also known as BMS-663513), MGA271, antagonists against TGF- β, such as metelilimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and T cells expressing Chimeric Antigen Receptors (CARs), such as cytotoxic T cells, for example adoptive transfer of T cells comprising a dominant negative TGF- β receptor, such as a dominant negative TGF- β type II receptor.

In a particular embodiment, the biological modifier is a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist or additional therapeutic agent is administered before or after administration of the bispecific antigen binding molecule. In some embodiments, the PD-1 axis binding antagonist or additional therapeutic agent is administered concurrently with the bispecific antigen binding molecule. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist (e.g., atelizumab (MPDL3280A), yw243.55.s70, MDX-1105, MEDI4736 (kovacizumab), and MSB0010718C (avizumab)), a PD-1 binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidumab), MEDI-0680(AMP-514), PDR001 (sibatuzumab), refgn 0 (cimiralizumab), and BGB-108), and a PD-L2 binding antagonist (e.g., an antibody or immunoadhesin).

Additionally or alternatively, a bispecific antigen binding molecule of the invention can be co-administered with one or more chemotherapeutic agents (e.g., as part of a method of treating a subject having a cell proliferative disorder, such as cancer).

In yet another aspect, the invention provides a method for treating HER2 positive cancer. In one embodiment, the method comprises administering to an individual having a HER2 positive cancer an effective amount of a bispecific antigen binding molecule of the invention, such as a 1Fab-IgG TDB molecule having a bivalent anti-HER 2 arm and a monovalent anti-CD 3 arm. In a particular embodiment, the anti-HER 2 arm possesses an acceptable toxicity profile when administered in an effective amount in a patient. In one embodiment, the anti-CD 3 arm of a 1Fab-IgG TDB molecule with an acceptable toxicity profile is a low affinity anti-CD 3 antigen binding moiety. In one embodiment, the anti-CD 3 antigen-binding moiety of the 1Fab-IgG TDB with an acceptable toxicity profile is 40G5 c.

In a particular embodiment, the HER2 positive cancer is HER2 positive breast cancer or HER2 positive gastric cancer. A HER2 positive cancer (e.g., HER2 positive breast cancer or HER2 positive gastric cancer) may be characterized by at least 200,000 copies per cell (e.g., at least 250,000 copies per cell of HER2, at least 300,000 copies per cell of HER2, at least 400,000 copies per cell of HER2, at least 500,000 copies per cell of HER2, at least 600,000 copies per cell of HER2, at least 700,000 copies per cell of HER2, at least 750,000 copies per cell of HER2, at least 800,000 copies per cell of HER2, at least 900,000 copies per cell of HER2, at least 1,000,000 copies per cell of HER2, at least 1,200,000 copies per cell of HER2, at least 1,500,000 copies per cell of HER2, at least 2,000,000,000 copies per cell of HER2, at least 2,500,000,000 copies per cell of HER2, at least 3,000,000,000 copies per cell of HER2 copies per cell, at least 200,4645, at least 200,000 copies per cell of HER2, or 500,000 to 1,000,000 HER2 copies per cell, such as 200,000 to 1,000,000 HER2 copies per cell (e.g., 200,000 to 250,000 HER2 copies per cell, 250,000 to 300,000 HER2 copies per cell, 300,000 to 400,000 HER2 copies per cell, 400,000 to 500,000 HER2 copies per cell, 500,000 to 750,000 HER2 copies per cell, or 750,000 to 1,000,000 HER2 copies per cell) or 1,000,000 to 3,000,000 HER2 copies per cell (e.g., 1,000,000 to 1,500,000 HER2 copies per cell, 1,500,000 to 2,000,000,000 HER2 copies per cell, 2,000,000 to 2,500,000 HER2 copies per cell, or 2,500,000 to 3,000,000 HER2 copies per cell (e.g., average number of HER 36 2,000 copies per cell).

In one embodiment, the anti-HER 2 bispecific antigen binding molecule is co-administered with one or more additional therapeutic agents targeting the HER pathway. In one embodiment, the HER pathway-targeting therapeutic agent is selected from an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, and/or a HER4 inhibitor. In one embodiment, with a compound selected from trastuzumabT-DM1

And pertuzumabCo-administering HER2 TDB (e.g. 1Fab-IgG TDB) with one or more additional therapeutic agents. In one embodiment, the anti-HER 2/CD3 bispecific antigen binding molecule is co-administered with trastuzumab. In one embodiment, an anti-HER 2/CD3 bispecific antigen binding molecule is co-administered with T-DM 1. In one embodiment, the anti-HER 2/CD3 bispecific antigen binding molecule is co-administered with pertuzumab. In one embodiment, the anti-HER 2/CD3 bispecific is co-administered with trastuzumab and pertuzumabA heterologous antigen binding molecule. In one embodiment, the anti-HER 2/CD3 bispecific antigen binding molecule is co-administered with T-DM1 and pertuzumab.

Such combination therapies noted above encompass both combined administration (where the two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration (where administration of the bispecific antigen binding molecule of the invention may occur prior to, concurrently with, and/or after administration of one or more additional therapeutic agents). In one embodiment, administration of the antibody and administration of the additional therapeutic agent occur within about 1 month, or within about 1,2, or 3 weeks, or within about 1,2,3,4,5, or 6 days of each other.

The bispecific antigen binding molecules of the invention (and/or any additional therapeutic agent) may be administered by any suitable means, including subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. For example, in some embodiments, the bispecific antigen binding molecule is administered subcutaneously. In other embodiments, the bispecific antigen binding molecule is administered intravenously. In some embodiments, a bispecific antigen binding molecule administered by subcutaneous injection exhibits less toxic response in a patient than the same bispecific antigen binding molecule administered by intravenous injection. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The bispecific antigen binding molecules of the present invention can be formulated, dosed, and administered in a manner consistent with excellent medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the condition, the site of agent delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The bispecific antigen binding molecule need not be, but is optionally, formulated with one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and administration routes described herein, or at about 1-99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether prophylactic or therapeutic purposes are employed to administer the bispecific antigen binding molecule, previous therapy, the patient's clinical history and response to the bispecific antigen binding molecule, and the discretion of the attending physician. The bispecific antigen binding molecule is suitably administered to the patient in one or a series of treatments.

As a general proposition, a therapeutically effective amount of a bispecific antigen binding molecule administered to a human will be in the range of about 0.01 to about 100mg/kg of patient body weight, whether by one or more administrations. In some embodiments, the bispecific antigen binding molecule is used in an amount of, for example, about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1mg/kg administered daily. In one embodiment, a bispecific antigen binding molecule described herein is administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, or about 1400mg on day 1 of a 21 day cycle. The dose may be administered as a single dose or as multiple doses (e.g. 2 or 3 doses), such as infusion. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of antibody will be in the range of about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg,2.0mg/kg,4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to a subject. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20 doses, or, for example, about 6 doses, of the antibody). A higher initial loading agent may be administered followed by a lower dose or doses. The progress of this therapy is readily monitored by conventional techniques and assays. In some embodiments of any of the foregoing aspects of the invention, the subject, patient, or individual is a human.

VI. kit

Provided herein are kits comprising one or more compositions described herein (e.g., a composition comprising any one or more bispecific antigen-binding molecules described herein and a pharmaceutically acceptable carrier) and a package insert (e.g., a cell proliferative disorder (e.g., cancer, such as breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, non-hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), acute Myeloid Leukemia (AML), Chronic Lymphoid Leukemia (CLL), Marginal Zone Lymphoma (MZL), Small Lymphocytic Leukemia (SLL), Lymphoplasmacytic Lymphoma (LL), Waldenstrom Macroglobulinemia (WM), Central Nervous System Lymphoma (CNSL), Burkitt's Lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma/leukemia, splenic diffuse erythroid small B-cell lymphoma, hairy cell leukemia variants, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric follicular lymphoma, primary skin follicular central lymphoma, large B cell lymphoma rich in T cells/histiocytes, primary DLBCL of the central nervous system, primary skin DLBCL (legged), Epstein Barr Virus (EBV) positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, Anaplastic Lymphoma Kinase (ALK) -positive large B-cell lymphoma, plasmacytic lymphoma, large B-cell lymphoma caused in HHV 8-associated multicenter Castleman (Castleman) disease, primary effusion lymphoma, non-classifiable large B-cell lymphoma having intermediate characteristics between diffuse large B-cell lymphoma and Burkitt lymphoma, or non-classifiable, large B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and classical hodgkin lymphoma. In some embodiments, the cancer is a HER2 positive cancer (e.g., HER2 positive breast cancer or HER2 positive gastric cancer). Additionally or alternatively, the kit can include a package insert for administering the composition to a subject to treat or delay progression of an autoimmune disorder.

Examples

The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced in view of the general description provided above.