阅读说明：本技术 融合构建体及其使用方法 (Fusion constructs and methods of use thereof ) 是由 海伦·萨布泽瓦里 西蒙·米特诺 陈昶宏 鲁图尔·R·沙阿 于 2019-07-09 设计创作，主要内容包括：本文提供了包含融合蛋白或其片段或变体的组合物,该融合蛋白或其片段或变体包含抗PD1抗体或其片段/变体和TGF-β阱。本文提供了包含融合蛋白或其片段或其变体的组合物,该融合蛋白或其片段或变体包含抗PD1抗体或其片段/变体和ADA2多肽。本文还提供了使用该组合物治疗癌症的方法。(Provided herein are compositions comprising a fusion protein, or fragment or variant thereof, comprising an anti-PD 1 antibody, or fragment/variant thereof, and a TGF- β trap. Provided herein are compositions comprising a fusion protein or fragment or variant thereof comprising an anti-PD 1 antibody or fragment/variant thereof and an ADA2 polypeptide. Also provided herein are methods of using the compositions to treat cancer.)

1. A fusion protein comprising:

a. an antibody or fragment of said antibody or variant of said antibody capable of binding programmed cell death protein-1 (PD-1); and

b. Transforming growth factor beta (TGF- β) cytokine traps; wherein one or more polypeptides of the fusion protein are linked by a linker.

2. The fusion protein of claim 1, wherein the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6.

3. The fusion protein of claim 1, wherein the linker comprises (Gly) n, wherein n is 6, 7, or 8.

4. The fusion protein of claim 1, wherein the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6.

5. The fusion protein of claim 1, wherein the linker comprises A (EAAAK)₄ALEA(EAAAK)₄A。

6. The fusion protein of claim 1, wherein the linker comprises a sequence set forth in any one of SEQ ID NOs 17-34.

7. The fusion protein of any one of claims 1 to 6, wherein the TGF- β cytokine trap comprises a transforming growth factor receptor (TGF β R) or a functional fragment thereof, an anti-TGF- β antibody or an antigen-binding fragment thereof, a TGF- β 1 inhibitory peptide, or a variant thereof.

8. The fusion protein of claim 7, wherein the TGF β R is transforming growth factor β receptor II (TGF β RII) or the functional fragment thereof.

9. The fusion protein according to claim 8, wherein the functional fragment of TGF β RII is a TGF β RII extracellular domain (ECD).

10. The fusion protein of claim 9, wherein the ECD binds TGF- β 1.

11. The fusion protein of claim 9, wherein the ECD binds TGF- β 3.

12. The fusion protein of claim 9, wherein the ECD binds TGF- β 1 and TGF- β 3.

13. The fusion protein of claim 12, wherein the ECD binds TGF- β 1 and TGF- β 3, but not TGF- β 2.

14. The fusion protein of any one of claims 1 to 13, wherein the TGF- β cytokine trap comprises a sequence at least 80% identical to the sequence set forth in SEQ ID No.14, SEQ ID No.141, or SEQ ID No. 142.

15. The fusion protein of any one of claims 1 to 13, wherein the TGF- β cytokine trap comprises the sequence set forth in SEQ ID No.14, SEQ ID No.141, or SEQ ID No. 142.

16. The fusion protein of any one of claims 1 to 15, wherein the TGF- β cytokine trap comprises the sequence set forth in SEQ ID No. 14.

17. The fusion protein of any one of claims 1-16, wherein the antibody (anti-PD 1) is an immunoglobulin g (igg) antibody.

18. The fusion protein of claim 17, wherein the IgG is IgG1, IgG2, IgG3, or IgG 4.

19. The fusion protein of claim 18, wherein the IgG4 comprises a mutation at position 108 of SEQ ID No. 146 or SEQ ID No. 292.

20. The fusion protein of claim 19, wherein the mutation is the S108P mutation.

21. The fusion protein of any one of claims 18 to 20, wherein the IgG4 is linked to the TGF- β cytokine trap by the linker.

22. The fusion protein of any one of claims 1 to 21, wherein the fragment of the antibody is a Fab, (Fab)₂、(Fab’)₂、Fv、(Fv)₂Or a scFv.

23. The fusion protein of any one of claims 1-22, wherein the antibody comprises a heavy chain variable region (V) of the antibody_H) And light chain variable region (V)_L)。

24. The fusion protein of claim 23, wherein the linker links the heavy chain variable region (V)_H) (iii) attaching to the TGF- β cytokine trap.

25. The fusion protein of claim 23, wherein the linker links the light chain variable region (V)_L) (iii) attaching to the TGF- β cytokine trap.

26. The fusion protein of any one of claims 23 to 25, wherein in the fusion protein the heavy chain variable region (V)_H) Is connected to the light chain variable region (V) by a second linker _L)。

27. The fusion protein of claim 26, wherein the second linker comprises a sequence set forth in any one of SEQ ID NOs 17-34.

28. The fusion protein of any one of claims 23 to 27, wherein the heavy chain variable region (V)_H) At least 80% identical to the sequence shown in any one of SEQ ID NOS 1-7 and 149-164.

29. The fusion protein of any one of claims 23 to 28, wherein the light chain variable region (V)_L) At least 80% identical to the sequence set forth in any one of SEQ ID NOS 8-13 and 148.

30. The fusion protein of any one of claims 23 to 29, wherein the heavy chain variable region (V)_H) Comprises the sequence shown in any one of SEQ ID NO 1-7 and 149-164.

31. The fusion protein of any one of claims 23 to 30, wherein the light chain variable region (V)_L) Comprises the sequence shown in any one of SEQ ID NO 8-13 and 148.

32. The fusion protein of any one of claims 23 to 29, wherein the heavy chain variable region (V)_H) (iv) is at least 90% identical to the sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 12.

33. The fusion protein of claim 32, wherein the heavy chain variable region (V) _H) Comprises the sequence shown in SEQ ID NO 6 and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 12.

34. The fusion protein according to any one of claims 1 to 33, wherein the fusion protein comprises the sequence shown as SEQ ID No. 15 and the sequence shown as SEQ ID No. 16.

35. The fusion protein according to any one of claims 1 to 33, wherein the fusion protein comprises the sequence shown as SEQ ID No. 15 and the sequence shown as SEQ ID No. 143.

36. The fusion protein of any one of claims 23 to 29, wherein the heavy chain variable region (VH) is at least 90% identical to the sequence set forth in SEQ ID No. 7 and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID No. 13.

37. The fusion protein of claim 36, wherein the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO. 7, and the light chain variable region (V)_L) Comprises the sequence shown as SEQ ID NO. 13.

38. The fusion protein of any one of claims 1-31 and 36-37, wherein the fusion protein comprises the sequence set forth in SEQ ID No. 296 and the sequence set forth in SEQ ID No. 145.

39. The fusion protein of any one of claims 1-31 and 36-37, wherein the fusion protein comprises the sequence set forth in SEQ ID No. 296 and the sequence set forth in SEQ ID No. 144.

40. The fusion protein of any one of claims 1-39, wherein the antibody further comprises a fragment crystallizable region (F)_C)。

41. The fusion protein of claim 40, wherein said F_CIs a person F_C1、F_C2、F_C3、F_C4 or a fragment thereof.

42. The fusion protein of claim 40, wherein said F_CFurther comprising one or more mutations.

43. The fusion protein of any one of claims 22-42, wherein the antibody comprises the scFv and the F_CAnd (3) fragment.

44. The fusion protein of any one of claims 7 to 43, wherein the TGF-beta cytokine trap comprises the anti-TGF-beta antibody, or the antigen-binding fragment thereof.

45. The fusion protein of claim 44, wherein the anti-TGF β antibody comprises the VH encoded by SEQ ID NO 166, 168, 169, 171, 173, 175, or 177, and the VL encoded by SEQ ID NO 165, 167, 170, 172, 174, 176, or 178.

46. The fusion protein of any one of claims 7 to 43, wherein the TGF β cytokine trap comprises the TGF β inhibitory peptide, or the variant thereof.

47. The fusion protein according to claim 46, wherein the TGF β inhibitory peptide comprises the sequence set forth in any one of SEQ ID No. 193-227.

48. A polynucleotide encoding the fusion protein of any one of claims 1 to 47 and 117 to 120.

49. An expression vector comprising a polynucleotide encoding the fusion protein of any one of claims 1 to 47 and 117 to 120, wherein the polynucleotide is operably linked to a promoter.

50. The expression vector of claim 49, wherein the promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter.

51. The expression vector of claim 50, wherein said inducible promoter is a small molecule ligand inducible gene switch based on two polypeptide ecdysone receptors.

52. The expression vector of any one of claims 49-51, wherein the vector is an adenoviral vector.

53. A pharmaceutical composition comprising:

(a) the fusion protein of any one of claims 1 to 47 and 117 to 120;

(b) a polynucleotide encoding the fusion protein of any one of claims 1 to 47 and 117 to 120; or

(c) The expression vector of any one of claims 49 to 52; and

(d) a pharmaceutically acceptable excipient.

54. A method of treating cancer, comprising: contacting the cells

(a) The fusion protein of any one of claims 1 to 47 and 117 to 120;

(b) A polynucleotide encoding the fusion protein of any one of claims 1 to 47 and 117-120; or

(c) The expression vector of any one of claims 49 to 52.

55. The method of claim 54, wherein the cell is a cancer cell.

56. The method of any one of claims 54-55, wherein the cell is a mammalian cell.

57. A method of treating a subject having cancer, the method comprising: administering a composition comprising a fusion protein comprising

(a) An antibody capable of binding programmed cell death protein-1 (PD-1) or a fragment or variant of said antibody; and

(b) transforming growth factor receptor (TGF-beta R) or a functional fragment thereof, an anti-TGF-beta antibody or antigen-binding fragment thereof, a TGF-beta 1 inhibitory peptide, or a variant thereof;

wherein one or more polypeptides of the fusion protein are linked by a linker.

58. The method of claim 57, wherein the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6.

59. The method of claim 57, wherein the linker comprises (Gly) n, wherein n is 6, 7, or 8.

60. The method of claim 57, wherein the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6.

61. The method of claim 57, wherein the linker comprises A (EAAAK)4ALEA (EAAAK) 4A.

62. The method of claim 57, wherein the linker comprises a sequence set forth in any one of SEQ ID NOs 17-34.

63. The method of any one of claims 57-62, wherein the transforming growth factor receptor protein is TGF β RII.

64. The method of claim 63, wherein the functional fragment of TGF β RII is a TGF β RII extracellular domain (ECD).

65. The method of any one of claims 57 to 64, wherein the TGF- β cytokine trap comprises a sequence that is at least 80% identical to the sequence set forth in SEQ ID No.14, SEQ ID No.141, or SEQ ID No. 142.

66. The method of any one of claims 57 to 64, wherein the TGF- β cytokine trap comprises the sequence shown in SEQ ID No.14, SEQ ID No.141, or SEQ ID No. 142.

67. The method of any one of claims 57-66, wherein the antibody is an immunoglobulin G (IgG) antibody.

68. The method of any one of claims 57-67, wherein the IgG is IgG1, IgG2, IgG3, or IgG 4.

69. The method of claim 68, wherein the IgG4 comprises a mutation at position 108 of SEQ ID NO 146 or SEQ ID NO 292.

70. The method of claim 69, wherein the mutation is the S108P mutation.

71. The method of claim 57, wherein the fragment of the antibody is a Fab, (Fab)2, (Fab') 2, Fv, (Fv)2 or scFv of the antibody.

72. The method of any one of claims 57-71, wherein the antibody or fragment of the antibody or variant of the antibody comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L)。

73. The method of claim 72, wherein the linker links the heavy chain variable region (V)_H) (iii) attaching to the TGF- β cytokine trap.

74. The method of claim 72, wherein the linker links the light chain variable region (V)_L) (iii) attaching to the TGF- β cytokine trap.

75. The method of any one of claims 72-74, wherein the heavy chain variable isZone (V)_H) Is connected to the light chain variable region (V) by a second linker_L)。

76. The method according to any one of claims 72 to 75, wherein the heavy chain variable region (V)_H) At least 80% identical to the sequence shown in any one of SEQ ID NOS 1-7 and 149-164.

77. The method of any one of claims 72-76, wherein the light chain variable region (V) _L) At least 80% identical to the sequence set forth in any one of SEQ ID NOS 8-13 and 148.

78. The method according to any one of claims 72 to 77, wherein the heavy chain variable region (V)_H) Comprises the sequence shown in any one of SEQ ID NO 1-7 and 149-164.

79. The method of any one of claims 72-78, wherein the light chain variable region (V)_L) Comprises the sequence shown in any one of SEQ ID NO 8-13 and 148.

80. The method according to any one of claims 72 to 77, wherein the heavy chain variable region (V)_H) (iv) is at least 90% identical to the sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 12.

81. The method of claim 80, wherein the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO 6 and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 12.

82. The method according to any one of claims 57-81, wherein the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 16.

83. The method of any one of claims 57-81, wherein the fusion protein comprises the sequence set forth in SEQ ID NO 15(VL5 Igg4) and the sequence set forth in SEQ ID NO 143.

84. The method of any one of claims 72 to 77, wherein the heavy chain variable region (VH) is at least 90% identical to the sequence set forth in SEQ ID NO:7 and the light chain variable region (V)_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 13.

85. The method of claim 84, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO 7 and the light chain variable region comprises the sequence set forth in SEQ ID NO 13.

86. The method according to any one of claims 57-79 and 84-85, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO: 145.

87. The method according to any one of claims 57-79 and 84-85, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO: 144.

88. The method of any one of claims 57-87, wherein the antibody further comprises a fragment crystallizable region (F)_C)。

89. The method of claim 88, wherein said F_CIs a person F_C1、F_C2、F_C3、F_C4 or a fragment thereof.

90. The method of claim 88, wherein said F_CFurther comprising one or more mutations.

91. The method of any one of claims 71 to 90, wherein the antibody comprises the scFv and the F _CAnd (3) fragment.

92. The method of any one of claims 57-91, wherein the cancer is a refractory cancer.

93. The method of any one of claims 57 to 92, wherein the subject is non-responsive to treatment with a PD-1 antibody or a CTLA-4 antibody.

94. The method of any one of claims 57-93, further comprising administering one or more additional anti-cancer agents.

95. The method of claim 94, wherein the additional anti-cancer agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

96. The method of claim 95, wherein the PD-1 inhibitor is an anti-PD-1 antibody or fragment or variant thereof.

97. The method of claim 95, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody or a fragment or variant thereof.

98. The method of any one of claims 57-97, further comprising administering one or more cytokines.

99. The method of any one of claims 57-98, wherein the subject is a mammalian subject.

100. The method of any one of claims 57-99, wherein the subject is a human.

101. The method of any one of claims 57-100, wherein the cancer is mesothelioma, glioblastoma, endometrial, colorectal, gastric, cervical, ovarian, pancreatic, prostate, breast, gastric, bladder, liver, hodgkin lymphoma, lung, skin, renal, or head and neck cancer.

102. The method of claim 101, wherein the skin cancer is squamous cell carcinoma of skin, melanoma, or basal cell carcinoma.

103. The method of claim 101, wherein the lung cancer is non-small cell lung cancer (NSLC) or Small Cell Lung Cancer (SCLC).

104. The method of claim 101, wherein the breast cancer is Triple Negative Breast Cancer (TNBC).

105. The method of any one of claims 57-104, further comprising administering an effective amount of a T cell engineered to express an exogenous receptor.

106. The method of claim 105, wherein the exogenous receptor is a chimeric antigen receptor.

107. The method of claim 106, wherein the chimeric antigen receptor is an engineered T cell receptor.

108. The method of any one of claims 106-107, wherein the chimeric antigen receptor comprises an antigen binding domain that binds to an epitope on CD19, BCMA, CD44, a-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor a, MUC-1, MUC-4, MUC-16, MAGE-a1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2.

109. The method of claim 108, wherein the antigen binding domain comprises a sequence selected from the group consisting of SEQ ID NOs 37-56.

110. The method according to claim 108, wherein the antigen binding domain comprises a sequence selected from the group consisting of SEQ ID NOs 35-36.

111. The method of any one of claims 107-110, wherein the effective amount of engineered T cells is at least 10²Individual cells/kg.

112. The method of any one of claims 107-111, wherein the effective amount of engineered T cells is at least 10⁴Individual cells/kg.

113. The method of any one of claims 107-112, wherein the effective amount of engineered T cells is at least 10⁵Individual cells/kg.

114. The method of any one of claims 107-113, wherein the engineered T cell further expresses a cytokine.

115. The method of claim 114, wherein the cytokine is a fusion protein comprising IL-15 and IL-15 ra.

116. A method of treating cancer in a subject in need thereof, comprising

(a) Administering a composition comprising a fusion protein comprising an antibody or fragment of said antibody or variant of said antibody that binds to programmed cell death protein-1 (PD-1); and transforming growth factor receptor (TGF β R) protein or functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker; and

(b) Administering to the subject one or more doses of an effective amount of engineered T cells, wherein the engineered T cells comprise a chimeric receptor and membrane-bound IL-15.

117. The fusion protein of any one of claims 1-33, wherein the fusion protein comprises the sequence set forth in SEQ ID No. 15 and the sequence set forth in SEQ ID No. 294.

118. The fusion protein of any one of claims 1-31 and 36-37, wherein the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO: 295.

119. The method according to any one of claims 57-81, wherein the fusion protein comprises the sequence set forth in SEQ ID NO. 15 and the sequence set forth in SEQ ID NO. 294.

120. The method according to any one of claims 57-79 and 84-85, wherein the fusion protein comprises the sequence set forth in SEQ ID NO 13 and the sequence set forth in SEQ ID NO 295.

Background

Recently, monoclonal antibody-based cancer immunotherapy based on the disruption of inhibitory signals delivered to the adaptive immune system has shown promise clinically. With FDA approval of CTLA-4 antibody inhibitors (e.g., ipilimumab) and PD-1 inhibitors (e.g., pembrolizumab, nivolumab), there are now more treatment options available for treating solid tumors including lung, renal cell, and ovarian cancers. However, in most indications (e.g., ovarian, gastric and colorectal cancers) that co-express PD-1/PD-L1 and TGF- β, no or little response to immune checkpoint inhibitors is observed. Thus, there is a continuing need in the art to obtain safer and more effective cancer treatments.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

Provided herein is a fusion protein comprising: (a) an antibody or fragment of said antibody or variant of said antibody capable of binding programmed cell death protein-1 (PD-1); and (b) transforming growth factor beta (TGF- β) cytokine traps; wherein one or more polypeptides of the fusion protein are linked by a linker.

In one embodiment, the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6. In one embodiment, the linker comprises (Gly) n, wherein n is 6, 7 or 8. In one embodiment, the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6. In one embodiment, the linker comprises A (EAAAK)₄ALEA(EAAAK)₄A. In one embodiment, the linker comprises the sequence set forth in any one of SEQ ID NOS 17-34. In one embodiment, the TGF- β cytokine trap comprises a transforming growth factor receptor (TGF- β R) or functional fragment thereof, an anti-TGF- β antibody or antigen-binding fragment thereof, a TGF- β 1 inhibitory peptide, or a variant thereof.

In one embodiment, said TGF β R is transforming growth factor β receptor II (TGF β RII) or said functional fragment thereof. In one embodiment, said functional fragment of TGF β RII is a TGF β RII extracellular domain (ECD). In one embodiment, the ECD binds TGF- β 1. In one embodiment, the ECD binds TGF- β 3. In one embodiment, the ECD binds to TGF- β 1 and TGF- β 3. In one embodiment, the ECD binds to TGF- β 1 and TGF- β 3, but not TGF- β 2. In one embodiment, the TGF- β cytokine trap comprises a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO.14, SEQ ID NO.141, or SEQ ID NO. 142. In one embodiment, the TGF- β cytokine trap comprises the sequence shown in SEQ ID NO 14, SEQ ID NO 141, or SEQ ID NO 142.

In one embodiment, the TGF- β cytokine trap comprises the sequence set forth in SEQ ID NO 14. In one embodiment, the antibody (anti-PD 1) is an immunoglobulin g (igg) antibody. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG 4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO:146 or SEQ ID NO: 292. In one embodiment, the mutation is the S108P mutation. In one embodiment, the IgG4 is linked to the TGF- β cytokine trap via the linker. In one embodiment, said fragment of said antibody is a Fab, (Fab) ₂、(Fab’)₂、Fv、(Fv)₂Or a scFv.

In one embodiment, the antibody comprises the heavy chain variable region (V) of the antibody_H) And light chain variable region (V)_L). In one embodiment, the linker links the heavy chain variable region (V)_H) (iii) attaching to the TGF- β cytokine trap. In one embodiment, the linker binds the light chain variable region (V)_L) (iii) attaching to the TGF- β cytokine trap. In one embodiment, in said fusion protein, said heavy chain variable region (V)_H) Is connected to the light chain variable region (V) by a second linker_L). In one embodiment, the second linker comprises a sequence as set forth in any one of SEQ ID NOs 17-34. In one embodiment, the heavy chain variable region (V)_H) At least 80% identical to the sequence shown in any one of SEQ ID NOS 1-7 and 149-164.

In one embodiment, the light chain variable region (V)_L) At least 80% identical to the sequence set forth in any one of SEQ ID NOS 8-13 and 148. In one embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in any one of SEQ ID NO 1-7 and 149-164. In one embodiment, the light chain variable region (V)_L) Comprises the sequence shown in any one of SEQ ID NO 8-13 and 148. In one embodiment, the heavy chain variable region (V) _H) And SEQ ID6 and the light chain variable region (V) is at least 90% identical_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 12. In one embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO 6 and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 12.

In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 16. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 143. In one embodiment, the heavy chain variable region (VH) is at least 90% identical to the sequence set forth in SEQ ID NO. 7 and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO. 13. In one embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO. 7, and the light chain variable region (V)_L) Comprises the sequence shown as SEQ ID NO. 13.

In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 296 and the sequence shown as SEQ ID NO. 145. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 144. In one embodiment, the antibody further comprises a fragment crystallizable region (F) _C). In one embodiment, said F_CIs a person F_C1、F_C2、F_C3、F_C4 or a fragment thereof. In one embodiment, said F_CFurther comprising one or more mutations. In one embodiment, the antibody comprises the scFv and the F_CAnd (3) fragment. In one embodiment, the TGF β cytokine trap comprises the anti-TGF β antibody or the antigen-binding fragment thereof. In one embodiment, the anti-TGF β antibody comprises a VH encoded by SEQ ID NO 166, 168, 169, 171, 173, 175, or 177, and a VL encoded by SEQ ID NO 165, 167, 170, 172, 174, 176, or 178.

In one embodiment, the TGF β cytokine trap comprises the TGF β inhibitory peptide or the variant thereof. In one embodiment, the TGF-beta inhibitory peptide comprises the sequence set forth in any one of SEQ ID NO. 193-227.

Provided herein is a polynucleotide encoding a fusion protein disclosed herein.

Provided herein is an expression vector comprising a polynucleotide encoding a fusion protein disclosed herein, wherein the polynucleotide is operably linked to a promoter. In one embodiment, the promoter is a constitutive promoter, a tissue specific promoter, or an inducible promoter. In one embodiment, the inducible promoter is a small molecule ligand inducible gene switch based on two polypeptide ecdysone receptors. In one embodiment, the vector is an adenoviral vector.

Provided herein is a pharmaceutical composition comprising: (a) a fusion protein disclosed herein, (b) a polynucleotide encoding a fusion protein disclosed herein, or (c) an expression vector disclosed herein, and (d) a pharmaceutically acceptable excipient.

Provided herein is a method of treating cancer, comprising: contacting a cell with (a) a fusion protein disclosed herein, (b) a polynucleotide encoding a fusion protein disclosed herein, or (c) an expression vector disclosed herein. In one embodiment, the cell is a cancer cell. In one embodiment, the cell is a mammalian cell.

Provided herein is a method of treating a subject having cancer, the method comprising: administering a composition comprising a fusion protein comprising (a) an antibody or fragment or variant of said antibody that binds to programmed cell death protein-1 (PD-1), and (b) a transforming growth factor receptor (TGF β R) or functional fragment thereof, an anti-TGF- β antibody or antigen-binding fragment thereof, a TGF- β 1 inhibitory peptide, or variant thereof; wherein one or more polypeptides of the fusion protein are linked by a linker.

In one embodiment, the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6. In one embodiment, the linker comprises (Gly) n, wherein n is 6, 7 or 8. In one embodiment, the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6. In one embodiment, the linker comprises a (eaaak)4alea (eaaak) 4A. In one embodiment, the linker comprises the sequence set forth in any one of SEQ ID NOS 17-34. In one embodiment, the transforming growth factor receptor protein is TGF β RII. In one embodiment, said functional fragment of TGF β RII is a TGF β RII extracellular domain (ECD). In one embodiment, the TGF- β cytokine trap comprises a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO.14, SEQ ID NO.141, or SEQ ID NO. 142. In one embodiment, the TGF- β cytokine trap comprises the sequence shown in SEQ ID NO 14, SEQ ID NO 141, or SEQ ID NO 142.

In one embodiment, the antibody is an immunoglobulin g (igg) antibody. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG 4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO:146 or SEQ ID NO: 292. In one embodiment, the mutation is the S108P mutation. In one embodiment, the fragment of the antibody is a Fab, (Fab)2, (Fab') 2, Fv, (Fv)2 or scFv of the antibody. In one embodiment, the antibody or fragment of said antibody or variant of said antibody comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L). In one embodiment, the linker links the heavy chain variable region (V)_H) (iii) attaching to the TGF- β cytokine trap. In one embodiment, the linker binds the light chain variable region (V)_L) (iii) attaching to the TGF- β cytokine trap.

In one embodiment, the heavy chain variable region (V)_H) Is connected to the light chain variable region (V) by a second linker_L). In one embodiment, the heavy chain variable region (V)_H) At least 80% identical to the sequence shown in any one of SEQ ID NOS 1-7 and 149-164. In one embodiment, the light chain variable region (V) _L) At least 80% identical to the sequence set forth in any one of SEQ ID NOS 8-13 and 148. In one embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in any one of SEQ ID NO 1-7 and 149-164.

In one implementationIn the scheme, the light chain variable region (V)_L) Comprises the sequence shown in any one of SEQ ID NO 8-13 and 148. In one embodiment, the heavy chain variable region (V)_H) (iv) is at least 90% identical to the sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 12. In one embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO 6 and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 12. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 16.

In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15(VL5 Igg4) and the sequence shown as SEQ ID NO. 143. In one embodiment, the heavy chain variable region (VH) is at least 90% identical to the sequence set forth in SEQ ID NO:7 and the light chain variable region (V)_L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 13. In one embodiment, the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 7 and the light chain variable region comprises the sequence set forth in SEQ ID NO. 13. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 296 and the sequence shown as SEQ ID NO. 145. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 144.

In one embodiment, the antibody further comprises a fragment crystallizable region (F)_C). In one embodiment, said F_CIs a person F_C1、F_C2、F_C3、F_C4 or a fragment thereof. In one embodiment, said F_CFurther comprising one or more mutations. In one embodiment, the antibody comprises the scFv and the F_CAnd (3) fragment. In one embodiment, the cancer is a refractory cancer. In one embodiment, the subject is non-responsive to treatment with a PD-1 antibody or a CTLA-4 antibody. In one embodiment, the method further comprises administering one or more additional anti-cancer agents. In one embodiment, the additional anticancer agentThe agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody or a fragment or variant thereof.

In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody or a fragment or variant thereof. In one embodiment, the method further comprises administering one or more cytokines. In one embodiment, the subject is a mammalian subject. In one embodiment, the subject is a human. In one embodiment, the cancer is mesothelioma, glioblastoma, endometrial, colorectal, gastric, cervical, ovarian, pancreatic, prostate, breast, gastric, bladder, liver, hodgkin lymphoma, lung, skin, renal, or head and neck cancer.

In one embodiment, the skin cancer is squamous cell carcinoma of the skin, melanoma, or basal cell carcinoma. In one embodiment, the lung cancer is non-small cell lung cancer (NSLC) or Small Cell Lung Cancer (SCLC). In one embodiment, the breast cancer is Triple Negative Breast Cancer (TNBC). In one embodiment, the method further comprises administering an effective amount of a T cell engineered to express an exogenous receptor. In one embodiment, the exogenous receptor is a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor is an engineered T cell receptor.

In one embodiment, the chimeric antigen receptor comprises an antigen binding domain that binds to an epitope on CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor alpha, MUC-1, MUC-4, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In one embodiment, the antigen binding domain comprises a sequence selected from SEQ ID NOS 37-56. In one embodiment, the antigen binding domain comprises a sequence selected from SEQ ID NOS 35-36.

In one embodiment, the effective amount of engineered T cells is at least 10²Is smallCell/kg. In one embodiment, the effective amount of engineered T cells is at least 10⁴Individual cells/kg. In one embodiment, the effective amount of engineered T cells is at least 10⁵Individual cells/kg. In one embodiment, the engineered T cell further expresses a cytokine. In one embodiment, the cytokine is a fusion protein comprising IL-15 and IL-15R α.

Provided herein is a method of treating cancer in a subject in need thereof, comprising (a) administering a composition comprising a fusion protein comprising an antibody or fragment of said antibody or variant of said antibody that binds to programmed cell death protein-1 (PD-1); and transforming growth factor receptor (TGF β R) protein or functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker; and (b) administering one or more doses of an effective amount of engineered T cells to the subject, wherein the engineered T cells comprise a chimeric receptor and membrane-bound IL-15.

In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 294. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 295. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 294. In one embodiment, the fusion protein comprises the sequence shown as SEQ ID NO. 13 and the sequence shown as SEQ ID NO. 295.

Disclosed herein, inter alia, are fusion proteins comprising: (a) an antibody or fragment of said antibody or variant of said antibody capable of binding programmed cell death protein-1 (PD-1); and (b) an Adenosine Deaminase (ADA) protein or functional fragment thereof;

wherein one or more polypeptides of the fusion protein are linked by a linker. In one embodiment, the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6. In some embodiments of the present invention, the substrate is,

the linker comprises (Gly) n, wherein n is 6, 7 or 8. In another embodiment, the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6. In certain embodiments, the linker comprises a (eaaak)4alea (eaaak) 4A. In certain embodiments, the linker comprises a sequence set forth in any one of SEQ ID NOS 17-34.

In one embodiment, the adenosine deaminase protein is adenosine deaminase 2(ADA2) or a mutant or variant thereof. In another embodiment, the Adenosine Deaminase (ADA) protein comprises any one of ADA2 mutant 1(SEQ ID NO:273), ADA2 mutant 2(SEQ ID NO:274), ADA2 mutant 3(SEQ ID NO: 275), ADA2 mutant 4(SEQ ID NO:276), ADA2 mutant 5(SEQ ID NO:277) or ADA2 mutant 6(SEQ ID NO:278), ADA2 mutant 7(SEQ ID NO:279) or wild-type ADA2(SEQ ID NO: 284). In other embodiments, the Adenosine Deaminase (ADA) protein comprises a sequence that is at least 80% identical to the sequence set forth in any one of SEQ ID NO:284 or 273-279. In certain embodiments, the Adenosine Deaminase (ADA) protein comprises a sequence as set forth in any one of SEQ ID NO 284 or 273-279.

In one embodiment, the antibody is an immunoglobulin g (igg) antibody. In certain embodiments, the IgG is IgG1, IgG2, IgG3, or IgG 4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO:146 or 292. In some embodiments, the mutation is the S108P mutation. In some examples, the fragment of the antibody is a Fab, (Fab)2, (Fab') 2, Fv, (Fv)2, or scFv of the antibody. In one embodiment, the antibody or fragment of said antibody or variant of said antibody comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L). In one embodiment, the linker binds the heavy chain variable region (V)_H) Linked to adenosine deaminase 2(ADA2) or a mutant or variant thereof. In another embodiment, the linker binds the light chain variable region (V)_L) Linked to adenosine deaminase 2(ADA2) or a mutant or variant thereof.

In one example, the heavy chain variable region (V)_H) Is connected to the light chain variable region (V) by a second linker_L). In one embodiment, the heavy chain variable region (V)_H) And any one of SEQ ID NOs 1-7 and 149-164The sequences shown are at least 80% identical. In another example, the light chain variable region (V) _L) At least 80% identical to the sequence set forth in any one of SEQ ID NOS 8-13 and 148. In a further embodiment, the heavy chain variable region (V)_H) Comprises the sequence shown in any one of SEQ ID NO 1-7 and 149-164. In a further embodiment, the light chain variable region (V)_L) Comprises the sequence shown in any one of SEQ ID NO 8-13 and 148. In one embodiment, the heavy chain variable region (VH) is at least 90% identical to the sequence set forth in SEQ ID NO. 6(VH6) and the light chain variable region (VL) is at least 90% identical to the sequence set forth in SEQ ID NO. 12(VL 5). In embodiments, the heavy chain variable region (V)_H) Comprises the sequence shown in SEQ ID NO 6 and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 12.

In one example, the fusion protein comprises the sequence shown as SEQ ID NO:12(VL5) and the sequence shown as SEQ ID NO:280(VH6 IgG4(mut) -ADA2 wt). In another example, the fusion protein comprises the sequence shown in SEQ ID NO:12(VL5) and the sequence shown in SEQ ID NO:281(VH6 igG4(mut) ADA2 mut 7). In one example, the heavy chain variable region (V)_H) At least 90% identical to the sequence shown in SEQ ID NO. 7(VH7) and the light chain variable region (V) _L) Is at least 90 percent identical to the sequence shown in SEQ ID NO. 13(VL 6). In one example, the heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO 7(VH7) and the light chain variable region (V)_L) Comprises the sequence shown in SEQ ID NO. 13(VL 6). In another example, the fusion protein comprises the sequence shown in SEQ ID NO. 13(VL6) and the sequence shown in SEQ ID NO. 282(VH7 igG4(mut) -ADA2 wt). In one example, the fusion protein comprises the sequence shown as SEQ ID NO 13(VL6) and the sequence shown as SEQ ID NO 283(VH7 igG4(mut) ADA2 mut 7).

Provided herein is a polynucleotide encoding a fusion protein. Further provided herein is an expression vector comprising a polynucleotide encoding the fusion protein of any of the above aspects, wherein the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some embodiments, the inducible promoter is a small molecule ligand inducible gene switch based on two polypeptide ecdysone receptors. In some embodiments, the vector is an adenoviral vector.

Provided herein is a method of treating cancer, comprising: contacting a cell with the fusion protein, the polynucleotide encoding the fusion protein, or the expression vector. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a mammalian cell.

Further provided herein is a pharmaceutical composition comprising the fusion protein; or a polynucleotide encoding said fusion protein; or the expression vector, and a pharmaceutically acceptable excipient.

Provided herein is a method of treating a subject having cancer, the method comprising administering a composition comprising a fusion protein comprising an antibody or fragment of said antibody or variant of said antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker. In some cases, the adenosine deaminase protein is adenosine deaminase 2(ADA 2). In other embodiments, the Adenosine Deaminase (ADA) protein comprises any one of ADA2 mutant 1(SEQ ID NO:273), ADA2 mutant 2(SEQ ID NO:274), ADA2 mutant 3(SEQ ID NO: 275), ADA2 mutant 4(SEQ ID NO:276), ADA2 mutant 5(SEQ ID NO:277) or ADA2 mutant 6(SEQ ID NO:278), ADA2 mutant 7(SEQ ID NO:279), or wild-type ADA2(SEQ ID NO: 284).

In some cases, the linker comprises (G4S) n, wherein n is 2, 3, 4, 5, or 6. In some embodiments, the linker is (Gly) n, wherein n is 6, 7, or 8. In some embodiments, the linker comprises (EAAAK) n, wherein n is 1, 2, 3, 4, 5, or 6. In some embodiments, the linker comprises A (EAAAK) ₄ALEA(EAAAK)₄A. In some cases, the linker comprises any one of SEQ ID NOS 17-34The sequence shown.

Provided herein is a method of treating a subject having cancer. In certain instances, the cancer is mesothelioma, glioblastoma, endometrial, colorectal, gastric, cervical, ovarian, pancreatic, prostate, breast, gastric, bladder, liver, hodgkin lymphoma, lung, skin, renal, or head and neck cancer. In some cases, the skin cancer is a squamous cell carcinoma of the skin, melanoma, or basal cell carcinoma. In other cases, the lung cancer is non-small cell lung cancer (NSLC) or Small Cell Lung Cancer (SCLC). In some cases, the breast cancer is Triple Negative Breast Cancer (TNBC).

In another embodiment, there is a method of treating a subject having cancer, the method comprising administering a composition comprising a fusion protein comprising an antibody or fragment of said antibody or variant of said antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker. In further embodiments, the method of treating a subject having cancer further comprises administering an effective amount of a T cell engineered to express an exogenous receptor.

In some cases, the exogenous receptor is a chimeric antigen receptor. In other cases, the chimeric antigen receptor is an engineered T cell receptor. In one instance, the chimeric antigen receptor comprises an antigen binding domain that binds to an epitope on CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor alpha, MUC-1, MUC-4, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In some embodiments, the antigen binding domain comprises a sequence selected from SEQ ID NOS 37-56. In other embodiments, the antigen binding domain comprises a sequence selected from SEQ ID NOS 35-36.

In one embodiment, the effective amount of engineered T cells is at least 10²Individual cells/kg. In another embodiment, the effective amount of engineered T cells is at least 10⁴Individual cells/kg. In a further embodiment, the effective amount of engineered T cells is at least 10⁵Individual cells/kg.

In further embodiments, the engineered T cell further expresses a cytokine. In another embodiment, the cytokine is a fusion protein comprising IL-15 and IL-15R α.

Provided herein is a method of treating cancer in a subject in need thereof comprising administering a composition comprising a fusion protein comprising an antibody or fragment of said antibody or variant of said antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker, and administering one or more doses of an effective amount of engineered T cells to the subject, wherein the engineered T cells comprise a chimeric receptor and membrane-bound IL-15.

Drawings

The features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1 is a schematic of PD-1/PD-L1 in immunosuppression.

FIG. 2 is a schematic representation of TGF-. beta.s in immunosuppression.

FIG. 3 shows the TGF- β related gene cluster (abundant in stage III/IV) associated with metastatic disease and poor prognosis in a subset of ovarian cancer patients.

FIG. 4A, FIG. 4B and FIG. 4C show the design of anti-PD 1-TGFRII fusion protein design. In other exemplary embodiments, ADA2 may be fused to anti-PD 1.

FIG. 5 is a graph showing the blocking of PD-1/PD-L1 interactions by anti-PD 1(VH6-VL5) IgG1-TGF β RII and anti-PD 1(VH6-VL5) IgG4-TGF β RII.

FIG. 6 is a graph showing neutralization of TGF- β 1 isotype signaling by anti-PD 1(VH6-VL5) IgG1-TGF β RII and anti-PD 1(VH6-VL5) IgG4-TGF β RII.

FIG. 7 is a graph showing neutralization of TGF-. beta.2 isoforms by anti-PD 1(VH6-VL5) IgG 1-TGF-. beta.RII and anti-PD 1(VH6-VL5) IgG 4-TGF-. beta.RII.

FIG. 8 is a graph showing neutralization of TGF- β 3 isotype signaling by anti-PD 1(VH6-VL5) IgG1-TGF β RII and anti-PD 1(VH6-VL5) IgG4-TGF β RII.

The graphs in figure 9A, figure 9B and figure 9C show that proliferation and IFN- γ production of stimulated PBMC is enhanced in a dose-dependent manner in the presence of anti-PD 1-TGFRII fusion protein compared to anti-PD 1 or a control antibody.

FIG. 9D and FIG. 9E are graphs showing, respectively, that the PD1 receptor is at CD8 when anti-PD 1-TGFRII fusion protein is added to co-cultures of PBMC and colorectal cancer (colorectal adenocarcinoma) cell lines⁺Occupancy on T cells and IFN- γ production.

The graphs in fig. 9F and 9G show, respectively, the occupancy of PD1 receptor on CD8+ T cells and IFN- γ production when anti-PD 1-TGFRII fusion protein was added to co-cultures of PBMC and head and neck cancer (pharyngeal cancer) cell lines.

The graphs in figure 9H and figure 9I depict TGF- β 1 and TGF- β 2 concentrations in PBMC and colorectal cancer (colorectal adenocarcinoma) cell co-culture supernatants, respectively, in the presence of anti-PD 1-TGFRII fusion protein.

The graphs in FIG. 9J and FIG. 9K depict TGF-. beta.1 and TGF-. beta.2 concentrations in PBMC and head and neck cancer (pharyngeal cancer) cell co-culture supernatants, respectively, in the presence of anti-PD 1-TGFRII fusion protein.

Figures 9L and 9M depict graphs depicting IFN- γ production in 3D spheroid cultures of colorectal cancer (colorectal adenocarcinoma) cells or head and neck cancer (pharyngeal cancer) cells and PBMCs, respectively, in the presence of anti-PD 1-TGFRII fusion protein, compared to anti-PD 1 alone.

FIGS. 10A-10D show the effect of anti-PD 1-TGFRII fusion protein treatment on T cell proliferation and activation in the presence of recombinant TGF-. beta.1.

FIGS. 11A-11F show expression of various cytokines by PBMC in the presence of recombinant TGF- β 1 and in the presence of anti-PD 1, anti-PD 1-TGFRII fusion protein, or control antibodies.

Figure 12A shows the effect of anti-PD 1-TGFRII fusion protein on tumor growth compared to anti-PD 1 alone in a humanized mouse model of colorectal cancer.

FIG. 12B shows that treatment with anti-PD 1-TGFRII fusion protein significantly increased CD8 in tumors in a humanized mouse model of colorectal cancer ⁺T cells and T_regThe ratio of.

Figure 12C shows the effect of anti-PD 1-TGFRII fusion protein treatment on perforin expression levels compared to anti-PD 1 treatment in a humanized mouse model of colorectal cancer.

Fig. 12D and 12E show the effect of anti-PD 1-TGFRII fusion protein treatment on TGF- β 1 and TGF- β 2 concentrations compared to anti-PD 1 treatment, respectively, in a humanized mouse model of colorectal cancer.

Figure 13A shows that treatment with anti-PD 1-TGFRII fusion protein significantly improved IFN γ production compared to anti-PD 1 treatment in an in vitro model of head and neck cancer. Fig. 13B-13G show that treatment with anti-PD 1-TGFRII fusion protein significantly increased T cell function as demonstrated by expression analysis of various pathway genes.

Figure 14A shows the effect of anti-PD 1-TGFRII fusion protein on tumor growth compared to anti-PD 1 alone in a humanized mouse model of head and neck cancer.

Figure 14B shows the survival of tumor-bearing mice in a humanized mouse model of head and neck cancer when treated with anti-PD 1-TGFRII fusion protein compared to treatment with anti-PD 1 alone or an isotype control.

FIG. 14C shows CD8 in tumors of mice treated with anti-PD 1-TGFRII fusion protein in a humanized mouse model of head and neck cancer ⁺Ratio of T cells to regulatory T cells.

FIGS. 14D and 14E show the effect of anti-PD 1-TGFRII fusion protein on TGF- β 1 and TGF- β 2 concentrations compared to anti-PD 1 alone in a humanized mouse model of head and neck cancer.

FIG. 14F shows the effect of anti-PD 1-TGFRII fusion protein on IFN- γ production in a humanized mouse model of head and neck cancer.

FIGS. 15A-15B show the effect of anti-PD 1(VH7/VL6) -TGFRII fusion protein on IFN-. gamma.production and TGF-. beta.1 concentration in samples from patients with primary colorectal cancer.

FIG. 15C shows gene expression analysis of primary colorectal cancer patient samples co-cultured with anti-PD 1(VH7/VL6) -TGFRII fusion protein.

FIG. 16 shows a graph depicting the results of cytotoxicity assays for anti-PD 1(VH7/VL6) -TGFRII fusion protein compared to anti-PD-L1-TGFRII fusion protein.

The graph in figure 17 depicts the results of cytotoxicity assays of anti-PD 1(VH7/VL6) -TGFRII fusion protein in combination with chimeric receptor antigen (CAR) T cells compared to anti-PD-1 in combination with CAR T cells compared to CAR T cells alone.

The graphs in fig. 18A and 18B depict the cytotoxicity assay results against PD1(VH6/VL5) -TGFRII fusion protein in combination with CD33 CAR-T and against PD1(VH7/VL6) -TGFRII fusion protein in combination with CD33 CAR-T, respectively.

The graphs in fig. 19A and 19B depict tumor cell lysis using anti-PD 1(VH6/VL5) -TGFRII fusion protein and anti-PD 1(VH7/VL6) -TGFRII fusion protein, respectively, when co-cultured with NK cells.

The graph in FIG. 20 depicts Biacore analysis of simultaneous binding of TGF-b1 and PD1 by an anti-PD 1(VH6/VL5) -TGFRII fusion protein using different linkers.

FIG. 21 is a graph showing the blocking of the PD-1/PD-L1 interaction by anti-PD 1 IgG4-ADA 2.

FIG. 22 is a graph showing ADA2 enzyme activity measured against anti-PD 1 hIgG1-ADA2 and anti-PD 1 hIgG4-ADA 2.

FIGS. 23A-23C are graphs showing the effect of various variants of anti-PD 1 and anti-PD 1-ADA2 fusion proteins on the PD-L1/PD-1 interaction.

FIGS. 24A-24F are graphs showing enzyme activity, as measured by ADA enzyme activity, against various variants of PD1-ADA2 fusion proteins.

FIG. 25 is a graph showing the enzyme activity against PD1-mutADA2 compared to PD1-wtADA2 as measured by ADA enzyme activity.

FIGS. 26A-26D are graphs depicting the effect of variants against PD1-wtADA2 on T cell proliferation.

Figure 26E is a graph depicting the production of IFN γ by anti-PD 1-wtADA2 compared to anti-PD 1 or isotype control.

FIGS. 27A-27B are graphs depicting the effectiveness of wtADA2 and mutADA2 in reversing adenosine-mediated suppression of T-cell proliferation.

Figure 28 is a graph depicting the effect of variants of anti-PD 1-ADA2 fusion proteins on blocking of PD1-PDL1 interactions.

FIG. 29 is a graph showing the enzyme activity against PD1-ADA2-scFv-Fc as measured by ADA enzyme activity.

Figure 30 is a bar graph showing enzyme activity, measured by ADA enzyme activity, against a variant of PD1-ADA 2.

FIGS. 31A-31B are graphs depicting the effect of anti-PD 1-wtADA2 on IFN- γ production and Tumor Infiltrating Lymphocyte (TIL) proliferation in tumors from patients with primary CRC.

Fig. 32 is a graph depicting the effect of anti-PD 1 compared to anti-PD 1-wtADA2 on tumor volume of a humanized mouse model of lung cancer.

FIGS. 33A-33C show the protocol design of the anti-PD 1-adenosine deaminase 2(ADA2) design, anti-PD 1-ADA 2.

Detailed Description

The following description and examples set forth in detail embodiments of the disclosure.

It is to be understood that this disclosure is not limited to the particular embodiments described herein, as such may vary. Those skilled in the art will recognize that there are variations and modifications of the present disclosure, which are within the scope of the present disclosure.

All terms should be interpreted as they would be understood by one skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Although various features of the disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment.

The following definitions supplement those in the art and are directed to the present application without being ascribed to any related or unrelated case, e.g., any commonly owned patent or application. Preferred materials and methods are described herein, but any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure. Thus, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definition of

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In this application, the use of "or" means "and/or" unless stated otherwise. As used herein, the terms "and/or" and "any combination thereof" and grammatical equivalents thereof are used interchangeably. These terms may be expressed, specifically referring to any combination. For illustrative purposes only, the following phrases "A, B and/or C" or "A, B, C or any combination thereof" may refer to "a alone; b alone; c alone; a and B; b and C; a and C; and A, B and C ". The term "or" may be used conjunctively or disjunctively unless the context specifically indicates a disjunctive use.

Furthermore, the use of the terms "including" and other forms, such as "comprises," "comprising," and "having," are not limiting.

Reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the disclosure.

As used in this specification and claims, the words "comprise" (and any form of comprise), "have" (and any form of have), "include" (and any form of include), or "contain" (and any form of contain) are inclusive or open-ended and do not exclude additional unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

As used herein, the term "about" and grammatical equivalents thereof with respect to a reference numerical value can include the numerical value itself and ranges of values plus or minus 10% of the numerical value.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, according to practice in the art. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, an amount of "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" with respect to a reference value can also include a range of values that is plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value. Alternatively, and particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where a particular value is described in the application and claims, unless otherwise stated, it is to be assumed that the term "about" means within an acceptable error range for the particular value.

"Polynucleotide" or "oligonucleotide" as used herein refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double-and single-stranded DNA, triple-stranded DNA, and double-and single-stranded RNA. It also includes polynucleotides in modified form (e.g., by methylation and/or by capping) and in unmodified form. The term is also intended to include molecules comprising non-naturally occurring or synthetic nucleotides and nucleotide analogs.

"transfection", "transformation" or "transduction" as used herein refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. The polynucleotide sequences and vectors disclosed or contemplated herein can be introduced into a cell by, for example, transfection, transformation, or transduction. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J (eds.), Methods in Molecular Biology, vol.7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature,346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al, mol. cell biol.,7:2031-2034 (1987)). After growth of the infectious particles in suitable packaging cells, the phage or viral vector can be introduced into a host cell, many of which are commercially available.

As used herein, "polypeptide," "peptide," and grammatical equivalents thereof refer to polymers of amino acid residues. The polypeptide may optionally comprise a glycosylated or otherwise modified protein typical for a given protein in a given cellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, alpha-amino-N-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, beta-phenylserine beta-hydroxyphenylalanine, phenylglycine, alpha-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N '-benzyl-N' -methyl-lysine, N-phenylglycine, N ', N' -dibenzyl-lysine, 6-hydroxylysine, ornithine, alpha-aminocyclopentanecarboxylic acid, alpha-aminocyclohexanecarboxylic acid, alpha-aminocycloheptane-carboxylic acid, alpha- (2-amino-2-norbornane) -carboxylic acid, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine and alpha-tert-butylglycine. The present disclosure further contemplates that expression of a polypeptide described herein in an engineered cell can be associated with post-translational modification of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, disulfide bond formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation (geranylation), glycosylphosphatidylinositol (glycosylation), lipidation (lipoylation), and iodination.

The term "identical" and grammatical equivalents thereof or "sequence identity," as used herein, in the context of the amino acid sequences of two nucleic acid sequences or polypeptides, means that the residues in the two sequences are identical when aligned for maximum correspondence over a specified comparison window. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically from about 50 to about 200, more typically from about 100 to about 150 contiguous positions, wherein one sequence can be compared to a reference sequence having the same number of contiguous positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by: the local homology algorithm of Smith and Waterman, adv.appl.math, 2:482 (1981); needleman and Wunsch, J.mol.biol.,48:443 (1970); similarity search methods of Pearson and Lipman, Proc.Nat.Acad.Sci.U.S.A.,85:2444 (1988); computerized implementations of these algorithms (including but not limited to CLUSTAL in the PC/Gene program of Intelligences, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is fully described in the following documents: higgins and Sharp, Gene,73: 237-; corpet et al, Nucleic Acids Res.,16:10881-10890 (1988); huang et al, Computer Applications in the Biosciences,8:155-165 (1992); and Pearson et al, Methods in Molecular Biology,24:307-331 (1994). Alignment is also typically performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof, e.g., as determined by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described with reference to a starting nucleic acid, e.g., they may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference nucleic acid or fragment thereof, e.g., as determined by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When a molecule is described as having a certain percentage of sequence identity to a larger molecule, this means that when two molecules are optimally aligned, the percentage of residues in the smaller molecule find matching residues in the larger molecule, according to the order in which the two molecules are optimally aligned.

The term "substantially identical" and grammatical equivalents thereof as applied to nucleic acid or amino acid sequences refers to nucleic acid or amino acid sequences comprising sequences having at least 90% or greater, at least 95%, at least 98%, and at least 99% sequence identity, as compared to a reference sequence using the above-described programs (e.g., BLAST), using standard procedures. For example, the BLASTN program (for nucleotide sequences) default to a word length (W) of 11, expected (E) of 10, M-5, N-4, and a comparison of the two strands. For amino acid sequences, the BLASTP program defaults to using a word length (W) of 3, an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, proc. natl. acad. sci. usa 89:10915 (1992)). The percent sequence identity is determined by comparing the two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by the following method: determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. In some embodiments, there is substantial identity over a region of the sequence of at least about 50 residues in length over a region of at least about 100 residues, and in some embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequence is substantially the same over the entire length of the coding region.

"homology" is typically inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The percent exact identity between sequences that can be used to establish homology varies with the nucleic acid and protein in question, but homology is routinely established using sequence identity as low as 25%. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percent sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and/or nucleic acid sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are "homologous" when their encoding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules may be referred to as "homologues". For example, any naturally occurring protein can be modified by any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

The term "isolated" and grammatical equivalents thereof as used herein refers to the removal of a nucleic acid from its natural environment. The term "purified" and grammatical equivalents thereof, as used herein, refers to molecules or compositions of increased purity, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, wherein "purity" is a relative term and not "absolute purity". However, it will be appreciated that the nucleic acid and protein may be formulated with diluents or adjuvants and still be isolated for practical purposes. For example, when used for introduction into a cell, the nucleic acid is typically mixed with an acceptable carrier or diluent. The term "substantially purified" and grammatical equivalents thereof as used herein refers to a nucleic acid sequence, polypeptide, protein, or other compound that is substantially free, i.e., greater than about 50% free, greater than about 70% free, greater than about 90% free, of polynucleotides, proteins, polypeptides, and other molecules with which the nucleic acid, polypeptide, protein, or other compound is naturally associated.

An "expression vector" or "vector" is any genetic element, such as a plasmid, chromosome, virus, transposon, which represents an autonomous unit of intracellular polynucleotide replication (i.e., capable of replicating under its own control) or is capable of replication by insertion into the host cell chromosome, to which another polynucleotide segment has been attached in order to effect replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, phages and cosmids. The vector may contain the polynucleotide sequences necessary to effect ligation or insertion of the vector into the desired host cell and to effect expression of the attached segment. Such sequences vary depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, the expression vector may be capable of direct expression of the nucleic acid sequence product encoded therein without the vector being linked to or integrated into a host cell DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome" that is capable of replicating in a host cell and is present as an extrachromosomal segment of DNA within the host cell in the presence of an appropriate selection pressure (see, e.g., Conese et al, Gene Therapy,11: 1735-. Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids utilizing EB Nuclear antigen 1(EBNA1) and the Epstein-Barr Virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7 and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of episomal vectors using T-antigen and the SV40 origin of replication in place of EBNA1 and oriP. The vector may also comprise a selectable marker gene.

As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or excluded in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in international patent application publications WO 1992/08796 and WO 1994/28143; wigler et al, Proc.Natl.Acad.Sci.USA,77:3567 (1980); o' Hare et al, Proc. Natl.Acad.Sci.USA,78:1527 (1981); mulligan & Berg, proc.natl.acad.sci.usa,78:2072 (1981); Colberre-Garapin et al, J.mol.biol.,150:1 (1981); santerre et al, Gene,30:147 (1984); kent et al, Science,237:901-903 (1987); wigler et al, Cell,11:223 (1977); szyballska & szyballski, proc.natl.Acad.Sci.USA,48:2026 (1962); lowy et al, Cell,22:817 (1980); and U.S. Pat. nos. 5,122,464 and 5,770,359.

The term "coding sequence" as used herein refers to a polynucleotide segment that encodes a protein or polypeptide. This region or sequence is bounded by a start codon near the 5 'end and a stop codon near the 3' end. Coding sequences may also be referred to as open reading frames.

The term "operably linked" as used herein refers to a physical and/or functional linkage of a DNA segment to another DNA segment, thereby allowing these segments to function in their intended manner. A DNA sequence encoding a gene product is operably linked to regulatory sequences, such as promoters, enhancers and/or silencers, in a manner that allows for the direct or indirect regulation of transcription of the DNA sequence. For example, a DNA sequence is operably linked to a promoter when it is linked downstream of the promoter's transcription initiation site, in proper reading frame with the transcription initiation site, and allows for transcriptional extension to occur through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence that encodes a gene product when the enhancer or silencer is linked to the DNA sequence in a manner that increases or decreases transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of a DNA sequence. If the signal sequence is expressed as a preprotein involved in the secretion of the polypeptide, the DNA for the signal sequence is operably linked to the DNA encoding the polypeptide. Ligation of the DNA sequence to the regulatory sequence is usually accomplished by ligation at appropriate restriction sites or by adapters or linkers inserted into the sequence using restriction endonucleases known to those skilled in the art.

The term "induce" and grammatical equivalents thereof as used herein refers to an increase in transcription, promoter activity and/or expression of a nucleic acid sequence by a transcriptional regulator relative to a certain basal transcription level.

The term "transcriptional regulator" refers to a biochemical element that is used under certain environmental conditions to prevent or inhibit transcription of a promoter-driven DNA sequence (e.g., a repressor or nuclear repressor protein), or under certain environmental conditions to allow or stimulate transcription of a promoter-driven DNA sequence (e.g., an inducer or enhancer).

The term "enhancer" as used herein refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located thousands of bases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from collections such as the ATCC and other commercial or individual sources). Many polynucleotides that include a promoter (such as the commonly used CMV promoter) also include an enhancer sequence. Enhancers can be located upstream, within, or downstream of a coding sequence. The term "Ig enhancer" refers to enhancer elements derived from enhancer regions located within immunoglobulin (Ig) loci (such enhancers include, for example, the heavy chain (μ)5 ' enhancer, the light chain (κ)5 ' enhancer, κ and μ intronic enhancers, and the 3 ' enhancer) (see generally Paul W.E (eds.), Fundamental Immunology, 3 rd edition, Raven Press, New York (1993), p. 353-363; and U.S. Pat. No. 5,885,827).

The term "promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. The promoter is located near the transcription start site of the gene, on the same strand of DNA and upstream (toward the 5' region of the sense strand). Some promoters are constitutive in that they are active in all cases in the cell, while others become active in response to a particular stimulus being regulated, e.g., inducible promoters. The term "promoter activity" and grammatical equivalents thereof refers to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity can be determined directly (e.g., by Northern blot analysis) by determining the amount of RNA transcript produced, or indirectly by determining the amount of product encoded by the linked nucleic acid sequence (e.g., a reporter nucleic acid sequence linked to the promoter).

An "inducible promoter" as used herein refers to a promoter whose activity is induced by the presence or absence of a transcriptional regulator (e.g., an biotic or abiotic factor). Inducible promoters are useful because the expression of the genes to which they are operably linked can be switched on or off at certain developmental stages of the organism or in specific tissues. Non-limiting examples of inducible promoters include alcohol regulated promoters, tetracycline regulated promoters, steroid regulated promoters, metal regulated promoters, pathogenesis regulated promoters, temperature regulated promoters, and light regulated promoters. Inducible promoters may be part of a gene switch or genetic switch.

As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte that plays an important role in cell-mediated immunity. They can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of a T Cell Receptor (TCR) on the cell surface.

As used herein, the term "antibody", also referred to as immunoglobulin (Ig), may be a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B cell and that binds the same epitope. In contrast, "polyclonal antibodies" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. The antibody may be from any animal source. The antibody may be IgG (including IgG1, IgG2, IgG3 and IgG4), IgA (including IgA1 and IgA2), IgD, IgE or IgM and IgY. In some embodiments, the antibody may be an intact antibody, including a single chain intact antibody. In some embodiments, the antibody may be a fragment of an antibody, which may include, but is not limited to, Fab ', F (ab')₂Fd (from V)_HAnd CH 1), Fv fragment (consisting of V)_HAnd V_LComposition), single chain variable fragment (scFv), single chain antibody, disulfide linked variable fragment (dsFv) and antibodies comprising V _LOr V_HA fragment of a domain. Intact antibodies typically consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (V)_H) A region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (V)_L) Zone and one C terminal constant (C)_L) And (4) a zone. The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. V_HRegion and V_LRegions have similar general structures, each region comprising four framework regions of relatively conserved sequence. The framework regions are connected by three Complementarity Determining Regions (CDRs). The three CDRs, termed CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding. These specific regions have been described by Kabat et al, J.biol.chem.252,6609-6616(1977) and Kabat et al, Sequences of proteins of immunological interest (1991), by Chothia et al, J.mol.biol.196:901-917(1987), and by MacCallum et al, J.mol.biol.262:732-745(1996), all of which are incorporated herein by reference in their entirety, wherein when compared to each other, an overlap or subset of amino acid residues is defined. Preferably, the term "CDR" is a CDR defined by Rabat based on sequence comparison. CDRH1, CDRH2 and CDRH3 represent heavy chains The CDRs, while CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

The terms "fragment of an antibody", "antibody fragment", "fragment of an antibody", "antigen-binding portion", or grammatical equivalents thereof, are used interchangeably herein to refer to one or more fragments or portions of an antibody that retain the ability to specifically bind an antigen (see generally Holliger et al, Nat. Biotech.,23(9):1126-1129 (2005)). For example, an antibody fragment desirably comprises one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Non-limiting examples of antibody fragments include (1) Fab fragments, which are composed of V_L、V_H、C_LAnd a CH1 domain; (2) a F (ab') 2 fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the stem region; (3) fv fragment consisting of a V of one arm of an antibody_LAnd V_HDomain composition; (4) single chain Fv (scFv), which are two domains of an Fv fragment connected by a linker (i.e., V)_LAnd V_H) A monovalent molecule of composition that enables the synthesis of both domains as a single polypeptide chain (see, e.g., Bird et al, Science,242:423-426 (1988); huston et al, Proc.Natl.Acad.Sci.USA,85: 5879-; and Osbourn et al, nat. Biotechnol.,16:778(1998)), and (5) diabodies, which are dimers of polypeptide chains, wherein each polypeptide chain comprises a V linked to a V through a peptide linker _LConnected V_HThe peptide linker is too short to allow V on the same peptide chain_HAnd V_LAre paired with each other to drive different V_H-V_LPairing between complementary domains on polypeptide chains occurs to generate dimeric molecules with two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, for example, U.S. patent 8,603,950.

"antigen recognition portion", "antigen recognition domain", "antigen binding domain" or "antigen binding region" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule, or a fragment thereof.

The term "conserved amino groupAcid substitutions "or" conservative mutations "refer to the substitution of one amino acid for another with common properties. A functional approach to define the common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). From such an analysis, groups of amino acids can be defined, wherein the amino acids within a group are preferentially exchanged with each other, so that they most closely resemble each other in their impact on the overall protein structure (Schulz, g.e. and Schirmer, r.h., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as lysine for arginine, and vice versa, so that a positive charge can be retained; glutamic acid for aspartic acid and vice versa, so that a negative charge can be maintained; serine replaces threonine so that free-OH can be maintained; glutamine replaces asparagine, making it possible to maintain free-NH ₂. Alternatively or additionally, a functional variant may comprise an amino acid sequence of a reference protein having at least one non-conservative amino acid substitution.

The term "non-conservative mutation" relates to an amino acid substitution between different groups, e.g., a lysine for tryptophan, or a phenylalanine for serine, etc. In such cases, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions may enhance the biological activity of a functional variant, such that the biological activity of the functional variant is increased as compared to the homologous parent protein.

The term "proliferative disease" as referred to herein refers to a unified concept in which hyperproliferation of cells and/or turnover of cellular matrix contributes significantly to the pathogenesis of diseases including cancer. In some embodiments, the proliferative disease is cancer.

As used herein, "patient" or "subject" refers to a mammalian subject diagnosed as having or suspected of having or developing a proliferative disorder, such as cancer. In some embodiments, the term "patient" refers to a mammalian subject with a higher than average likelihood of developing a proliferative disorder, such as cancer. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients may be male and/or female. By "patient in need" or "subject in need" is meant herein a patient diagnosed as having or suspected of having a disease or disorder, such as, but not limited to, cancer.

By "administering" herein is meant providing one or more of the compositions described herein to a patient or subject. By way of example and not limitation, administration (e.g., injection) of the composition may be by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more of these approaches may be used. Parenteral administration may be, for example, by bolus injection or by gentle perfusion over time. Alternatively or simultaneously, administration may be by the oral route. In addition, it may also be administered by surgical deposition of cell pellets or pills or placement of a medical device. In embodiments, the compositions of the present disclosure may comprise an engineered cell or host cell expressing a nucleic acid sequence described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferative disorder. The pharmaceutical composition may comprise a target cell population as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

As used herein, the terms "treat," "treating," or grammatical equivalents thereof refer to obtaining a desired pharmacological and/or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures the disease and/or adverse symptoms caused by the disease. In some embodiments, the term "treating" may include "preventing" a disease or condition.

As used herein, "treatment period" refers to a treatment cycle, e.g., a course of administration of a therapeutic agent that may be repeated (e.g., on a regular schedule). In embodiments, the dosing regimen may have one or more periods of no therapeutic agent administration between treatment periods. For example, the treatment period can include one dose of the fusion protein administered in combination (prior to, concurrently with, or subsequent to) a second therapeutic agent, e.g., CAR-T cells.

The term "co-administration" or "co-providing" as used herein refers to the delivery of two (or more) different treatments to a subject during the subject's illness, e.g., after the subject has been diagnosed with the condition, and before the condition has been cured or eliminated or the treatment has otherwise ceased. In some embodiments, delivery of one therapy is still ongoing at the beginning of delivery of the second therapy, so there is overlap in dosing. This is sometimes referred to herein as "simultaneous" or "parallel delivery". In other embodiments, the delivery of one therapy ends before the delivery of another therapy begins. In some embodiments of either case, the treatment is more effective as a result of the combined administration. For example, the second treatment is more effective, e.g., the same effect is observed with less of the second treatment than would be observed with the second treatment administered in the absence of the first treatment, or the second treatment alleviates symptoms to a greater extent, or a similar condition is observed with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than the reduction observed when one treatment is delivered in the absence of the other treatment. The effects of both treatments may be partially additive, fully additive or greater than additive. The delivery may be such that the effect of the delivered first treatment may still be detected when the second treatment is delivered.

In some embodiments, the first treatment and the second treatment can be administered simultaneously (e.g., at the same time) in the same or separate compositions, or sequentially. Sequential administration refers to administration of one treatment prior to administration of an additional (e.g., second) treatment (e.g., immediately prior to administration, less than 5, 10, 15, 30, 45, 60 minutes prior to 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 or more hours prior to administration, 4, 5, 6, 7, 8, 9 or more days, 1, 2, 3, 4, 5, 6, 7, 8 or more weeks prior to administration). The order of administration of the first and second treatments may also be reversed.

The terms "therapeutically effective amount," "therapeutic amount," "immunologically effective amount," "anti-neoplastic effective amount," "tumor inhibiting effective amount," or grammatical equivalents thereof, refer to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compositions described herein to elicit a desired response in one or more subjects. The precise amount of the composition of the present disclosure to be administered can be determined by a physician considering individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

Alternatively, the pharmacological and/or physiological effect of administration of one or more of the compositions described herein to a patient or subject may be "prophylactic", i.e., the effect prevents, in whole or in part, a disease or a symptom thereof. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

Programmed cell death proteins and other checkpoint inhibitors

Programmed cell death protein 1, also known as PD-1 or CD279 (cluster of differentiation 279), is an immune checkpoint protein. The PD-1/PD-L1 signaling axis may promote tumor-mediated immune escape. In some cases, PDL-1 may be overexpressed by tumor cells, helper cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), Antigen Presenting Cells (APCs), in the tumor microenvironment. In some cases, PD-1 may be upregulated by "depleted" T cells and may signal upon binding to its ligands (PDL-1, PDL2 and CD80) to inhibit effector T cell function. Blockade of the PD-1/PD-L1 pathway by anti-PD-1 or anti-PD-L1 could restore function of depleted T cells and promote killing of tumor cells (fig. 1).

In some embodiments, a fusion protein comprising a PD-1 inhibitor and a TGF- β trap may include, but is not limited to, full-length nivolumab (anti-PD-1), MK-3945 (anti-PD-1), pembrolizumab (anti-PD-1), pidilizumab (anti-PD-1), REGN2810 (anti-PD-1), AMP-224 (anti-PD-1), MEDI0680 (anti-PD-1), PDR001 (anti-PD-1), CT-001 (anti-PD-1), or a functional fragment or variant thereof. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab.

Fusion proteins

In some embodiments, a fusion protein provided herein, or a fragment or variant thereof, comprises a PD-1 inhibitor or antibody fused to a cytokine trap via a linker.

In some embodiments, the PD-1 inhibitor may be an antibody or a fragment of the antibody or a variant of the antibody that targets PD-1. In some embodiments, a fusion protein comprising premrolizumab can be fused to a cytokine trap (e.g., a TGF- β trap). In some embodiments, a fusion protein comprising nivolumab may be fused to a cytokine trap (e.g., a TGF- β trap).

In some embodiments, a fusion protein or fragment or variant thereof described herein comprises a cytokine trap and an antibody targeting an immune checkpoint gene, a fragment or variant thereof, as described above. In some embodiments, an antibody or fragment of the antibody or variant of the antibody that targets an immune checkpoint such as cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and programmed cell death ligand-1 (PDL1) may be fused to a TGF- β trap via a linker. In some embodiments, the PD-L1 inhibitor is atelizumab.

In some embodiments, a fusion protein provided herein or a fragment or variant thereof comprises a PD1 inhibitor or antibody fused to an adenosine deaminase (e.g., ADA2) or a functional variant or derivative thereof as described herein.

Cytokine trap

Cytokines have an impact on many biological processes. Inhibition of cytokines can have clinical benefit, for example, in cancer. Several cytokines have been shown to be causative agents of various diseases. Such cytokines include, but are not limited to, IL-1, IL-4, Il-6, TNF- α, TGF- β, and various isoforms thereof. The term "cytokine trap" as used herein refers to a blocker or neutralizer of the effect of cytokines. Examples of such cytokine traps may include, but are not limited to, extracellular domains of cytokine receptors, antibodies that bind cytokines, and peptides that bind cytokines (e.g., inhibitory peptides). In one embodiment, the cytokine is TGF- β. In one embodiment, the cytokine is TGF- β 1. In one embodiment, the cytokine is TGF- β 3. In one embodiment, the cytokines are TGF- β 1 and TGF- β 3. In further embodiments, a cytokine trap (e.g., a TGF- β trap) targeted to TGF- β may include the extracellular domain of TGF- β RII or variants thereof (e.g., SEQ ID NOs 141 and 142), anti-TGF- β antibodies, and inhibitory peptides of TGF- β 1, TGF- β 2, and/or TGF- β 3.

Transforming growth factor

Transforming growth factor-beta (TGF- β) is a group of multifunctional peptides that can control proliferation, differentiation and other functions in many cell types. TGF-. beta.may act synergistically with TGF-. alpha.in inducing transformation. It may also act as a negative autocrine growth factor. Dysregulation of TGF- β activation and signaling can lead to apoptosis. Many cells can synthesize TGF- β and almost all cells have specific receptors for this peptide. TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3 all may function through the same receptor signaling system. TGF-. beta.1 may play an important role in controlling the immune system, and may exhibit different activities on different types of cells or cells at different stages of development. Most immune cells (or leukocytes) secrete TGF- β 1. TGF-. beta.1 is a peptide of 112 amino acid residues derived from the C-terminus of a precursor protein by proteolytic cleavage. TGF-. beta.is a small secreted polypeptide that can signal through a type II serine/threonine kinase dimer receptor (TGF. beta. RII) that recruits and phosphorylates a type I dimer receptor (TGF. beta. RI). TGF β RI can phosphorylate and activate SMAD, a transcription factor that may regulate genes involved in cell proliferation, differentiation, apoptosis, and growth. Many advanced cancers are known to overexpress TGF- β and TGF β R, promoting the formation of invasive tumors. Inhibition of the TGFB signaling pathway may be a key therapeutic strategy for the treatment of cancer.

Some T cells (e.g., regulatory T cells) may release TGF-. beta.1 to inhibit the effects of other T cells. The activity of TGF-. beta.1 may prevent IL-1 and IL-2 dependent proliferation of activated T cells, as well as the activation of resting helper T cells and cytotoxic T cells. Similarly, TGF- β 1 can inhibit the secretion and activity of many other cytokines including, but not limited to, interferon- γ, tumor necrosis factor α (TNF- α), and various interleukins. It can also decrease the expression level of cytokine receptors such as IL-2 receptor, to down-regulate the activity of immune cells. However, TGF-. beta.1 may also increase the expression of certain cytokines in T cells and promote their proliferation, particularly in the case of immature cells (FIG. 2).

TGF-. beta.1 may have a similar effect on B cells, which may vary depending on the differentiation state of the cells. It can inhibit B cell proliferation and can stimulate B cell apoptosis, and can play a role in controlling the expression of antibodies, transferrin and MHC class II proteins on immature and mature B cells.

The effects of TGF-. beta.1 on macrophages and monocytes may be primarily inhibitory; the cytokine can inhibit proliferation of these cells and prevent them from producing reactive oxygen species (e.g., superoxide (O) ₂ ^-) And nitrogen (e.g., Nitric Oxide (NO)) intermediates. However, like other cell types, TGF-. beta.1 may also have an adverse effect on cells of myeloid origin. For example, TGF- β 1 may act as a chemotactic agent, directing an immune response to certain pathogens; macrophages and monocytes may respond in a chemotactic manner to low levels of TGF- β 1. In addition, the effect of TGF- β 1 may increase the expression of monocyte cytokines including IL-1 α, IL-1 β and TNF- α and phagocyte killing by macrophages (FIG. 2).

Transforming growth factor-beta III (TGF-beta 3) is a subset of the cytokine family, responsible for a variety of functions, including cell proliferation, embryogenesis, immune system regulation, and differentiation.

Transforming growth factor-beta receptor II (TGF. beta. RII)

The TGF-. beta.receptor (TGF. beta.R) is a single pass serine/threonine kinase receptor. They may exist in several different isoforms, which may be homodimeric or heterodimeric. The number of characterized ligands in the TGF- β superfamily can far exceed the number of known receptors, suggesting a confounding interaction between ligands and receptors. Three TGF-beta superfamily receptors (TGF. beta.R) that are specific for TGF-beta can be distinguished by their structural and functional properties. TGF β RI (ALK5) and TGF β RII may have similar ligand binding affinities and can only be distinguished from each other by peptide mapping. Both TGF-. beta.RI and TGF-. beta.RII have high affinity for TGF-. beta.1 and low affinity for TGF-. beta.2. TGF-. beta.RIII (β -glycan) may have high affinity for both homodimeric TGF-. beta.1 and TGF-. beta.2, and additionally for heterodimeric TGF-. beta.1, 2. TGF-beta receptors may also bind TGF-beta 3. By "TGF-beta RII" or "TGF-beta receptor II" is meant a polypeptide having a wild-type human TGF-beta receptor type 2 isoform A sequence (e.g., the amino acid sequence of NCBI reference sequence (RefSeq) accession NP-001020018 (SEQ ID NO:289)), or a wild-type human TGF-beta receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq accession NP-003233 (SEQ ID NO:290)) or a polypeptide having a sequence substantially identical to the amino acid sequence of SEQ ID NO:289 or SEQ ID NO: 290. The tgfbetarii may retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of the TGF β binding activity of the wild-type sequence. The expressed polypeptide of TGF β RII lacks a signal sequence.

TGF- β 1 may reduce MHC II efficacy in astrocytes and dendritic cells, which in turn reduces activation of appropriate helper T cell populations. TGF- β 1 may promote tumor growth as the cancer progresses and, in some embodiments, does not inhibit inflammatory cell responses, but may promote regulatory T cell function. TGF-. beta.1 may be produced by tumor cells, tumor-associated fibroblasts, regulatory T cells and immature myeloid cells. TGF-. beta.1 may inhibit T cell priming and promote a depleted phenotype. TGF-. beta.1 may inhibit the anti-tumor activity of innate immune cell populations including natural killer cells, macrophages and dendritic cells. TGF-beta receptor II may be upregulated by tumor-associated myeloid cells and may promote metastasis.

TGF-beta trap fusion proteins or fragments or variants thereof

Provided herein is a fusion protein, or fragment or variant thereof, comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor or an antibody, and a cytokine trap that can neutralize a cytokine (e.g., TGF- β). In certain instances, the cytokine trap may be a TGF- β trap (also referred to as TGF- β RII or a fragment or variant thereof) comprising SEQ ID No. 142. Examples of TGF- β traps may include, but are not limited to, the extracellular domain (ECD) of a receptor (e.g., TGF- β RII) or a functional variant or derivative thereof, a TGF- β inhibitory peptide (e.g., SEQ ID NO.193-227), or an anti-TGF- β antibody. In some embodiments, the anti-TGF-beta antibody comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) 166, 168, 169, 171, 173, 175, or 177, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to a sequence selected from any one of SEQ ID NOs. In some embodiments, the anti-TGF-beta antibody comprises a light chain variable region (V)_L) The light chain variable region (V)_L) 165, 167, 170, 172, 174, 176 or 178, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to a sequence selected from any one of SEQ ID NOs. In certain embodiments, the TGF- β trap may specifically bind to or have high affinity for TGF- β 1 or TGF- β 02 or TGF- β 13. In other embodiments, the TGF- β 2 trap may specifically bind to or have high affinity for TGF- β 31, TGF- β 2, and TGF- β 3. In other embodiments, the TGF- β trap may specifically bind to or have high affinity for TGF- β 1 and TGF- β 3. In further embodiments, the TGF- β trap may have a low affinity for TGF- β 2 or may not bind TGF- β 2.

The fusion proteins provided herein, or fragments or variants thereof (e.g., PD-1 inhibitors or antibodies fused to cytokine traps such as TGF- β traps), can elicit synergistic anti-tumor effects by simultaneously blocking the interaction between, for example, PD-L1 on tumor cells and PD-1 on immune cells, and neutralizing TGF- β, for example, in the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune escape mechanisms and the targeted depletion of TGF- β in the tumor microenvironment by single molecule entities. This consumption may be achieved by one or more of: (1) anti-PD-1 targeting of tumor cells; (2) binding of TGF- β traps (e.g., TGFbRII) to TGF- β in the tumor microenvironment; and/or (3) disruption of bound TGF-. beta.by PD-L1 receptor-mediated endocytosis. The fusion proteins provided herein, or fragments or variants thereof (e.g., PD-1 inhibitors or antibodies fused to cytokine traps such as TGF- β traps) can also promote natural killer cell-mediated killing of tumor cells.

(i) TGF-beta RII trap fusion proteins or fragments or variants thereof

In some embodiments, the fusion protein or fragment or variant thereof further comprises a cytokine trap and a PD-1 inhibitor or an anti-PD-1 antibody or fragment or variant thereof. In some embodiments is a fusion protein or fragment or variant thereof comprising a cytokine trap (e.g., a TGF- β trap) fused to a PD-1 inhibitor, optionally via a cleavable or non-cleavable linker. In some embodiments, the cytokine trap (e.g., TGF- β trap) is a cytokine receptor (e.g., TGF β RII). In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of a receptor (e.g., TGF β RII) or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of TGF β RII comprises a polypeptide sequence as set forth in SEQ ID NO: 14. In some embodiments, the cytokine receptor sequence in the fusion proteins described herein, or fragments or variants thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 14. In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of a receptor (e.g., TGF β RII) or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of a TGF β RII comprises a polypeptide sequence as set forth in SEQ ID NO: 141. In some embodiments, the cytokine receptor sequence in the fusion proteins described herein, or fragments or variants thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 141. In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of a receptor (e.g., TGF β RII) or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of a TGF β RII comprises a polypeptide sequence as set forth in SEQ ID NO: 142. In some embodiments, the cytokine receptor sequence in the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 142. In some embodiments, the cytokine receptor sequence in a fusion protein described herein, or a fragment or variant thereof, binds TGF-beta 1 and/or TGF-beta 3, but not TGF-beta 2. In certain embodiments, the cytokine receptor sequence in the fusion proteins described herein, or fragments or variants thereof, binds only TGF- β 1. In certain embodiments, the cytokine receptor sequence in the fusion proteins described herein, or fragments or variants thereof, binds only TGF- β 3. In certain embodiments, the cytokine receptor sequences in the fusion proteins described herein, or fragments or variants thereof, bind only TGF- β 1 and/or TGF- β 3, but have low or no affinity for TGF- β 2.

In some embodiments, the PD-1 antibody is fused to TGF β RII or a fragment thereof (e.g., the ECD of TGF β RII). In some embodiments, the PD-1 antibody moiety is fused to TGF β RII or a fragment thereof (e.g., the ECD of TGF β RII) via a linker. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGF β RII. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGF β RII via a linker.

In some embodiments, the PD-1 antibody fragment or variant is a Fab, Fab of the PD-1 antibody₂、(Fab’)₂、Fv、(Fv)₂、scFv、scFv-F_C、F_CA diabody, a triabody, or a minibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. In some embodiments, the single domain antibody is a V of a PD-1 antibody_NAROr V_HAnd (4) H fragment.

Non-limiting exemplary fusion proteins are shown in FIGS. 4A-4C. In some embodiments, a fusion protein comprising an anti-PD-1 antibody, or fragment or variant thereof, fused to a TGF- β trap may elicit a synergistic anti-tumor effect by simultaneously blocking the interaction between PD-L1 on tumor cells and PD-1 on immune cells, and neutralizing TGF- β in the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune escape mechanisms and the targeted depletion of TGF- β in the tumor microenvironment by single molecule entities. This consumption is achieved as follows: (1) PD-1 targeting of tumor cells; (2) binding of TGF- β traps (e.g., TGF β RII) to TGF- β in the tumor microenvironment; and (3) disruption of bound TGF-. beta.by PD-L1 receptor-mediated endocytosis.

In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is associated with a heavy chain variable region (V) of a PD-1 antibody or fragment/variant thereof_H) And (4) fusing. In other embodiments, the TGF- β trap is fused to IgG of the PD-1 antibody (e.g., fig. 4A). In certain aspects, the IgG is IgG1, IgG2, IgG3, or IgG 4. In embodiments, the IgG is IgG 4. In another embodiment, the IgG4 is SEQ ID NO:146 (wild type), SEQ ID NO:291, SEQ ID NO:292, or SEQ ID NO:147 (S108P). In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked to a heavy chain variable region (V) of a PD-1 antibody or fragment/variant thereof via a linker_H) And (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to V of a PD-1 antibody or fragment/variant thereof_HOfAnd (4) performing regional fusion. V of PD-1 antibody or fragment or variant thereof_HExamples of sequences include, but are not limited to, SEQ ID NOS 1-7 and 149-164. V of PD-1 antibody or fragment or variant thereof_LExamples of sequences include, but are not limited to, SEQ ID NOS 8-13 and 148. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is associated with a light chain variable region (V) of a PD-1 antibody or fragment/variant thereof_L) And (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to V of a PD-1 antibody or fragment/variant thereof _LThe constant region of (a) is fused. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked to a light chain variable region (V) of a PD-1 antibody or fragment/variant thereof via a linker_L) And (4) fusing. In one aspect, the TGF- β trap (e.g., TGF β RII) is fused to the N-terminus or C-terminus of the VL or VH chain or fragment/variant thereof via a linker.

The terms "anti-PD 1(VL/VH) -TGF β RII" or "anti-PD 1(VH/VL) -TGF β RII" are used interchangeably and refer to the particular VL or VH used in the fusion protein. In one embodiment, the term "anti-PD 1(VL/VH) -TGF β RII" or "anti-PD 1(VH/VL) -TGF β RII" refers to a TGF- β trap (e.g., TGF β RII) fused to the heavy chain constant region of anti-PD 1, or to a TGF- β trap (e.g., TGF β RII) fused to the light chain constant region of anti-PD 1.

In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to a Fab of a PD-1 antibody or fragment/variant thereof. In some embodiments, the TGF- β trap (e.g., TGF β RII) is fused to the Fab of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with a Fab of a PD-1 antibody or fragment/variant thereof₂And (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked to a Fab of a PD-1 antibody or fragment/variant thereof via a linker ₂And (4) fusing. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is conjugated to a PD-1 antibody or fragment/variant thereof (Fab')₂And (4) fusing. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is linked to a PD-1 antibody or fragment/variant thereof (Fab')₂And (4) fusing. In one aspect, a TGF-beta trap (e.g., TGF-beta RII) is linked to a Fab or (Fab')₂The N-terminus or the C-terminus of (1) is fused.

In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to an Fv of a PD-1 antibody or fragment/variant thereof. In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused via a linker to an Fv of a PD-1 antibody or fragment/variant thereof. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with a PD-1 antibody or fragment/variant thereof (Fv)₂And (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to a PD-1 antibody or fragment/variant thereof (Fv)₂And (4) fusing. In one aspect, a TGF-beta trap (e.g., TGF-beta RII) is linked via a linker to an Fv or (Fv) of a PD-1 antibody or fragment/variant thereof₂The N-terminus or the C-terminus of (1) is fused.

In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to an scFv of a PD-1 antibody or fragment/variant thereof. In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to an scFv of a PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with scFv-F of a PD-1 antibody or fragment/variant thereof _CAnd (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF- β RII) is linked to scFv-F of a PD-1 antibody or fragment/variant thereof via a linker_CAnd (4) fusing. In one aspect, the TGF- β trap (e.g., TGF β RII) is fused via a linker to the N-terminus or C-terminus of an scFv or scFv-Fc of a PD-1 antibody or fragment/variant thereof.

In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with F of a PD-1 antibody or fragment/variant thereof_CAnd (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked to F of a PD-1 antibody or fragment/variant thereof via a linker_CAnd (4) fusing. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is contacted with the C-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to the C-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting. In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is contacted with the N-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to the N-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting.

In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to a diabody of a PD-1 antibody or fragment/variant thereof. In some embodiments, the TGF- β trap (e.g., TGF β RII) is fused to the diabody of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to a triabody of a PD-1 antibody or fragment/variant thereof. In some embodiments, the TGF- β trap (e.g., TGF β RII) is fused to the triabody of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to a minibody of a PD-1 antibody or fragment/variant thereof. In some embodiments, a TGF- β trap (e.g., TGF β RII) is fused to a minibody of a PD-1 antibody or fragment/variant thereof via a linker. In one aspect, the TGF- β trap (e.g., TGF β RII) is fused via a linker to the N-terminus or C-terminus of a minibody of a PD-1 antibody or fragment/variant thereof.

In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with V of a PD-1 antibody or fragment/variant thereof_NARAnd (4) fusing. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to V of a PD-1 antibody or fragment/variant thereof_NARAnd (4) fusing. In all embodiments described, the TGF-beta trap (e.g., TGF-beta RII) is linked to the V of the PD-1 antibody or fragment/variant thereof via a linker_NARThe N-terminus or the C-terminus of (1) is fused.

In some embodiments, a TGF-beta trap (e.g., TGF-beta RII) is combined with V of a PD-1 antibody or fragment/variant thereof_HAnd H is fused. In some embodiments, a TGF- β trap (e.g., TGF β RII) is linked via a linker to V of a PD-1 antibody or fragment/variant thereof_HAnd H is fused. In all embodiments described, the TGF-beta trap (e.g., TGF-beta RII) is linked to the V of the PD-1 antibody or fragment/variant thereof via a linker_HH is fused at the N-terminus or C-terminus.

In some embodiments, the PD-1 antibody moiety is fused to TGF β RII or a fragment or variant thereof (e.g., the ECD of TGF β RII). In some embodiments, the PD-1 antibody moiety is fused to TGF β RII or a fragment or variant thereof (e.g., the ECD of TGF β RII) via a linker. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGF β RII. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGF β RII via a linker. Non-limiting exemplary fusion proteins are shown in FIGS. 4A-4C.

In some embodiments, the PD-1 antibody fragment is a Fab, Fab of the PD-1 antibody₂、(Fab’)₂、Fv、(Fv)₂、scFv、scFv-F_C、F_CA diabody, a triabody, or a minibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. In some embodiments, the single domain antibody is a V of a PD-1 antibody_NAROr V_HAnd (4) H fragment.

In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_H) Comprises one or more polypeptide sequences as shown in any one of SEQ ID NO 1-7. In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_H) Comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identity to any one of the polypeptide sequences as represented in SEQ ID Nos 1-7 and 149-164.

In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_L) Comprising one or more polypeptide sequences as set forth in any one of SEQ ID NOs 8-13 and 148. In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof _L) Comprising one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identity to any one of the polypeptide sequences set forth in SEQ ID Nos. 8-13 and 148.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprises the polypeptide sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO 12. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 12.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 15, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 16.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 16.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 15, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 143.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 143.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 15, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 294.

In embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 15 and the sequence shown as SEQ ID NO. 294.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO. 7, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 13. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V) _L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 7, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 13.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 296, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 145.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 145.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 296, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 144.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 144.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 296, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 295.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO:296 and the sequence shown as SEQ ID NO: 295.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 12, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 16.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 12, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 143.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 13, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 145. In some embodiments, the fusion protein comprises the sequence set forth as SEQ ID NO: 145.

In some embodiments, the fusion protein comprises a linker and a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO 16 and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 15. In some embodiments, the fusion protein comprises a linker and a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO 16, and the light chain variable region (V) _L) At least 50 percent, at least 55 percent and the sequence shown as SEQ ID NO. 15,At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 1. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 1.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 2. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 2.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 3. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 3.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 4. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 4.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 5. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 5.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 6. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 6.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 7. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 7.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprises the polypeptide sequence shown as SEQ ID NO. 5, and the light chainVariable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 5, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:149 and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:149, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:157, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The heavy chain canVariable region (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO:157, and the light chain variable region (V) _L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:158 and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 158, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 5. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 5.

In some embodiments, the fusion protein comprises a heavy chain variable region(V_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 150. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 150.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 151. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 80% of the sequence shown as SEQ ID NO. 15195%, at least 99%, at least 99.5% or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 152. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 152.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO 153. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 153.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 154. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 154.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence as shown in SEQ ID NO: 155. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65% of the sequence shown as SEQ ID NO. 155At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 156. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 156.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 159. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) And S159 the sequence depicted by EQ ID NO:159 is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 160. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 160.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 161. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 161.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 162. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 162.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 163. In some embodiments, the fusion eggThe white light comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 163.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 164. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 164.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a light chain variable region (V) _L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 9. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 9.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises the sequence as shown in SEQ IDPolypeptide sequence shown in NO. 10. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 10.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 11. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 11.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 12. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 12.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 13. In some embodiments, the fusion protein comprises a light chain variable region (V) _L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 13.

In some embodiments, the fusion protein comprises a light chain variableZone (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 148. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 148.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 15. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 15.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 16. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 16.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 143. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 143.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO: 144. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 144.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO: 145. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 145.

(ii) anti-TGF-beta antibody-fusion protein or fragment or variant thereof

In other embodiments, the cytokine trap is an antibody, antibody fragment, or antibody variant directed against TGF- β. In such embodiments, such antibodies may include pan-neutralizing anti-TGF antibodies or anti-receptor antibodies that block receptor binding to TGF β 1, 2, and/or 3. In certain embodiments, the antibody fragment or variant is a Fab, Fab of a TGF-beta antibody₂、(Fab’)₂、Fv、(Fv)₂、scFv、scFv-F_C、F_CA diabody, a triabody, or a minibody. In one embodiment, the anti-TGF-beta antibody, or fragment or variant thereof, binds to TGF-beta 1, TGF-beta 02, and TGF-beta 13. In certain embodiments, the anti-TGF-beta 2 antibody, or fragment or variant thereof, binds to TGF-beta 31. In certain embodiments, the anti-TGF-beta 4 antibody, or fragment or variant thereof, binds to TGF-beta 53. In certain embodiments, the anti-TGF-beta 6 antibody, or fragment or variant thereof, binds to TGF-beta 1 and TGF-beta 2. In certain embodiments, the anti-TGF-beta antibody, or fragment or variant thereof, binds to TGF-beta 1 and TGF-beta 3. Examples of VH sequences of TGF- β antibodies or fragments or variants thereof include, but are not limited to, SEQ ID nos. 166, 168, 169, 171, 173, 175, and 177. Examples of VL sequences of TGF- β antibodies or fragments or variants thereof include, but are not limited to, SEQ ID NOs 165, 167, 170, 172, 174, 176, and 178. In some embodiments, the anti-TGF-beta antibody comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least one amino acid sequence selected from any one of SEQ ID NOs 166, 168, 169, 171, 173, 175, or 177,At least 99%, at least 99.5% or 100% identical. In some embodiments, the anti-TGF-beta antibody comprises a light chain variable region (V)_L) The light chain variable region (V)_L) 165, 167, 170, 172, 174, 176 or 178, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical to a sequence selected from any one of SEQ ID NOs.

(iii) TGF-beta antagonistic peptide-fusion proteins or fragments or variants thereof

In certain embodiments, the cytokine trap may include a TGF- β antagonist peptide or a TGF- β inhibitor peptide. Such peptides can be generated de novo using phage display. In one embodiment, such peptides may be derived from a TGF- β isoform or segment of a TGF- β receptor, examples of TGF- β antagonist peptides may include, but are not limited to, SEQ ID NO. 193-227. Examples of TGF- β inhibitory peptides may include, but are not limited to, SEQ ID NO 193-227. In one embodiment, the peptide may be linked to the V of the PD-1 antibody or fragment/variant thereof via a linker _HAnd/or V_LAnd (4) fusing. In another embodiment, one peptide may be fused to a PD-1 antibody or fragment/variant thereof. In further embodiments, more than one peptide may be fused to a PD-1 antibody or fragment/variant thereof. When more than one peptide is used, the peptides may be fused to form concatemers with or without the use of a linker. When more than one peptide is used, concatemers may be formed using the same peptide. Alternatively, a combination of two or more different peptides may be used to form a concatemer.

Adenosine:

adenosine is a key immunomodulator in the tumor microenvironment. Extracellular adenosine inhibits inflammatory responses upon binding to the subtype adenosine receptor A2A (A2AR), which is predominantly expressed in most immune cells. Several tumors express high levels of CD39 and CD73, which are extracellular nucleotidases responsible for converting ATP and ADP to AMP and AMP to adenosine, respectively. Thus, adenosine promotes the inhibitory activity of regulatory T cells by inducing expression of Foxp3, CD39, and CD 73. In addition, hypoxia induces the accumulation of extracellular adenosine in the tumor microenvironment through the induction of CD39 and CD 73. In addition, high levels of extracellular adenosine in the tumor microenvironment are maintained by hypoxia-inducible factor (HIF) -dependent inhibition of the nucleotide transporter ENT-1 and by inhibition of adenosine kinase, which prevents the relocation of adenosine in the intracellular space and the formation of AMP, respectively. Thus, targeted reduction of extracellular adenosine in the tumor microenvironment is understood to enhance immune cell function and promote tumor cell killing.

In embodiments provided herein are fusion proteins comprising adenosine deaminase (e.g., ADA2) to target reduction of extracellular adenosine in a tumor microenvironment.

Adenosine deaminase

Adenosine deaminase (also known as adenosine aminohydrolase, or ADA) ADA irreversibly deaminates adenosine, converting it to the related nucleoside inosine by substituting an amino group for a keto group. Inosine can then be enucleated and glycosylated (removed from the ribose) with another enzyme called Purine Nucleoside Phosphorylase (PNP), which is converted to hypoxanthine. ADA is required for the breakdown of adenosine from food and the renewal of nucleic acids in tissues. Its main function in humans is to develop and maintain the immune system. However, ADA has also been found to be associated with epithelial cell differentiation, neurotransmission and pregnancy maintenance. ADA has 2 isoforms: ADA1 and ADA 2.

ADA1 is found in most body cells, particularly lymphocytes and macrophages, where ADA1 is not only present in cytosol and nucleus, but also on the cell membrane in extracellular form attached to dipeptidyl peptidase-4 (also known as CD 26). ADA1 is primarily involved in intracellular activity and exists in both small (monomeric) and large (dimeric) forms. The small to large format interconversions are regulated by "conversion factors" in the lungs.

ADA2 was first identified in human spleen. And subsequently found in other tissues, including macrophages, where it coexists with ADA 1. These two isoforms regulate the ratio of adenosine to deoxyadenosine. ADA2 is present mainly in human plasma and serum and exists mainly in homodimeric form. ADA2 is the predominant form present in human plasma, and is increasing in many diseases, especially those associated with the immune system: such as rheumatoid arthritis, psoriasis and sarcoidosis. In most cancers, plasma ADA2 isoform is also increased. ADA2 is not ubiquitous, but coexists with ADA1 in monocyte-macrophages.

ADA2 fusion proteins or fragments or variants thereof

Provided herein is a fusion protein, or fragment or variant thereof, comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor or an antibody, and an adenosine deaminase (e.g., ADA2) that can neutralize adenosine. The fusion proteins provided herein, or fragments or variants thereof (e.g., PD-1 inhibitors or antibodies fused to adenosine deaminase (e.g., ADA 2)), can elicit a synergistic anti-tumor effect by simultaneously blocking the interaction between PD-L1, e.g., on tumor cells, and PD-1 on immune cells, and neutralizing adenosine, e.g., in the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune escape mechanisms and the targeted depletion of adenosine by single molecule entities in the tumor microenvironment. This consumption may be achieved by one or more of: (1) anti-PD-1 targeting of tumor cells; (2) binding of adenosine deaminase (e.g., ADA2) to adenosine in the tumor microenvironment; and (3) disruption of bound adenosine by PD-L1 receptor-mediated endocytosis.

In some embodiments, the adenosine deaminase (e.g., ADA2) is part of a fusion protein or fragment or variant thereof that further comprises a PD-1 inhibitor or antibody or fragment or variant thereof. In some embodiments is a fusion protein or fragment or variant thereof comprising an adenosine deaminase (e.g., ADA2) optionally fused to a PD-1 inhibitor via a cleavable or non-cleavable linker. In some embodiments, the adenosine deaminase is adenosine deaminase 2(ADA 2). In some embodiments, the fusion proteins described herein comprise an adenosine deaminase (e.g., ADA2) described herein or a functional variant or derivative thereof. Examples of ADA2 and variants are described in WO 2016061286, which is incorporated herein by reference in its entirety. In some embodiments, the TGF- β cytokine trap comprises any one of ADA2 mutant 1, ADA2 mutant 2, ADA2 mutant 3, ADA2 mutant 4, ADA2 mutant 5, ADA2 mutant 6, or ADA2 mutant 7. In some embodiments, the fusion proteins provided herein, or fragments or variants thereof, comprise a PD-1 inhibitor or antibody fused to an ADA protein, or functional fragment thereof, via a linker. In some embodiments, the PD-1 inhibitor may be an antibody that targets PD-1 or a fragment or variant of the antibody.

In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 284. In some embodiments, the fusion protein or fragment or variant thereof described herein comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 273. In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 274. In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 37275. In some embodiments, the fusion protein or fragment or variant thereof described herein comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 276. In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 277. In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 278. In some embodiments, the fusion protein described herein, or a fragment or variant thereof, comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID No. 279.

In some embodiments, a fusion protein or fragment or variant thereof described herein comprises an adenosine deaminase (e.g., ADA2) as described above and an antibody, fragment or variant thereof that targets an immune checkpoint gene. In some embodiments, an antibody or fragment or variant of the antibody that targets an immune checkpoint such as cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1) may be fused to an adenosine deaminase (e.g., ADA2) via a linker.

In some embodiments, the PD-1 antibody moiety is fused to an adenosine deaminase (e.g., ADA2) or fragment thereof. In some embodiments, the PD-1 antibody moiety is fused to an adenosine deaminase (e.g., ADA2) via a linker. In some embodiments, the PD-1 antibody portion is fused to at least one domain of ADA 2. In some embodiments, the PD-1 antibody moiety is fused to at least one domain of ADA2 via a linker.

In some embodiments, the PD-1 antibody fragment or variant is a Fab, Fab of the PD-1 antibody₂、(Fab’)₂、Fv、(Fv)₂、scFv、scFv-F_C、F_CA diabody, a triabody, or a minibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody.In some embodiments, the single domain antibody is a V of a PD-1 antibody _NAROr V_HAnd (4) H fragment.

Non-limiting exemplary fusion proteins are shown in FIGS. 33A-33C. In some embodiments, a fusion protein comprising an anti-PD-1 antibody or fragment or variant thereof fused to an adenosine deaminase (e.g., ADA2) can elicit a synergistic anti-tumor effect by simultaneously blocking the interaction between PD-L1 on tumor cells and PD-1 on immune cells and targeted reduction of extracellular adenosine in the tumor microenvironment.

In some embodiments, adenosine deaminase (e.g., ADA2) is associated with the heavy chain variable region (V) of a PD-1 antibody or fragment/variant thereof_H) And (4) fusing. In other embodiments, the adenosine deaminase is fused to an IgG of a PD-1 antibody (e.g., fig. 4 a). In certain aspects, the IgG is IgG1, IgG2, IgG3, or IgG 4. In embodiments, the IgG is IgG 4. In another embodiment, the IgG4 is SEQ ID NO:146 (wild type), SEQ ID NO:291, SEQ ID NO:292, or SEQ ID NO:147 (S108P). In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof via a linker_H) And (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the V of the PD-1 antibody or fragment/variant thereof via a linker _HThe constant region of (a) is fused. In some embodiments, adenosine deaminase (e.g., ADA2) is combined with the light chain variable region (V) of a PD-1 antibody or fragment/variant thereof_L) And (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the light chain variable region (V) of the PD-1 antibody or fragment/variant thereof via a linker_L) And (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the V of a PD-1 antibody or fragment/variant thereof via a linker_LThe constant region of (a) is fused. In one aspect, the adenosine deaminase is conjugated to the V of the PD-1 antibody or fragment/variant thereof via a linker_HOr V_LThe N-terminus or the C-terminus of (1) is fused.

In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a Fab of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to the Fab of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, adenosine deaminase (e.g., ADA2) is combined with a Fab of a PD-1 antibody or fragment/variant thereof₂And (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to a Fab of a PD-1 antibody or fragment/variant thereof via a linker₂And (4) fusing. In some embodiments, adenosine deaminase (e.g., ADA2) is conjugated to a PD-1 antibody or fragment/variant thereof (Fab') ₂And (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to a (Fab' of a PD-1 antibody or fragment/variant thereof via a linker₂And (4) fusing. In one aspect, the adenosine deaminase is linked to a Fab or Fab of the PD-1 antibody or fragment/variant thereof via a linker₂The N-terminus or the C-terminus of (1) is fused.

In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to an Fv of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to the Fv of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, adenosine deaminase (e.g., ADA2) is combined with a PD-1 antibody or fragment/variant thereof (Fv)₂And (4) fusing. In some embodiments, adenosine deaminase (e.g., ADA2) is linked to a PD-1 antibody or fragment/variant thereof via a linker (Fv)₂And (4) fusing. In one aspect, the adenosine deaminase is linked via a linker to the Fv or (Fv) of the PD-1 antibody or fragment/variant thereof₂The N-terminus or the C-terminus of (1) is fused.

In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to an scFv of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to the scFv of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, adenosine deaminase (e.g., ADA2) is combined with scFv-F of PD-1 antibody or fragment/variant thereof _CAnd (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked via a linker to scFv-F of a PD-1 antibody or fragment/variant thereof_CAnd (4) fusing. In one aspect, the adenosine deaminase is linked to an scFv or scFv-F of a PD-1 antibody or fragment/variant thereof via a linker_CThe N-terminus or the C-terminus of (1) is fused.

In some embodiments, adenosine deaminase (e.g., ADA2) is combined with F of a PD-1 antibody or fragment/variant thereof_CAnd (4) fusing. In some embodimentsIn one embodiment, adenosine deaminase (e.g., ADA2) is linked via a linker to F of a PD-1 antibody or fragment/variant thereof_CAnd (4) fusing. In some embodiments, adenosine deaminase (e.g., ADA2) is associated with the C-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the C-terminal F of the PD-1 antibody or fragment/variant thereof via a linker_CAnd (4) connecting. In some embodiments, adenosine deaminase (e.g., ADA2) is conjugated to the N-terminal F of a PD-1 antibody or fragment/variant thereof_CAnd (4) connecting. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the N-terminal F of the PD-1 antibody or fragment/variant thereof via a linker_CAnd (4) connecting.

In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a diabody of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a diabody of a PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a triabody of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a triabody of the PD-1 antibody or fragment/variant thereof via a linker. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to a minibody of a PD-1 antibody or fragment/variant thereof. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to the minibody of the PD-1 antibody or fragment/variant thereof via a linker.

In some embodiments, adenosine deaminase (e.g., ADA2) is conjugated to V of a PD-1 antibody or fragment/variant thereof_NARAnd (4) fusing. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the V of a PD-1 antibody or fragment/variant thereof via a linker_NARAnd (4) fusing. In one aspect, the adenosine deaminase is conjugated to the V of the PD-1 antibody or fragment/variant thereof via a linker_NARThe N-terminus or the C-terminus of (1) is fused.

In some embodiments, adenosine deaminase (e.g., ADA2) is conjugated to V of a PD-1 antibody or fragment/variant thereof_HAnd H is fused. In some embodiments, the adenosine deaminase (e.g., ADA2) is linked to the V of a PD-1 antibody or fragment/variant thereof via a linker_HAnd H is fused. In one aspect, adenosine deaminase is prepared byV of PD-1 antibody or fragment/variant thereof by a linker_HH is fused at the N-terminus or C-terminus.

In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_H) Comprises one or more polypeptide sequences as shown in any one of SEQ ID NO 1-7. In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_H) Comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identity to any one of the polypeptide sequences as represented in SEQ ID Nos 1-7 and 149-164.

In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_L) Comprising one or more polypeptide sequences as set forth in any one of SEQ ID NOs 8-13 and 148. In some embodiments, the heavy chain variable region (V) of the PD-1 antibody or fragment/variant thereof_L) Comprising one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identity to any one of the polypeptide sequences set forth in SEQ ID Nos. 8-13 and 148.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprises the polypeptide sequence shown in SEQ ID NO 6, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 12. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO 6, and the light chain variable region (V) _L) At least 50 percent and at least 55 percent of the sequence shown in SEQ ID NO. 12At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 12, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 280.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 12 and the sequence shown as SEQ ID NO. 280.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 12, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 281.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 12 and the sequence shown as SEQ ID NO. 281.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO. 7, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 13. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50% and at least 5% of the sequence shown as SEQ ID NO. 75%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 13.

In some embodiments, the fusion protein comprises a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 13, and a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 282.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO. 13 and the sequence shown as SEQ ID NO. 282.

In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 13, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 283.

In some embodiments, the fusion protein comprises the sequence shown as SEQ ID NO 13 and the sequence shown as SEQ ID NO 283.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 1. In some embodiments, the fusion protein comprises heavyVariable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 1.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 2. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 2.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 3. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 3.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 4. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 4.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises the polypeptide shown as SEQ ID NO. 5And (4) sequencing. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 5.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 6. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 6.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 7. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 7.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H)，The heavy chain variable region (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 150. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 150.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 151. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 151.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 152. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 152.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO 153. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 153.

At one endIn some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 154. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 154.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence as shown in SEQ ID NO: 155. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 155.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 156. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 156.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 9% of the sequence shown as SEQ ID NO 157 9%, at least 99.5% or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 159. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 159.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V) _H) Comprises a polypeptide sequence shown as SEQ ID NO. 160. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 160.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 161. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70% of the sequence shown as SEQ ID NO. 161At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 162. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V) _H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 162.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 163. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 163.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 164. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 164.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) And SEQ ID NO. 8Is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 9. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 9.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 10. In some embodiments, the fusion protein comprises a light chain variable region (V) _L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 10.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 11. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 11.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 12. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 12.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 13. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 13.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 148. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 148.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 15. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 15.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO: 280. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 280.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 281. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO 281.

In some embodiments, the fusion protein comprises a polypeptide sequence as set forth in SEQ ID NO 282. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID No. 282.

In some embodiments, the fusion protein comprises the polypeptide sequence shown as SEQ ID NO: 283. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 283.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO. 5, and the variable region of the light chain (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 5, and the light chain variable region (V)_L) And SEQ ID NO. 8The indicated sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:149 and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:149, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:157, and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO:157, and the light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 8。

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) Comprising the polypeptide sequence shown in SEQ ID NO:158 and the light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence shown in SEQ ID NO. 158, and the light chain variable region (V) _L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 8.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 5. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 5.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149.

In some embodimentsThe fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a heavy chain variable region (V)_H) The variable region of the heavy chain (V)_H) Comprises a polypeptide sequence shown as SEQ ID NO. 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V) _H) The variable region of the heavy chain (V)_H) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO. 158.

In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) Comprises a polypeptide sequence shown as SEQ ID NO. 8. In some embodiments, the fusion protein comprises a light chain variable region (V)_L) The light chain variable region (V)_L) At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% identical to the sequence shown in SEQ ID NO. 8Or 100% identical.

Connecting body

In some embodiments, the linker comprises one or more polypeptide sequences as set forth in any one of SEQ ID NOs 17-34. In some embodiments, the linker can be a flexible linker. Flexible linkers can be used when the linked domains require some degree of movement or interaction. The flexible linker may be composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The flexible linker may have a sequence consisting essentially of stretches of Gly and Ser residues ("GS" linkers). Non-limiting examples of flexible linkers can have the sequence (Gly-Ser) n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. By adjusting the copy number "n", the length of this exemplary GS linker can be optimized to achieve proper isolation of functional domains or to maintain the necessary inter-domain interactions. In addition to the GS linker, other flexible linkers can be used for recombinant fusion proteins. In some embodiments, the flexible linker may have the sequence (Gly) n, where n may be 6, 7, or 8. In some cases, the flexible linker may also be rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In some cases, a linker described herein can be a rigid linker. Rigid linkers can be used to maintain a fixed distance between the domains of the fusion proteins described herein, or fragments or variants thereof. Non-limiting examples of rigid connectors may be: α -helix forming linker, Pro-rich sequence, (XP) n, X-Pro backbone, (EAAAK) n (n ═ 1-6). In some cases, a rigid linker may exhibit a relatively stiff structure by adopting an alpha-helical structure or by comprising multiple Pro residues. In some embodiments, an immune checkpoint inhibitor, such as a PD-1 inhibitor, and a cytokine trap (e.g., a TGF- β trap) that can neutralize a cytokine (e.g., TGF- β) in a fusion protein or fragment or variant thereof described herein, can be separated by an intervening sequence encoding an intervening linker polypeptide. In some embodiments, an immune checkpoint inhibitor, such as a PD-1 inhibitor, and ADA2 (or a mutant thereof) in a fusion protein or fragment or variant thereof described herein, can be separated by an intervening sequence encoding an intervening linker polypeptide. In certain embodiments, the linker polypeptide comprises the sequences disclosed in the following table:

TABLE 1 linker amino acid sequences and polynucleotide sequences

In some embodiments, the linker can be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some cases, the linker can link the functional domains together (as in flexible and rigid linkers) or release the free functional domains in vivo, as in an in vivo cleavable linker. In some embodiments, the linker can improve biological activity, increase expression yield, and achieve a desired pharmacokinetic profile. In some embodiments, the linker may further comprise a hydrazone, a peptide, a disulfide bond, or a thioether (thioester).

In some cases, a linker sequence described herein can include a flexible linker. Flexible linkers can be used when the linked domains require some degree of movement or interaction. The flexible linker may be composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The flexible linker may have a sequence consisting essentially of stretches of Gly and Ser residues ("GS" linkers). An example of a flexible linker may have the sequence (G4S) n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the flexible linker may have the sequence (Gly) n, where n may be 6, 7, or 8. In some cases, the flexible linker may also be rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu may be used to increase solubility. By adjusting the copy number "n", the length of these non-limiting exemplary linkers can be optimized to achieve proper separation of functional domains, or to maintain the necessary inter-domain interactions. In addition to GS linkers, other flexible linkers can also be used for the fusion proteins described herein or fragments or variants thereof. In some cases, the flexible linker may also be rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu may be used to increase solubility.

The flexible linkers included in the linker sequences described herein may be rich in small or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. When a fusion protein domain or fragment or variant thereof requires a particular movement or interaction, a flexible linker may be a suitable choice. In addition, although flexible connectors have no rigid structure, they can be used as inert connectors to maintain the distance between functional domains. The length of the flexible linker can be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein or fragment or variant thereof.

In some cases, a linker described herein can further comprise a rigid linker. Rigid linkers can be used to maintain a fixed distance between the domains of the fusion proteins described herein, or fragments or variants thereof. Examples of rigid connectors may be (to name a few): α -helix forming linker, Pro-rich sequence, (XP) n, X-Pro backbone, (EAAAK) n (n ═ 1-6). In some cases, a rigid linker may exhibit a relatively stiff structure by adopting an alpha-helical structure or by comprising multiple Pro residues.

In some embodiments, the linker described herein can be a cleavable linker. In other cases, the linker is not cleavable. The non-cleavable linker can covalently join together the functional domains of the fusion protein or fragments or variants thereof to act as one molecule in the whole in vivo process or in vitro process. The linker may also be cleavable in vivo. Cleavable linkers can be introduced to release the free functional domain in vivo. To name a few, the cleavable linker can be cleaved by the presence of a reducing agent, a protease. For example, reduction of disulfide bonds can be used to generate cleavable linkers. In the case of disulfide linkers, cleavage can occur by a cleavage event that exchanges with a disulfide bond of a thiol such as glutathione. In other cases, in vivo cleavage of the linker in the recombinant fusion protein may also be performed by proteases that may be expressed in vivo in specific cells or tissues or that are confined to certain cellular compartments under pathological conditions (e.g., cancer or inflammation). In some cases, a cleavable linker may allow for targeted cleavage. For example, the specificity of many proteases may provide for slower linker cleavage in the constrained compartment. The cleavable linker may also comprise a hydrazone, a peptide, a disulfide bond or a thioether (thioester). For example, hydrazones can confer serum stability. In other cases, the hydrazone may allow cleavage in the acidic compartment. The acidic compartment may have a pH of up to 7. The linker may also include a thioether. The thioether may be non-reducible. Thioethers can be designed for intracellular proteolytic degradation.

In some cases, the linker can be an engineered linker. For example, the linker can be designed to include a chemical property, such as hydrophobicity. In some cases, at least two different linker polypeptide sequences may encode the same polypeptide linker sequence. The method of designing the linker may be computational. In some cases, the computational method may include graphical techniques. Computational methods can be used to search for suitable peptides from a library of three-dimensional peptide structures derived from a database. For example, a Brookhaven Protein Data Bank (PDB) can be used to span the spatial distance between selected amino acids of the linker. In some cases, the polypeptide linker may further comprise one or more GS linker sequences, such as (G4S) n, (GS) n, (SG) n, (GSG) n, and (SGSG) n, where n may be any number from zero to fifteen.

Methods of treating cancer

Also provided herein is a method of treating cancer with a fusion protein comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor, and a cytokine trap (e.g., TGF- β trap) that can neutralize a cytokine (e.g., TGF- β). Also provided herein is a method of treating cancer with a fusion protein comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor, and an adenosine deaminase protein. The development of monoclonal antibodies that target blocking immune checkpoint pathways such as the PD1 and PD-L1/2 signaling pathways, as well as the CTLA-4 and CD80/86 signaling pathways, has revolutionized cancer therapy, which has shown sustained clinical activity in a variety of cancer indications including, but not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, MSI high colorectal cancer, Merkel cell carcinoma, and hodgkin lymphoma. Despite the persistent response, the response rate is still very low and several patients develop resistance leading to disease progression. Furthermore, checkpoint inhibitors fail to show any substantial clinical response in several indications such as ovarian, gastroesophageal, prostate, pancreatic and many others.

The failure of checkpoint blockade can be attributed to the complexity of immunosuppressive factors present in the tumor microenvironment. These factors may include, but are not limited to, inhibitory cells, such as myeloid derived inhibitory cells, Tumor Associated Macrophages (TAMs); inhibitory cytokines and growth factors, such as TGF-beta and interleukin 10 (IL-10); and metabolic derivatives such as adenosine and indoleamine 2, 3-dioxygenase (IDO) by-products. The anti-PD 1-TGF β RII fusion proteins provided herein are examples of therapies that can target two negative inhibitory pathways in the tumor microenvironment. These pathways may include tumor cell-mediated intracellular interactions, of which the PD-1/PD-L1 interaction may play a major role, and immunosuppressive cytokine-mediated extracellular interactions, of which TGF- β may be a major member.

In some embodiments, the cancer is, but is not limited to, glioblastoma, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, and renal cancer. In addition, the fusion proteins described herein may be useful for indications such as in non-small cell cancers (NSCLs) and melanomas where checkpoint blockade is less responsive and TGF- β is highly expressed.

Cancers include, but are not limited to, B cell cancers, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary, small or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancers of blood tissues, and the like. Other non-limiting examples of types of cancers suitable for use in the methods encompassed by the present disclosure include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, cervical cancer, and non-malignant tumors of the human brain, Lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (hodgkins and non-hodgkins), multiple myeloma, waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the methods of the present disclosure is an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, larynx cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways, including but not limited to serous, endometrioid, mucinous, clear cell, brenner-type (brenner), or undifferentiated. In some embodiments, the present disclosure is used for the treatment, diagnosis and/or prognosis of lymphoma or a subtype thereof (including but not limited to mantle cell lymphoma).

In certain embodiments, an anti-PD 1-TGF β RII fusion protein provided herein is an example of a therapy that can be used to treat cancer with an average response rate of about 0, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to standard therapy (including but not limited to chemotherapy, and current clinical trial therapy). In certain embodiments, the anti-PD 1-ADA2 fusion proteins provided herein are examples of therapies that can be used to treat cancers with an average response rate of about 0, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to standard therapies (including but not limited to chemotherapy, and current clinical trial therapies). Such cancers include, but are not limited to, hodgkin's lymphoma, melanoma, non-small cell lung cancer (NSCLC), solid tumors with high microsatellite instability (MSI) or mismatch repair (MMR) deficiency, CSCC, RCC, CRC, melanoma, Merkel cell carcinoma, bladder cancer, RCC, hepatocellular carcinoma (HCC), head and neck cancer (H & N), cervical cancer, gastric cancer, Small Cell Lung Cancer (SCLC), endometrial cancer, mesothelioma, ovarian cancer, Triple Negative Breast Cancer (TNBC), breast cancer, colorectal cancer (CRC), pancreatic cancer, prostate cancer.

Combination therapy

In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a biomolecule, such as an antibody. For example, the treatment may involve administering the fusion protein in combination with an antibody directed against a tumor associated antigen (including but not limited to an antibody that binds EGFR, HER2/ErbB2, and/or VEGF). In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab (ramucirumab), trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (vectib), nimotuzumab (nimotuzumab), zalutumumab (zalutumumab), or cetuximab (ERBITUX). In some embodiments, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment may involve administering a fusion protein provided herein, or a fragment or variant thereof, in combination with a small molecule that acts as an inhibitor against tumor-associated antigens, including but not limited to EGFR, HER2(ErbB2), and/or VEGF. In some embodiments, the fusion protein or fragment or variant thereof is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), Sunitinib (SUTENT), lapatinib (lapatanib), vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR) and pazopanib (GW 786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor. In another embodiment, the additional therapeutic agent is a T-decrease _REGChemotherapy or other inhibitors of cell number. In certain embodiments, the therapeutic agent is cyclophosphamide or an anti-CTLA 4 antibody. In another embodiment, the additional therapeutic agent reduces the presence of myeloid-derived suppressor cells. In a further embodiment, the additional therapeutic agent is carboplatin paclitaxel (carbopaxol). In advance ofIn one embodiment, the additional therapeutic agent is ibrutinib.

In some embodiments, the methods may further comprise one or more checkpoint inhibitors in combination with a fusion protein described herein or a fragment or variant thereof. In some embodiments, the additional checkpoint inhibitor may be an anti-CTLA-4 antibody. anti-CTLA-4 antibodies (e.g., ipilimumab) showed persistent anti-tumor activity and prolonged survival in late stage melanoma participants, and were therefore approved by the U.S. Food and Drug Administration (FDA) in 2011. See Hodi et al, Improved survival with ipilimumab in properties with metastatic melanoma.N Engl J Med. (2010)8 months and 19 days; 363(8):711-23. In some embodiments, the one or more checkpoint inhibitors may be an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody may be full-length atelizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), or a fragment or variant thereof. In some embodiments, the one or more checkpoint inhibitors may be any one or more of a CD27 inhibitor, a CD28 inhibitor, a CD40 inhibitor, a CD122 inhibitor, a CD137 inhibitor, an OX40 (also referred to as CD134) inhibitor, a GITR inhibitor, an ICOS inhibitor, or any combination thereof. In some embodiments, the one or more checkpoint inhibitors may be any one or more of an A2AR inhibitor, a B7-H3 (also known as CD276) inhibitor, a B7-H4 (also known as VTCN1) inhibitor, a BTLA inhibitor, an IDO inhibitor, a KIR inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, or any combination thereof.

In certain embodiments, the additional therapeutic agent comprises a second immunotherapeutic agent. In some embodiments, the additional immunotherapeutic agent includes, but is not limited to, colony stimulating factor, interleukins, antibodies that block immune suppression function (e.g., anti-CTLA-4 antibodies, anti-CD 28 antibodies, anti-CD 3 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies), antibodies that enhance immune cell function (e.g., anti-GITR antibodies, anti-OX-40 antibodies, anti-CD 40 antibodies, or anti-4-1 BB antibodies), toll-like receptors (e.g., TLR4, TLR7, TLR9), soluble ligands (e.g., GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4-1BB ligand, or 4-1BB ligand-Fc), or B7 family members (e.g., CD80, CD 86). In some embodiments, the additional immunotherapeutic agent targets CTLA-4, CD28, CD3, PD-L1, TIGIT, GITR, OX-40, CD-40, or 4-1 BB.

In some embodiments, the additional therapeutic agent is an additional immune checkpoint inhibitor. In some embodiments, the additional immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD 28 antibody, an anti-TIGIT antibody, an anti-LAG 3 antibody, an anti-TIM 3 antibody, an anti-GITR antibody, an anti-4-1 BB antibody, or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody selected from BMS935559(MDX-1105), alexizumab (atexolizumab) (MPDL3280A), devolizumab (MEDI4736), and avizumab (MSB 0010718C). In some embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody selected from ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from BMS-986016 and LAG 525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from MEDI6469, MEDI0562, and MOXR 0916. In some embodiments, the additional therapeutic agent is an anti-4-1 BB antibody selected from PF-05082566. In some embodiments, the fusion protein or fragment or variant thereof may be administered in combination with a biomolecule selected from the group consisting of: cytokines, Adrenomedullin (AM), angiogenin (Ang), BMP, BDNF, EGF, Erythropoietin (EPO), FGF, GDNF, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), Stem Cell Factor (SCF), GDF9, HGF, HDGF, IGF, migration stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF- α, TGF- β, TNF- α, VEGF, PlGF, γ -IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

Cytokine

In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant or combination thereof. In some cases, the cytokine is an interleukin. In some cases, the interleukin is at least one of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and functional variants and fragments thereof. In some embodiments, the cytokine may be membrane bound or secreted. In embodiments, the cytokine is soluble IL-15, soluble IL-15/IL-15 Ra complex (e.g., ALT-803). In some cases, the interleukin may comprise membrane-bound IL-15(mbiL-15) or a fusion of IL-15 and IL-15 Ra. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15, which can be co-expressed with the modified immune effector cells described herein. In some embodiments, the mbIL-15 comprises a full length IL-15 (e.g., a native IL-15 polypeptide), or a fragment or variant thereof, fused in-frame to a full length IL-15 ra, fragment or variant thereof. In some cases, the IL-15 is indirectly linked to IL-15R α via a linker. In some cases, the mbiL-15 is as described in Hurton et al, "thermal IL-15 additives activity and promoters a stem-cell memory subset in molecular-specific T cells," PNAS 2016. In some cases, the cytokine is expressed in the same immune effector cell as the CAR.

In some embodiments, mbIL-15 is expressed with a cell tag such as HER1t, HER-1t-1, CD20t-1, or CD20 as described herein. mbiL-15 may be expressed in-frame with HER1t, HER-1t-1, CD20t-1 or CD 20.

In some embodiments, mbIL-15 may be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, an inducible promoter can be a small molecule ligand inducible gene switch based on a two-polypeptide ecdysone receptor, e.g.A gene switch.

In addition toIn one aspect, the interleukin can comprise IL-12. In some embodiments, the IL-12 is a single chain IL-12(scIL-12), protease sensitive IL-12, destabilized IL-12, membrane bound IL-12, embedded IL-12. In some cases, IL-12 variants are described in WO2015/095249, WO2016/048903, WO2017/062953, all of which are incorporated herein by reference. In some embodiments, the cytokines described above may be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, an inducible promoter can be a small molecule ligand inducible gene switch based on a two-polypeptide ecdysone receptor, e.g. A gene switch.

In some embodiments, a fusion protein as described herein can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, an inducible promoter can be a small molecule ligand inducible gene switch based on a two-polypeptide ecdysone receptor, e.g.A gene switch.

Gene switch

Provided herein are gene switch polypeptides, polynucleotides encoding ligand-inducible gene switch polypeptides, and methods and systems comprising these polypeptides and/or polynucleotides. The term "gene switch" refers to a combination of a response element associated with a promoter, e.g., with an ecdysone receptor (EcR) -based system, which regulates the expression of a gene incorporating the response element and promoter in the presence of one or more ligands. Tightly regulated inducible gene expression systems or gene switches can be used for a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and modulation of traits in transgenic plants and animals. Such inducible gene expression systems may include ligand inducible heterologous gene expression systems.

Early forms of EcR-based gene switches used Drosophila melanogaster (Drosophila melanogaster) EcR (dmecr) and mouse (Mus musculus) rxr (mmrxr) polypeptides, and showed that these receptors transactivated reporter genes in mammalian cell lines and transgenic mice in the presence of steroids, pertussis a (Christopherson et al, 1992; No et al, 1996). Subsequently, Suhr et al, 1998 showed that the non-steroidal ecdysone agonist tebufenozide induces high levels of counter-activation of the reporter gene in mammalian cells by the silkworm (Bombyx mori) EcR (BmEcR) in the absence of an exogenous heterodimer partner.

International patent applications PCT/US97/05330(WO 97/38117) and PCT/US99/08381(WO99/58155) disclose methods of modulating the expression of a foreign gene wherein a DNA construct comprising the foreign gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor which binds to the ecdysone response element in the presence of its ligand, optionally in the presence of a receptor capable of acting as a silencing partner, to induce gene expression. In this example, the ecdysone receptor was isolated from Drosophila melanogaster. Generally, such systems require the presence of a silencing partner, preferably a Retinoid X Receptor (RXR), to provide optimal activation. In mammalian cells, insect ecdysone receptors (ecrs) are capable of heterodimerization with mammalian Retinoid X Receptors (RXRs) for modulating the expression of target or heterologous genes in a ligand-dependent manner. International patent application PCT/US98/14215(WO 99/02683) discloses that ecdysone receptors isolated from silkworm moths are functional in mammalian systems, without the need for a foreign dimer partner.

Us patent 6,265,173 discloses that individual members of the steroid/thyroid superfamily of receptors can be combined with drosophila melanogaster supercoiled receptor (USP) or fragments thereof comprising at least a USP dimerization domain for use in gene expression systems. U.S. Pat. No. 5,880,333 discloses the drosophila melanogaster EcR and supercoiled (USP) heterodimer system for use in plants, wherein the transactivation domain and the DNA binding domain are located on two different hybrid proteins. In each of these cases, the transactivation domain and the DNA binding domain (such as the native EcR in international patent application PCT/US98/14215 or the modified EcR in international patent application PCT/US 97/05330) are incorporated into a single molecule, and the other heterodimerization partner (USP or RXR) is used in its native state.

International patent application PCT/US01/0905 discloses an ecdysone receptor-based inducible gene expression system in which the transactivation domain and the DNA binding domain are separated from each other by placing them on two different proteins, resulting in a substantial decrease in background activity in the absence of ligand and a significant increase in activity over background in the presence of ligand. This two-hybrid system is a significantly improved inducible gene expression regulation system compared to the two systems disclosed in applications PCT/US97/05330 and PCT/US 98/14215. It is believed that this two-hybrid system takes advantage of the following ability of a pair of interacting proteins: they position the transcriptional activation domain more favorably relative to the DNA binding domain so that the transactivation domain more efficiently activates the promoter when the DNA binding domain binds to a DNA binding site on a gene (see, e.g., U.S. patent 5,283,173). This two-hybrid gene expression system comprises two gene expression cassettes; the first encodes a DNA binding domain fused to a nuclear receptor polypeptide, and the second encodes a transactivation domain fused to a different nuclear receptor polypeptide. In the presence of the ligand, it is thought that a conformational change is induced which facilitates the interaction of the antibody with the TGF- β cytokine trap, leading to dimerization of the DNA binding domain and the transactivation domain. Since the DNA binding domain and the transactivation domain are located on two different molecules, the background activity is greatly reduced in the absence of ligand.

In addition, the two-hybrid system avoids some side effects due to over-expression of RXR that may occur when using unmodified RXR as a switching partner.

Ecdysone receptors (ecrs) are members of the nuclear receptor superfamily and are classified as subfamily 1, group H (referred to herein as "group H nuclear receptors"). The members of each group have 40-60% amino acid identity in the E (ligand binding) domain (Laudet et al, A Unified Nomenclature System for the nucleic acid Receptor subunit, 1999; Cell 97: 161-163). Other members of this nuclear receptor subfamily 1, group H, in addition to the ecdysone receptor, include: ubiquitin Receptor (UR), orphan receptor 1(OR-1), steroid hormone nuclear receptor 1(NER-1), RXR interacting protein-15 (RIP-15), liver x receptor beta (LXR beta), steroid hormone receptor-like protein (RLD-1), liver x receptor (LXR alpha), Farnesoid X Receptor (FXR), receptor interacting protein 14(RIP-14), and farnesoid receptor (HRR-1).

In some cases, an inducible promoter ("IP") can be a small molecule ligand inducible gene switch based on the two-polypeptide ecdysone receptor, such as that of Intrexon CorporationA gene switch. In some cases, the gene switch may be selected from ecdysone-based receptor components, as described in, but not limited to, any of the systems described below: PCT/US2001/009050(WO 2001/070816); U.S. Pat. nos. 7,091,038; 7,776,587, respectively; 7,807,417, respectively; 8,202,718, respectively; PCT/US2001/030608(WO 2002/029075); us patent 8,105,825; 8,168,426, respectively; PCT/US52002/005235(WO 2002/066613); U.S. application 10/468,200 (U.S. publication 20120167239); PCT/US2002/005706(WO 2002/066614); us patent 7,531,326; 8,236,556, respectively; 8,598,409, respectively; PCT/U52002/005090(WO 2002/066612); us patent 8,715,959 (us publication 20060100416); PCT/US2002/005234(WO 2003/027266); us patent 7,601,508; 7,829,676, respectively; 7,919,269, respectively; 8,030,067, respectively; PCT/U52002/005708(WO 2002/066615); U.S. application 10/468,192 (U.S. publication 20110212528); PCT/US2002/005026(WO 2003/027289); us patent 7,563,879; 8,021,878, respectively; 8,497,093, respectively; PCT/US2005/015089(WO 2005/108617); us patent 7,935,510; 8,076,454, respectively; PCT/U52008/011270(WO 2009/045370); U.S. application 12/241,018 (U.S. publication 20090136465); PCT/US2008/011563(WO 2009/048560); U.S. application 12/247,738 (U.S. publication 20090123441); PCT/US2009/005510(WO 2010/042189); U.S. application 13/123,129 (U.S. publication 20110268766); PCT/US2011/029682(WO 2011/119773); U.S. application 13/636,473 (U.S. publication 20130195800); PCT/US2012/027515(WO 2012/122025); WO 2018/132494(PCT/US 2018/013196); and U.S. patent 9,402,919; each of which is incorporated by reference in its entirety.

As used herein, the term "ligand" as applied to a ligand-activated ecdysone receptor-based gene switch is a small molecule (such as a diacylhydrazine compound) with varying solubility that has the ability to activate the gene switch to stimulate gene expression (i.e., wherein ligand-induced expression of a polynucleotide (e.g., mRNA, miRNA, etc.) and/or polypeptide is provided). Examples of such ligands include, but are not limited to, the following: WO 2004/072254(PCT/US 2004/003775); WO 2004/005478(PCT/US 2003/021149); WO 2005/017126(PCT/US 2004/005149); WO 2004/078924(PCT/US 2004/005912); WO 2008/153801(PCT/US 2008/006757); WO 2009/114201(PCT/US 2009/001639); WO 2013/036758(PCT/US 2012/054141); WO 2014/144380(PCT/US 2014/028768); and WO 2016/044390(PCT/US 2015/050375); each of which is incorporated by reference herein in its entirety.

Examples of ligands also include, but are not limited to: ecdysteroids such as ecdysone, 20-hydroxyecdysterone, ponasterone a, etc., 9-cis-retinoic acid, synthetic analogs of retinoic acid, N' -diacylhydrazines such as those disclosed in U.S. Pat. nos. 6,258,603, 6,013,836, 5,117,057, 5,530,028, 5,378,726, 7,304,161, 7,851,220, 8,748,125, 9,272,986, 7,456,315, 7,563,928, 8,524,948, 9,102,648, 9,169,210, 9,255,273, and 9,359,289; oxadiazolines, as described in U.S. patents 8,669,072 and 8,895,306; dibenzoylalkylcyanohydrazines, such as those disclosed in european application 2,461,809; N-alkyl-N, N' -diarylhydrazines such as those disclosed in U.S. patent 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in european application 234,994; N-aroyl-N-alkyl-N' -aroylhydrazines such as those described in U.S. patent 4,985,461; aminoketones such as those described in U.S. Pat. nos. 7,375,093, 8,129,355, and 9,802,936; each of the above is incorporated herein by reference, as well as other similar materials, including 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetyl harpagide, oxysterol, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5- α -6- α -epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, farnesol (framesol), bile acids, 1-diphosphonate, juvenile hormone III, and the like. Examples of diacylhydrazine ligands that may be used in the present invention include RG-115819(3, 5-dimethyl-benzoic acid N- (1-ethyl-2, 2-dimethyl-propyl) -N' - (2-methyl-3-methoxy-benzoyl) -hydrazide-), RG-115932((R) -3, 5-dimethyl-benzoic acid N- (1-tert-butyl) -N '- (2-ethyl-3-methoxy-benzoyl) -hydrazide) and RG-115830(3, 5-dimethyl-benzoic acid N- (1-tert-butyl) -N' - (2-ethyl-3-methoxy-benzoyl) -hydrazide). See, for example, WO 2008/153801(PCT/US2008/006757) and WO 2013/036758(PCT/US2012/054141), both of which are incorporated herein by reference in their entirety.

For example, the ligand of the ecdysone receptor-based gene switch may be selected from any suitable ligand. Naturally occurring ecdysone or ecdysone analogs (e.g., 20-hydroxyecdysone, velvetidone a, pertussis ecdysone B, pertussis ecdysone C, 26-iodopertussis ecdysone a, hyssoproctone, or 26-methanesulfonyl hyssoproctone) and non-steroid inducing agents can be used as ligands for the gene switch of the present invention. U.S. Pat. No. 6,379,945 describes an insect steroid receptor ("HEcR") isolated from Heliothis virescens (Heliothis virescens) that is capable of acting as a gene switch that is reactive to both steroids and certain non-steroid inducing agents. In this and many other systems that are responsive to both steroid and non-steroid inducers, non-steroid inducers have distinct advantages over steroids for several reasons, including, for example: low production cost, metabolic stability, absence from insects, plants or mammals, and environmental acceptability. U.S. Pat. No. 6,379,945 describes the use of two dibenzoylhydrazines, 1, 2-dibenzoyl-1-tert-butylhydrazine and tebufenozide (N- (4-ethylbenzoyl) -N '- (3, 5-dimethylbenzoyl) -N' -tert-butylhydrazine) as ligands for an ecdysone-based gene switch. Other dibenzoylhydrazines are also included as ligands in the present invention, such as those disclosed in U.S. patent 5,117,057. The use of tebufenozide as a chemical ligand for the ecdysone receptor from Drosophila melanogaster (Drosophila melanogaster) is also disclosed in us patent 6,147,282. Other non-limiting examples of ecdysone ligands are 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, 1, 2-diacylhydrazine, N '-substituted-N, N' -disubstituted hydrazine, dibenzoylalkylhydrazines, N-substituted-N-alkyl-N, N-diarylhydrazines, N-substituted-N-acyl-N-alkyl, carbohydrazide or N-aroyl-N '-alkyl-N' -arylhydrazines. (see U.S. patent 6,723,531).

In one embodiment, the ligand of the ecdysone-based gene switch system is a diacylhydrazine ligand or a chiral diacylhydrazine ligand. The ligand used in the gene switch system may be a compound of formula I

Wherein A is alkoxy, arylalkoxy or aryloxy; b is optionally substituted aryl or optionally substituted heteroaryl; and R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

In another embodiment, the ligand may be an enantiomerically enriched compound of formula II

Wherein a is alkoxy, arylalkoxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl; b is optionally substituted aryl or optionally substituted heteroaryl; and R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; provided that R1 is not equal to R2; wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly S; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

In certain embodiments, the ligand may be an enantiomerically enriched compound of formula III

Wherein a is alkoxy, arylalkoxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl; b is optionally substituted aryl or optionally substituted heteroaryl; and R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; provided that R1 is not equal to R2; wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly R; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

In one embodiment, the ligand may be (R) -3, 5-dimethyl-benzoic acid N- (1-tert-butyl) -N' - (2-ethyl-3-methoxy-benzoyl) -hydrazide in at least 95% enantiomeric excess, or a pharmaceutically acceptable salt, hydrate, crystalline form, or amorphous form thereof.

When used with an ecdysone-based gene switch system, the diacylhydrazine ligand of formula I and the chiral diacylhydrazine ligand of formula II or III provide a means for externally temporally modulating the expression of a therapeutic polypeptide or therapeutic polynucleotide of the invention. See U.S. patent nos.: 8,076,517, respectively; 8,884,060, respectively; and 9,598,355; each of which is fully incorporated herein by reference.

The ligands used in the present invention may form salts. The term "salt" as used herein denotes acid and/or base salts formed with inorganic and/or organic acids and bases. In addition, when a compound of formula I, II or III contains both a basic moiety and an acidic moiety, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e. non-toxic, physiologically acceptable) salts are used, but other salts are also useful, for example in isolation or purification steps which may be employed in the preparation process. Salts of compounds of formula I, II or III can be formed, for example, by reacting the compound with an amount (e.g., equivalent) of an acid or base in a medium (such as the medium in which the salt precipitates) or an aqueous medium, followed by lyophilization.

Ligands containing basic moieties can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetate salts (e.g., formed with acetic acid or trihaloacetic acids such as trifluoroacetic acid), adipate salts, alginate salts, ascorbate salts, aspartate salts, benzoate salts, benzenesulfonate salts, bisulfate salts, borate salts, butyrate salts, citrate salts, camphorate salts, camphorsulfonate salts, cyclopentanepropionate salts, digluconate salts, dodecylsulfate salts, ethanesulfonate salts, fumarate salts, glucoheptonate salts, glycerophosphate salts, hemisulfate salts, heptanoate salts, hexanoate salts, hydrochloride salts (formed with hydrochloric acid), hydrobromide salts (formed with hydrogen bromide), hydroiodide salts, 2-hydroxyethanesulfonate salts, lactate salts, maleate salts (formed with maleic acid), methanesulfonate salts (formed with methanesulfonic acid), 2-naphthalenesulfonate salts, nicotinate salts, nitrate salts, oxalate salts, pectate salts, persulfate salts, 3-phenylpropionate salts, Phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (e.g., with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, tosylates, undecanoates, and the like.

Ligands containing acidic moieties can form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts, such as sodium, lithium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as benzathine, dicyclohexylamine, abamectin (formed from N, N-bis (dehydroabietyl) ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and salts with amino acids such as arginine, lysine and the like.

Non-limiting examples of ligands for inducible gene expression systems also include those that utilize the FK506 binding domain, which is FK506, cyclosporin a or rapamycin. FK506, rapamycin, and analogs thereof are disclosed in U.S. patents 6,649,595, 6,187,757, 7,276,498, and 7,273,874.

In some embodiments, the diacylhydrazine ligand for inducible gene expression is administered in a unit daily dose of about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 120 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 5 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 10 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 15 mg. In some embodiments, the diacylhydrazine ligand is administered at a unit daily dose of about 20mg per day.

In some embodiments, combination therapy with two or more therapeutic agents may use agents that act through different mechanisms of action, although this is not required. Combination therapy with agents having different mechanisms of action may cause additive or synergistic effects. Combination therapy may allow for lower doses of each agent than are used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agents. Combination therapy can reduce the likelihood of development of drug resistant cancer cells. In some embodiments, the combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.

In certain embodiments, the methods or treatments further comprise administering at least one additional therapeutic agent in addition to administering the fusion protein or fragment or variant thereof described herein. The additional therapeutic agent may be administered prior to, concurrently with, and/or after administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Therapeutic agents that can be administered in combination with the fusion proteins described herein, or fragments or variants thereof, include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments involve administering an agent described herein in combination with a chemotherapeutic agent or in combination with a mixture of chemotherapeutic agents. In some embodiments, the methods can further comprise one or more antineoplastic agents, such as cisplatin, capecitabine, or 5-fluorouracil, in combination with the fusion proteins, or fragments or variants thereof, described herein. Treatment with the agent may be performed before, simultaneously with, or after administration of chemotherapy. Co-administration may include co-administration in a single pharmaceutical formulation or using separate formulations, or sequential administration in either order, but typically is carried out over a period of time such that all active agents can exert their biological activities simultaneously. The preparation and dosing regimen for such chemotherapeutic agents can be used according to manufacturer's instructions or determined empirically by the skilled artisan. Preparation and dosing regimens for such Chemotherapy are also described in The Chemotherapy Source Book, 4 th edition, 2008, m.c. per eds, Lippincott, Williams & Wilkins, philiadelphia, PA.

Useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, orlistatin (auristatin), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono (platinum), di (platinum), and tri-nuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycin (duocarmycin), etoposide, fluorinated pyrimidines, ionophores, lexitropin (lexitrophin), nitrosoureas, pravastatin (platinol), purine antimetabolites, puromycin (puromycin), radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids (vinca alkaloids), and the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic agent, a topoisomerase inhibitor, or an angiogenesis inhibitor.

The fusion proteins provided herein, or fragments or variants thereof, can be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy, and/or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated). For example, a panel of tumor antigens can be used, for example, in most cancer patients.

Fusion proteins associated with chimeric receptors

In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chimeric receptor, such as a chimeric antigen receptor or an engineered T cell receptor. For example, treatment may include the simultaneous administration of the fusion protein and the chimeric receptor. In one embodiment, the treatment may comprise co-administering the fusion protein with a modified effector cell comprising a chimeric receptor. In one embodiment, the treatment may comprise sequential administration of the modified effector cells comprising the chimeric receptor, followed by administration of the fusion protein. In another embodiment, treatment may comprise sequential administration of the fusion protein followed by administration of the modified effector cells comprising the chimeric receptor. In one aspect, there may be a lag of at least 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 24 hours between administrations. In another aspect, there can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 75, 90 or more days between administrations.

In some embodiments, the modified immune effector cell is a modified immune cell comprising a T cell and/or a natural killer cell. T cells or T lymphocytes are a subset of leukocytes that are involved in cell-mediated immunity. Exemplary T cells include T helper cells, cytotoxic T cells, TH17 cells, memory stem cell-like T cells (TSCMs), naive T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells. In certain aspects, embodiments described herein include making and/or expanding modified immune effector cells (e.g., T cells, tregs, NK cells, or NK T cells) comprising transfecting the cells with an expression vector comprising a DNA (or RNA) construct encoding a chimeric receptor.

In some embodiments, described herein include modified effector cells comprising a chimeric receptor expressed on the surface of a cell. In some cases, the chimeric receptor comprises an antigen-binding region that is capable of recognizing and binding a tumor antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). In some cases, the antigen binding region comprises an antibody or binding fragment, e.g., Fab ', F (ab')₂、F(ab’)₃、scFv、sc(Fv)₂dsFv, diabodies, minibodies, and nanobodies or binding fragments thereof. In some cases, the antigen-binding region comprises an scFv. In some cases, the chimeric receptor comprises an scFv (e.g., a Chimeric Antigen Receptor (CAR)). In some cases, the chimeric antigen receptor comprises a pattern recognition receptor. In other cases, the chimeric receptor comprises an engineered T Cell Receptor (TCR).

Further provided herein is an immune effector cell comprising a cell signature that functions as a kill switch, a selectable marker, a biomarker, or a combination thereof. In some embodiments, the cell tag comprises HER1t, HER1t-1, CD20t-1, or CD 20. In some cases, the cell tag comprises HER1t, and the HER1t comprises the polypeptide sequence of SEQ ID NO: 68. In some cases, the cell tag comprises HER1t-1, and the HER1t-1 comprises the polypeptide sequence of SEQ ID NO: 69.

Chimeric Antigen Receptor (CAR)

Chimeric Antigen Receptors (CARs) are engineered receptors that specifically transplant foreign bodies onto immune effector cells. In some cases, the CAR comprises an extracellular domain (ectodomain) comprising an antigen binding domain, a stem region, a transmembrane domain, and an intracellular domain (endodomain). In some cases, the intracellular domain further comprises one or more intracellular signaling domains. In some cases, a CAR described herein comprises an antigen binding domain, a stalk region, a transmembrane domain, one or more co-stimulatory domains, and a signaling domain for T cell activation.

The antigen binding domain may comprise a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and/or an antigen binding fragment thereof. Complementarity Determining Regions (CDRs) are short amino acid sequences found in the variable domains of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that are complementary to the antigen, thus providing the receptor with specificity for that particular antigen. Each polypeptide chain of an antigen receptor may contain three CDRs (CDR1, CDR2, and CDR 3). In some cases, the antigen binding domain comprises F (ab') ₂Fab', Fab, Fv or scFv. In some cases, the antigen binding domain is an scFv. In some cases, the antigen binding domain is a Fab. In some cases, the antigen binding domain is a Fab. In some cases, the antigen binding domain is F (ab')₂. In some cases, the antigen binding domain is an Fv.

In some embodiments, the CARs described herein comprise an antigen binding domain that binds to an epitope on CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor alpha, mucins such as MUC-1, MUC-4, or MUC-16, MAGE-a1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In one embodiment, the CAR described herein comprises an antigen binding domain capable of binding to an epitope on MUC 16. In some embodiments, the CARs described herein comprise an antigen binding domain that binds to an epitope on CD19 or CD 33. In some cases, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD 19. In some cases, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD 33. In further embodiments, the CARs described herein comprise a self-antigen or antigen-binding region that binds to an epitope on HLA-a2, Myelin Oligodendrocyte Glycoprotein (MOG), factor viii (fviii), MAdCAM1, SDF1, or collagen type II.

In some embodiments, the CARs and methods described herein can be used to treat hyperproliferative diseases, such as cancer, autoimmune diseases, or to treat infections, such as viral, bacterial, or parasitic infections. In some aspects, the CAR targets an antigen that is elevated in a cancer cell, an autoimmune cell, or a cell infected with a virus, bacterium, or parasite. Pathogens that may be targeted include, but are not limited to, plasmodium, trypanosoma, Aspergillus (Aspergillus), Candida (Candida), hepatitis a, hepatitis b, hepatitis c, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or ebola pathogens. Autoimmune diseases may include graft versus host disease, rheumatoid arthritis, lupus, celiac disease, crohn's disease, sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris or autoimmune uveitis.

The pathogen recognized by the CAR can be essentially any kind of pathogen, but in some embodiments, the pathogen is a fungus, a bacterium, or a virus. Exemplary viral pathogens include viral pathogens of the following families: adenoviridae, EB virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HPV viridae, HSV viridae, HHV viridae, hepatitis viridae, picornaviridae, herpesviridae, hepadnaviridae, flaviviridae, retroviridae, orthomyxoviridae, paramyxoviridae, papovaviridae, polyomaviruses, rhabdoviridae, and togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, ebola, and rubella. Exemplary pathogenic fungi include candida, aspergillus, cryptococcus, histoplasma, pneumocystis, and stachybotrys. Exemplary pathogenic bacteria include streptococcus, pseudomonas, shigella, campylobacter, staphylococcus, helicobacter, escherichia coli, rickettsia, bacillus, bordetella, chlamydia, spirochete, and salmonella. In some embodiments, the pathogen receptor Dectin-1 can be used to generate CARs that recognize carbohydrate structures on the cell wall of fungi, such as aspergillus. In another embodiment, the CAR can be made based on antibodies that recognize viral determinants (e.g., glycoproteins from CMV and ebola) to disrupt viral infection and pathology.

In some embodiments, the antigen binding domain is linked to the transmembrane domain using a "stem region" or "spacer" or "hinge" region. In some cases, a "stem domain" or "stem region" includes any oligonucleotide or polypeptide that functions to link a transmembrane domain to a cytoplasmic domain in an extracellular domain or polypeptide chain. In some embodiments, it is flexible enough to allow the antigen binding domains to be oriented in different directions, thereby facilitating antigen recognition. In some cases, the stem region comprises a hinge region from IgG 1. In alternative cases, the stem region comprises the CH2CH3 region of an immunoglobulin and optionally part of CD 3. In some cases, the stem region comprises a CD8 a hinge region, an IgG4-Fc 12 amino acid hinge region (ESKYGPPCPPCP), or an IgG4 hinge region, as described in WO/2016/073755.

The transmembrane domain may be from a natural or synthetic source. Where the source is native, the domain may be derived from any membrane bound or transmembrane protein. Suitable transmembrane domains may include transmembrane regions of the α, β or zeta chain of the T cell receptor; or a transmembrane region from CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. Alternatively, the transmembrane domain may be synthetic and may comprise hydrophobic residues, such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at one or both termini of the synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker of 2 to 10 amino acids in length in some embodiments may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In some embodiments, the linker is a glycine-serine linker.

In some embodiments, the transmembrane domain comprises a CD8 a transmembrane domain or a CD3 ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 a transmembrane domain. In other embodiments, the transmembrane domain comprises a CD3 zeta transmembrane domain.

The intracellular domain may comprise one or more co-stimulatory domains. Exemplary co-stimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more or two or more of the co-stimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof. In some cases, a CAR described herein comprises one or more or two or more of the co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, OX40(CD134), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more or two or more of the costimulatory domains selected from CD8, CD28, 4-1BB (CD137), or a fragment or combination thereof. In some cases, a CAR described herein comprises one or more or two or more of the co-stimulatory domains selected from CD28, 4-1BB (CD137), or a fragment or combination thereof. In some cases, a CAR described herein comprises co-stimulatory domains CD28 and 4-1BB (CD137), or respective fragments thereof. In some cases, a CAR described herein comprises co-stimulatory domains CD28 and OX40(CD134), or respective fragments thereof. In some cases, a CAR described herein comprises co-stimulatory domains CD8 and CD28, or respective fragments thereof. In some cases, a CAR described herein comprises co-stimulatory domain CD28 or a fragment thereof. In some cases, a CAR described herein comprises co-stimulatory domain 4-1BB (CD137) or a fragment thereof. In some cases, a CAR described herein comprises co-stimulatory domain OX40(CD134) or a fragment thereof. In some cases, a CAR described herein comprises co-stimulatory domain CD8 or a fragment thereof.

In some embodiments, the endodomain further comprises a signaling domain for T cell activation. In some cases, the signaling domain for T cell activation comprises a domain derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, or CD66 d. In some cases, the signaling domain for T cell activation comprises a domain derived from CD3 ζ.

In some embodiments, the CAR described herein is administered to a subject with one or more additional therapeutic agents, including but not limited to cytokines described herein. In further embodiments, immune effector cells expressing a CAR described herein express membrane-bound IL-15 ("mIL-15 or mbIL-15"). In an aspect of the invention, the mbIL-15 comprises a fusion protein between IL-15 and IL-15 ra. In further embodiments, the mbIL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID No. 69. In some cases, the CAR and cytokine are expressed in separate vectors. In particular cases, the vector may be a lentiviral vector, a retroviral vector, or a Sleeping Beauty transposon.

CD 19-specific CAR

CD19 is a cell surface glycoprotein of the immunoglobulin superfamily and is found primarily in malignant B lineage cells. In some cases, CD19 has also been detected in solid tumors such as pancreatic, liver, and prostate cancers.

In some embodiments, described herein include a CD 19-specific CAR, wherein the antigen binding domain comprises a F (ab')₂Fab', Fab, Fv or scFv. In some cases, the antigen binding domain recognizes an epitope on CD 19. In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by FMC 63. In some embodiments, the scFv and/or VH/VL domain is derived from FMC 63. FMC63 is typically a mouse monoclonal IgG1 antibody directed against Nalm-l and-16 cells expressing human-derived CD19 (Ling, N.R. et al (1987) Leucocyte typing III.302). In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, described herein include CD 19-specific CAR-T cells comprising an scFv antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, the CD 19-specific CAR-T cells described herein comprise an anti-CD 19 antibody described in US 20160152723. In some embodiments, the CD 19-specific CAR-T cells described herein comprise an anti-CD 19 antibody described in WO 2015/123642. In some embodiments, the CD 19-specific CAR-T cells described herein comprise an anti-CD 19 scFv derived from clone FMC63 (Nicholson et al Construction and characterization of a functional CD19 specific chain Fv fragment for immunology of B line leukaemia and lymphoma. mol. immunol.,34:1157-1165, 1997).

In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19(Kite Pharma, Inc.). In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, described herein include CD 19-specific CAR-T cells comprising a scFv antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19, which is also recognized by KTE-C19. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, the CD 19-specific CAR-T cells described herein comprise an anti-CD 19 antibody described in WO2015187528, or a fragment or derivative thereof.

In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL 019. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, CD 19-specific CAR-T cells comprising an scFv antigen-binding domain are included as described herein, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL 019. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (celectis). In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART 19. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, CD 19-specific CAR-T cells comprising a scFv antigen-binding domain are included as described herein, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART 19. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, described herein include CD 19-specific CAR-T cells comprising an scFv antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some cases, the antigen binding domain recognizes an epitope on CD19 that is also recognized by rituximab (Amgen), certolizumab (colleximabravtansine) (ImmunoGen Inc./Sanofi-aventis), MOR208(Morphosys AG/Xencor Inc.), MEDI-551 (medimum), denudinuzumab (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplimumab (tagumomapapptox) (National Cancer Institute), XmAb 5871(Amgen/Xencor, med.), MDX-1342 (Amgen/Xencor, Inc.), or AFM11 (Affimed). In some cases, the CD 19-specific CAR further comprises a transmembrane domain selected from the group consisting of a CD8 a transmembrane domain or a CD3 zeta transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some embodiments, described herein include CD 19-specific CAR-T cells, wherein the antigen-binding domain comprises a F (ab')₂Fab', Fab, Fv or scFv. In some cases, the antigen binding domain recognizes an epitope on CD 19. In some cases, the antigen binding domain recognizes an epitope on CD19 that is also recognized by rituximab (Amgen), certolizumab (colleximabravtansine) (ImmunoGen Inc./Sanofi-aventis), MOR208(Morphosys AG/Xencor Inc.), MEDI-551 (medimum), denudinuzumab (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplimumab (tagumomapapptox) (National Cancer Institute), XmAb 5871(Amgen/Xencor, med.), MDX-1342 (Amgen/Xencor, Inc.), or AFM11 (Affimed). In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

In some cases, CD 19-specific CAR-T cells described herein comprise a scFv antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by FMC63, rituximab (Amgen), crotuzumab (ImmunoGen Inc./Sanofi-aventis), MOR208(Morphosys AG/Xencor Inc.), MEDI-551 (medimumne), dinning chart mab (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taprimomab (National Cancer Institute), XmAb 5871(Amgen/Xencor, Inc.), MDX-1342(Medarex), or AFM11 (impacted). In some cases, the CD 19-specific CAR-T cell further comprises a transmembrane domain selected from the CD8 a transmembrane domain or the CD3 ζ transmembrane domain; one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40(CD134), or a fragment or combination thereof; and the signaling domain from CD3 ζ.

CD 33-specific CAR

"CD 33" is a 67kDa single transmembrane glycoprotein and is a member of the sialic acid binding immunoglobulin-like lectin (Siglecs) superfamily. CD33 is characterized by a V-group Ig-like domain responsible for sialic acid binding and a C2-group Ig-like domain in its extracellular domain. Alternative splicing of CD33 mRNA results in a shorter isoform (CD33m) lacking the V-group Ig-like domain and the disulfide bond linking the V-group and C2-group Ig-like domains. In healthy subjects, CD33 is expressed primarily as a myeloid differentiation antigen found on normal pluripotent bone marrow precursors, unipotent colony forming cells, monocytes, and mature granulocytes. CD33 is expressed on more than 80% of myeloid leukemia cells, but not on normal hematopoietic stem cells or mature granulocytes. (Andrews, R. et al, The L4F3 anti is expressed by units and multi-potential colony-forming cells but by The r precursors, Blood,68(5):1030-5 (1986)). CD33 has been reported to be expressed on malignant myeloid cells, activated T cells and activated NK cells, and is found on at least a portion of the blast cells of the vast majority of AML patients (Pollard, J. et al, correction of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin binding chemistry in childhood AML, Blood,119(16):3705-11 (2012)). In addition to widespread expression on AML blasts, CD33 can also be expressed on stem cells that lead to AML.

In embodiments, the antigen binding portion of a CAR described herein is specific for CD33 (CD33 CAR). When expressed on the cell surface, CD 33-specific CARs redirect T cell specificity to human CD 33. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a target antigen-specific monoclonal anti-CD 33 antibody linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is M195, M2H12, DRB2 and/or My 9-6. In embodiments, the scFv is humanized, e.g., hM 195. In some embodiments, the antigen-binding portion may comprise a directionally linked VH and VL, e.g., from N-terminus to C-terminus, VH-linker-VL, or VL-linker-VH.

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:35 (hM195 VL).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:36 (hM195 VH).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:37 (M2H12 VH).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:38 (M2H12 VL).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:39 (DRB2 VH).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:40 (DRB2 VL).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:41 (My9-6 VH).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:42 (My9-6 VL).

MUC 16-specific CAR

MUC16 is a large carbohydrate antigen, also known as CA-125. MUC16 is encoded by the MUC16 gene located on human chromosome 19. MUC16 is a highly glycosylated multidomain type I transmembrane protein comprising 3 domains. The C-terminal domain comprises a plurality of extracellular SEA (echinospasmin, enterokinase and agrin) modules with self-proteolytic activity. SEA has two proteolytic sites in the vicinity of the Transmembrane (TM) domain. The cleaved large domain, designated CA-125, is released into the circulation at acidic pH. CA-125 is commonly used as a disease biomarker for ovarian cancer. The highly conserved truncated outer-membrane-bound protein domain is called the MUCl6ecto domain. An antibody to MUC16 was identified that specifically binds the extracellular domain of MUC16 retained on the surface of tumor cells. By "MUC 16 overexpression" of a cell of interest (e.g., a cancer cell) is meant that the cell of interest expresses higher levels of MUC16 protein and/or mRNA as compared to a control cell (e.g., a non-cancer cell, a normal cell, etc.).

In embodiments, the antigen binding portion of a CAR described herein is specific for MUC16 (MUC16 CAR). When expressed on the cell surface, MUC 16-specific CARs redirect the specificity of T cells to human MUC 16. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising the light chain variable domain (VL) and the heavy chain variable domain (VH) of a target antigen-specific monoclonal anti-MUC 16 antibody linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is MUC16-1scFv (SEQ ID NOS: 43-44), MUC16-2 scFv (SEQ ID NOS: 45-46), MUC16-3 scFv (SEQ ID NOS: 47-48), MUC16-4 scFv (SEQ ID NOS: 49-50), MUC16-5 scFv (SEQ ID NOS: 51-52), MUC16-6 scFv (SEQ ID NOS: 53-54), or MUC16-7 scFv (SEQ ID NOS: 55-56). In embodiments, the scFv is humanized.

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:43 (MUC 16-1).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:44 (MUC 16-1).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:45 (MUC 16-2).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:46 (MUC 16-2).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:47 (MUC 16-3).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:48 (MUC 16-3).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:49 (MUC 16-4).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:50 (MUC 16-4).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:51 (MUC 16-5).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:52 (MUC 16-5).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:53 (MUC 16-5).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:54 (MUC 16-6).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:55 (MUC 16-7).

In embodiments, a CAR described herein comprises an antigen binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:56 (MUC 16-7).

In some embodiments, the antigen-binding portion may comprise a directionally linked VH and VL, e.g., from N-terminus to C-terminus, VH-linker-VL, or VL-linker-VH.

Engineered T Cell Receptor (TCR)

In some embodiments, the chimeric receptor comprises an engineered T cell receptor. The T Cell Receptor (TCR) consists of two chains (α β or γ δ) that pair on the T cell surface to form a heterodimeric receptor. In some cases, α β TCRs are expressed on most T cells in vivo and are known to be involved in the recognition of specific MHC-restricted antigens. Each α and β chain is composed of two domains: a constant domain (C) that anchors the protein to the cell membrane and associates with an invariant subunit of the CD3 signaling transducer; and a variable domain (V) that confers antigen recognition through six loops called Complementarity Determining Regions (CDRs). In some cases, each V domain comprises three CDRs; for example, CDR1, CDR2, and CDR3, with CDR3 as the hypervariable region. These CDRs interact with complexes formed by binding of antigenic peptides to proteins encoded by the major histocompatibility complex (pepmchc), e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, or HLA-DRB1 complexes. In some cases, the constant domain further comprises a junction region connecting the constant domain to the variable domain. In some cases, the beta strand further comprises a shorter diversity region that forms part of the junction region.

In some cases, such TCRs are reactive to a particular tumor antigen (e.g., NY-ESO, Mage a3, Titin). In other cases, such TCRs are reactive to a particular neoantigen expressed within a patient's tumor (i.e., patient-specific mutations, somatic mutations, non-synonymous mutations expressed by the tumor). In some cases, the engineered TCR may be affinity-enhanced.

In some embodiments, the TCR is described using the international Immunogenetics (IMGT) TCR nomenclature, and is linked to the IMGT public database of TCR sequences. For example, there may be several types of alpha chain variable (V α) regions and several types of beta chain variable (V β) regions, distinguished by their framework, CDR1, CDR2, and CDR3 sequences. Thus, the va type may be designated in IMGT nomenclature as a unique TRAV number. For example, "TRAV 21" defines the TCR va region with unique framework and CDR1 and CDR2 sequences as well as CDR3 sequences, which CDR3 sequences are defined in part by amino acid sequences conserved between TCRs but also include amino acid sequences that vary between TCRs. Similarly, "TRBV 5-1" defines a TCR ν β region with unique framework and CDR1 and CDR2 sequences, as well as CDR3 sequences that are only partially defined.

In some cases, the beta strand diversity region is indicated by the abbreviation TRBD in the IMGT nomenclature.

In some cases, the unique sequences defined by IMGT nomenclature are well known and accessible to those working in the TCR field. For example, they can be found in IMGT public database and "T cell Receptor facebook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

In some embodiments, for example, the α β heterodimeric TCR is transfected as a full-length chain having a cytoplasmic domain and a transmembrane domain. In some cases, the TCRs contain an introduced disulfide bond between residues of the respective constant domains, as described, for example, in WO 2006/000830.

In some cases, the TCRs described herein are in single chain form, e.g., see WO 2004/033685. The single chain forms include α β TCR polypeptides of the type V α -L-V β, V β -L-V α, V α -C α -L-V β, V α -L-V β -C β, V α -C α -L-V β -C β, wherein V α and V β are TCR α and β variable regions, respectively, C α and C β are TCR α and β constant regions, respectively, and L is a linker sequence. In certain embodiments, the single chain TCRs of the invention may have an introduced disulfide bond between residues of the respective constant domains, as described in WO 2004/033685.

The TCRs described herein can be associated with a detectable label, a therapeutic agent, or a PK modifying moiety.

Exemplary detectable labels for diagnostic purposes include, but are not limited to, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

Therapeutic agents that may be associated with the TCRs described herein include immunomodulators, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents. To ensure that toxic effects are exerted at the desired location, the toxin may be within a liposome attached to the TCR, such that the compound is released in a controlled manner. In some cases, controlled release minimizes disruption during transport in vivo and ensures that the toxin has maximal effect after binding of the TCR to the relevant antigen presenting cell.

In some embodiments, other suitable therapeutic agents include, for example:

a. small molecule cytotoxic agents, such as compounds having the ability to kill mammalian cells having a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or convert under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rebeccamycin (rachelmycin), calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphor II, temozolomide, topotecan, tritetrazol (citrate gluconate), reoxidin e (auristatin e), vincristine, and doxorubicin;

b. A peptide cytotoxin, i.e., a protein or fragment thereof having the ability to kill mammalian cells. For example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, and rnase;

c. radionuclides, i.e., unstable elemental isotopes that decay with the emission of one or more of alpha or beta particles or gamma rays. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213; chelators may be utilized to facilitate the association of these radionuclides with high affinity TCRs or multimers thereof;

d. an immunostimulant, i.e., an immune effector molecule that stimulates an immune response. For example, cytokines, such as IL-2 and IFN-gamma,

e. superantigens and mutants thereof;

tcr-HLA fusions;

g. chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, and the like;

h. antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD 3, anti-CD 28, or anti-CD 16);

i. alternative protein scaffolds with antibody-like binding properties

j. A complement activator; and

k. heterologous protein domains, allogeneic protein domains, viral/bacterial peptides.

Dosage form

The appropriate dosage of the fusion protein and modified immune effector cells used will depend on the age and weight of the subject and the particular drug used. The dosage and treatment regimen of the fusion protein and the modified immune effector cells can be determined by the skilled artisan.

In certain embodiments, the fusion protein is administered by injection (e.g., subcutaneously or intravenously) at a dose of 1 to 30mg/kg, e.g., about 5 to 25mg/kg, about 10 to 20mg/kg, about 1 to 5mg/kg, or about 3 mg/kg. In some embodiments, the fusion protein is administered at a dose of about 1mg/kg, about 3mg/kg, about 5mg/kg, about 10mg/kg, about 20mg/kg, about 30mg/kg, or about 40 mg/kg. In some embodiments, the fusion protein is administered at a dose of about 1-3mg/kg or about 3-10 mg/kg. In some embodiments, the fusion protein is administered at a dose of about 0.5-2, 2-4, 2-5, 5-15, or 5-20 mg/kg. The dosing schedule may vary from, for example, once a week to once every 2, 3, or 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 10 to 20mg/kg every other week. In another embodiment, the fusion protein is administered at a dose of about 1mg/kg once every two weeks, about 3mg/kg once every two weeks, 10mg/kg once every two weeks, 3mg/kg once every four weeks, or 5mg/kg once every four weeks.

In other embodiments, the fusion protein is administered by injection (e.g., subcutaneously or intravenously) at a dose (e.g., flat dose) of about 200mg to 500mg, e.g., about 250mg to 450mg, about 300mg to 400mg, about 250mg to 350mg, about 350mg to 450mg, or about 300mg or about 400 mg. In some embodiments, the fusion protein is administered at a dose of about 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, or about 500 mg. In some embodiments, the fusion protein is administered at a dose of 200 or 300 mg. In some embodiments, the fusion protein is administered at a dose of about 250-450mg or about 300-400 mg. In some embodiments, the fusion protein is administered at a dose of about 200-300mg, 250-350mg, 300-400mg, 350-450mg, or 400-500 mg. The dosing schedule may vary from, for example, once a week to once every 2, 3, 4, 5, or 6 weeks. In one embodiment, the fusion protein is administered at a dose of about 300mg to 400mg once every three weeks or once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 300mg once every three weeks. In one embodiment, the fusion protein is administered at a dose of about 400mg once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 300mg once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 400mg once every three weeks. The fusion protein may be administered one or more times, e.g., once, twice, three times, four times, five times, six times, seven times, or more. In one embodiment, the fusion protein is administered six times. The fusion protein can be administered at least 5 days, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 25, 30, 35, or 40 days after administration of the CAR-expressing cell, e.g., MUC16, CD33, CD19, or BCMA-specific CAR-expressing cell. In some embodiments, the fusion protein can be administered about 8 days or about 15 days after administration of the CAR-expressing cell, e.g., a MUC 16-specific CAR-expressing cell or a CD 33-specific CAR-expressing cell.

The fusion protein may be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. For example, the fusion protein may be administered by intravenous infusion at a rate of greater than 20mg/min, such as 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve a dose of about 35 to 440mg/m2, typically about 70 to 310mg/m2, and more typically about 110 to 130mg/m 2. In embodiments, the fusion protein may be administered by intravenous infusion at a rate of less than 10mg/min, preferably less than or equal to 5mg/min, to achieve a dose of about 1 to 100mg/m2, preferably about 5 to 50mg/m2, about 7 to 25mg/m2, and more preferably about 10mg/m 2.

The fusion protein may be administered by intravenous infusion at a rate of greater than 20mg/min, for example 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve a dose of about 35 to 440mg/m2, typically about 70 to 310mg/m2, more typically about 110 to 130mg/m 2. In embodiments, an infusion rate of about 110 to 130mg/m2 reaches a level of about 3 mg/kg. In other embodiments, the fusion protein may be administered by intravenous infusion at a rate of less than 10mg/min, e.g., less than or equal to 5mg/min, to achieve a dose of about 1 to 100mg/m2, e.g., about 5 to 50mg/m2, about 7 to 25mg/m2, or about 10mg/m 2. In some embodiments, the antibody is infused over a period of about 30 minutes.

Dose of modified effector cells

In some embodiments, an amount of the modified effector cells is administered to a subject in need thereof, and the amount is determined based on efficacy and potential to induce cytokine-related toxicity. In some cases, the amount of modified effector cells comprises about 10⁵To about 10⁹Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁵To about 10⁸Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁵To about 10⁷Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁶To about 10⁹Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁶To about 10⁸Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁷To about 10⁹Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁵To about 10⁶Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁶To about 10⁷Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10 ⁷To about 10⁸Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁸To about 10⁹Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁹Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁸Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁷Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁶Individual modified effector cells/kg. In some cases, the amount of modified effector cells comprises about 10⁵Individual modified effector cells/kg.

In some embodiments, the modified effector cell is a modified T cell. In some cases, the modified T cell is a CAR-T cell. In some cases, the amount of CAR-T cells comprises about 10⁵To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁵To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁵To about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10 ⁶To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁶To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁷To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁵To about 10⁶Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁶To about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁷To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁸To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁸Individual CAR-T cells/kg. In some casesIn some cases, the amount of CAR-T cells comprises about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁶Individual CAR-T cells/kg. In some cases, the amount of CAR-T cells comprises about 10⁵Individual CAR-T cells/kg.

In some embodiments, the CAR-T cell is a CD 19-specific CAR-T cell. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁵To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10 ⁵To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁵To about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁶To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁶To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁷To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁵To about 10⁶Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁶To about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁷To about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁸To about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁹Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁸Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁷Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10 ⁶Individual CAR-T cells/kg. In some cases, the amount of CD 19-specific CAR-T cells comprises about 10⁵Individual CAR-T cells/kg.

In some embodiments, the modified T cell is an engineered TCR T cellAnd (4) cells. In some cases, the amount of engineered TCR T cells comprises about 10⁵To about 10⁹Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁵To about 10⁸Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁵To about 10⁷Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁶To about 10⁹Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁶To about 10⁸Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁷To about 10⁹Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁵To about 10⁶Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁶To about 10⁷Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁷To about 10⁸Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁸To about 10⁹Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10 ⁹Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁸Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁷Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁶Individual TCR cells/kg. In some cases, the amount of engineered TCR cells comprises about 10⁵Individual TCR cells/kg.

It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It will be further understood that the specific dosage regimen for any particular subject will be adjusted over time according to the needs of the individual and the professional judgment of the administrator administering or supervising the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Expression and purification system for fusion protein

The fusion proteins described herein, or fragments or variants thereof, can be produced from a cell as follows: culturing a host cell transformed with an expression vector comprising one or more polynucleotides encoding the fusion protein or fragment or variant thereof under conditions and for a length of time sufficient to allow expression of the fusion protein or fragment or variant thereof. For example, a polypeptide expressed in E.coli can be refolded from inclusion bodies (see, e.g., Hou et al (1998) Cytokine10: 319-30). Bacterial expression systems and methods of use thereof are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning-A Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells can vary according to many factors and can be readily optimized as desired. The fusion proteins described herein, or fragments or variants thereof, can be expressed in mammalian cells or other Expression systems including, but not limited to, yeast, baculovirus and in vitro Expression systems (see, e.g., Kaszubska et al (2000) Protein Expression and Purification 18: 213-220).

After expression, the fusion protein or fragment or variant thereof may be purified or isolated. The term "purified" or "isolated" as applied to any fusion protein or fragment or variant thereof described herein may refer to a polypeptide or protein that has been isolated or purified from naturally associated components (e.g., proteins or other naturally occurring organisms or organic molecules), such as other proteins, lipids, and nucleic acids in a prokaryote expressing the protein. Typically, a polypeptide is purified when it constitutes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% by weight of the total protein in the sample.

The fusion proteins described herein, or fragments or variants thereof, can be isolated or purified in a variety of ways depending on what other components are present in the sample. Standard purification methods include electrophoresis, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity and reverse phase HPLC chromatography. For example, the fusion protein or fragment or variant thereof can be purified using a standard anti-fusion protein antibody affinity column. Ultrafiltration and diafiltration techniques, as well as protein concentration, are also useful. See, for example, Scopes (1994) "Protein Purification, 3 rd edition," Springer-Verlag, New York City, N.Y.. The degree of purification necessary may vary depending on the intended use. In some cases, the expressed fusion protein or fragment or variant thereof does not require purification.

Methods for determining the yield or purity of a purified fusion protein or fragment or variant thereof can include, for example, Bradford assay, ultraviolet spectrophotometry, biuret protein assay, Lowry protein assay, amido black protein assay, High Pressure Liquid Chromatography (HPLC), Mass Spectrometry (MS), and gel electrophoresis (e.g., using a protein stain such as coomassie blue or colloidal silver stain). Once expressed, purified, or after post-expression purification, any of a variety of desired properties of the fusion proteins described herein, or fragments or variants thereof, can be determined using in vitro or in vivo assays, such as any of those described herein. For example, the fusion proteins described herein, or fragments or variants thereof, can be assayed for their ability to inhibit PD-1 and capture TGF- β, for example.

Various techniques can be used to generate the fusion proteins described herein or fragments or variants thereof. For example, polynucleotides encoding the fusion proteins described herein, or fragments or variants thereof, can be inserted into an expression vector containing transcriptional and translational regulatory sequences, which can include, for example, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. In some embodiments, the regulatory sequences may include a promoter and transcription initiation and termination sequences. In addition, the expression vector may include more than one replication system, allowing it to be maintained in two different organisms, for example, expression in mammalian or insect cells, and cloning and amplification in prokaryotic hosts.

Several possible carriersThe system can be used to express a fusion protein or a fragment or variant thereof from a nucleic acid in a mammalian cell (e.g., a host cell). For example, one type of vector relies on the integration of a desired gene sequence into the genome of a host cell. Cells with stably integrated DNA can be selected by the simultaneous introduction of a drug resistance gene such as E.coli gpt (Mullgan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5neo (Southern and Berg (1982) Mol Appl Genet1: 327). The selectable marker gene can be linked to the DNA gene sequence to be expressed or can be introduced into the same Cell by co-transfection (Wigler et al (1979) Cell 16: 77). Another class of vectors utilizes DNA elements that confer the ability of extrachromosomal plasmids to replicate autonomously. These vectors may be derived from animal viruses such as bovine papilloma virus (Sarver et al (1982) Proc Natl Acad Sci USA,79:7147), polyoma virus (Deans et al (1984) Proc Natl Acad Sci USA81:1292) or SV40 virus (Lusky and Botchan (1981) Nature293: 79). The expression vector may be introduced into a cell (e.g., a host cell) in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is primarily determined by the cell type targeted, as described below. A non-limiting exemplary method may include CaPO ₄Precipitation, liposome fusion, lipofection, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

Suitable host cells for expression of the fusion protein or fragments or variants thereof may include, but are not limited to, yeast, bacteria, insects, plants, and mammalian cells as described above. Of interest are bacteria such as E.coli, fungi such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris), insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cell lines (e.g., primary mammalian cells). In some embodiments, the fusion protein or fragment or variant thereof may be expressed in Chinese Hamster Ovary (CHO) cells or a suitable myeloma cell line such as (NS 0). Suitable cell lines also include, for example, BHK-21 (baby hamster kidney) cells; 293 (human embryonic kidney) cells; HMEpC (human mammary epithelial cells); 3T3 (mouse embryonic fibroblasts) cells.

The methods described herein provide for the expression and purification of fusion proteins, or fragments or variants thereof, in various cell-based expression systems, such as protein production in bacterial, mammalian, insect, yeast, and chymadomonas cells. Protein expression may be constitutive or inducible, using an inducer, such as copper sulfate, a sugar such as galactose, methanol, methylamine, thiamine, tetracycline or IPTG. After expression of the fusion protein or fragment or variant thereof in the host cell, the host cell is lysed to release the fusion protein or fragment or variant thereof for purification. Methods for lysing various host cells are described in "Sample Preparation-Tools for Protein Research" EMD Bioscience and in the Current Protocols in Protein Sciences (CPPS). A non-limiting exemplary purification method is affinity chromatography, such as ion-metal affinity chromatography using nickel, cobalt or zinc affinity resins for e.g. histidine-tagged fusion proteins or fragments or variants thereof. Clontech describes a method for purifying histidine-tagged proteins using its Talonx cobalt resin, which Novagen describes in its pET systems Manual (10 th edition). Another non-limiting exemplary purification strategy is by immunoaffinity chromatography, e.g., a myc-tagged fusion protein or fragment or variant thereof can be affinity purified using an anti-myc antibody-conjugated resin. Enzymatic digestion with serine proteases such as thrombin and enterokinase cleaves and releases the fusion protein or fragment or variant thereof from the histidine or myc tag, thereby releasing the fusion protein or fragment or variant thereof from the affinity resin, while the histidine tag and myc tag remain attached to the affinity resin.

Methods for introducing and expressing polynucleotides encoding fusion proteins or fragments or variants thereof into cells (e.g., host cells) are known in the art. In the case of expression vectors, vectors comprising polynucleotides encoding fusion proteins or fragments or variants thereof can be readily introduced into host cells, such as mammalian cells, bacterial, yeast or insect cells, by any method known in the art. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In embodiments, the method used to introduce the polynucleotide into the host cell is calcium phosphate transfection or Polyethyleneimine (PEI) transfection.

Biological methods for introducing polynucleotides encoding fusion proteins or fragments or variants thereof into host cells can include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells (e.g., human cells). Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patents 5,350,674 and 5,585,362.

In some embodiments, the expression vector may have the necessary 5 ' upstream and 3 ' downstream regulatory elements, such as a promoter sequence, ribosome recognition and binding TATA box, and 3 ' UTR AAUAAA transcription termination sequence, for efficient gene transcription and translation in its respective host cell. The expression vector may have additional sequences, such as 6X-histidine, V5, thioredoxin, glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose binding peptide, metal binding peptide, HA and "secretion" signals (bee melittin, alpha-factor, PHO, Bip), introduced into the expressed fusion protein or fragment or variant thereof. In addition, enzymatic digestion sites may be introduced after these sequences to facilitate their enzymatic removal after they are not needed. These additional sequences can be used to detect fusion protein expression, protein purification by affinity chromatography, to increase the solubility of the recombinant protein in the host cytoplasm, and/or to secrete the fusion protein into the culture medium, into the periplasm of prokaryotic bacteria, or into the spheroplasts of yeast cells. Expression of the fusion protein may be constitutive in the host cell or may be induced, for example, with copper sulfate, sugars such as galactose, methanol, methylamine, thiamine, tetracycline, baculovirus infection, and (isopropyl- β -D-thiogalactopyranoside) IPTG (a stable synthetic analogue of lactose).

Non-limiting examples of expression vectors and host cells can include pET vectors (Novagen), pGEX vectors (Amersham Pharmacia), and pMAL vectors (New England labs. Inc.) for expressing proteins in E.coli host cells such as BL21, BL21(DE3), and AD494(DE3) pLysS, Rosetta (DE3), and Origami (DE3) (Novagen); strong CMV promoter based pcDNA3(Invitrogen) and pCIneo vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication-defective adenovirus vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech), pAd/CMV/V5-DEST, pAd-DEST vectors (Invitrogen) for adenovirus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN and pLAPSN retroviral vectors for use with the Retro-XTM system from Clontech for retroviral mediated gene transfer and expression in mammalian cells; pLenti4V5-DEST.TM., pLenti6/V5-DEST and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; adeno-associated virus expression vectors, such as pAAV-MCS, pAAV-IRES-hrGFP and pAAV-RC vector (Stratagene), for adeno-associated virus-mediated gene transfer and expression in mammalian cells; BACpak6 baculovirus (Clontech) and pFastBacTM-HT (Invitrogen) for expression in Spodoptera frugiperda (Spodoptera frugiperda)9(S9) and Sfl1 insect cell lines; pMT/BiP/V5-His (Invitrogen) for expression in Drosophila Schneider S2 cells; pichia expression vectors pPICZ α, pPICZ, pFLD α and pFLD (invitrogen) for expression in pichia pastoris and vectors pMET α and pMET for expression in pichia methanolica (p. methanolica); pYES2/GS and pYD1(Invitrogen) vectors for expression in Saccharomyces cerevisiae. Griesbeck C, et al, 2006Mol, Biotechnol.34:213-33 and Fuhrmann M.2004, Methods Mol Med.94:191-5 describe the latest developments in large-scale expression of heterologous proteins in Chlamydomonas reinhardtii (Chlamydomonas reinhardtii).

In addition to cell-based expression systems, cell-free expression systems are also contemplated. Compared to traditional cell-based expression methods, cell-free expression systems have several advantages, including ease of modification of reaction conditions to facilitate protein folding, reduced sensitivity to product toxicity, and applicability to high-throughput strategies (e.g., rapid expression screening or large-scale protein production) because of reduced reaction volumes and shorter processing times. Cell-free expression systems may use plasmids or linear DNA. Moreover, the increase in translation efficiency has resulted in protein yields of more than one milligram per milliliter of reaction mixture.

In one embodiment, a continuous cell-free translation system can be used to produce the fusion protein or a fragment or variant thereof. Spirin A S. et al, Science 242:1162(1988) describe a continuous cell-free translation system capable of producing proteins in high yields. The method uses a continuous flow design of feed buffer containing amino acids, Adenosine Triphosphate (ATP) and Guanosine Triphosphate (GTP) throughout the reaction mixture and continuously removes the translated polypeptide product. The system uses E.coli lysate to provide a cell-free continuous feed buffer. The continuous flow system is compatible with both prokaryotic and eukaryotic expression vectors. Chang G, et al, Science 310:1950-3(2005) describe large scale cell free production.

Other commercially available Cell-Free Expression Systems include expressway Cell-Free Expression Systems (Invitrogen), which utilize an e.coli-based in vitro system for efficient coupled transcription and translation reactions to produce active recombinant proteins in amounts up to milligrams in a tube reaction format; rapid Transfer System (RTS) (Roche Applied Science), which also uses an E.coli-based in vitro System; and TNT Coupled Reticulocyte Lysate Systems (Promega), which uses an in vitro rabbit Reticulocyte-based system.

In other exemplary methods of production, the fusion proteins described herein, or fragments or variants thereof, can be synthesized de novo, in whole or in part, using chemical methods. For example, the constituent amino acid sequences of a fusion protein or fragment or variant thereof can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high performance liquid chromatography, and then chemically linked to form the desired polypeptide. The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing.

The methods for detecting and/or measuring the amount of endotoxin present in a sample (before and after purification) may be based on commercially available kits. For example, QCL-1000Chromogenic kit (BioWhittaker), a Limulus Amoebocyte Lysate (LAL) -based kit such as those available from Associates of Cape Cod Incorporated may be used Chromo-LAL and CSE kits to determine the concentration of endotoxin in a protein sample. In some embodiments, a variety of commercially available reagents can be used to remove endotoxin from a fusion protein preparation, including but not limited to ProteoSpin^TM Endotoxin Removal Kits(Norgen Biotek Corporation)、Detoxi-Gel Endotoxin Removal Gel(Thermo Scientific；Pierce Protein Research Products)、Endotoxin Removal Kit (Mirus) or Acrodisc^TM-E film (Pall Corporation).

In some embodiments, the fusion proteins described herein, or fragments or variants thereof, may be modified after expression and purification. The modification may be covalent or non-covalent. Such modifications can be introduced into the fusion protein or fragment or variant thereof by, for example, reacting targeted amino acid residues of the fusion protein or fragment or variant thereof with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. Suitable modification sites can be selected using any of a variety of criteria including, for example, structural analysis or amino acid sequence analysis of the fusion protein or fragment or variant thereof.

In some exemplary methods of production, the fusion proteins described herein, or fragments or variants thereof, can be conjugated to a heterologous moiety. In some embodiments, the heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, or a luminescent label. Suitable heterologous polypeptides may include, for example, an antigen tag (e.g., FLAG, polyhistidine, Hemagglutinin (HA), glutathione-S-transferase (GST), or Maltose Binding Protein (MBP)) for purification of the antibody or fragment or variant thereof. Heterologous polypeptides may also include polypeptides that can be used as diagnostic or detectable markers, for example, luciferase, Green Fluorescent Protein (GFP), or Chloramphenicol Acetyl Transferase (CAT). When the heterologous moiety is a polypeptide, the moiety can be incorporated into a fusion protein described herein, or a fragment or variant thereof, to yield a fusion protein comprising the heterologous moiety.

Promoters

"promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. The promoter is located near the transcription start site of the gene, on the same strand of DNA and upstream (toward the 5' region of the sense strand). Some promoters are constitutive in that they are active in all cases in the cell, while others become active in response to a particular stimulus being regulated, e.g., inducible promoters. Still other promoters are tissue-specific or activated promoters, including but not limited to T cell-specific promoters.

The term "promoter activity" and grammatical equivalents thereof refers to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity can be determined directly (e.g., by Northern blot analysis) by determining the amount of RNA transcript produced, or indirectly by determining the amount of product encoded by the linked nucleic acid sequence (e.g., a reporter nucleic acid sequence linked to the promoter).

An "inducible promoter" as used herein refers to a promoter whose activity is induced by the presence or absence of a transcriptional regulator (e.g., an biotic or abiotic factor). Inducible promoters are useful because the expression of the genes to which they are operably linked can be switched on or off at certain developmental stages of the organism or in specific tissues. Examples of inducible promoters are alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters and light-regulated promoters.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence to which it is operably linked. However, other constitutive promoter sequences may also be used, including, but not limited to, monkey virus 40(SV40) early promoter, Mouse Mammary Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

In some embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some embodiments, the inducible promoter is a small molecule ligand inducible gene switch based on two polypeptide ecdysone receptors.

Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Typically these promoter elements are located in the region 30-110bp upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is typically flexible, thus preserving promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements may increase to 50bp apart before activity begins to decline. It appears that the individual elements may act synergistically or independently to activate transcription, depending on the promoter.

Reporter gene

Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the host cell and that encodes a polypeptide whose expression is manifested by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is determined at a suitable time after introduction of the polypeptide encoding the fusion protein or fragment or variant thereof into the host cell. Suitable reporter genes may include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al, FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a polynucleotide construct with a minimal 5' flanking region that shows the highest expression level of the reporter gene is identified as a promoter. Such promoter regions may be linked to a reporter gene and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Variants

The term "fragment" or "variant" refers to variants and derivatives of the fusion proteins described herein, which comprise one or more binding moieties to, for example, PD-1 and/or TGF- β. In certain embodiments, amino acid sequence variants of the fusion proteins are contemplated. For example, in some embodiments, amino acid sequence variants of the fusion proteins described herein are expected to improve the binding affinity and/or other biological properties of the fusion protein. Exemplary methods for making amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the fusion protein, 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 fusion protein.

Any combination of deletions, insertions, and substitutions of the various domains can be made to arrive at the final fusion protein, provided that the final fusion protein has the desired properties, e.g., PD-1 inhibition and TGF- β trap. In some embodiments, fusion protein variants having one or more amino acid substitutions are provided. In some embodiments, the sites of interest for substitutional mutagenesis may include CDRs and framework regions. In some embodiments, amino acid substitutions may be introduced into the variable domain of a target binding protein of interest and the product screened for a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC). Both conservative and non-conservative amino acid substitutions are contemplated for making antibody variants (e.g., PD-1 antibody variants).

In another example of substitutions used to generate variant fusion proteins, one or more hypervariable region residues of the parent antibody may be substituted. Typically, the variants are then selected based on an improvement in the desired property compared to the parent antibody, e.g., increased affinity, decreased immunogenicity, increased pH dependence of binding. For example, affinity matured variant antibodies can be generated using, for example, phage display-based affinity maturation techniques, such as those described herein and known in the art. Substitutions may be made in the hypervariable regions (HVRs) of the parent antibody to generate variants, which are then selected based on binding affinity, i.e. by affinity maturation. In some embodiments of affinity maturation, diversity can be introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library can then be created. The library can then be screened to identify any antibody variants with the desired affinity. Another method of introducing diversity may involve HVR targeting methods, in which several HVR residues (e.g., 4-6 residues at a time) may be randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Substitutions may be at one, two, three, four or more positions within the parent antibody sequence.

In some embodiments, a fragment or variant of a fusion protein described herein may comprise a VL domain and a VH domain having amino acid sequences corresponding to the amino acid sequences of a naturally occurring VL or VH domain, respectively, but which have been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VL or VH domain (particularly in the framework sequence) with one or more amino acid residues present at corresponding positions in the VL or VH domain of a conventional 4-chain antibody from a human. This may be done in a manner known in the art and which may be apparent to those skilled in the art, for example, based on the further description herein. It is noted that such humanized fusion proteins or fragments or variants thereof are obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VL and/or VH domains as starting material.

In some embodiments, a fragment or variant of a fusion protein as described herein comprises VL and VH domains having amino acid sequences corresponding to the amino acid sequences of naturally occurring VL or VH domains, respectively, but which have been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequences of naturally occurring VL or VH domains from conventional 4-chain antibodies with one or more amino acid residues present at corresponding positions in the VL or VH domains of heavy chain antibodies. Such "camelization" may preferably be inserted at amino acid positions forming and/or present at the VH-VL interface and/or at so-called camelid tag residues (see e.g. WO 94/04678, and Davies and Riechmann (1994 and 1996)). In some embodiments, the VH sequence used as a starting material or starting point for generating or designing a camelised single domain may be a VH sequence from a mammal, such as a human, such as a VH3 sequence. It should be noted that such camelized fusion proteins or fragments or variants thereof may in certain embodiments be obtained in any suitable manner known in the art and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VL and/or VH domains as starting material.

For example, "humanization" and "camelization" can both be performed as follows: nucleotide sequences encoding naturally occurring VL and/or VH domains are provided, respectively, and then one or more codons in the nucleotide sequences are altered in such a way that the new polynucleotide sequences encode a "humanized" or "camelized" fusion protein, or fragment or variant thereof, respectively. The polynucleotide may then be expressed to provide the desired binding capacity (e.g., PD-1). Alternatively, in other embodiments, the fusion protein or fragment or variant thereof comprises a "humanized" or "camelized" antibody synthesized de novo from the amino acid sequence of a naturally occurring antibody comprising a VL and/or VH domain using known peptide synthesis techniques. In some embodiments, the fusion protein or fragment or variant thereof comprises a "humanized" or "camelized" antibody synthesized de novo from the amino acid sequence or nucleotide sequence of a naturally occurring antibody comprising a VL and/or VH domain, respectively, using known peptide synthesis techniques, designing the nucleotide sequence encoding the desired humanized or camelized antibody, respectively, of the disclosure, and then synthesized de novo using known nucleic acid synthesis techniques, followed by expression of the nucleic acid thus obtained using known expression techniques, to provide the desired antibody.

Other suitable methods and techniques for obtaining a fusion protein or fragment or variant thereof starting from naturally occurring sequences of VL or VH domains include, for example, combining one or more portions of naturally occurring VL or VH sequences, such as one or more Framework (FR) sequences and/or Complementarity Determining Region (CDR) sequences, and/or one or more synthetic or semi-synthetic sequences, and/or naturally occurring sequences of the CH2 domain, and naturally occurring sequences of the CH3 domain (which contain amino acid substitutions that are more favorable for heterodimer formation relative to homodimers) in a suitable manner to provide a fusion protein or fragment or variant thereof.

Antibody engineering

In some embodiments, it may be desirable to change certain amino acids containing exposed side chains to another amino acid residue as follows to provide greater chemical stability of the final antibody (e.g., PD-1 antibody). Deamidation of asparagine can occur at either the N-G or D-G sequences and results in the production of isoaspartic acid residues which introduce kinks (kink) into the polypeptide chain and reduce its stability (isoaspartic acid action). In some embodiments, the antibody of the fusion protein of the present disclosure does not comprise an asparagine isomerization site.

For example, asparagine (Asn) residues can be changed to Gln or Ala to reduce the likelihood of forming isoaspartic acid within any Asn-Gly sequence, particularly within the CDR. Similar problems may occur with the Asp-Gly sequence. Reissner and Aswad (2003) cell. mol. Life Sci.60: 1281. The formation of isoaspartic acid can reduce or completely eliminate the binding of the antibody to its target antigen. See, Presta (2005) J.allergy Clin.Immunol.116:731,734. In one embodiment, asparagine may be changed to glutamine (Gln). It may also be desirable to alter the amino acids adjacent to asparagine (Asn) or glutamine (Gln) residues to reduce the likelihood of deamidation, which occurs more frequently when small amino acids are adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J.Chromatog.662: 261. In addition, any methionine residue in the CDRs (typically Met exposed to solvent) can be changed to Lys, Leu, Ala or Phe to reduce the likelihood of methionine sulfur being oxidized, which can reduce antigen binding affinity and can also contribute to molecular heterogeneity in the final antibody preparation. As above. In one embodiment, methionine may be changed to alanine (Ala). In addition, it may be desirable to change any Asn-Pro combination seen in the CDRs to Gln-Pro, Ala-Pro or Asn-Ala in order to prevent or minimize potential fragile Asn-Pro peptide bonds. Antibodies having such substitutions can then be screened to ensure that the substitutions do not reduce the affinity or specificity or other desired biological activity of the antibody to human PD-L1 to an unacceptable level.

The antibodies of the fusion proteins disclosed herein may also be conjugated to a chemical moiety, such as a radionuclide or other detectable label. Radionuclide packageComprises⁹⁹Tc、⁹⁰Y、¹¹¹In、³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹¹C、¹⁵O、¹³N、¹⁸F、³⁵S、⁵¹Cr、⁵⁷To、²²⁶Ra、⁶⁰Co、⁵⁹Fe、⁵⁷Se、¹⁵²Eu、⁶⁷Cu、²¹⁷Ci、²¹¹At、²¹²Pb、⁴⁷Sc、¹⁰⁹Pd、²³⁴Th、⁴⁰K、¹⁵⁷Gd、⁵⁵Mn、⁵²Tr and⁵⁶fe. The fluorescent or chemiluminescent label may comprise a fluorophore such as a rare earth chelate, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrins, phycocyanins, allophycocyanins, o-phthalaldehyde, fluorescamine, fluorescein, and/or fluorescein, and the like,¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal, isoluminal, aromatic acridinium ester, imidazole, acridinium salt, oxalate, aequorin, 2, 3-dihydrophthalhydrazide, biotin/avidin, spin labeling, and stable free radicals.

Any method known in the art for conjugating antibody molecules to various moieties can be used, including Hunter et al, (1962) Nature 144: 945; david et al, (1974) Biochemistry 13: 1014; pain et al, (1981) j.immunol.meth.40: 219; and Nygren, J., (1982) Histochem.and Cytochem.30: 407. Methods for conjugating antibodies are conventional and well known in the art.

Pharmaceutical composition

The present disclosure provides a composition comprising a fusion protein described herein, or a fragment or variant thereof, and a carrier (e.g., a pharmaceutically acceptable carrier) therefor. The composition is desirably a physiologically acceptable (e.g., pharmaceutically acceptable) composition comprising a carrier, preferably a physiologically (e.g., pharmaceutically) acceptable carrier, and the fusion protein or fragment or variant thereof. Any suitable carrier may be used in the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will depend in part on the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Ideally, in the case of a replication-defective adenovirus vector, the pharmaceutical composition preferably does not contain a replication-competent adenovirus. The pharmaceutical composition optionally may be sterile.

Suitable compositions include aqueous and non-aqueous isotonic sterile solutions, which may contain antioxidants, buffers, and bacteriostats, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The compositions may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules and tablets. Preferably, the carrier is a buffered saline solution. In some cases, the fusion protein or fragment or variant thereof is part of a composition formulated to protect the fusion protein or fragment or variant thereof from damage prior to administration. For example, the composition can be formulated to reduce loss of the fusion protein or fragment or variant thereof on a device used to prepare, store, or administer the fusion protein or fragment or variant thereof, such as a glass vessel, syringe, or needle. The composition may be formulated to reduce photosensitivity and/or temperature sensitivity of the fusion protein or fragment or variant thereof. To this end, the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such a composition will extend the shelf life of the fusion protein or fragment or variant thereof and facilitate its administration. Formulations of compositions comprising fusion proteins or fragments or variants thereof are further described, for example, in U.S. patent 6,225,289, U.S. patent 6,514,943, and international patent application publication WO 2000/034444.

The compositions may also be formulated to enhance transduction efficiency. In addition, one of ordinary skill in the art will appreciate that the fusion protein or fragment or variant thereof can be present in the composition with other therapeutic or bioactive agents. For example, factors that control inflammation, such as ibuprofen or steroids, may be part of the composition to reduce swelling and inflammation associated with in vivo administration of the fusion protein or fragment or variant thereof. Antibiotics, i.e., microbicides and fungicides, may be present to treat existing infections and/or reduce the risk of future infections, such as those associated with application programs.

In some cases, pharmaceutical compositions comprising the fusion proteins or fragments or variants thereof described herein are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The appropriate formulation will depend on the chosen route of administration. A summary of The pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, 19 th edition (Easton, Pa., Mack Publishing Company, 1995); hoover, John e., Remington's Pharmaceutical Sciences, Mack Publishing co, Easton, Pennsylvania 1975; liberman, h.a. and Lachman, l. eds, Pharmaceutical document Forms, Marcel Decker, New York, n.y., 1980; and Pharmaceutical document Forms and Drug Delivery Systems, 7 th edition (Lippincott Williams & Wilkins 1999).

The pharmaceutical compositions are optionally prepared in a conventional manner, such as, for example only, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

In certain embodiments, the composition may further comprise one or more pH adjusting agents or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

In other embodiments, the composition may further comprise one or more salts in an amount necessary to bring the osmolality of the composition within an acceptable range. Such salts include those having a sodium, potassium or ammonium cation and a chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anion; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

The pharmaceutical compositions described herein are administered by any suitable route of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraarticular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal routes of administration. In some cases, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intraarticular, intraperitoneal, or intracranial) administration.

The pharmaceutical compositions described herein are formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a subject to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melting formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. In some embodiments, the pharmaceutical composition is formulated as a capsule. In some embodiments, the pharmaceutical composition is formulated as a solution (e.g., for IV administration). In some cases, the pharmaceutical composition is formulated as an infusion. In some cases, the pharmaceutical composition is formulated as an injection.

The pharmaceutical solid dosage forms described herein optionally comprise a fusion protein or fragment or variant thereof as described herein and one or more pharmaceutically acceptable additives such as compatible carriers, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, permeation enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof.

In other aspects, a film coating may be provided around the composition using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20 th edition (2000). In some embodiments, the composition is formulated into granules (e.g., for administration by capsule) and some or all of the granules may be coated. In some embodiments, the composition is formulated into granules (e.g., for administration by capsule) and some or all of the granules may be microencapsulated. In some embodiments, the composition may be formulated as granules (e.g., for administration by capsule) and some or all of the granules are not microencapsulated and uncoated.

In certain embodiments, the compositions provided herein may further comprise one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing materials, such as phenylmercuric borate (merfen) and thimerosal (thiomersal); stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

"defoamers" reduce foaming during processing, which can cause coagulation of the aqueous dispersion, bubbles in the finished film, or generally impaired processing. Exemplary anti-foaming agents include silicon emulsions or sorbitan sesquioleate (sorbitan sesquooleate).

"antioxidants" include, for example, Butylated Hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite, and tocopherol. In certain embodiments, antioxidants enhance chemical stability, if desired.

The formulations described herein may benefit from antioxidants, metal chelators, thiol-containing compounds, and other common stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol; (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1mM to about 10mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) a combination thereof.

"Adhesives" impart cohesive properties and include, for example, alginic acid and its salts; cellulose derivatives, e.g. carboxymethyl cellulose, methyl cellulose (e.g. cellulose acetate)) Hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. hydroxypropyl cellulose)) Ethyl cellulose (e.g. cellulose acetate)) And microcrystalline cellulose (e.g. cellulose acetate)) (ii) a Microcrystalline dextrose; amylose starch; magnesium aluminum silicate; a gluconic acid; bentonite; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; polyvinyl polypyrrolidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, sugars, e.g. sucrose (e.g. sucrose)) Glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g. glucose, sorbitol, xylitol, mannitol, sorbitol, xylitol, sorbitol, or mixtures thereof) And lactose; natural or synthetic gums, e.g. acacia, tragacanth, ghatti, psyllium seed gum, polyvinylpyrrolidone (e.g. gum arabicCL、CL、XL-10), larch arabinogalactan,Polyethylene glycol, wax, sodium alginate, and the like.

The "carrier" or "carrier material" includes any excipient commonly used in pharmacy and should be selected with regard to compatibility with the compounds disclosed herein, such as ibrutinib and anticancer compounds, and the release profile properties of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.

Pharmaceutically compatible carrier materials may include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. See, for example, Remington: The Science and Practice of Pharmacy, nineteenth edition (Easton, Pa.: Mack Publishing Company,1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H.A., and Lachman, L.ed., Pharmaceutical document such as Marcel Decker, New York, N.Y.,1980, and Pharmaceutical document Forms and Drug Delivery Systems, seventh edition (Lippincott Williams & Wilkins 1999).

"dispersants" and/or "viscosity modifiers" include materials that control the diffusion and uniformity of the drug through a liquid medium or a granulation process or a mixing process. In some embodiments, these agents also contribute to the effectiveness of coating or eroding the substrate. Exemplary diffusion promoters/dispersants include, for example, hydrophilic polymers, electrolytes, 60 or 80, PEG, polyvinylpyrrolidone (PVP; trade name: SEQ ID NO.)) And carbohydrate-based dispersants such as hydroxypropyl cellulose (e.g., HPC-SL, and HPC-L), hydroxypropylmethyl cellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone/vinyl acetate copolymer (S630), 4- (1,1,3, 3-tetramethylbutyl) -phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g.,andthey are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic)Also known as PoloxamineIt is a tetrafunctional block copolymer obtained by sequentially adding propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, n.j.), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene glycol such as polyethylene glycol which may have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums such as tragacanth and acacia, guar gum, xanthan gum (including xanthan gum), sugars, cellulose preparations such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated lost foam, and the like Sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosan, and combinations thereof. Plasticizers such as cellulose or triethylcellulose may also be used as dispersants. Particularly useful dispersing agents in liposomal dispersions and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural phosphatidylcholine from eggs, natural phosphatidylglycerol from eggs, cholesterol and isopropyl myristate.

Combinations of one or more corrosion promoters with one or more diffusion promoters may also be used in the compositions of the present invention.

The term "diluent" refers to a chemical compound used to dilute a compound of interest prior to delivery. Diluents can also be used to stabilize the compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which may also provide pH control or maintenance) are utilized in the art as diluents, including but not limited to phosphate buffered saline solutions. In certain embodiments, the diluent increases the volume of the composition to facilitate compression or to create sufficient volume for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Calcium hydrogen phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray dried lactose; pregelatinized starches, compressible sugars, e.g.(Amstar); mannitol, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, sugar fructose; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrate (dextrate); hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

"fillers" include compounds such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

"Lubricants" and "glidants" are compounds that prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil Higher fatty acids and their alkali metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc salts, stearic acid, sodium stearate, glycerin, talc, waxes,boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax^TMSodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium lauryl sulfate or sodium lauryl sulfate, colloidal silica such as Syloid^TM，Starches such as corn starch, silicone oils, surfactants, and the like.

A "plasticizer" is a compound used to soften the microencapsulated material or film coating to make it less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. In some embodiments, plasticizers may also be used as dispersing or wetting agents.

"solubilizers" include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium lauryl sulfate, docusate sodium, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrin, ethanol, N-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-.

"stabilizers" include compounds such as any antioxidant, buffer, acid, preservative, and the like.

"suspending agents" include, for example, polyvinylpyrrolidone, such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol (for example, polyethylene glycol may have a molecular weight of from about 300 to about 6000, or from about 3350 to about 4000, or from about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as tragacanth and acacia, guar gum, xanthan gum (including xanthan gum), sugars, celluloses, such as carboxymethylcellulose sodium, methylcellulose, carboxymethylcellulose sodium, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, Polyethoxylated sorbitan monolaurate, povidone, and the like.

"surfactants" include, for example, sodium lauryl sulfate, docusate sodium, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide such as (BASF) and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol (octoxynol)10, octoxynol 40. In some implementationsIn embodiments, surfactants may be included to enhance physical stability or for other purposes.

"viscosity enhancing agents" include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, gum arabic, chitosan, and combinations thereof.

"wetting agents" include compounds such as oleic acid, glycerol monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, docusate sodium, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, tween 80, vitamin E TPGS, ammonium salts, and the like.

Kit/article of manufacture

In certain embodiments, disclosed herein are kits and articles of manufacture for use with one or more of the methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers, e.g., vials, tubes, and the like, each container comprising a separate element for use in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from a variety of materials, such as glass or plastic.

The articles provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

The kit typically includes a label and/or instructions for use listing the contents, and a package insert with instructions for use. A set of instructions is also typically included.

In some embodiments, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers or other characters comprising the label are attached, molded or etched onto the container itself; a label is associated with a container when the label is present within a receptacle or carrier that also holds the container (e.g., as a package insert). In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions for use of the contents, such as in the methods described herein.

Examples

These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

Example 1 fusion proteins against PD 1-TGF-. beta.trap

anti-PD 1 VH and VL were synthesized and formatted in an IgG, scFv-Fc or scFv configuration, with TGF β RII fused at the C-terminus by linker (G4S) 2. anti-PD 1 trap fusion proteins were transiently expressed in Expi293 cells according to the manufacturer's protocol. To purify the fusion protein, the transfected supernatant was loaded onto a protein L column using AKTA AVANT. The column was washed with PBS, followed by elution of the protein with IgG elution buffer (Pierce). The eluted protein buffer was then exchanged into PBS using a PD-10 column (GE Healthcare).

Surface plasmon resonance

Surface Plasmon Resonance (SPR) analysis was performed using a Biacore3000, CM5 chip, amine coupling kit, 10X HBS-P running buffer and glycine for regeneration (GE Healthcare). For the anti-PD 1 kinetic assay, purified PD-1Fc fusion proteins were immobilized on CM5 chips using a pre-determined ligand immobilization procedure. Purified anti-PD 1 trap fusion proteins were diluted to a range of final concentrations in HBS-P running buffer and injected. This was allowed to dissociate, followed by a pulse of 10mM glycine (pH 1.5) to regenerate the chip surface.

To investigate whether the anti-PD 1 trap fusion protein could bind both PD1 and TGF β RII, 100nM of anti-PD 1 trap fusion protein was injected onto a CM5 chip immobilized with PD1 for 3 min and then dissociated for 3 min. Subsequently, different isoforms of TGF- β were injected to check for binding of TGF- β to PD 1-linked anti-PD 1 trap fusion protein.

To analyze the binding affinity of anti-PD 1-TGF β RII to TGF- β isoforms, anti-PD 1-TGF β RII was immobilized on a CM5 chip, TGF- β protein was then diluted to a range of final concentrations in HBS-P running buffer and injected to measure the binding affinity of TGF- β to immobilized anti-PD 1-TGF β RII. The affinity of the anti-PD 1(VH6-VL5 or VH7-VL6) -TGF-. beta.RII fusion protein for immobilized PD-1 is shown in Table 2 (FIGS. 6-8). Table 3 shows the affinity of TGF- β isotypes for immobilized anti-PD-1 IgG4 antibody (VH6-VL5) -TGF β RII fusion proteins. As observed, the fusion protein binds TGF-beta 1 and TGF-beta 3 with high affinity, and TGF-beta 2 with lower affinity.

TABLE 2 affinity of anti-PD 1-TGF-. beta.RII for immobilized PD-1

TABLE 3 affinity of TGF-. beta.for immobilized anti-PD 1(VH6-VL5) IgG 4-TGF-. beta.RII

The binding affinity of various anti-PD-1 variants fused to TGF β RII was assessed by Surface Plasmon Resonance (SPR) analysis using Biacore 3000. PD-1 fused to the human Fc region was immobilized on sensor chip CM 5. Various fusion proteins were injected at different concentrations in the solution phase and the data were analyzed using BIAevaluation to calculate the KD of the fusion protein. Variants such as anti-PD 1(VH6-VL5) -TGF β RII fusion proteins and anti-PD 1(VH7-VL6) -TGF β RII fusion proteins were found to have similar binding affinities to PD1 compared to the corresponding anti-PD 1 alone.

TABLE 5 affinity of two anti-PD 1-TGF-. beta.RII fusion proteins compared to the corresponding anti-PD 1 antibody alone

PD-1/PDL-1 blocking assay

To investigate whether an anti-PD 1-TGF-. beta.RII fusion protein could function as an anti-PD 1 antibody by blocking the PD-1/PD-L1 interaction, a PD-1/PD-L1 blocking bioassay kit from Promega was used. Briefly, PD-L1 aAPC/CHO-K1 cells were thawed one day prior to assay initiation and allowed to thaw at 37 deg.C with 5% CO₂The next recovery was performed overnight for 16 to 20 hours. To generate dose response curves, serial dilutions of anti-PD 1TGF β RII proteins were prepared in the range of 100nM to 0.098 nM. The plate was then removed from the incubator and the cell culture medium was discarded. The antibody of interest was then added directly to the plate containing PD-L1 aAPC/CHO-K1 cells. Following this step, PD-1 effector cells were thawed and added to 96-well plates. The plates were incubated at 37 ℃ with 5% CO₂Incubate for 6 hours. The mixture was subjected to Bio-Glo^TMLuciferase substrate reagent (Promega) was added to each well and Relative Luciferase Units (RLU) were measured on a Glomax 96 microplate luminometer. IC determination of anti-PD 1 TGF-beta-RII proteins by fitting RLU concentration data to a four parameter logistic equation₅₀. These values were used as a measure of the blocking potency of PD-1/PD-L1 (FIG. 5).

HEK-Blue TGF-. beta.assay

To examine the efficacy of anti-PD 1 TGF-. beta.RII fusion proteins to neutralize TGF-. beta.function, a stable cell line with an induced reporter gene (HEK-Blue) was used^TMTGB-beta reporter cell line, Invitrogen). The stable cell line allows for the detection of biologically active TGF- β by activating the TGF- β/SMAD pathway, leading to the production of Secreted Embryonic Alkaline Phosphatase (SEAP), which can be quantified. Briefly, TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3 were diluted to predetermined ECs₅₀Concentration, and addition to flat bottom 96-well plates. To generate dose response curves, serial dilutions of anti-PD 1TGF β RII proteins were added to plates containing different isoforms of TGF- β. The plates were incubated at 37 ℃ for 30 minutes. Then HEK-Blue^TMTGF-beta cells at 0.7X 10⁶Final concentration of individual cells/mL was added to the plate. The plates were then incubated at 37 ℃ with 5% CO₂The incubation was continued for a total of 20 hours. After incubation, an aliquot of the supernatant was removed and addedTo contain QUANTI-Blue^TMSubstrate (Invitrogen) used to quantify secreted SEAP in plates. The plates were incubated at 37 ℃ for 1 hour. Plates were then read on a SpectraMax Plus plate reader at 620 to 655 nm. IC was determined by fitting absorbance-concentration data to a four parameter logistic equation (GraphPad Prism) ₅₀. The efficacy results for various anti-PD 1 tgfbetarii fusion proteins compared to controls are shown in fig. 5-8.

Tables 6 and 7 further list the various fusion protein constructs tested and the inhibition of TGF-. beta.1 induced reporter gene activity.

Table 6: IC of various fusion constructs on TGF-beta 1 induced reporter Activity₅₀Summary of inhibition

Fusion proteins	IC50(pM)
		anti-PD 1(VL5-VH6). IgG4	9.0
anti-PD 1(VL5-VH6), IgG4(S108P), TGFbRII	9.7
		anti-PD 1(VL5-VH6) ScFv-Fc	4.3
anti-PD 1(VL6-VH7), IgG4.TGFbRII	5.6
		anti-PD 1(VL6-VH7), IgG4(S108P), TGFbRII	9.3
anti-PD 1(nVL1-nVH3), IgG4-TGFbRII	10.7
		anti-PD 1(nVL1-nVH3). scFv-Fc-TGFbRII	6.5
anti-PD 1(nVL1-nVH7), IgG4-TGFbRII	15.1
		anti-PD 1(nVL1-nVH8), IgG4-TGFbRII	54.5
anti-RSV IgG4-TGFbRII	8.6
		anti-RSV S108P IgG4-TGFbRII	6.9
anti-RSV scFv-Fc4	8.9

Table 7: summary of TGF-. beta.1 neutralizing Activity of various fusion constructs

PD1 binding of various anti-PD 1 IgG4-TGFbRII fusion constructs

PD1-Fc antigen and reference antigen were immobilized on the surface of Biacore CM 5. Various anti-PD 1 IgG4-TGFbRII concentrations were serially injected in serial 6-12 repeated serial dilutions onto PD1-Fc antigen and reference surface (table 8). For 11, model: langmuir with mass transfer, kinetic data were evaluated. As observed in Table 7, the binding affinity (K) of anti-PD 1 IgG4-TGFbRII to PD1-Fc antigen _D) At 10¹²To 10¹⁰Between the ranges.

Table 8: PD1 binding affinity analysis of PD1 IgG4-TGFbRII fusion protein

Simultaneous PD1 and TGF-beta 1 binding of various anti-PD 1 IgG 4-TGF-beta RII fusion constructs

To investigate whether anti-PD 1-TGFRII fusion proteins with different linkers (table 9) could bind both PD1 and TGF- β 1, the following constructs were made and tested in Biacore experiments. The PD1-Fc antigen was immobilized on the surface of a Biacore CM5 chip. anti-PD 1(VL5-VH6), IgG 4-linker-TGFbRII was injected onto the sensor surface, followed by TGF β 1 injection onto the sensor surface. As shown in figure 20, all anti-PD 1(VL5-VH6), IgG 4-linker-TGFRII constructs bound both PD1-Fc antigen and TGF β 1.

Table 9: anti-PD 1(VL5-VH6) with various linkers IgG 4-linker-TGFRII constructs

Example 2 anti-PD 1-TGF-. beta.trap promotes T cell activation in vitro culture

To assess the ability of the anti-PD 1-TGF- β trap to promote T cell function, Peripheral Blood Mononuclear Cells (PBMCs) purified in vitro by Ficoll-Hypaque from leukapheresis products (obtained from normal healthy donors according to protocols approved by the Institutional Review Board (IRB)) were used. PBMCs were labeled with the cell proliferation dye CellTrace violet and stimulated with anti-CD 3 and anti-CD 28. At the end of the incubation, the supernatant was collected and stored until used for IFN- γ release assay. Cells were labeled with fluorescently conjugated antibodies to assess T cell proliferation.

PBMCs stimulated in the presence of anti-PD 1-TGF- β trap showed enhanced proliferation and IFN- γ production in a dose-dependent manner compared to anti-PD 1 or anti-RSV control antibodies, indicating that the fusion molecules target both the PD-1/PD-L1 pathway and the TGF- β pathway (fig. 9A-9C). The data also demonstrate the additive enhancement of the fusion molecule compared to anti-PD 1 alone.

In vitro models of colorectal cancer, anti-PD 1-TGF-RII showed superior anti-tumor responses compared to anti-PD 1 alone

The effectiveness of the anti-PD 1-TGFRII fusion protein was evaluated in an in vitro model of colorectal cancer. PBMCs purified from healthy donors were co-cultured with cancer cell line HT-29 for 5 days. Cells were stimulated with anti-CD 3 in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol. Cells were collected and analyzed by flow cytometry for target occupancy on T cells. TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3 levels in cell culture supernatants were quantified by Luminex according to the manufacturer's protocol.

Fig. 9D shows that, similar to anti-PD 1, anti-PD 1-TGFRII binds to PD1 on the surface of CD8+ T cells. As shown in fig. 9E, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. Figure 9h TGF- β 1 produced in HT-29PBMC co-culture system was completely neutralized after anti-PD 1-TGFRII treatment even at the lowest dose tested. FIG. 9I TGF-. beta.2 produced in HT-29PBMC co-culture system was partially neutralized after anti-PD 1-TGFRII treatment. Overall, the results of this in vitro model show an excellent anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

In vitro models of head and neck cancer, anti-PD 1-TGFRII showed superior anti-tumor responses compared to anti-PD 1 alone

The effectiveness of an anti-PD 1-TGFRII fusion protein was evaluated in an in vitro model of head and neck cancer. PBMCs purified from healthy donors were co-cultured with the cancer cell line Detroit 562 for 5 days. Cells were stimulated with anti-CD 3 in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol. Cells were collected and analyzed by flow cytometry for target occupancy on T cells. TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3 levels in cell culture supernatants were quantified by Luminex according to the manufacturer's protocol.

Fig. 9F shows that, similar to anti-PD 1, anti-PD 1-TGFRII binds to PD1 on the surface of CD8+ T cells. As shown in fig. 9G, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. Figure 9j TGF- β 1 produced in Detroit 562PBMC co-culture system was completely neutralized after anti-PD 1-TGFRII treatment even at the lowest dose tested. FIG. 9K TGF-. beta.2 produced in Detroit 562PBMC co-culture system was partially neutralized after anti-PD 1-TGFRII treatment. Overall, the results of this in vitro model show an excellent anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

In an in vitro 3D tumor sphere model of colorectal cancer, anti-PD 1-TGF-RII showed superior anti-tumor response compared to anti-PD 1 alone

The effectiveness of the anti-PD 1-TGFRII fusion protein was evaluated in an in vitro 3D tumor sphere model of colorectal cancer. 3D tumor spheroids were generated by co-culturing the colorectal cancer cell line HT-29 and a fibroblast cell line. PBMCs purified from healthy donors were then added to the spheroid culture. Cells were stimulated with anti-CD 3 for 5 days in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol.

As shown in fig. 9L, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. Overall, the results of the 3D tumor sphere model show superior anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

In an in vitro 3D tumor spheroid model of head and neck cancer, anti-PD 1-TGF-RII showed superior anti-tumor response compared to anti-PD 1 alone

The effectiveness of the anti-PD 1-TGFRII fusion protein was evaluated in an in vitro 3D tumor sphere model of colorectal cancer. 3D tumor spheroids were generated by co-culturing Detroit 562 cell line and fibroblast cell line. PBMCs purified from healthy donors were then added to the spheroid culture. Cells were stimulated with anti-CD 3 for 5 days in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol.

As shown in figure 9M, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. Overall, the results of the 3D tumor sphere model show superior anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

Example 3 in vitro anti-PD 1-TGF-beta trap has superior activity compared to anti-PD 1 in the presence of recombinant TGF-beta 1

To evaluate the ability of anti-PD 1-TGF- β traps to promote T cell function in an environment with high levels of TGF- β, PBMCs were stimulated as described above in the presence of recombinant TGF- β 1. At the end of the incubation, the supernatant was collected and stored until used for cytokine analysis. Cells were labeled with fluorescently conjugated antibodies and T cell proliferation and upregulation of IL-2 receptor alpha or CD25 were assessed as a readout for activation.

Recombinant TGF- β 1 inhibited T cell activation, proliferation, and cytokine and chemokine production (fig. 10 and 11). This inhibition was significantly reversed by anti-PD 1-TGF- β 1 trap in a dose-dependent manner compared to anti-PD 1. The data indicate the ability of the fusion protein to neutralize large amounts of exogenous TGF- β 1 and disrupt the PD-1/PD-L1 pathway, thereby enhancing the T cell immune response. Thus, blocking of PD-1/PD-L1 and neutralization of TGF- β by the fusion molecule may be an attractive immunotherapy for eliciting potent anti-tumor responses in cancer indications that fail checkpoint inhibitor therapy.

Example 4 in a humanized mouse model of colorectal cancer, anti-PD 1-TGF-RII showed superior anti-tumor response compared to anti-PD 1 alone

The ability of the anti-PD 1-TGFRII fusion protein to inhibit colorectal cancer tumors was evaluated in a humanized mouse model. HT-29 cancer cells were administered to humanized NOG mice. Tumor-bearing mice were randomized and administered twice weekly with anti-PD 1-TGFRII fusion protein, anti-PD 1 antibody alone, or isotype control. As shown in fig. 12A, the anti-PD 1-TGFRII fusion protein significantly inhibited tumor growth compared to anti-PD 1 and isotype control. Analysis of tumors at the end of the study indicated that mice treated with the anti-PD 1-TGFRII fusion protein had a higher frequency of Tumor Infiltrating Lymphocytes (TILs). Analysis of tumors from anti-PD 1-TGFRII treated mice also showed a higher ratio of CD8+ T cells to regulatory T cells (Tregs).

Example 5 anti-PD 1-TGFRII showed superior anti-tumor response compared to anti-PD 1 alone in an in vitro model of head and neck cancer

The effectiveness of an anti-PD 1-TGFRII fusion protein was evaluated in an in vitro model of head and neck cancer. PBMCs purified from healthy donors were co-cultured with the cancer cell line Detroit 562 for 48 hours. Cells were stimulated with anti-CD 3 in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol. Cell pellets from the co-cultures were collected by centrifugation at the end of the culture period and RNA was purified using Qiagen RNAeasy micro kit according to the manufacturer's protocol. Purified RNA was used for gene expression analysis using nano-strings (nanostring) according to the manufacturer's instructions. Gene expression was analyzed using Nanostring nCounter software.

As shown in figure 13A, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. anti-PD 1-TGFRII fusion protein treatment resulted in significant upregulation of interferon pathway genes (fig. 13B) as well as cytotoxic genes (fig. 13C) compared to anti-PD 1 treatment, highlighting the improved cytotoxic function in the presence of tumor compared to anti-PD 1 antibody. Furthermore, anti-PD 1-TGFRII fusion protein treatment resulted in significant down-regulation of genes associated with tumor metastasis and angiogenic pathways (fig. 13D). Overall, the results of this in vitro model show an excellent anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

Example 6 anti-PD 1-TGFRII showed superior anti-tumor response compared to anti-PD 1 alone in a humanized mouse model of head and neck cancer

The ability of the anti-PD 1-TGFRII fusion protein to inhibit head and neck cancer was evaluated in a humanized mouse model. The D562 cell line was administered to humanized NSG mice. Tumor-bearing mice were randomized and administered twice weekly with anti-PD 1-TGFRII fusion protein, anti-PD 1 antibody alone, or isotype control. As shown in figure 14, the anti-PD 1-TGFRII fusion protein significantly inhibited tumor growth (figure 14A), improved survival of tumor-bearing mice (figure 14B), and increased IFN γ levels (figure 14F) compared to anti-PD 1 and isotype controls. Treatment with anti-PD 1-TGFRII fusion protein significantly improved CD8 in tumors ⁺T cell to Treg ratio (fig. 14C).

Example 7 anti-PD 1-TGFRII showed superior anti-tumor response compared to anti-PD 1 alone in primary colorectal cancer (CRC) patient samples

The effectiveness of an anti-PD 1-TGFRII fusion protein was evaluated in primary colorectal cancer patient samples. PBMCs from CRC patients were co-cultured with dissociated tumor cells. Cells were stimulated in the presence of isotype control, anti-PD 1, or anti-PD 1-TGFRII fusion proteins. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol. TGF-. beta.levels were evaluated by Luminex following the manufacturer's protocol. Cell pellets from the co-cultures were collected by centrifugation at the end of the culture period and RNA was purified using Qiagen RNAeasy micro kit according to the manufacturer's protocol. Purified RNA was used for gene expression analysis using nano-strings (nanostring) according to the manufacturer's instructions. Gene expression was analyzed using Nanostring nCounter software.

As shown in figure 15A, the anti-PD 1-TGFRII fusion protein promoted higher levels of IFN- γ production compared to anti-PD 1 or isotype controls. The anti-PD-TGFRII fusion protein completely neutralized TGF- β 1 produced by patient PBMCs and dissociated tumor cells (fig. 15B). anti-PD 1-TGFRII fusion protein treatment resulted in significant up-regulation of cytotoxic genes compared to anti-PD 1 treatment (fig. 15C), highlighting the improved cytotoxic function in the presence of tumor compared to anti-PD 1 antibody. Furthermore, anti-PD 1-TGFRII fusion protein treatment resulted in significant down-regulation of genes associated with tumor metastasis and angiogenic pathways (fig. 15C). As expected, the TGF- β signaling pathway was significantly down-regulated in the anti-PD 1-TGFRII fusion protein treatment group. Overall, the results of this patient-derived sample show an excellent anti-tumor response of the anti-PD 1-TGFRII fusion protein compared to the anti-PD 1 antibody.

Example 8 anti-PD 1-TGFRII exhibits increased cytotoxicity compared to anti-PD-L1-TGFRII in an ovarian cancer in vitro model

The effectiveness of anti-PD 1-TGFRII was compared to anti-PD-L1-TGFRII in an in vitro model of ovarian cancer. PBMC purified from healthy donors were co-cultured with the cancer cell line SK-OV-3. Immune cells were stimulated in the presence of isotype control, anti-PD 1-TGFRII fusion protein or anti-PD-L1-TGFRII fusion protein. Killing of tumor cells throughout the culture period was evaluated using the Incucyte viable cell analysis system.

As shown in figure 16, the presence of anti-PD 1-TGFRII fusion protein showed increased killing of tumor cells compared to anti-PD-L1-TGFRII fusion protein. Overall, anti-PD 1-TGFRII showed improved cytotoxicity compared to anti-PD-L1-TGFRII.

Example 9 combination of anti-PD 1-TGF-RII with CAR-T significantly enhanced 3D tumor sphere killing compared to either anti-PD 1 in combination or CAR-T alone

anti-PD 1-TGFRII fusion protein in combination with CAR-T cells was evaluated in an in vitro 3D tumor sphere model of ovarian cancer. Note that in this example, CAR-T cells refer to T cells engineered to express MUC16 CAR. 3D tumor spheroids were generated by co-culturing the ovarian cancer cell line SK-OV-3 and a fibroblast cell line. CAR-T cells were then added to 3D tumor sphere cultures in the presence of anti-PD 1-TGFRII fusion protein or anti-PD 1. Target-specific killing of 3D tumor spheres throughout the period was assessed using the Incucyte viable cell analysis system.

As shown in figure 17, the combination of anti-PD 1-TGFRII fusion protein and CAR-T cells showed increased killing of 3-D tumor spheres compared to the combination of anti-PD 1 and CAR-T cells or CAR-T cells alone. Overall, the results of this killing assay indicate that the addition of anti-PD-1-TGF-RII to CAR-T cells enhances the anti-tumor response of CAR-T cells.

Example 10 combination of anti-PD 1-TGF-RII with CD33 specific CAR-T cells

CD33 CAR-T cells were co-cultured with MOLM-13AML tumor cell line in a cytotoxicity assay and cultures were incubated in the IncuCyte S3 live cell analyzer. anti-PD 1 or anti-PD 1-TGFRII were added to the indicated cultures at equimolar concentrations. Sytox Green was added directly at the beginning of the culture to allow identification and enumeration of dying cells in the culture. Analysis was performed using IncuCyte S3 software. Data presented in the graph are mean ± SD of triplicate wells scanned 5 times per well. As shown in figure 18A, the combination of anti-PD 1(VH6/VL5) -TGFRII fusion protein with CD33 CAR T cells showed increased killing compared to CAR T cells in combination with anti-PD 1(VH6/VL5) or CAR T cells alone.

As shown in figure 18B, the combination of anti-PD 1(VH7/VL6) -TGFRII fusion protein with CD33 CAR-T cells showed increased killing compared to CAR-T cells and anti-PD 1 combination or CAR-T cells alone. Overall, the results of these cytotoxicity assays demonstrated that anti-PD 1(VH6/VL5) -TGFRII fusion protein and anti-PD 1(VH7/VL6) -TGFRII fusion protein enhance the anti-tumor immune response of CAR-T cells.

Example 11 combination of anti-PD 1-TGF-RII with CD19 CAR-T cells enhances tumor cell killing

CD19 CAR-T cells were co-cultured with tumor cells expressing CD19 antigen on the cell surface in the presence of anti-PD 1-TGFRII fusion protein or anti-PD 1. The combination of anti-PD 1-TGFRII fusion protein with CD19 CAR-T cells showed increased tumor cell killing compared to the combination of anti-PD 1 and CAR-T cells or CAR-T cells alone.

Example 12 combination of anti-PD 1-TGF-RII with BCMA-specific CAR-T improves the cytotoxic potential of CAR-T cells

BCMA-specific CAR-T cells were co-cultured with BCMA antigen expressing tumor cell lines in the presence or absence of anti-PD 1-TGFRII fusion protein or anti-PD 1. The combination of the anti-PD 1-TGFRII fusion protein with CAR-T cells improves the cytotoxic potential of CAR-T cells compared to the combination of anti-PD 1 and CAR-T cells or CAR-T cells alone.

Example 13 combination of anti-PD 1-TGF-RII with PSMA-specific CAR-T enhances killing of tumor cells by CAR-T cells

PSMA-specific CAR-T cells were co-cultured with PSMA antigen-expressing tumor cell lines in the presence or absence of anti-PD 1-TGFRII fusion protein or anti-PD 1. The combination of the anti-PD 1-TGFRII fusion protein with CAR-T cells improved the cytotoxicity of CAR-T cells compared to the combination of anti-PD 1 and CAR-T cells or CAR-T cells alone.

Example 14 anti-PD 1-TGFRII improves NK cell target cell lysis

The ability of anti-PD 1(VH7/VL6) -TGFRII and anti-PD 1(VH6/VL5) -TGFRII to promote natural killer cell-mediated killing of tumor cells was evaluated in an in vitro model of ovarian cancer. Effector NK cells were purified from healthy donors and co-cultured overnight with SK-OV-3 target cell lines at different E: T cell ratios in the presence of anti-RSV-TFGBRII (control), anti-PD 1(VH6/VL5) and anti-PD 1(VH7/VL6), anti-PD 1(VH7/VL6) -TGFRII and anti-PD 1(VH6/VL5) -TGFRII. Propidium iodide was added to the culture to identify dead cells, and cell death was determined using Nexcelom Celigo. As observed in fig. 19A and 19B, the anti-PD 1-TGFBRII fusion protein promoted higher tumor cell killing compared to anti-PD 1 or TGFBRII (anti-RSV-TGFBRII) alone.

Example 15: anti-PD 1-ADA2 fusion protein

anti-PD 1 VH and VL were synthesized and formatted in an IgG, scFv-Fc or scFv configuration, with adenosine deaminase 2(ADA2) fused at the C-terminus via linker (G4S) 2. anti-PD 1-adenosine deaminase fusion protein was transiently expressed in Expi293 cells and purified using AKTA AVANT system according to the manufacturer's protocol.

Surface plasmon resonance

Surface Plasmon Resonance (SPR) analysis was performed using a Biacore3000, CM5 chip, amine coupling kit, 10X HBS-P running buffer and glycine. For the anti-PD 1 kinetic assay, the recombinant PD-1Fc protein was immobilized on the chip using a pre-determined ligand immobilization procedure. The purified anti-PD 1-adenosine deaminase fusion protein was diluted to a range of final concentrations in running buffer and injected. Allowing it to dissociate and subsequently regenerating the chip surface.

TABLE 10 affinity of anti-PD 1-ADA2 for immobilized PD1

PD-1/PDL-1 blocking assay

To investigate whether the anti-PD 1-ADA2 fusion protein could act as an anti-PD 1 antibody by blocking the PD-1/PD-L1 interaction, a PD-1/PD-L1 blocking bioassay kit from Promega was used as described in example 1. These values were used as a measure of the blocking potency of PD-1/PD-L1 (FIG. 21).

ADA enzyme Activity assay

To determine the enzymatic activity against the PD1 ADA2 fusion protein, a colorimetric Adenosine Deaminase (ADA) activity kit (Abcam) was used according to the manufacturer's recommendations. Time points of 2 minutes and 30 minutes were selected to calculate ADA enzyme activity over time. As shown in fig. 22, the anti-PD 1-ADA2 fusion protein exhibited enzymatic activity over time.

Example 16: PD1 binding affinity analysis against PD1 IgG 4S 108P-wtADA2 and-mutADA 2

PD1-Fc antigen and reference were immobilized on a Biacore CM5 surface and then 6-12 replicates of serial dilution concentrations of anti-PD 1 IgG 4S 108P-wtADA2 and-mutADA 2 were serially injected onto PD1-Fc antigen and reference surface. For the 1:1 model: langmuir with mass transfer, kinetic data were evaluated. The results are shown in Table 11.

Table 11: binding affinities of anti-PD 1(VH6/VL5) and (VH7/VL6) IgG 4S 108P and anti-PD 1-wtADA2 and-mutADA 2

Example 17: anti-PD 1-ADA2 fusion protein effectively blocks PD-L1/PD1 signaling

The ability of the anti-PD 1-ADA2 fusion protein to block the PD1/PD-L1 interaction was evaluated using the reporter bioassay mentioned in example 12.

As shown in FIGS. 23A-23B, anti-PD 1(VH7/VL6) -wtADA2 and-mut 7ADA2, anti-PD 1(VH6/VL5) -wtADA2 and-mut 7ADA2 blocked the PD1/PD-L1 interaction with similar efficacy.

Example 18: ADA2 enzymatic Activity

The ability of the anti-PD 1-ADA2 fusion protein to enzymatically degrade adenosine was evaluated in the in vitro ADA enzyme activity assay mentioned in example 12.

As shown in FIGS. 24A-24F, at lower adenosine concentrations, mut7ADA2 had higher enzyme activity compared to wtADA 2.

Example 19: the anti-PD 1-mut7-ADA2 has higher enzyme activity than the anti-PD 1-wtADA2

The Michaelis Menten constant (Km) was measured against PD1(VH6/VL5) -wtADA2 and against PD1(VH6/VL5) -mut7ADA2 using the fluorescent ADA2 enzyme assay kit. As observed in FIG. 25, anti-PD 1-mut7ADA2 has a higher Km than anti-PD 1-wtADA 2.

Example 20: anti-PD 1-wtADA2 reverses adenosine-mediated suppression of T-cell proliferation

The ability of anti-PD 1-wtADA2 to promote T cell proliferation was assessed in vitro using normal donor PBMC. CellTrace purple-labeled PBMC co-cultured in medium containing 1mM adenosine were stimulated in the presence of increasing concentrations of anti-PD 1(VH6/VL5) -wtADA2 and anti-PD 1(VH6/VL 5). The supernatant was collected for cytokine analysis. T cell proliferation of cells was determined by flow cytometry. The data show that anti-PD 1(VH6/VL5) -wtADA2 significantly reversed adenosine-mediated CD4 compared to anti-PD 1(VH6/VL5) or isotype control⁺(FIG. 26A) and CD8⁺Inhibition of T cells (fig. 26B). As observed in FIGS. 26C and D, both anti-PD 1 and anti-PD 1-wtADA2 similarly occupied CD4⁺(FIG. 26C) and CD8⁺PD1 receptor on T cells (fig. 26D).

The ability of anti-PD 1(VH7/VL6) -wtADA2 to promote T cell function was further assessed by stimulating PBMCs in adenosine-containing media in the presence of increasing concentrations of anti-D1 (VH7/VL6), anti-PD 1(VH7/VL6) -wtADA2, or isotype controls. Culture supernatants were collected and assayed for IFN γ production. Figure 26E shows that anti-PD 1(VH7/VL6) -wtADA2 treatment induced significantly more IFN- γ compared to anti-PD 1 or isotype control.

Example 21: WtADA2 and mutADA2 showed comparable reversal of adenosine-mediated suppression of T cell proliferation

The effectiveness of wtADA2 and mutADA2 in reversing adenosine-mediated suppression of T cell proliferation was assessed by stimulating labeled PBMCs in adenosine-containing medium in the presence of increasing concentrations of anti-PD 1-wtADA2, anti-PD 1-mutADA2, anti-RSV-wtADA 2 and anti-RSV-mutADA 2. Cells were stained and then analyzed for proliferation by flow cytometry. The wtADA2 and mutADA fusion proteins were equally effective in reversing adenosine-mediated suppression of CD 4T cells (fig. 27A) and CD8T cells (fig. 27B).

Example 22: blockade of PD1-PDL1 interaction by anti-PD 1(VH6/VL5) -ADA2

The ability of various forms of the anti-PD 1-ADA2 fusion protein to block the PD1/PD-L1 interaction was evaluated using the reporter bioassay mentioned in example 12.

As shown in FIG. 28, anti-PD 1(VH6/VL5) scFv-Fc-ADA2, hIgG1-ADA2, and scFv-ADA2 all showed functional activity as measured by PD1/PD-L1 blocking bioassay.

Example 23: ADA enzyme Activity against PD1-ADA2-scFv-Fc

The ability of various forms of the anti-PD 1-ADA2 fusion protein to enzymatically degrade adenosine was evaluated in the in vitro ADA enzyme activity assay mentioned in example 12.

As shown in FIG. 29, anti-PD 1(VH7/VL6) scFv-Fc-ADA2 and (VH6/VL5) scFv-Fc-ADA2 have similar ADA2 enzyme activity comparable to the control.

Example 24: ADA enzyme Activity against various PD1-ADA2 and against PD1-mutADA2 constructs

ADA enzyme activity assays were performed as described above. As shown in FIG. 30, mutADA2 had higher enzyme activity at 10nM adenosine compared to wtADA 2.

Example 25: in primary CRC patient tumors, anti-PD 1-ADA2 promotes IFN- γ production and Tumor Infiltrating Lymphocyte (TIL) proliferation

PBMCs purified from CRC patients were co-cultured with matched dissociated tumor cells. CellTrace purple-labeled cells were stimulated with anti-CD 3 and anti-CD 28 in the presence of an isotype control, anti-PD 1(VH6/VL5) or anti-PD 1(VH6/VL5) -wtADA2 fusion protein. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol and cells were evaluated for T cell proliferation. For gene expression analysis, cell pellets were collected by centrifugation and RNA was purified using Qiagen RNAeasy micro kit according to the manufacturer's protocol. Purified RNA was used for gene expression analysis using nano-strings (nanostring) according to the manufacturer's instructions. Gene expression was analyzed using Nanostring nCounter software.

As shown in figure 31A, the anti-PD 1-wtADA2 fusion protein promoted higher levels of IFN- γ production and T cell proliferation compared to anti-PD 1 or isotype control (figure 31B). anti-PD 1-wtADA2 fusion protein treatment resulted in significant upregulation of IFN- γ signaling and chemokine signaling genes as well as cytotoxic genes compared to anti-PD 1 treatment (fig. 31C), highlighting the improved cytotoxic function of T cells in the presence of tumor compared to anti-PD 1 antibody.

Example 26: effect of anti-PD 1-wtADA2 fusion protein in humanized mouse model of Lung cancer

NSG mice humanized with peripheral blood mononuclear cells were intrapancreatically (injected orthotopically) inoculated with the pancreatic cancer cell line panc.08. fluc.egfp. Mouse body weights were measured weekly and tumor weights were measured at the end of the study on day 38. Mice were randomized into treatment groups and treated twice weekly with HBSS (vehicle control), anti-PD 1(VH6/VL5) and anti-PD 1(VH6/VL5) -wtADA 2. As can be seen in figure 32, mice treated with anti-PD-wtADA 2 had significantly smaller tumors than mice treated with anti-PD 1 or isotype control.

As can be seen in figure 32, mice treated with anti-PD-wtADA 2 had significantly smaller tumors than mice treated with anti-PD 1 or isotype control.

Example 27: anti-PD 1(VH6/VL5) -wtADA2 treatment down-regulates adenosine pathways, angiogenesis and up-regulates cytotoxicity and cytokine genes

PBMCs purified from CRC patients were co-cultured with matched dissociated tumor cells. CellTrace purple-labeled cells were stimulated with anti-CD 3 and anti-CD 28 in the presence of an isotype control, anti-PD 1(VH6/VL5) or anti-PD 1(VH6/VL5) -wtADA2 fusion protein. Culture supernatants were collected and IFN- γ levels were quantified by MSD according to the manufacturer's protocol and cells were evaluated for T cell proliferation. For gene expression analysis, the culture was repeated, cell pellets were collected by centrifugation, and RNA was purified using Qiagen RNAeasy micro kit according to the manufacturer's protocol. Purified RNA was used for gene expression analysis using nano-strings (nanostring) according to the manufacturer's instructions. Gene expression was analyzed using Nanostring nCounter software.

The anti-PD 1-wtADA2 fusion protein promoted higher levels of IFN- γ production and T cell proliferation compared to anti-PD 1 or isotype controls. anti-PD 1-wtADA2 fusion protein treatment also resulted in significant upregulation of IFN- γ signaling and chemokine signaling genes as well as cytotoxic genes compared to anti-PD 1 treatment (data not shown), highlighting the improved cytotoxic function of T cells in the presence of tumor compared to anti-PD 1 antibody.

Sequence of

Provided herein is a representative list of certain sequences included in the embodiments provided herein.

TABLE 4 non-limiting exemplary polypeptide and nucleotide sequences

TABLE 12 exemplary anti-TGF β VH and VL sequences

Table 12 provides exemplary TGF β receptor sequences for the anti-PD 1 fusion proteins described herein. In any of the embodiments illustrated with a fusion protein such as a tgfbetarii ECD, an anti-tgfbeta antibody (e.g., scFv, Fab) may be used instead.

TABLE 13 exemplary TGF-beta 1 inhibitory peptides and nucleotide sequences

Table 13 provides exemplary TGF β 1 inhibitory peptides for the anti-PD 1 fusion proteins described herein. In any of the embodiments illustrated with a fusion protein such as a TGF β RII ECD, a TGF β 1 inhibitory peptide may be used instead.

TABLE 14 exemplary ADA2 sequences

Table 14 provides exemplary ADA2 sequences for the anti-PD 1 fusion proteins described herein. In any embodiment illustrating a fusion protein with, for example, a tgfbetarii ECD, the ADA2 sequence may be used instead.

TABLE 15 exemplary anti-PD 1 VH/VL pair

Table 15 provides exemplary anti-PD 1 VH/VL pairs for the fusion proteins described herein.

Exemplary anti-PD 1 VL	Exemplary anti-PD 1 VH
		anti-PD 1 VL5(SEQ ID NO:12)	anti-PD 1 VH6(SEQ ID NO:6)
anti-PD 1 VL6(SEQ ID NO:13)	anti-PD 1 VH7(SEQ ID NO:7)
		anti-PD 1 VL1(SEQ ID NO:8)	anti-PD 1 VH5(SEQ ID NO:5)
anti-PD 1 nVL1(SEQ ID NO:8)	anti-PD 1 nVH3(SEQ ID NO:149)
		anti-PD 1 nVL1(SEQ ID NO:8)	anti-PD 1 nVH7(SEQ ID NO:157)
anti-PD 1 nVL1(SEQ ID NO:8)	anti-PD 1 nVH8(SEQ ID NO:158)

TABLE 16 exemplary anti-PD 1-TGFbRII ECD fusion protein sequences

Table 16 provides exemplary anti-PD 1-TGFbRII ECD fusion protein sequences. The anti-PD 1 fusion protein comprises a sequence encoding the anti-PD 1 variable region of the light chain and a sequence encoding the anti-PD 1 variable region of the heavy chain. ECD stands for extracellular domain. "wt" refers to the wild-type sequence and "mut" refers to the mutant sequence.

TABLE 17 exemplary anti-PD 1-ADA2 fusion protein sequences

Table 17 provides exemplary anti-PD 1-ADA2 fusion protein sequences the anti-PD 1 fusion protein comprises sequences encoding the anti-PD 1 variable region of the light chain and the anti-PD 1 variable region of the heavy chain. "wt" refers to the wild-type sequence, and "mut" refers to the mutant sequence