阅读说明：本技术 胶原蛋白定位的免疫调节分子及其方法 (Collagen-localized immunomodulatory molecules and methods thereof ) 是由 K·D·维特拉普 N·穆明 J·帕尔梅里 M·钦 于 2019-07-26 设计创作，主要内容包括：本公开内容提供了免疫调节融合蛋白,其包含与免疫调节结构域可操作地连接的胶原蛋白结合结构域。本公开内容还涉及使用其例如治疗癌症的组合物和方法。(The present disclosure provides immunomodulatory fusion proteins comprising a collagen binding domain operably linked to an immunomodulatory domain. The disclosure also relates to compositions and methods of using the same, for example, to treat cancer.)

1. An immunomodulatory fusion protein, comprising:

(i) an immunomodulatory domain;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

(iii) optionally, the presence of a linker,

wherein the immunomodulatory domain is operably linked to the collagen binding domain with or without the linker.

2. The immunomodulatory fusion protein of claim 1, wherein the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D。

3. The immunomodulatory fusion protein of any of claims 1-2, wherein the collagen-binding domain has a MW of about 5-100kDa, about 10-80kDa, about 20-60kDa, about 30-50kDa, or about 10kDa, about 20kDa, about 30kDa, about 40kDa, about 50kDa, about 60kDa, about 70kDa, about 80kDa, about 90kDa, or about 100 kDa.

4. The immunomodulatory fusion protein of any of claims 1-3, wherein the collagen binding domain comprises one or more leucine rich repeats that bind collagen.

5. The immunomodulatory fusion protein of claim 4, wherein the collagen binding domain comprises two, three, four, five, six, seven, eight, nine, or ten leucine-rich repeats that bind collagen.

6. The immunomodulatory fusion protein of any of claims 1-5, wherein the collagen binding domain comprises one or more leucine-rich repeats from a class II human proteoglycan member of the leucine-rich small proteoglycan (SLRP) family.

7. The immunomodulatory fusion protein of claim 6, wherein the SLRP is selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, aspergilloprotein, PRELP, osteonectin/osteomodulin, opticalmodulin, osteonectin/mimecan, podocan, basement membrane glycans, and entactin.

8. The immunomodulatory fusion protein of claim 7, wherein the SLRP is a basement membrane glycan.

9. The immunomodulatory fusion protein of any of claims 1-5, wherein the collagen binding domain comprises a human SLRP.

10. The immunomodulatory fusion protein of claim 9, wherein the SLRP is selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, aspergilloprotein, PRELP, osteonectin/osteomodulin, opticalmodulin, osteochanoglycan/mimecan, podocan, basement membrane glycans, and entactin.

11. The immunomodulatory fusion protein of claim 10, wherein the SLRP is a basement membrane glycan.

12. The immunomodulatory fusion protein of claim 11, wherein the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID No. 107.

13. The immunomodulatory fusion protein of any of claims 1-3, wherein the collagen-binding domain comprises a human type I glycoprotein with an Ig-like domain or binds to the extracellular portion of collagen.

14. The immunomodulatory fusion protein of claim 13, wherein the type I glycoprotein competes with basement membrane glycans for binding to type I collagen.

15. The immunomodulatory fusion protein of any of claims 13-14, wherein the human type I glycoprotein is selected from the group consisting of LAIR1, LAIR2, and glycoprotein IV.

16. The immunomodulatory fusion protein of claim 14, wherein the human type I glycoprotein is LAIR 1.

17. The immunomodulatory fusion protein of claim 13, wherein the human type I glycoprotein is LAIR1 and the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID No. 98.

18. The immunomodulatory fusion protein of any of claims 1-3, wherein the collagen binding domain comprises a LAIR1 variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

19. The immunomodulatory fusion protein of any of claims 1-3, wherein the collagen binding domain comprises a LAIR1 variant having increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

20. The immunomodulatory fusion protein of any of claims 1-3, wherein the collagen binding domain comprises a LAIR1 variant having reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

21. The immunomodulatory fusion protein of any of claims 1-20, wherein the immunomodulatory domain comprises a polypeptide that activates, enhances, or promotes a response of an immune cell.

22. The immunomodulatory fusion protein of any of claims 1-20, wherein the immunomodulatory domain comprises a polypeptide that inhibits, reduces, or suppresses a response of an immune cell.

23. The immunomodulatory fusion protein of any of claims 21-22, wherein the immune cell is a lymphoid cell selected from the group consisting of innate lymphoid cells, T cells, B cells, NK cells, and combinations thereof.

24. The immunomodulatory fusion protein of any of claims 21-23, wherein the immune cell is a myeloid cell selected from the group consisting of monocytes, neutrophils, granulocytes, mast cells, macrophages, dendritic cells, and combinations thereof.

25. The immunomodulatory fusion protein of any of claims 21-24, wherein the response of the immune cell comprises production of cytokines, production of antibodies, production of antigen-specific immune cells, increased effector function and/or cytotoxicity, and combinations thereof.

26. The immunomodulatory fusion protein of any of claims 21-25, wherein the immunomodulatory domain comprises one or more selected from cytokines, chemokines, activating ligands/receptors, inhibitory ligands/receptors, or combinations thereof.

27. The immunomodulatory fusion protein of claim 26, wherein the immunomodulatory domain comprises one or more cytokines.

28. The immunomodulatory fusion protein of claim 27, wherein the cytokine is a human gamma shared chain receptor interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-13, IL-15/IL-15RA, IL-21, and combinations thereof.

29. The immunomodulatory fusion protein of claim 28, wherein the cytokine is IL-2.

30. The immunomodulatory fusion protein of claim 27, wherein the cytokine is a human IL-12 family member selected from IL-12(p35), IL-12(p40), IL-12(p35)/IL-12(p40), IL-23, IL-27, IL-35, and combinations thereof.

31. The immunomodulatory fusion protein of claim 30, wherein the cytokine is a single chain fusion of IL-12(p35)/IL-12(p 40).

32. The immunomodulatory fusion protein of claim 27, wherein the cytokine is a human IL-1 family member selected from the group consisting of IL-1, IL-18, IL-33, and combinations thereof.

33. The immunomodulatory fusion protein of claim 32, wherein the cytokine is IL-18.

34. The immunomodulatory fusion protein of claim 27, wherein the cytokine is selected from the group consisting of TNF α, INF α, IFN- γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

35. The immunomodulatory fusion protein of claim 26, wherein the immunomodulatory domain comprises one or more chemokines.

36. The immunomodulatory fusion protein of claim 35, wherein the chemokine is selected from the group consisting of LIF, MIP-2, MIP-1 a, MIP-1 β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin (Eotaxin), RANTES, LIX, and combinations thereof.

37. The immunomodulatory fusion protein of claim 35, wherein the chemokine is selected from the group consisting of CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

38. The immunomodulatory fusion protein of claim 26, wherein the immunomodulatory domain comprises one or more activating ligands/receptors.

39. The immunomodulatory fusion protein of claim 38, wherein the activating ligand/receptor is selected from the TNF superfamily, CD28 receptor superfamily, B7 ligand family, and T cell receptor.

40. The immunomodulatory fusion protein of claim 39, wherein the activating ligand/receptor is a TNF superfamily ligand selected from TNF α, CD40L, 4-1BBL, OX40, and combinations thereof.

41. The immunomodulatory fusion protein of claim 39, wherein the activating ligand/receptor is a TNF superfamily receptor and the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of an anti-TNFR 1 antibody, an anti-TNFR 2 antibody, an anti-CD 40 antibody, an anti-4-1 BB antibody, and an anti-OX 40 antibody.

42. The immunomodulatory fusion protein of claim 39, wherein the activating ligand/receptor is a CD28 superfamily member or a B7 family member selected from ICOS ligand, CD80 and CD86, and combinations thereof.

43. The immunomodulatory fusion protein of claim 39, wherein the activating ligand/receptor is a member of the CD28 superfamily and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-ICOS antibody and an anti-CD 28 antibody.

44. The immunomodulatory fusion protein of claim 39, wherein the activating ligand/receptor is a T cell receptor and the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of an anti-CD 3 γ antibody, an anti-CD 3 δ antibody, an anti-CD 3 ζ antibody and an anti-CD 3 ε antibody.

45. The immunomodulatory fusion protein of claim 26, wherein the immunomodulatory domain comprises one or more inhibitory ligands/receptors.

46. The immunomodulatory fusion protein of claim 45, wherein the inhibitory ligand/receptor is selected from the group consisting of the CD28 receptor superfamily, the TNF superfamily, and checkpoint inhibitors.

47. The immunomodulatory fusion protein of claim 46, wherein the inhibitory ligand/receptor is a member of the CD28 superfamily and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA 4 antibody.

48. The immunomodulatory fusion protein of claim 46, wherein the inhibitory ligand/receptor is a TNF superfamily member and the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of an anti-TIGIT antibody and an anti-BTLA antibody.

49. The immunomodulatory fusion protein of claim 46, wherein the inhibitory ligand/receptor is a checkpoint inhibitor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD 47 antibody and an anti-SIRPa antibody.

50. The immunomodulatory fusion protein of any of claims 1-49, wherein the immunomodulatory domain is operably linked to the collagen binding domain by a linker.

51. The immunomodulatory fusion protein of claim 50, wherein the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers.

52. The immunomodulatory fusion protein of claim 50, wherein the linker provides the fusion protein with sufficient molecular weight to reduce diffusion from tissue.

53. The immunomodulatory fusion protein of claim 50, wherein the linker allows spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement.

54. The immunomodulatory fusion protein of any of claims 51-53, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein 1-1000, 10-900, 30-800, 40-700, 50-600, 100-500, or 200-400.

55. The immunomodulatory fusion protein of claim 50, wherein the linker is human serum albumin or a fragment thereof.

56. The immunomodulatory fusion protein of claim 50, wherein the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

57. The immunomodulatory fusion protein of any of claims 1-56, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

58. The immunomodulatory fusion protein of claim 50, wherein the fusion protein is ≧ 60 kDa.

59. The immunomodulatory fusion protein of claim 50, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

60. An immunomodulatory fusion protein, comprising:

(i) at least one cytokine;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain <10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the cytokine is operably linked to the collagen binding domain via the linker and wherein the fusion protein is ≧ 60 kDa.

61. The immunomodulatory fusion protein of claim 60, wherein the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D。

62. The immunomodulatory fusion protein of any of claims 60-61, wherein the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, apocrin, PRELP, osteoadhesin/osteomodulin, optically active proteins, osteoglyceroglycans/mimecan, podocan, basement membrane glycans, and entactin.

63. The immunomodulatory fusion protein of claim 62, wherein the SLRP is a basement membrane glycan.

64. The immunomodulatory fusion protein of claim 63, wherein the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107.

65. The immunomodulatory fusion protein of any of claims 60-61, wherein the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV.

66. The immunomodulatory fusion protein of claim 65, wherein the collagen binding domain is LAIR 1.

67. The immunomodulatory fusion protein of claim 66, wherein the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98.

68. The immunomodulatory fusion protein of claim 66, wherein the collagen binding domain comprises a LAIR1 variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

69. The immunomodulatory fusion protein of claim 66, wherein the collagen binding domain comprises a LAIR1 variant having increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

70. The immunomodulatory fusion protein of claim 66, wherein the collagen binding domain comprises a LAIR1 variant having reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

71. The immunomodulatory fusion protein of any of claims 60-70, wherein the cytokine is a human gamma shared chain receptor interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-13, IL-15/IL-15RA, IL-21, and combinations thereof.

72. The immunomodulatory fusion protein of claim 71, wherein the cytokine is IL-2.

73. The immunomodulatory fusion protein of any of claims 60-70, wherein the cytokine is a human IL-12 family member selected from the group consisting of IL-12(p35), IL-12(p40), IL-12(p35)/IL-12(p40), IL-23, IL-27, IL-35, and combinations thereof.

74. The immunomodulatory fusion protein of claim 73, wherein the cytokine is a single chain fusion of IL-12(p35)/IL-12(p 40).

75. The immunomodulatory fusion protein of claim 74, comprising a second cytokine, wherein the second cytokine is IL-2.

76. The immunomodulatory fusion protein of any of claims 60-70, wherein the cytokine is a human IL-1 family member selected from the group consisting of IL-1, IL-18, IL-33, and combinations thereof.

77. The immunomodulatory fusion protein of any of claims 60-70, wherein the cytokine is selected from the group consisting of TNF α, INF α, IFN- γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

78. The immunomodulatory fusion protein of any of claims 60-77, wherein the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement.

79. The immunomodulatory fusion protein of any of claims 60-77, wherein the linker is human serum albumin or a fragment thereof.

80. The immunomodulatory fusion protein of any of claims 60-77, wherein the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

81. The immunomodulatory fusion protein of any of claims 60-80, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

82. The immunomodulatory fusion protein of any of claims 60-80, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

83. An immunomodulatory fusion protein, comprising:

(i) at least one chemokine;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the chemokine is operably linked to the collagen binding domain by the linker and wherein the fusion protein is ≧ 60 kDa.

84. The immunomodulatory fusion protein of claim 83, wherein the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D。

85. The immunomodulatory fusion protein of any of claims 83-84, wherein the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, apocrin, PRELP, osteoadhesin/osteomodulin, optically active proteins, osteoglyceroglycans/mimecan, podocan, basement membrane glycans, and entactin.

86. The immunomodulatory fusion protein of claim 85, wherein the SLRP is a basement membrane glycan.

87. The immunomodulatory fusion protein of claim 86, wherein the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107.

88. The immunomodulatory fusion protein of any of claims 83-84, wherein the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV.

89. The immunomodulatory fusion protein of claim 88, wherein the collagen binding domain is LAIR 1.

90. The immunomodulatory fusion protein of claim 89, wherein the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98.

91. The immunomodulatory fusion protein of claim 89, wherein the collagen binding domain comprises a LAIR1 variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

92. The immunomodulatory fusion protein of claim 89, wherein the collagen binding domain comprises a LAIR1 variant having increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

93. The immunomodulatory fusion protein of claim 89, wherein the collagen binding domain comprises a LAIR1 variant having reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

94. The immunomodulatory fusion protein of any of claims 83-93, wherein the chemokine is selected from the group consisting of LIF, MIP-2, MIP-1 a, MIP-1 β, CXCL1, CXCL9, CXCL10, MCP-1, eotaxin, RANTES, LIX, and combinations thereof.

95. The immunomodulatory fusion protein of any of claims 83-93, wherein the chemokine is selected from the group consisting of CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

96. The immunomodulatory fusion protein of any of claims 83-95, wherein the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement.

97. The immunomodulatory fusion protein of any of claims 83-95, wherein the linker is human serum albumin or a fragment thereof.

98. The immunomodulatory fusion protein of any of claims 83-95, wherein the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

99. The immunomodulatory fusion protein of any of claims 83-98, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

100. The immunomodulatory fusion protein of any of claims 83-98, wherein the fusion protein binds type I and/or type IV collagen after in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

101. An immunomodulatory fusion protein, comprising:

(i) an agonist antibody that binds an activating ligand/receptor, the agonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction; and

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa;

wherein the collagen binding domain is operably linked to the C-terminus of the Fc domain or mutant Fc domain.

102. The immunomodulatory fusion protein of claim 101, wherein the collagen binding domain is directed against type I and/or type IV collagen K _DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D。

103. The immunomodulatory fusion protein of any of claims 101-102, wherein the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, apocrin, PRELP, osteoadhesin/osteomodulin, opticalmodulin, osteocampan/mimecan, podocan, perlecan, and nidogen.

104. The immunomodulatory fusion protein of claim 103, wherein the SLRP is a basement membrane glycan.

105. The immunomodulatory fusion protein of claim 104, wherein the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID No. 107.

106. The immunomodulatory fusion protein of any of claims 101-102, wherein the collagen binding domain is selected from the group consisting of LAIR1, LAIR2, and glycoprotein IV.

107. The immunomodulatory fusion protein of claim 106, wherein the collagen binding domain is LAIR 1.

108. The immunomodulatory fusion protein of claim 107, wherein the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO: 98.

109. The immunomodulatory fusion protein of claim 107, wherein the collagen binding domain comprises a LAIR1 variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

110. The immunomodulatory fusion protein of claim 107, wherein the collagen binding domain comprises a LAIR1 variant having increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

111. The immunomodulatory fusion protein of claim 107, wherein the collagen binding domain comprises a LAIR1 variant having reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

112. The immunomodulatory fusion protein of any one of claims 101-111, wherein the agonist antibody is selected from the group consisting of an anti-TNFR 1 antibody, an anti-TNFR 2 antibody, an anti-CD 40 antibody, an anti-4-1 BB antibody, and an anti-OX 40 antibody.

113. The immunomodulatory fusion protein of any of claims 101-111, wherein the agonist antibody is selected from the group consisting of an anti-ICOS antibody and an anti-CD 28 antibody.

114. The immunomodulatory fusion protein of any of claims 101-111, wherein the antibody is selected from the group consisting of an anti-CD 3 γ antibody, an anti-CD 3 δ antibody, an anti-CD 3 ζ antibody and an anti-CD 3 epsilon antibody.

115. The immunomodulatory fusion protein of any of claims 101-114, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

116. The immunomodulatory fusion protein of any of claims 101-114, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

117. An immunomodulatory fusion protein, comprising:

(i) an antagonist antibody that binds an inhibitory ligand/receptor, the antagonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction; and

(ii) A collagen binding domain, wherein the collagen binding domain specifically binds to type IAnd/or type IV collagen and in K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa;

wherein the collagen binding domain is operably linked to the C-terminus of the Fc domain or variant Fc domain.

118. The immunomodulatory fusion protein of claim 117, wherein the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D。

119. The immunomodulatory fusion protein of any of claims 117-118, wherein the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondroprotein, apocrin, PRELP, osteoadhesin/osteomodulin, opticalmodulin, osteochannan/mimecan, podocan, perlecan, and nidogen.

120. The immunomodulatory fusion protein of claim 119, wherein the SLRP is a basement membrane glycan.

121. The immunomodulatory fusion protein of claim 120, wherein the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID No. 107.

122. The immunomodulatory fusion protein of any of claims 117-118, wherein the collagen binding domain is selected from the group consisting of LAIR1, LAIR2, and glycoprotein IV.

123. The immunomodulatory fusion protein of claim 122, wherein the collagen binding domain is LAIR 1.

124. The immunomodulatory fusion protein of claim 111, wherein the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO: 98.

125. The immunomodulatory fusion protein of claim 123, wherein the collagen binding domain comprises a LAIR1 variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

126. The immunomodulatory fusion protein of claim 123, wherein the collagen binding domain comprises a LAIR1 variant having increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98.

127. The immunomodulatory fusion protein of claim 123, wherein the collagen binding domain comprises a LAIR1 variant having reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

128. The immunomodulatory fusion protein of any one of claims 117-127, wherein the antagonist antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA 4 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD 47 antibody, and an anti-sirpa antibody.

129. The immunomodulatory fusion protein of any of claims 117-128, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

130. The immunomodulatory fusion protein of any of claims 117-128, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

131. An immunomodulatory fusion protein, comprising:

(i) human IL-2;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein IL-2 is operably linked to the basement membrane glycan or LAIR1 through the linker and wherein the fusion protein is ≧ 60 kDa.

132. An immunomodulatory fusion protein, comprising:

(i) single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-stranded fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

133. An immunomodulatory fusion protein, comprising:

(i) human CCL-3;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein CCL-3 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

134. An immunomodulatory fusion protein, comprising:

(i) human CCL-4;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein CCL-4 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

135. An immunomodulatory fusion protein, comprising:

(i) human CCL-5;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

Wherein CCL-5 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

136. An immunomodulatory fusion protein, comprising:

(i) human eotaxin;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein eotaxin is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

137. The immunomodulatory fusion protein of any of claims 131-136, wherein the basement membrane glycan comprises the amino acid sequence shown in SEQ ID No. 107.

138. The immunomodulatory fusion protein of any of claims 131-136, wherein the collagen binding domain LAIR1 comprises the amino acid sequence set forth in SEQ ID No. 98 or comprises one or more amino acid substitutions, additions or deletions relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID No. 98, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions.

139. The immunomodulatory fusion protein of any of claims 131-138, wherein the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement.

140. The immunomodulatory fusion protein of any of claims 131-138, wherein the linker is human serum albumin or a fragment thereof.

141. The immunomodulatory fusion protein of any of claims 131-138, wherein the linker comprises an Fc domain or a mutated Fc domain with reduced FcR interaction.

142. The immunomodulatory fusion protein of any of claims 131-141, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

143. The immunomodulatory fusion protein of any of claims 131-142, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

144. An immunomodulatory fusion protein, comprising:

(i) an agonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction, wherein the agonist antibody is selected from the group consisting of an anti-CD 3 antibody, an anti-4-1-BB antibody, an anti-CD 40 antibody, and an anti-OX 40 antibody; and

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant;

wherein a basement membrane glycan or LAIR1 is operably linked to the C-terminus of the Fc domain or mutant Fc domain.

145. The immunomodulatory fusion protein of claim 144, wherein the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration.

146. The immunomodulatory fusion protein of any of claims 144-145, wherein the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

147. A pharmaceutical composition comprising an immunomodulatory fusion protein according to any one of the preceding claims and a pharmaceutically acceptable carrier.

148. A nucleic acid comprising a nucleotide sequence encoding the immunomodulatory fusion protein of any of claims 1-146.

149. An expression vector comprising the nucleic acid of claim 148.

150. A cell transformed with the expression vector of claim 149.

151. A method for producing an immunomodulatory fusion protein, the method comprising maintaining a cell according to claim 150 under conditions that allow expression of the immunomodulatory fusion protein.

152. The method of claim 151, further comprising obtaining the immunomodulatory fusion protein.

153. A method for activating, enhancing or promoting an immune cell response in a subject comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any one of claims 1-146 or the pharmaceutical composition of claim 147.

154. A method of inhibiting, reducing, or suppressing an immune cell response in a subject, comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any one of claims 1-146 or the pharmaceutical composition of claim 147.

155. The method of any one of claims 153-154, wherein the immune cells are lymphoid cells selected from the group consisting of innate lymphoid cells, T cells, B cells, NK cells, and combinations thereof.

156. The method of any one of claims 153-154, wherein the immune cell is a myeloid cell selected from the group consisting of a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell, and a combination thereof.

157. The method of any one of claims 153-156, wherein the response of the immune cell comprises cytokine production, antibody production, antigen-specific immune cell production, increased effector function and/or cytotoxicity, and combinations thereof.

158. The method of any one of claims 153-157, wherein the response of the immune cell occurs in a tumor microenvironment.

159. A method for reducing or inhibiting tumor growth, comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any one of claims 1-146 or the pharmaceutical composition of claim 147.

160. A method for treating cancer in a subject, comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any one of claims 1-146 or the pharmaceutical composition of claim 147.

161. The method of any one of claims 159-160, wherein an anti-tumor immune response is induced in the subject following administration of the immunomodulatory fusion protein or the pharmaceutical composition.

162. The method of claim 161, wherein the anti-tumor immune response is a T cell response comprising production of IFN γ and/or IL-2 by one or both of CD4+ T cells and CD8+ T cells.

163. The method of any one of claims 159-162, wherein infiltration of immune cells into the tumor microenvironment is increased following administration of the immunomodulatory fusion protein or the pharmaceutical composition.

164. The method of any one of claims 159-163, wherein following administration of the immunomodulatory fusion protein or the pharmaceutical composition, the amount of regulatory T (treg) cells in the tumor microenvironment is reduced or the T cell depletion in the tumor microenvironment is reduced.

165. The method of any one of claims 153-164 wherein the immunomodulatory fusion protein or pharmaceutical composition is administered intratumorally.

166. A kit for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof, the kit comprising a container comprising the immunomodulatory protein of any one of claims 1-146 and optionally a pharmaceutically acceptable carrier or the pharmaceutical composition of claim 147, and a package insert comprising instructions for administering the fusion protein or pharmaceutical composition.

167. A kit for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof, the kit comprising a container comprising the immunomodulatory fusion protein of any one of claims 1-146 and optionally a pharmaceutically acceptable carrier or the pharmaceutical composition of claim 147, and a package insert comprising instructions for administering the antibody or pharmaceutical composition alone or in combination with another agent.

168. Use of an immunomodulatory fusion protein according to any one of claims 1-146 and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition according to claim 147 for the preparation of a medicament for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof.

169. An immunomodulatory fusion protein according to any one of claims 1-146 and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition according to claim 147 for use in the preparation of a medicament for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof.

170. An immunomodulatory fusion protein according to any one of claims 1-146 and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition according to claim 147 for use as a medicament.

171. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any of claims 1-146 or the pharmaceutical composition of claim 147, and an effective amount of a second composition comprising a tumor antigen-targeting antibody, or antigen-binding fragment thereof, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

172. The method of claim 156, wherein the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen.

173. The method of any one of claims 171-172, wherein the tumor antigen targeting antibody specifically binds to human HER-2/neu, EGFR, VEGFR, CD20, CD33, or CD 38.

174. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any of claims 146 or the pharmaceutical composition of claim 147, and an effective amount of a second composition comprising a cancer vaccine, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

175. The method of claim 174, wherein the cancer vaccine is a population of cells immunized in vitro with a tumor antigen and administered to the subject.

176. The method of claim 174, wherein the cancer vaccine is a peptide comprising one or more tumor associated antigens.

177. The method of claim 174, wherein the cancer vaccine is an amphiphilic peptide conjugate comprising a tumor-associated antigen, a lipid, and optionally a linker, wherein the amphiphilic peptide conjugate binds albumin under physiological conditions.

178. The method of any one of claims 174-177, wherein the cancer vaccine further comprises an adjuvant.

179. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any of claims 1-146 or the pharmaceutical composition of claim 147, and an effective amount of a second composition comprising an immune checkpoint inhibitor, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

180. The method of claim 179, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds PD-1, PD-L1, CTLA-4, LAG3, or TIM 3.

181. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the immunomodulatory fusion protein of any of claims 1-146 or the pharmaceutical composition of claim 147, and an effective amount of a second composition comprising adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

182. The method of claim 181, wherein the adoptive cell therapy comprises immune effector cells comprising a Chimeric Antigen Receptor (CAR) molecule that binds a tumor antigen.

183. The method of any of claims 181-182, wherein the CAR molecule comprises an antigen binding domain, a transmembrane domain and an endodomain comprising a costimulatory domain and/or a primary signaling domain.

184. The method of claim 183, wherein the antigen binding domain binds to a tumor antigen associated with the disease.

185. The method of claim 183, wherein the tumor antigen is selected from the group consisting of CD19, EGFR, Her2/neu, CD30, and BCMA.

186. The method of any one of claims 182-185, wherein the immune effector cell is a T cell, such as a CD8+ T cell.

187. The method of any one of claims 182-185, wherein the immune effector cell is a Natural Killer (NK) cell.

188. The method of any one of claims 182-185, wherein the immunomodulatory fusion protein or the pharmaceutical composition is administered intratumorally.

189. The method of any one of claims 171-188, wherein the immunomodulatory fusion protein or the pharmaceutical composition and the second composition are administered simultaneously or sequentially.

190. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein comprising:

(i) human IL-2;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein IL-2 is operably linked to a basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa,

Thereby reducing or inhibiting tumor growth or treating cancer in the subject.

191. A method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein comprising:

(i) single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-chain fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa,

thereby reducing or inhibiting tumor growth or treating cancer in the subject.

192. The method of claim 190, further comprising administering a second composition comprising an effective amount of an immunomodulatory fusion protein comprising:

(i) single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) A linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-chain fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60 kDa.

193. The method of any one of claims 190-192, further comprising administering a second composition comprising an effective amount of a tumor antigen targeting antibody or antigen binding fragment thereof.

194. The method of any one of claims 190-193, further comprising administering a second composition comprising an effective amount of a composition comprising a cancer vaccine.

195. The method of any one of claims 190-194, further comprising administering a second composition comprising an effective amount of a second composition comprising an immune checkpoint inhibitor.

196. The method of claim 195, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds PD-1, PD-L1, CTLA-4, LAG3, or TIM 3.

197. The method of any one of claims 190-196, further comprising administering a second composition comprising an effective amount of a second composition comprising an adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject.

198. The method of claim 197, wherein the adoptive cell therapy comprises an immune effector cell comprising a Chimeric Antigen Receptor (CAR) molecule that binds a tumor antigen.

199. The method of claim 198, wherein the immune effector cell is a T cell (e.g., a CD8+ T cell) or an NK cell.

200. The method of any one of claims 190-199, wherein the immunomodulatory fusion protein or the pharmaceutical composition is administered intratumorally.

201. The method of any one of claims 190-200, wherein the immunomodulatory fusion protein or the pharmaceutical composition and the second composition are administered simultaneously or sequentially.

Background

Although immunotherapy has altered oncology with a long lasting curative response in a few patients, immune-related adverse events (irAE) have limited its broadest application (Michot et al, 2016, Eur J Cancer,54: 139-. It is desirable to limit the most effective immune activation events to tumor tissue while sparing non-tumor healthy tissue. The accepted goal of new immunotherapies is to "heat" immunologically "cold" tumors, driving inflammation and immune cell infiltration (Chen and Mellman 2017, Nature,541: 321-. Various methods of tumor localization have been proposed: linking immunomodulators to tumor targeting modules in immunocytokines (Hutmacher and Neri 2018, Adv Drug Deliv Rev); systemic active masking agents with proteolytic activation of tumor localization (Thomas and Daugherty 2009, Protein Sci 18: 2053-; intratumoral injection of agents (Singh and overhijk 2015, Nat Commun 8: 1447; Ager et al 2017, Cancer Immunol Res 5: 676-; injecting solid biological material around the tumor to capture the agent (Park et al, 2018, Sci Transl Med,10: ear 1916); conjugated to solid particles (Kwong et al, 2013, Cancer Res 73:1547-1558) or conjugated with alkaline charged peptides to drive some non-specific adhesion of the agent to the tumor extracellular matrix (Ishihara et al, 2017, Sci Transl Med 9: eaan 0401; Ishihara et al, 2018, Mol Cancer The 17: 2399-2411). A related but unique approach is to localize growth factors in tissues to drive tissue regeneration (Nishi et al, 1998, Proc Natl Acad Sci 95: 7018-.

Each of the current methods described above presents serious problems. Immunocytokines systemically expose immune cells to immunomodulators (Tzeng et al, 2015, Proc Natl Acad Sci 112: 3320-3325). The masking agent may not be masked outside the target tissue, and the masking agent may complicate production and immunogenicity. Intratumoral injection usually results in rapid diffusion out of the tumor cavity. Conjugation of peptides at random sites is difficult to replicate, may negatively impact contrast activity, may not completely prevent tumor exit, and leads to significant CMC problems due to the heterogeneous products of the random conjugation process.

Thus, there remains a need for new immunotherapeutic approaches to promote tumor localization and increase effectiveness while preventing systemic toxicity.

Disclosure of Invention

The present disclosure is based, at least in part, on the following findings: immunomodulatory domains (e.g., cytokines, anti-immunoreceptor antibodies, anti-tumor associated antigen antibodies, etc.) can be conjugated to the collagen binding domain, resulting in enhanced anti-tumor effectiveness relative to the unconjugated immunomodulatory domain. Without wishing to be bound by theory, the collagen localization of the immunomodulatory domain results in enhanced anti-tumor effectiveness, as T cells are encapsulated in collagen-rich regions around tumors, thus making these sites desirable for targeting immunomodulatory agents. Almost half of human tumors display an immune rejection phenotype in which CD8+ T cells are apparently trapped in a collagen-rich proliferative matrix (Mariatasan et al, Nature,2018,554: 544-. Given the primary importance of CD8+ T cells in the effectiveness of immunotherapy, it is desirable to localize immunomodulators to this collagen-rich, CD8+ T cell-rich tumor compartment. Specificity is important because previous agents are retained by non-specific electrostatic interactions in small unstructured peptides (Martino et al, Science,2014,343:885-888), bind promiscuously to most negatively charged extracellular matrix components, rather than binding within specific collagen-rich target chambers. Such unstructured positively charged peptides also result in relatively poor retention kinetics, with in some cases leakage of half of the injected conjugate payload into the systemic circulation (Ishihara et al, Mol Cancer ther.2018,17: 2399-.

Thus, provided herein are immunomodulatory fusions with structural proteins having specific affinity for collagen, resulting in greater retention in specific collagen-rich target compartments. In some aspects described herein, the immunomodulatory fusion protein comprises a cytokine, wherein following intratumoral administration in a preclinical animal model, the collagen binding domain increases tumor retention and prevents systemic exposure to the cytokine, thereby reducing toxicity associated with the treatment. Furthermore, immunomodulatory fusion proteins have increased anti-tumor effectiveness and reduced toxicity when combined with one or more other immunotherapies (e.g., tumor targeting antibodies, checkpoint blockades, cancer vaccines, and T cell therapies) compared to equivalent fusion proteins lacking a collagen binding domain.

As provided herein, these immunomodulatory fusion proteins exhibit a durable and systemic anti-tumor response, enabling local immunization against injected tumors, and systemic immunization against contralateral non-injected tumors for effective treatment. The novel adjuvant administration of immunomodulatory fusion proteins may also improve survival by preventing metastasis following surgical resection of residual primary tumors, further demonstrating that immunomodulatory fusion proteins promote systemic anti-tumor immunity. Thus, immunomodulatory fusion proteins of the disclosure can be used to treat metastatic tumors and/or mediate distal effects (abscopal effects) in a therapeutic (e.g., anti-cancer) modality.

Also provided herein are variant collagen binding domains that have altered (e.g., increased or decreased) binding affinity for collagen. By disclosing the selection of variant collagen binding domains with different collagen binding affinities, the disclosure herein provides the option of selecting immunomodulatory fusion proteins with different binding affinities for a collagen-rich compartment (e.g., a collagen-expressing tumor).

The collagen binding compositions and methods provided herein allow tumor and payload independent local targeting of active therapies. Collagen-binding compositions have also been shown to have increased effectiveness, with concomitant reduction in toxicity associated with systemic immunotherapy.

In some aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) an immunomodulatory domain;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

(iii) optionally, the presence of a linker,

Wherein the immunomodulatory domain is operably linked to the collagen binding domain with or without the linker.

In some aspects, the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D. In some aspects, the collagen binding domain has a MW of about 5-100kDa, about 10-80kDa, about 20-60kDa, about 30-50kDa, or about 10kDa, about 20kDa, about 30kDa, about 40kDa, about 50kDa, about 60kDa, about 70kDa, about 80kDa, about 90kDa, or about 100 kDa.

In some aspects, the immunomodulatory fusion protein comprises a collagen binding domain comprising one or more leucine-rich repeats that bind collagen. In some aspects, the collagen-binding domain comprises two, three, four, five, six, seven, eight, nine, or ten leucine-rich repeats that bind collagen. In some aspects, the collagen binding domain comprises one or more leucine-rich repeats from a class II human proteoglycan member of the leucine-rich small proteoglycan (SLRP) family. In some aspects, the SLRP is selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondrin, aposporin, PRELP, osteoaggrecan/osteomodulin, optically active proteins, osteocampan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan.

In some aspects, the immunomodulatory fusion protein comprises a collagen binding domain comprising a human SLRP. In some aspects, the SLRP is selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, chondrin, aposporin, PRELP, osteoaggrecan/osteomodulin, optically active proteins, osteocampan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan. In some aspects, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107.

In some aspects, the immunomodulatory fusion protein comprises a collagen-binding domain comprising a human type I glycoprotein having an Ig-like domain or binds to the extracellular portion of collagen. In some aspects, the type I glycoprotein competes with the basement membrane glycan for binding to type I collagen. In some aspects, the human type I glycoprotein is selected from LAIR1, LAIR2, and glycoprotein IV. In some aspects, the human type I glycoprotein is LAIR 1. In some aspects, the human type I glycoprotein is LAIR1 and the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to a LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant has a reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

In any of the preceding aspects, the immunomodulatory domain comprises a polypeptide that activates, enhances, or promotes a response of an immune cell. In other aspects, the immunomodulatory domain comprises a polypeptide that inhibits, reduces, or suppresses a response of an immune cell.

In some aspects, the immune cell is a lymphoid cell selected from the group consisting of innate lymphoid cells, T cells, B cells, NK cells, and combinations thereof. In other aspects, the immune cell is a myeloid-lineage cell selected from the group consisting of a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell, and a combination thereof.

In some aspects, the response of the immune cell comprises cytokine production, antibody production, antigen-specific immune cell production, increased effector function and/or cytotoxicity, and combinations thereof.

In any of the preceding aspects, the immunomodulatory domain comprises one or more selected from a cytokine, a chemokine, an activating ligand/receptor, an inhibitory ligand/receptor, or a combination thereof. In some aspects, the immunomodulatory domain comprises one or more cytokines.

In some aspects, the cytokine is a human gamma shared chain receptor interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-13, IL-15/IL-15RA, IL-21, and combinations thereof. In some aspects, the cytokine is IL-2.

In some aspects, the cytokine is a human IL-12 family member selected from the group consisting of IL-12(p35), IL-12(p40), IL-12(p35)/IL-12(p40), IL-23, IL-27, IL-35, and combinations thereof. In some aspects, the cytokine is a single chain fusion of IL-12(p35)/IL-12(p 40).

In other aspects, the cytokine is a human IL-1 family member selected from the group consisting of IL-1, IL-18, IL-33, and combinations thereof. In some aspects, the cytokine is IL-18.

In still other aspects, the cytokine is selected from the group consisting of TNF α, INF α, IFN- γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

In some aspects, the immunomodulatory domain comprises one or more chemokines. In some aspects, the chemokine is selected from the group consisting of LIF, MIP-2, MIP-1 α, MIP-1 β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin (Eotaxin), RANTES, LIX, and combinations thereof. In other aspects, the chemokine is selected from the group consisting of CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

In any of the preceding aspects, the immunomodulatory domain comprises one or more activating ligands/receptors. In some aspects, the activating ligand/receptor is selected from the TNF superfamily, the CD28 receptor superfamily, the B7 ligand family, and the T cell receptor. In other aspects, the activating ligand/receptor is a TNF superfamily ligand selected from the group consisting of TNF α, CD40L, 4-1BBL, OX40, and combinations thereof. In still other aspects, the activating ligand/receptor is a TNF superfamily receptor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-TNFR 1 antibody, an anti-TNFR 2 antibody, an anti-CD 40 antibody, an anti-4-1 BB antibody, and an anti-OX 40 antibody. In other aspects, the activating ligand/receptor is a member of the CD28 superfamily or a member of the B7 family selected from ICOS ligands, CD80, and CD86, and combinations thereof. In still other aspects, the activating ligand/receptor is a member of the CD28 superfamily and the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of an anti-ICOS antibody and an anti-CD 28 antibody. In a further aspect, the activating ligand/receptor is a T cell receptor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-CD 3 γ antibody, an anti-CD 3 δ antibody, an anti-CD 3 ζ antibody and an anti-CD 3 epsilon antibody.

In some aspects, the activating ligand/receptor is selected from the TNF superfamily, the CD28 receptor superfamily, the B7 ligand family, the T cell receptor, the killer cell Ig-like receptor, the leukocyte Ig-like receptor, the CD94/NKG2 receptor family, and the Fc receptor. In other aspects, the activating ligand/receptor is a killer cell Ig-like receptor ligand and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-KIR 2DS1 antibody, an anti-KIR 2DS2 antibody, an anti-KIR 2DS3 antibody, an anti-KIR 2DS4 antibody, an anti-KIR 2DS5 antibody, and an anti-KIR 3DS1 antibody. In a further aspect, the activating ligand/receptor is a leukocyte Ig-like receptor and the immunomodulatory domain comprises an anti-LIRA 2 antibody or antigen-binding fragment thereof. In other aspects, the activating ligand/receptor is a member of the CD94/NKG2 receptor family selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 (subtype 1), ULBP5 (subtype 2), and ULBP 6. In still other aspects, the activating ligand/receptor is a member of the CD94/NKG2 receptor family and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-CD 94/NKG2D antibody, an anti-CD 94/NKG2C antibody, an anti-CD 94/NKG2E antibody, and an anti-CD 94/NKG2H antibody. In a further aspect, the activating ligand/receptor is an Fc receptor family member and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-Fc γ RI antibody, an anti-Fc γ RIIC antibody, an anti-Fc γ RIIIA antibody, an anti-Fc γ RIIIB antibody, an anti-Fc epsilon RI antibody, an anti-Fc epsilon RII antibody, an anti-Fc α R antibody, and an anti-Fc μ R antibody.

In any of the preceding aspects, the immunomodulatory domain comprises one or more inhibitory ligands/receptors. In some aspects, the inhibitory ligand/receptor is selected from the group consisting of the CD28 receptor superfamily, the TNF superfamily, and checkpoint inhibitors. In other aspects, the inhibitory ligand/receptor is a member of the CD28 superfamily and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA 4 antibody. In a further aspect, the inhibitory ligand/receptor is a TNF superfamily member and the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of an anti-TIGIT antibody and an anti-BTLA antibody. In some aspects, the inhibitory ligand/receptor is a checkpoint inhibitor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD 47 antibody, and an anti-sirpa antibody.

In some aspects, the inhibitory ligand/receptor is selected from the group consisting of the CD28 receptor superfamily, the TNF superfamily, the Siglec family, the CD94/NKG2A family, the leukocyte Ig-like receptor family, the killer Ig-like receptor ligand, the Fc receptor, the adenosine pathway molecule, and the checkpoint inhibitor. In other aspects, the inhibitory ligand/receptor comprises a Siglec family member and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-Siglec 1 antibody, an anti-Siglec 2 antibody, an anti-Siglec 3 antibody, an anti-Siglec 4a antibody, an anti-Siglec 5 antibody, an anti-Siglec 6 antibody, an anti-Siglec 7 antibody, an anti-Siglec 8 antibody, an anti-Siglec 9 antibody, an anti-Siglec 10 antibody, an anti-Siglec 11 antibody, and an anti-Siglec 12 antibody. In still other aspects, the inhibitory ligand/receptor comprises a CD94/NKG2 receptor family inhibitory receptor or inhibitory ligand and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-CD 94/NKG2A antibody and an anti-CD 94/NKG2B antibody. In some aspects, the inhibitory ligand/receptor comprises a leukocyte Ig-like receptor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-LIRB 1 antibody, an anti-LIRB 2 antibody, an anti-LIRB 3 antibody, an anti-LIRB 4 antibody. In other aspects, the inhibitory ligand/receptor comprises a killer cell Ig-like receptor ligand and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-KIR 2DL1 antibody, an anti-KIR 2DL2 antibody, an anti-KIR 2DL3 antibody, an anti-KIR 2DL4 antibody, an anti-KIR 2DL5A antibody, an anti-KIR 2DL5B antibody, an anti-KIR 3DL1 antibody, an anti-KIR 3DL2 antibody, and an anti-KIR 3DL3 antibody. In still other aspects, the inhibitory ligand/receptor comprises an Fc receptor and the immunomodulatory domain comprises an anti-fcyriib antibody or antigen-binding fragment. In some aspects, the inhibitory ligand/receptor comprises an adenosine pathway molecule and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-CD 39 antibody and an anti-CD 73 antibody. In other aspects, the inhibitory ligand/receptor comprises a checkpoint inhibitor and the immunomodulatory domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD 47 antibody, and an anti-sirpa antibody.

In any of the preceding aspects, the immunomodulatory domain is operably linked to the collagen binding domain by a linker. In some aspects, the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers. In some aspects, the linker provides the fusion protein with sufficient molecular weight to reduce diffusion from tissue. In some aspects, the linker allows spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement. In some aspects, the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein 1-1000, 10-900, 30-800, 40-700, 50-600, 100-. In some aspects, the linker is human serum albumin or a fragment thereof. In other aspects, the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

In any of the preceding aspects, the immunomodulatory fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein is ≧ 60 kDa. In some aspects, the immunomodulatory fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In some aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) at least one cytokine;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at 500nM or less, and wherein the glue isIsoelectric Point pI of Protein binding Domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and kDa; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the cytokine is operably linked to the collagen binding domain through the linker and wherein the fusion protein is>60 kDa. In some aspects, the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D. In some aspects, the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, cartilage protein, apocrin, PRELP, osteonectin/osteomodulin, optically active proteins, osteochanoglan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan. In some aspects, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107. In other aspects, the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV. In some aspects, the collagen binding domain is LAIR 1. In some aspects, the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant is relative to a LAIR comprising the amino acid sequence of SEQ ID No. 98 1 has a reduced binding affinity for collagen.

In some aspects, the cytokine is a human gamma shared chain receptor interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-13, IL-15/IL-15RA, IL-21, and combinations thereof. In some aspects, the cytokine is IL-2. In some aspects, the cytokine is a human IL-12 family member selected from the group consisting of IL-12(p35), IL-12(p40), IL-12(p35)/IL-12(p40), IL-23, IL-27, IL-35, and combinations thereof. In some aspects, the cytokine is a single chain fusion of IL-12(p35)/IL-12(p 40). In some aspects, the immunomodulatory fusion protein comprises a second cytokine. In some aspects, the second cytokine is IL-2.

In other aspects, the cytokine is a human IL-1 family member selected from the group consisting of IL-1, IL-18, IL-33, and combinations thereof. In still other aspects, the cytokine is selected from the group consisting of TNF α, INF α, IFN- γ, GM-CSF, FLT3L, G-CSF, M-CSF, and combinations thereof.

In some aspects, the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement. In some aspects, the linker is human serum albumin or a fragment thereof. In other aspects, the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) at least one chemokine;

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and kDa; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the chemokine is operably linked to the collagen binding domain by the linker, and wherein the fusion protein is ≧ 60 kDa. In some aspects, the collagen binding domain is directed against type I and/or type IV collagen K _DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D. In some aspects, the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, cartilage protein, apocrin, PRELP, osteonectin/osteomodulin, optically active proteins, osteochanoglan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan. In some aspects, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107. In other aspects, the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV. In some aspects, the collagen binding domain is LAIR 1. In some aspects, the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In that In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant has a reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

In some aspects, the chemokine is selected from the group consisting of LIF, MIP-2, MIP-1 α, MIP-1 β, CXCL1, CXCL9, CXCL10, MCP-1, eotaxin, RANTES, LIX, and combinations thereof. In some aspects, the chemokine is selected from the group consisting of CCL3, CCL4, CCL5, eotaxin, and combinations thereof.

In some aspects, the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement. In some aspects, the linker is human serum albumin or a fragment thereof. In other aspects, the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In yet other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) an agonist antibody that binds to an activating ligand/receptor, the agonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction; and

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_D≤500nM binds type I collagen, and wherein the isoelectric point pI of the collagen binding domain<10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

wherein the collagen binding domain is operably linked to the C-terminus of the Fc domain or mutant Fc domain. In some aspects, the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen _D. In some aspects, the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, cartilage protein, apocrin, PRELP, osteonectin/osteomodulin, optically active proteins, osteochanoglan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan. In some aspects, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107. In other aspects, the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV. In some aspects, the collagen binding domain is LAIR 1. In some aspects, the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant has a reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

In some aspects, the agonist antibody is selected from an anti-TNFR 1 antibody, an anti-TNFR 2 antibody, an anti-CD 40 antibody, an anti-4-1 BB antibody, and an anti-OX 40 antibody. In other aspects, the agonist antibody is selected from an anti-ICOS antibody and an anti-CD 28 antibody. In some aspects, the agonist antibody is selected from an anti-CD 3 γ antibody, an anti-CD 3 δ antibody, an anti-CD 3 ζ antibody, and an anti-CD 3 epsilon antibody.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In a further aspect, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) an antagonist antibody that binds an inhibitory ligand/receptor, the antagonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction; and

(ii) a collagen binding domain, wherein the collagen binding domain specifically binds type I and/or type IV collagen and is expressed as K_DBinds collagen type I at ≦ 500nM, and wherein the isoelectric point pI of the collagen binding domain <10 and the Molecular Weight (MW) is more than or equal to 5 kDa; and

wherein the collagen binding domain is operably linked to the C-terminus of the Fc domain or mutant Fc domain. In some aspects, the collagen binding domain is directed against type I and/or type IV collagen K_DLess than the collagen binding domain for a K of an extracellular matrix component selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D. In some aspects, the collagen binding domain comprises a human SLRP selected from the group consisting of basement membrane glycans, decorin, biglycan, fibromodulin, cartilage protein, apocrin, PRELP, osteonectin/osteomodulin, optically active proteins, osteochanoglan/mimecan, podocan, basement membrane glycans, and entactin. In some aspects, the SLRP is a basement membrane glycan. In some aspects, the base filmThe glycan comprises the amino acid sequence shown in SEQ ID NO: 107. In other aspects, the collagen binding domain is selected from LAIR1, LAIR2, and glycoprotein IV. In some aspects, the collagen binding domain is LAIR 1. In some aspects, the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant has a reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

In some aspects, the antagonist antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA 4 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD 47 antibody, and an anti-sirpa antibody.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) human IL-2;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein IL-2 is operably linked to the basement membrane glycan or LAIR1 through the linker and wherein the fusion protein is ≧ 60 kDa.

In a further aspect, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) Single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-stranded fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

In a further aspect, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) human CCL-3;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein CCL-3 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

In other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) human CCL-4;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

Wherein CCL-4 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

In some aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) human CCL-5;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein CCL-5 is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

In other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) human eotaxin;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein eotaxin is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is ≧ 60 kDa.

In any of the preceding aspects, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107.

In any of the preceding aspects, the LAIR1 comprises the amino acid sequence set forth in SEQ ID NO 98. In some embodiments, the LAIR1 is a variant comprising one or more amino acid substitutions, additions or deletions, optionally two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions, additions or deletions, relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has increased binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In other further embodiments, the LAIR1 variant has a reduced binding affinity for collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

In any of the preceding aspects, the linker is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on collagen fibers, and/or to provide the fusion protein with sufficient molecular weight to reduce diffusion from tissue, and/or to allow spatial separation of the immunomodulatory domain from collagen fibers to facilitate receptor/ligand engagement. In some aspects, the linker is human serum albumin or a fragment thereof. In other aspects, the linker comprises an Fc domain or a mutant Fc domain with reduced FcR interaction.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In yet other aspects, the present disclosure provides an immunomodulatory fusion protein comprising:

(i) an agonist antibody comprising an Fc domain or a mutant Fc domain with reduced FcR interaction, wherein the agonist antibody is selected from the group consisting of an anti-CD 3 antibody, an anti-4-1-BB antibody, an anti-CD 40 antibody, and an anti-OX 40 antibody; and

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant;

wherein a basement membrane glycan or LAIR1 is operably linked to the C-terminus of the Fc domain or mutant Fc domain.

In some aspects, the fusion protein is of sufficient mass to reduce size-dependent escape by diffusion or convection following in vivo administration. In some aspects, the fusion protein binds type I and/or type IV collagen following in vivo administration, thereby reducing systemic exposure of the immunomodulatory fusion protein.

In some aspects, the present disclosure provides a pharmaceutical composition comprising an immunomodulatory fusion protein disclosed herein, and a pharmaceutically acceptable carrier.

In other aspects, the disclosure provides a nucleotide sequence encoding an immunomodulatory fusion protein disclosed herein. In some aspects, the present disclosure provides an expression vector comprising a nucleic acid disclosed herein. In other aspects, the present disclosure provides a cell transformed with an expression vector disclosed herein.

In another aspect, the disclosure provides a method for producing an immunomodulatory fusion protein, the method comprising maintaining a cell described herein under conditions that allow expression of the immunomodulatory fusion protein. In a further aspect, the method comprises obtaining the immunomodulatory fusion protein.

In other aspects, the present disclosure provides a method of activating, enhancing or promoting an immune cell response in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition disclosed herein.

In a still further aspect, the present disclosure provides a method of inhibiting, reducing or suppressing an immune cell response in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition disclosed herein.

In any preceding aspect, the immune cell is a lymphoid cell selected from the group consisting of innate lymphoid cells, T cells, B cells, NK cells, and combinations thereof. In other aspects, the immune cell is a myeloid-lineage cell selected from the group consisting of a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell, and a combination thereof. In some aspects, the response of the immune cell comprises cytokine production, antibody production, antigen-specific immune cell production, increased effector function and/or cytotoxicity, and combinations thereof. In some aspects, the immune cell occurs in a tumor microenvironment.

In other aspects, the present disclosure provides a method for reducing or inhibiting tumor growth comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition disclosed herein.

In a further aspect, the present disclosure provides a method for treating cancer in a subject comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition disclosed herein.

In any of the preceding aspects, an anti-tumor immune response is induced in the subject following administration of the immunomodulatory fusion protein or the pharmaceutical composition. In some aspects, the anti-tumor immune response is a T cell response comprising IFN γ and/or IL-2 produced by one or both of CD4+ T cells and CD8+ T cells.

In any of the preceding aspects, infiltration of immune cells into the tumor microenvironment is increased following administration of the immunomodulatory fusion protein or the pharmaceutical composition.

In any preceding aspect, the amount of regulatory t (treg) cells in the tumor microenvironment is decreased following administration of the immunomodulatory fusion protein or the pharmaceutical composition. In any of the preceding aspects, the T cell depletion in the tumor microenvironment is reduced following administration of the immunomodulatory fusion protein or the pharmaceutical composition.

In any of the preceding aspects, the immunomodulatory fusion protein or pharmaceutical composition is administered intratumorally.

In any of the preceding aspects, the immunomodulatory fusion protein or pharmaceutical composition is administered by viral vector, electroporation, transplantation of cells expressing the immunomodulatory fusion protein, or replicon.

In other aspects, the disclosure provides a kit for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof, the kit comprising a container comprising an immunomodulatory protein described herein and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition described herein, and a package insert comprising instructions for administering the fusion protein or pharmaceutical composition.

In a further aspect, the present disclosure provides a kit for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof, the kit comprising a container comprising an immunomodulatory fusion protein described herein and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition described herein, and a package insert comprising instructions for administering the antibody or pharmaceutical composition, alone or in combination with another agent.

In some aspects, the disclosure provides the use of an immunomodulatory fusion protein described herein and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition described herein for the preparation of a medicament for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof.

In other aspects, the disclosure provides an immunomodulatory fusion protein described herein and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition described herein for use in the preparation of a medicament for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof.

In a further aspect, the present disclosure provides an immunomodulatory fusion protein described herein and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition described herein for use as a medicament.

In other aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition described herein and an effective amount of a second composition comprising a tumor antigen-targeting antibody or antigen-binding fragment thereof, thereby reducing or inhibiting tumor growth or treating cancer in the subject. In some aspects, the tumor antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor neoantigen. In other aspects, the tumor antigen targeting antibody specifically binds to human HER-2/neu, EGFR, VEGFR, CD20, CD33, or CD 38.

In still other aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition described herein and an effective amount of a second composition comprising a cancer vaccine, thereby reducing or inhibiting tumor growth or treating cancer in the subject. In some aspects, the cancer vaccine is a population of cells that are immunized with a tumor antigen in vitro and administered to the subject. In other aspects, the cancer vaccine is a peptide comprising one or more tumor associated antigens. In some aspects, the cancer vaccine is an amphiphilic peptide conjugate comprising a tumor associated antigen, a lipid, and optionally a linker, wherein the amphiphilic peptide conjugate binds albumin under physiological conditions. In some aspects, the cancer vaccine further comprises an adjuvant.

In some aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition described herein and an effective amount of a second composition comprising an immune checkpoint inhibitor, thereby reducing or inhibiting tumor growth or treating cancer in the subject. In some aspects, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds PD-1, PD-L1, CTLA-4, LAG3, or TIM 3.

In a further aspect, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein or pharmaceutical composition described herein and an effective amount of a second composition comprising an adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject. In some aspects, the adoptive cell therapy comprises an immune effector cell comprising a Chimeric Antigen Receptor (CAR) molecule that binds a tumor antigen. In some aspects, the CAR molecule comprises an antigen binding domain, a transmembrane domain, and an endodomain comprising a costimulatory domain and/or a primary signaling domain. In some aspects, the antigen binding domain binds to the tumor antigen associated with the disease. In some aspects, the tumor antigen is selected from the group consisting of CD19, EGFR, Her2/neu, CD30, and BCMA. In some aspects, the immune effector cell is a T cell, e.g., a CD8+ T cell. In some aspects, the immune effector cell is a Natural Killer (NK) cell.

In any of the foregoing methods, the immunomodulatory fusion protein or the pharmaceutical composition is administered intratumorally. In some aspects, the immunomodulatory fusion protein or the pharmaceutical composition and the second composition are administered simultaneously or sequentially.

In other aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein comprising:

(i) human IL-2;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein IL-2 is operably linked to a basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa,

thereby reducing or inhibiting tumor growth or treating cancer in the subject.

In some aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunomodulatory fusion protein comprising:

(i) Single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-chain fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa,

thereby reducing or inhibiting tumor growth or treating cancer in the subject.

In some aspects, the present disclosure provides a method for reducing or inhibiting tumor growth or treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a first composition comprising an immunomodulatory fusion protein comprising:

(i) human IL-2;

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein IL-2 is operably linked to a basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa, and a second composition comprising an effective amount of an immunomodulatory fusion protein comprising:

(i) Single-chain fusions of human IL-12(p35)/IL-12(p 40);

(ii) human basement membrane glycan, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N is 1-1000, 10-900, 30-800, 40-700, 50-600, 100-,

wherein the single-chain fusion of IL-12(p35)/IL-12(p40) is operably linked to the basement membrane glycan or LAIR1 through the linker, and wherein the fusion protein is >60kDa,

thereby reducing or inhibiting tumor growth or treating cancer in the subject.

In some aspects, the method further comprises administering a second (or third, or fourth) composition comprising an effective amount of a tumor antigen-targeting antibody or antigen-binding fragment thereof. In other aspects, the method further comprises administering a second composition comprising an effective amount of a composition comprising a cancer vaccine. In still other aspects, the method further comprises administering a second composition comprising an effective amount of a second composition comprising an immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof that binds PD-1, PD-L1, CTLA-4, LAG3, or TIM 3.

In another aspect, the method further comprises administering a second composition comprising an effective amount of a second composition comprising an adoptive cell therapy, thereby reducing or inhibiting tumor growth or treating cancer in the subject. In some aspects, the adoptive cell therapy comprises an immune effector cell comprising a Chimeric Antigen Receptor (CAR) molecule that binds a tumor antigen. In some aspects, the immune effector cell is a T cell (e.g., a CD8+ T cell) or an NK cell.

In any of the preceding aspects, the immunomodulatory fusion protein or the pharmaceutical composition is administered intratumorally.

In any of the preceding aspects, the immunomodulatory fusion protein or the pharmaceutical composition and the second composition are administered simultaneously or sequentially.

Drawings

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

FIG. 1A provides a graph showing the binding of Gauss luciferase alone (Gluc) or fused to a collagen-binding polypeptide (basement Membrane glycan-Gluc, ColG s3a/s3b-Gluc, ColH s3-Gluc) to type I collagen as a function of concentration. Binding was determined by ELISA.

FIG. 1B provides a graph showing the binding of Gauss luciferase alone (Gluc) or fused to a collagen-binding polypeptide (basement Membrane glycan-Gluc, ColG s3a/s3B-Gluc, ColH s3-Gluc) to type IV collagen as a function of concentration. Binding was determined by ELISA.

FIG. 1C provides a graph showing His-tagged mouse LAIR-1(mLAIR1-His) and His-tagged biotinylated basement membrane glycans (Lwt-HIS-b) binding to type I collagen as a function of concentration. Binding was determined by ELISA using an anti-HIS antibody conjugated to horseradish peroxidase (HRP).

Figure 1D provides a graph showing competitive binding to type I collagen between His-tagged mouse LAIR-1(mLAIR1) and His-tagged biotinylated basement membrane glycans as a function of mLAIR1 concentration. Basement membrane glycans bound to type I collagen were determined by competition ELISA using streptavidin conjugated to horseradish peroxidase (HRP) in the presence of various concentrations of mLAIR 1.

Figure 2A provides a graph of quantification of fluorescently labeled basement membrane glycan or basement membrane glycan-MSA over time versus tumor fluorescence as compared to fluorescently labeled MSA after intratumoral injection in B16F10-Trp2KO tumor as determined by in vivo fluorescence imaging.

FIG. 2B provides a plot of the quantitative fluorescence in percent injected dose from B16F10-Trp2KO tumor-bearing mouse sera injected with fluorescently labeled basement membrane glycan-MSA or should labeled MSA. Serum fluorescence was determined by fluorescence imaging of microaneurysm heparin coated tubes containing mouse blood samples.

Figure 3A provides the Mantel-Cox survival curves for B16F10 melanoma-bearing mice treated with PBS (control) (i.tu.), MSA-IL2(i.tu.), basement membrane glycan-MSA-IL 2(i.tu.), or basement membrane glycan (i.tu.). On days 6 and 12, mice (n ═ 5 or 7 per treatment group) were treated as indicated (arrows). Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P < 0.002).

Fig. 3B provides Mantel-Cox survival curves for B16F10 melanoma-bearing mice treated intratumorally with PBS (n-7, i.tu.), anti-TYRP 1 antibody (TA99) (i.p.), in combination with MSA-IL2 (n-17, i.tu.), basement membrane glycan-MSA-IL 2 (n-17, i.tu.), or with basement membrane glycan (n-17, i.tu.). Mice were treated on days 6, 12 and 18 as indicated (arrows). Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P <0.03), ((P <0.002), ((P <0.0002), () and (P <0.0001), n.s., without significance.

Figure 3C provides Mantel-Cox survival curves for B16F10 melanoma-bearing mice treated with PBS (control) (i.tu.) or with anti-TYRP 1 antibody (TA99) (i.p.) in combination with intratumoral (i.tu.), paratumoral (peri.tu) (i.e., adjacent to the tumor) or subcutaneous (s.c. caudal) administration near the caudal root of the tail, of basement membrane glycan-MSA-IL 2. Mice were treated on days 6, 12 and 18 as indicated (arrows). Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P <0.03), ((P <0.002), ((P <0.0002), () and (P <0.0001), n.s., without significance.

Fig. 3D provides Mantel-Cox survival curves for B16F10 melanoma-bearing mice treated with PBS (control) by both (i.tu.) and inguinal tumor draining lymph nodes (i.tdln), with anti-TYRP 1 antibody (TA99) (i.p.) in combination with basement membrane glycan-MSA-IL 2(i.tu.) and PBS (i.tdln), or with anti-TYRP 1 antibody (TA99) (i.p.) in combination with basement membrane glycan-MSA-IL 2(i.tdln) and PBS (i.tu). On days 6 and 12, mice (n ═ 7 per treatment group) were treated as indicated (arrows). Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P < 0.002).

Figure 4 provides B16F10 melanoma-bearing mouse BatF3 treated with PBS (control) (i.tu.) or anti-TYRP 1 antibody (TA99) (i.p.) in combination with basement membrane glycan-MSA-IL 2(i.tu) and immune cell-depleting or cytokine-neutralizing antibodies as indicated^-/-Or the Mantel-Cox survival curve of wild-type (WT) mice. Mice (n ═ 5 per treatment group) were treated as indicated on days 6, 12, and 18. Survival statistics were determined by log rank Mantel-Cox test. Significance is shown by (P)<0.03)、**(P<0.002)、***(P<0.0002)、****(P<0.0001) means, n.s., no significance.

Figure 5A provides a plot of the quantification of IFN γ + cells in live CD45+ CD3+ CD8+ T cells derived from mice excised on day 10 (treated as described in figure 3B), stimulated with irradiated B16F10 or 4T1 cells for 12 hours in the presence of brefeldin a, followed by splenocytes stained for surface markers and intracellular IFN γ (n ═ 5 mice per treatment group). Data analysis was performed by one-way ANOVA combined with Tukey multiple comparison test.

Figure 5B provides a graph of the mean tumor area and the percent survival (right) of the contralateral (untreated) (left panel) and ipsilateral (treated) (middle panel) lesions from treated B16F10 melanoma-bearing mice monitored over time (n-7/group). On day 0, mice were inoculated with B16F10 cells on the right flank (ipsilateral) and B16F10 cells on the left flank (contralateral). Ipsilateral tumors were treated intratumorally with TA99(i.p.) on days 6 and 12. Tumor area (mean + s.d.) and survival (right) of contralateral (untreated) and ipsilateral (treated) lesions were monitored over time (left panel) (n-7/group). For each group, tumor area is shown until mice reach the standard of euthanasia. Survival statistics were determined by log rank Mantel-Cox test. Assumed significance was with P < 0.03; p < 0.002; p < 0.0002; p < 0.0001; n.s., no significance.

Fig. 6A provides a graph of quantification of B16F10 melanoma tumor-bearing mouse body weight change following treatment with PBS (i.tu.) (n ═ 6), basement membrane glycans (i.tu.) (n ═ 7), IL12-MSA (i.tu.) (n ═ 7), IL 12-MSA-basement membrane glycans (i.tu.) (n ═ 7), or IL12-MSA (i.p.) (n ═ 7). Mice were treated on days 6 and 12 as indicated (arrows).

Figure 6B provides survival curves of mice vaccinated with B16F10 melanoma on day 0 and treated with PBS (control), basement membrane glycans (i.tu.), IL12-MSA (i.tu), IL12-MSA (i.p.), or IL 12-MSA-basement membrane glycans (i.tu) on days 6 and 12.

Figure 7 provides graphs representing changes in body weight over time (left panel) and corresponding survival rates (right panel) for B16F10 tumor-bearing mice treated with intra-tumor (i.tu.) injections of PBS (n-5), MSA-IL2 and IL12-MSA (n-5), or basement-membrane glycan-MSA-IL 2 and IL 12-MSA-basement-membrane glycan (n-5) on days 5 and 11 as compared to baseline. The arrows indicate the treatment time. Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P <0.03), ((P <0.002), ((P <0.0002), () and (P <0.0001), n.s., without significance.

Figure 8A provides B16F10 melanoma-bearing mouse BatF3 treated with PBS (control) (i.tu.) or a combination of basement membrane glycan-MSA-IL 2(i.tu.) with IL 12-MSA-basement membrane glycan (i.tu.) and immune cell depleting or cytokine neutralizing antibodies as indicated ^-/-Or the Mantel-Cox survival curve of wild-type (WT) mice. Mice (n ═ 5 per treatment group) were treated as indicated on days 6, 12, and 18. Survival statistics were determined by log rank Mantel-Cox test. Significance is shown by (P)<0.03)、**(P<0.002)、***(P<0.0002)、****(P<0.0001) means, n.s., no significance.

Figure 8B provides B16F10 melanoma-bearing mouse BatF3 treated with PBS (control) (i.tu.) or a combination of basement membrane glycan-MSA-IL 2(i.tu.) and IL 12-MSA-basement membrane glycan (i.tu.) and immune cell depleting antibodies as indicated^-/-Or the Mantel-Cox survival curve of wild-type (WT) mice. Mice (n ═ 5 per treatment group) were treated as indicated on days 6, 12, and 18. Survival statistics were determined by log rank Mantel-Cox test. Significance is shown by (P)<0.03)、**(P<0.002)、***(P<0.0002)、****(P<0.0001) means, n.s., no significance.

Figure 8C provides a heatmap showing fold change in immune cells in tumor infiltrates of B16F10 melanoma tumor-bearing mice versus treatment with PBS intra-tumoral treatment with a combination of IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 (basement membrane glycan form) or a combination of IL12-MSA + MSA-IL2(MSA form).

Figures 8D-8E provide graphs of the quantification of tumor infiltrating CD8+ T cells (figure 8D) and their surface PD-1's corresponding Median Fluorescence Intensity (MFI) (figure 8E) isolated from B16F10 melanoma-bearing mice on day 11 after tumor cell injection following treatment as shown in figure 8C on day 5 after tumor cell injection.

Figure 9 provides graphs representing body weight change from baseline (left panel) and corresponding survival (right panel) over time for B16F10 melanoma tumor-bearing mice treated with intratumoral (i.tu.) injection with PBS (n-5), anti-PD-1 antibody in combination with MSA-IL2 and IL12-MSA (n-5), or anti-PD-1 antibody in combination with basement membrane glycan-MSA-IL 2 and IL 12-MSA-basement membrane glycan (n-5) on days 5 and 11. The arrows indicate the treatment time. Body weight change and comparative statistics were determined by one-way ANOVA in combination with Tukey multiple comparison test. Survival statistics were determined by log rank Mantel-Cox test. Significance was expressed as (P <0.03), ((P <0.002), ((P <0.0002), () and (P <0.0001), n.s., without significance.

Figures 10A-10B provide graphs showing the tumor area (left) and percent survival (right) for EMT6 tumor-bearing mice (figure 10A) or MC38 tumor-bearing mice (figure 10B) and treated as indicated (arrows) on days 5, 11, and 17 as indicated (arrows). Body weight change and comparative statistics were determined by one-way ANOVA in combination with Tukey multiple comparison test. Survival statistics were determined by log rank Mantel-Cox test. Significance shown as P < 0.03; p < 0.002; p < 0.0002; p < 0.0001; n.s., no significance.

Figure 11 provides a graph representing body weight change from baseline (left panel, mean + s.d.), corresponding tumor area (middle panel, mean + s.d.) and survival (right panel) for B16F10 melanoma tumor-bearing mice treated with intra-tumor (i.tu.) injections of either PBS (n-12) or IL-12 (for IL12-MSA, n-10; for IL 12-MSA-basement membrane glycan, n-10), or cancer vaccine alone (n-7), or cancer vaccine and IL-12 (for IL12-MSA, n-7; for IL 12-MSA-basement membrane glycan, n-7) as shown (arrows) on days 5, 11 and 17. Shows that until the mice reach the standard of euthanasia (>100mm²) The tumor area of (a). The body weight change statistics shown in the graphs were determined by one-way ANOVA in combination with Tukey multiple comparison test. Survival statistics adjacent to legend determined by log rank Mantel-Cox testAmount of the compound (A). Assumed significance is given by<0.03；**，P<0.002；***，P<0.0002；****，P<0.0001 represents; n.s., no significance.

Figure 12 provides graphs representing body weight change from baseline (left panel, mean + s.d.), corresponding tumor area (middle panel, mean + s.d.) and percent survival (right panel) for B16F10 melanoma tumor-bearing mice treated with intra-tumor (i.tu.) injections of either CAR-T and IL12 (for IL12-MSA, n-9; for IL 12-MSA-basement-membrane glycan, n-5) as shown (arrows) on days 5 and 11 and with PBS (n-11) or IL-12 (for IL12-MSA, n-9; for IL 12-MSA-basement-membrane glycan, n-5), or CAR-T (n-11) alone. Mice were inoculated with B16F10 cells on day 0 and were lymphodepleted by whole body irradiation on day 4. On day 5, CAR-T treatment was administered by a single bolus tail vein injection (i.v.). Shows that until the mice reach the standard of euthanasia ( >100mm²) The tumor area of (a). The body weight change statistics shown in the graphs were determined by one-way ANOVA in combination with Tukey multiple comparison test. Survival statistics adjacent to the legend were determined by the log rank Mantel-Cox test. Assumed significance is given by<0.03；**，P<0.002；***，P<0.0002；****，P<0.0001 represents; n.s., no significance.

Figure 13 provides graphs showing total body weight (left panel), primary tumor growth and weight (middle panel), and survival rate (right panel) of 4T1 breast cancer tumor-bearing mice treated with intratumoral (i.tu.) injection of IL-12 (against IL12, n-5; against IL 12-MSA-basement membrane glycan, n-5) and intraperitoneal (i.p.) injection of anti-PD-1 on days 7 and 13. The arrows indicate treatment time and the crosses indicate surgical time. On day 0, mice were seeded with 4T1-Luc cells in the mammary fat pad. New adjuvant therapy was administered on days 7 and 13 and primary tumors were surgically excised on day 16. Post-operative mice were monitored for metastasis by In Vivo Imaging (IVIS). For each group, tumor area is shown until the primary tumor is resected. The body weight change statistics shown in the graphs were determined by one-way ANOVA in combination with Tukey multiple comparison test. Survival statistics adjacent to the legend were determined by the log rank Mantel-Cox test. Assumed significance was with P < 0.03; p < 0.002; p < 0.0002; p < 0.0001; n.s., no significance.

Figure 14A provides a graph representing the average tumor area of 4T1 breast cancer tumor-bearing mice treated intratumorally with a combination of basement membrane glycan-GLuc or basement membrane glycan-CCL 3, basement membrane glycan-CCL 4, and basement membrane glycan-CCL 5 on days 7 and 13. The IFN α were administered intraperitoneally on days 9 and 15. Tumor growth was monitored every other day (mean + SEM).

Figure 14B provides a graph representing the average tumor area of B16F10 melanoma-bearing mice treated intratumorally with a combination of basement membrane glycan-GLuc or basement membrane glycan-CCL 3, basement membrane glycan-CCL 4, and basement membrane glycan-CCL 5 on days 7 and 13. The IFN α were administered intraperitoneally on days 9 and 15. Tumor growth was monitored every other day (mean + SEM).

Figure 14C provides a graph showing the effect of various concentrations of the fusion proteins, basement membrane glycan-gluc (Lum gluc), basement membrane glycan-CCL 3(Lum CCL3), basement membrane glycan-CCL 5(Lum CCL5), on 4T1 breast tumor cell proliferation in vitro. Proliferation was determined by measuring the WST-1 proliferation reagent by absorbance at 450 nm.

Fig. 14D provides graphs showing the effect of various concentrations of the fusion proteins, basement membrane glycan-gluc (Lum gluc), basement membrane glycan-CCL 3(Lum CCL3), basement membrane glycan-CCL 5(Lum CCL5) on B16F10 melanoma tumor cell proliferation in vitro. Proliferation was determined by measuring the WST-1 proliferation reagent by absorbance at 450 nm.

Figure 15 provides a graph representing the mean tumor area of tumor lesions in B16F10 melanoma-bearing mice primed at day 5 after tumor cell injection and challenged subcutaneously in the tail root (s.c.) on days 11 and 17 after cancer vaccine treatment. Cancer vaccines were administered alone or in combination with the intratumoral administration of CCL 11-basement membrane glycans, TNF α, IFN γ or basement membrane glycans on days 11, 17, 23 and 29 as indicated. Tumor area was monitored every other day (mean + SD).

Figures 16A-16B provide tumor areas representing single tumor lesions in B16F10 melanoma-bearing mice treated with either basement membrane glycan (Lwt) (i.tu.) (figure 16B) or CCL 11-basement membrane glycan (11L) (i.tu.) (figure 16A) on days 5, 11, and 17 after tumor cell injection using a combination of tumor-targeting antibody 2.5F-Fc (i.p.) and MSA-IL2(i.p.) on days 5, 11, and 17. Tumor area was monitored every other day

Figure 17A provides a graph showing binding of agonist antibody-basement membrane glycan fusion protein to type I collagen as a function of concentration. Binding was determined by ELISA.

FIG. 17B provides the use of Alexa following intratumoral injection into 4T1 tumors over time as determined by in vivo fluorescence imaging 647 fluorescence-labeled mouse anti-FITC antibody alone or fused to a basement membrane glycan (4420). In vivo fluorescence measurements are provided in units of total radiant efficiency (p/s)/(μ W/cm 2).

Figure 18A provides a graph showing the binding of the subset of His-tagged basement membrane glycan-IgG-binding fusion proteins as shown to collagen type I (left panel) or collagen type IV (right panel) as a function of concentration. Binding was determined by ELISA.

Figure 18B provides a graph representing the binding of His-tagged basement membrane glycan-IgG binding fusion protein to mouse IgG2a isotype control (clone C1.18.4) as a function of concentration. Binding was determined by ELISA. anti-His (clone ab1187) was used to test each construct.

Figure 19 provides 3D microscopic images of mouse macroreticular tissues from OVCA433 human ovarian tumor-bearing mice, showing the specific accumulation of Alexa Fluor 647-labeled basement membrane glycans (yellow) around the micro-colonies (red) of OVCA433 human ovarian tumor cells expressing RFP in mouse macroreticular tissues, collagen was imaged to grey by SHG microscopy. The labeled basement membrane glycans were injected intraperitoneally in tumor-bearing mice.

Figure 20A provides a graph representing expression of IL-12 fusion proteins from self-replicating RNA in B16F10 cells as indicated, alone or fused to a fluorescent protein (mCherry), as determined by flow cytometry.

Figure 20B provides a graph representing expression of IL-12 fusion proteins from self-replicating RNA in B16F10 cells, alone or fused to a fluorescent protein (mCherry), as indicated, as determined by IL-12 ELISA.

Figure 21A provides a graph showing tumor volume (mean + SD) for tumor-bearing mice treated with intratumoral injection of PBS (n ═ 4) or with intratumoral collagen-anchored cytokine-based basement membrane glycans-MSA-IL 2 and IL 12-MSA-basement membrane glycans, and intraperitoneal TA99 and anti-PD-1 (n ═ 5) on days 25, 31, 37, 43, 49, 55, and 61. For each group, it is shown until mice reach standard of euthanasia (a)>1200mm³) The tumor volume of (a).

Figure 21B provides Mantel-Cox survival curves for tumor-bearing mice treated with intratumoral PBS (n ═ 10), with intratumoral basement membrane glycan-MSA-IL 2 and IL 12-MSA-basement membrane glycan and intraperitoneal TA99 and anti-PD-1 (n ═ 14), with intratumoral basement membrane glycan-MSA-IL 2 and IL 12-MSA-basement membrane glycan and intraperitoneal anti-PD-1 (n ═ 10), with intratumoral MSA-IL2 and IL12-MSA and intraperitoneal TA99 and anti-PD-1 (n ═ 9), or with intratumoral MSA-IL2 and IL12-MSA and intraperitoneal anti-PD-1 (n ═ 8) on days 25, 31, 37, 43, 49, 55, and 61. The arrows indicate the treatment time. Total survival maps enumerate the burden of death from tumors: ( >1200mm³) Or treatment-related weight loss (>20%) of mice; the latter is indicated by blue "x" for each mouse. Survival was compared by log rank Mantel-Cox test. P<0.03，***P<0.0002，****P<0.0001。

Fig. 22A provides a schematic for measuring LAIR binding capacity in B16F10 tumors.

Figure 22B provides a graph showing the weight of the resected tumor and its extracellular matrix.

Figure 22C provides a graph showing hydroxyproline content of B16F10 cellular components compared to matrix components.

Figure 22D provides a graph showing LAIR-fluorescence depletion by placing a B16F 10-derived matrix component in 1mL of AF 647-labeled LAIR.

Figure 22E provides a graph showing the correlation between hydroxyproline content of the matrix component and LAIR binding capacity of the B16F 10-derived matrix component.

FIG. 23A provides a graph showing that on day 6 and day 6B16F10 melanoma tumors treated on day 13 with PBS control (i.tu) (n ═ 5) or LAIR-MSA-IL2(i.tu) TA99(i.p.) (n ═ 7) (day 0 inoculated with 1x10⁶Individual cells).

Figure 23B provides B16F10 melanoma-bearing mice (inoculated with 1x10 on day 0) showing treatment with PBS control (i.tu) (n ═ 5) or LAIR-MSA-IL2(i.tu) T + a99(i.p.) (n ═ 7) on days 6 and 13⁶Individual cells) of the Mantel-Cox survival curve.

Figure 24A provides a sequence alignment of low affinity collagen binders lair.30.w.a, lair.30.w.b, lair.30.w.c, and lair.30.w.d compared to wild type lair (lair).

Fig. 24B-E provide a description of the crystal structure of wild-type LAIR (PDB 4ETY) shown as grey band, selected mutated amino acid residues of the low affinity collagen conjugates lair.30.w.a (fig. 24B), lair.30.w.b (fig. 24C), lair.30.w.c (fig. 24D) and lair.30.d.d (fig. 24E) highlighted as bonded spheres.

Figure 24F provides a graph showing binding of WT LAIR, WT LAIR-MSA, MSA-IL-2 (non-specific binding control) and mutant LAIR-MSA fusions to collagen type 1 in an ELISA assay (n ═ 2). The binding affinity (Kd) of each LAIR construct calculated from a nonlinear single point binding fit is also shown.

Figure 25A provides a sequence alignment of the low affinity collagen conjugates lair.30.w.e and lair.30.w.f compared to wild-type LAIR.

Fig. 25B-C provide a description of the crystal structure of wild-type LAIR (PDB 4ETY) shown as grey band, with selected mutated amino acid residues of the low affinity collagen conjugates lair.30.w.e (fig. 25B) and lair.30.w.f (fig. 25C) highlighted as bonded spheres.

Figure 25D provides a graph showing binding of WT LAIR, WT LAIR-MSA, MSA-IL-2 (non-specific binding control) and mutant LAIR-MSA fusions to collagen type 1 in an ELISA assay (n ═ 2). The binding affinity (Kd) of each LAIR construct modeled from a nonlinear single point binding fit is also shown.

Figure 26A provides a sequence alignment of the high affinity collagen conjugate lair.30.2.k1.b compared to wild type LAIR.

Figure 26B provides a description of the crystal structure of wild type LAIR (PDB 4ETY) shown as grey band, with selected mutated amino acid residues of the high affinity collagen conjugate lair.30.2.k1.B highlighted as bonded spheres.

FIGS. 26C-D provide flow cytometry plots showing protein display (goat anti-chicken AF488) of CRP-XL-biotin binding (streptavidin-AF 647) incubated in 100nM (FIG. 26C) or 0.01nM (FIG. 26D) of CRP-XL-biotin to yeast with wild-type LAIR (black) or LAIR30.2.K1.B (cyan).

Fig. 26E-F provides flow cytometry plots showing protein display (goat anti-chicken AF488) of yeast with LAIR30.2.k1.b (fig. 26E) or wild-type LAIR (fig. 26F) at different time points (competition 0, 16 and 40 hours) after competition with excess non-biotinylated CRP-XL, the remaining surface CRP-XL biotin signal (streptavidin-AF 647).

Fig. 26G provides a graph showing the median fluorescence intensity of CRP-XL-biotin bound over time in the kinetic dissociation experiments depicted in fig. 26E-F. The estimated off-rates modeled as one-phase exponential decay fits are also shown.

Detailed Description

Provided herein are immunomodulatory fusion proteins comprising an immunomodulatory domain operably linked to a collagen binding domain. Such fusion proteins are localized to the immunomodulatory domain (e.g., cytokines, antibodies) such that they do not diffuse systemically. Systemic dissemination of the immunomodulatory domain can result in reduced effectiveness due to rapid clearance from the target site (e.g., tumor), and/or toxicity due to effects on non-target cells other than the tumor. Thus, attachment of the immunomodulatory domain to the collagen binding protein localizes the immunomodulatory domain to prevent systemic spread, thereby retaining the immunomodulatory domain at the target site and reducing potential off-target effects that may lead to toxicity.

Definition of

Unless otherwise indicated, the terms used in the claims and the specification are defined as follows. In case of direct conflict with the terminology used in the parent provisional patent application, the terminology used in this application shall control.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "about" will be generally understood by one of ordinary skill and will vary to some extent depending on the context in which it is used. If there are terms that would be unclear to an ordinary artisan given the context of use, "about" means plus or minus 10% of the particular value.

As used herein, the term "agonist" refers to any molecule (e.g., an antibody or antigen-binding fragment thereof) that partially or fully promotes, increases, or activates the biological activity of a native polypeptide disclosed herein. Suitable agonist molecules include in particular agonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules and the like. In some embodiments, activation is observed in a dose-dependent manner in the presence of an agonist. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured using a negative control under comparable conditions. Also disclosed herein are methods of identifying agonists suitable for use in the methods of the present disclosure. For example, such methods include, but are not limited to, binding assays, such as enzyme-linked immunosorbent assays (ELISAs), fortes Systemic and Radioimmunoassay (RIA). These assays determine the ability of an agonist to bind a polypeptide of interest (e.g., a receptor or ligand), thereby indicating that the agonist promotesThe ability to increase or activate the activity of the polypeptide. The effectiveness of an agonist can also be determined using a functional assay, such as the ability of an agonist to activate or promote the function of a polypeptide. For example, a functional assay may comprise contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. Usually by its EC₅₀The value (the concentration required to activate 50% of the agonist response) defines the potency of the agonist. EC (EC)₅₀The lower the value, the higher the potency of the agonist and the lower the concentration required to activate the maximum biological response.

The term "albumin" refers to a protein having the same or very similar three-dimensional structure as human albumin (SEQ ID NO:42) and having a longer serum half-life. Exemplary albumin proteins include human serum albumin (HAS; SEQ ID NOS: 42 and 43), primate serum albumin (e.g., chimpanzee serum albumin), gorilla serum albumin or macaque serum albumin, rodent serum albumin (e.g., hamster serum albumin), guinea pig serum albumin, mouse serum albumin and rat serum albumin, bovine serum albumin (e.g., cow serum albumin), horse serum albumin (e.g., horse serum albumin or donkey serum albumin), rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin and pig serum albumin.

The term "ameliorating" refers to any therapeutically beneficial outcome in the treatment of a disease state (e.g., cancer), including preventing, lessening the severity or progression thereof, alleviating, or curing.

"amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by their well known three letter symbols or by one letter symbol recommended by the IUPAC-IUB Biochemical nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single letter codes.

"amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "replacement" amino acid residue. "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While insertions typically consist of insertions of one or two amino acid residues, larger "peptide insertions" may now be made, for example insertions of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue may be naturally occurring or non-naturally occurring as described above. "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

As used herein, the term "antagonist" refers to any molecule (e.g., an antibody or antigen-binding fragment thereof) that partially or completely blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Suitable antagonist molecules include in particular antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules and the like. In some embodiments, inhibition is observed in a dose-dependent manner in the presence of the antagonist. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, less than the signal measured using a negative control under comparable conditions, At least about 85%, at least about 90%, at least about 95%, or at least about 100%. Also disclosed herein are methods of identifying antagonists suitable for use in the methods of the present disclosure. For example, such methods include, but are not limited to, binding assays, such as enzyme-linked immunosorbent assays (ELISAs), fortesSystemic and Radioimmunoassay (RIA). These assays determine the ability of the antagonist to bind to the polypeptide of interest (e.g., a receptor or ligand), thereby indicating the ability of the antagonist to inhibit, neutralize, or block the activity of the polypeptide. The effectiveness of an antagonist can also be determined using a functional assay, such as the ability of an antagonist to inhibit the function of a polypeptide or agonist. For example, a functional assay may comprise contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. Usually through its IC₅₀The value (concentration required to inhibit 50% of the agonist response) defines the potency of the antagonist. IC (integrated circuit)₅₀The lower the value, the higher the potency of the antagonist and the lower the concentration required to inhibit the maximum biological response.

As used herein, the term "antibody" refers to a complete antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" includes polyclonal, monoclonal, chimeric or chimeric antibodies, humanized antibodies, primate antibodies, deimmunized antibodies and fully human antibodies. The antibody can be prepared in or derived from any of a variety of species, for example, a mammal, such as a human, a non-human primate (e.g., chimpanzee, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, and a mouse. The antibody may be a purified or recombinant antibody.

As used herein, the term "antibody fragment," "antigen-binding fragment," or similar terms refer to an antibody fragment that retains the ability to bind to one or more target antigens and promotes, induces, and/or increases the activity of the target antigens. Such fragments include, for example,single-chain antibody, single-chain Fv fragment (scFv), Fd fragment, Fab 'fragment, or F (ab')₂And (3) fragment. scFv fragments are single polypeptide chains comprising the variable regions of the heavy and light chains of the antibody from which the scFv was derived. In addition, antibodies, minibodies, trisomes and diabodies are also included in the definition of antibody and may be used in the methods described herein. See, e.g., Todorovska et al, (2001) JImmunol Methods248(1)47-66; hudson and Kortt (1999) J Immunol Methods231(1):177-189；Poljak(1994)Structure 2(12)1121- & ltSUB & gt 1123-; rondon and Marasco (1997) Annual Review of Microbiology51:257 and 283, the entire disclosure of each of which is incorporated herein by reference.

As used herein, the term "antibody fragment" also includes, for example, single domain antibodies, such as camelized single domain antibodies. See, e.g., Muydermans et al, (2001) Trends Biochem Sci26230-; nuttall et al, (2000) Curr Pharm Biotech1253, 263; reichmann et al, (1999) J Immunol Meth 23125-38; PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. patent No. 6,005,079, which are incorporated herein by reference in their entirety. In some embodiments, the present disclosure provides single domain antibodies comprising VH domains with modifications such that single domain antibodies are formed.

The "B7 family" refers to activating and inhibitory ligands. The B7 family contains at least activating ligands B7-1 and B7-2, and inhibitory ligands B7-H1, B7-H2, B7-H3 and B7-H4. B7-1 and B7-2 bound to CD28, B7-H1 (i.e., PD-L1) bound to PD-1 and B7-H2 bound to ICOS. B7-H3 and B7-H4 bind to unknown receptors. In addition, B7-H3 and B7-H4 have been shown to be upregulated on tumor cells and tumor infiltrating cells. The complete hB7-H3 and hB7-H4 sequences can be found in GenBank accession numbers Q5ZPR3 and AAZ17406(SEQ ID NOS: 49 and 50), respectively.

As used herein, the term "Chimeric Antigen Receptor (CAR)" refers to an artificial transmembrane protein receptor comprising (i) an extracellular domain capable of binding at least one predetermined CAR ligand or antigen, or a predetermined CAR ligand and antigen; (ii) an intracellular segment comprising one or more cytoplasmic domains derived from a signal transduction protein that is different from a polypeptide from which the extracellular domain is derived; and (iii) a transmembrane domain. "Chimeric Antigen Receptors (CARs) are sometimes referred to as" chimeric receptors "," T-bodies ", or" Chimeric Immunoreceptors (CIRs) ".

The phrase "CAR ligand" used interchangeably with "CAR antigen" refers to a natural or synthetic molecule (e.g., small molecule, protein, peptide, lipid, carbohydrate, nucleic acid) or a portion thereof or fragment thereof that can specifically bind to a CAR (e.g., the extracellular domain of a CAR). In some embodiments, the CAR ligand is a tumor associated antigen or a fragment thereof. In some embodiments, the CAR ligand is a tag.

An "intracellular signaling domain" refers to any oligopeptide or polypeptide domain known to function in transmitting signals, which causes activation or inhibition of a biological process in a cell, e.g., activation of an immune cell, e.g., a T cell or NK cell. Examples include ILR chains, CD28, and/or CD3 ζ.

As used herein, "cancer antigen" refers to (i) a tumor-specific antigen, (ii) a tumor-associated antigen, (iii) cells expressing a tumor-specific antigen, (iv) cells expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) autologous tumor cells, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen presenting cell or material associated with cancer.

As used herein, "cancer vaccine" refers to a treatment that induces the immune system to attack cells having one or more tumor-associated antigens. The vaccine can treat existing cancer (e.g., a therapeutic cancer vaccine) or prevent cancer progression in certain individuals (e.g., a prophylactic cancer vaccine). The memory cells produced by the vaccine will recognize the tumor cells bearing the antigen, thereby preventing tumor growth.

As used herein, the term "chemokine" refers to a member of the family of small cytokines or signaling proteins that induce directed chemotaxis. Chemokines are divided into four subfamilies: CXC, CC, (X) C and CX 3C.

As used herein, the term "collagen" refers to the major structural protein located within the extracellular space and maintains the mechanical integrity of many different tissues. The molecular structure of collagen determines its function. More than 20 collagens have been identified, the most common of which is type I.

As used herein, the term "collagen binding domain" refers to a polypeptide or portion thereof that binds to collagen. The collagen binding domain may be part of a larger fusion protein, a bioactive agent, or a drug. The binding of a composition, polypeptide or portion thereof, fusion protein, or drug or bioactive agent to collagen can be determined by methods well known in the art (e.g., collagen binding assays; see, e.g., Turecek et al, (2002) Semin Thromb Hemost 28(2): 149-. In some embodiments, the collagen binding domain is determined by its ability to compete with known or reference collagen binding proteins for binding to collagen. In some embodiments, the collagen binding domain is derived from a naturally occurring collagen binding protein or collagen receptor. Collagen binding proteins and collagen receptors comprising a collagen binding domain are well known in the art (see, e.g., Svensson et al, (2001) Osteoharthritis Cartilage 9Suppl A: S23-28; Leitinger and Hohennester E (2007) Matrix Biol 26(3): 146-. In some embodiments, the collagen binding domain is derived from a prokaryotic collagen binding protein. Prokaryotic collagen binding proteins are well known in the art (see, e.g., Symersky et al, (1997) Nat Struct Biol 4: 833-. In some embodiments, the collagen binding domain comprises one or more mutations that increase its affinity for collagen.

As used herein, "combination therapy" includes the administration of each drug or therapy in a sequential or simultaneous manner in a regimen that will provide the beneficial effects of the combination, as well as the coadministration of these agents or therapies in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of these active agents, or in multiple separate capsules for each agent. Combination therapy also includes combinations where the individual elements may be administered at different times and/or by different routes, but which may act in combination to provide beneficial effects through synergistic or pharmacokinetic and pharmacodynamic effects of each drug or tumor treatment methods of the combination therapy.

As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as a TCR/CD3 linkage, results in up-or down-regulation of T cell proliferation and/or key molecules.

"cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)" is a T cell surface molecule and is a member of the immunoglobulin superfamily. The protein down regulates the immune system by binding to CD80 and CD 86. As used herein, the term "CTLA-4" includes human CTLA-4(hCTLA-4), variants, subtypes, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found in GenBank accession number P16410(SEQ ID NO: 46).

A polypeptide or amino acid sequence "derived from" a given polypeptide or protein refers to the source of the polypeptide. Preferably, the polypeptide or amino acid sequence derived from a particular sequence has substantially the same amino acid sequence as the sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or can be determined by one of ordinary skill in the art to originate from the sequence. A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, for example one or more amino acid residues have been replaced by another amino acid residue or have one or more amino acid residue insertions or deletions.

The polypeptide may comprise a non-naturally occurring amino acid sequence. Such variants must have less than 100% sequence identity or similarity to the starting molecule. In certain embodiments, a variant will have an amino acid sequence that has from about 75% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100%, (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule.

In certain embodiments, there is an amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to the sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., identical residues) to the starting amino acid residue, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In certain embodiments, the polypeptide consists of, or consists essentially of, or comprises an amino acid sequence selected from the list of sequence summaries. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the list of sequence summaries. In certain embodiments, the polypeptide comprises a contiguous amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a contiguous amino acid sequence selected from the summary of sequences table. In certain embodiments, the polypeptide comprises an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer in these numbers) contiguous amino acid sequences selected from the amino acid sequences in the summary of sequences tables.

In certain embodiments, the peptides of the present disclosure are encoded by a nucleotide sequence. The nucleotide sequences of the present disclosure may be used in a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody production for e.g. passive immunization, PCR, generation of primers and probes, etc. In certain embodiments, the nucleotide sequence of the present disclosure comprises a sequence selected from SEQ ID NOs: 3. 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, consisting essentially of, or consisting of. In certain embodiments, the nucleotide sequence comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence set forth in the summary of sequences table. In certain embodiments, the nucleotide sequence comprises a contiguous nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a contiguous nucleotide sequence set forth in the summary of sequences table. In certain embodiments, the nucleotide sequence comprises a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer in these numbers) consecutive nucleotides of the nucleotide sequences set forth in the summary of sequences table.

It will also be understood by those of ordinary skill in the art that polypeptides suitable for use in the immunomodulatory fusion proteins disclosed herein can be altered such that their sequence differs from the native sequence that it naturally occurs or from which it is derived, but retains the desired activity of the native sequence. For example, nucleotide or amino acid substitutions may be made that result in conservative substitutions or changes at "non-essential" amino acid residues. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Polypeptides suitable for use in the immunomodulatory fusion proteins disclosed herein can comprise conservative amino acid substitutions at one or more amino acid residues (e.g., at essential or non-essential amino acid residues). A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace a non-essential amino acid residue in a binding polypeptide with another amino acid residue from the same side chain family. In certain embodiments, a string of amino acids may be replaced with a structurally similar string that differs in the order and/or composition of the side chain family members. Alternatively, in certain embodiments, mutations can be introduced randomly along all or part of a coding sequence, for example by saturation mutagenesis, and the resulting mutants can be introduced into binding polypeptides of the disclosure and screened for their ability to bind to a desired target.

As used herein, the term "effector cell" or "effector immune cell" refers to a cell involved in an immune response, e.g., promoting an immune effector response. In some embodiments, the immune effector cell specifically recognizes the antigen. Examples of immune effector cells include, but are not limited to, Natural Killer (NK) cells, B cells, monocytes, macrophages, T cells (e.g., Cytotoxic T Lymphocytes (CTLs)). In some embodiments, the effector cell is a T cell. As used herein, the term "immune effector function" or "immune effector response" refers to the function or response of an immune effector cell that promotes an immune response to a target.

As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of its two heavy chains. In some embodiments, the term "Fc domain" refers to a portion of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not comprise an Fv domain. In some embodiments, the term "Fc domain" refers to a portion of a single immunoglobulin (Ig) heavy chain that further comprises an Fv domain. Thus, the Fc domain may also be referred to as "Ig" or "IgG". In certain embodiments, the Fc domain begins at the hinge region upstream of the papain cleavage site and terminates at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, the Fc domain comprises a complete Fc domain (i.e., the hinge domain, CH2 domain, and CH3 domain). In certain embodiments, the Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, the Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, the Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains, as well as such peptides comprising only, for example, the hinge, CH2, and CH3 domains. The Fc domain may be from any species and/or any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The human IgG1 constant region can be found in Uniprot P01857 and SEQ ID NO 114. The Fc domain of human IgGl can be found in SEQ ID NO: 115. Fc domains include native Fc and Fc variant molecules. As with Fc variants and native Fc, the term Fc domain includes molecules in monomeric or multimeric form, whether digested from intact antibodies or otherwise produced. Numbering of the amino acid residues of the Fc domain is assigned according to the definition of Kabat. See, for example, Sequences of Proteins of Immunological Interest (Table of Contents, Introduction and Constant Region Sequences sections), 5 th edition, Bethesda, MD: NIH vol.1:647-723 (1991); kabat et al, "Introduction" Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, NIH, 5 th edition, Bethesda, MD vol.l: xiii-xcvi (1991); chothia & Lesk, J.mol.biol.196:901-917 (1987); chothia et al, Nature 342:878-883(1989), each of which is incorporated herein by reference for all purposes.

As described herein, one of ordinary skill in the art will appreciate that any Fc domain can be modified such that its amino acid sequence differs from the amino acid sequence of a native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., fcgamma binding).

Fc domains suitable for use in the immunomodulatory fusion proteins disclosed herein may be derived from different immunoglobulin molecules. For example, the Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term "gly-ser polypeptide linker" or "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser (Gly4Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n ═ 3, i.e., Ser (Gly4Ser) 3. In certain embodiments, n ═ 4, i.e., Ser (Gly4Ser) 4. In certain embodiments, n-5. In certain embodiments, n is 6. In certain embodiments, n ═ 7. In certain embodiments, n is 8. In certain embodiments, n is 9. In certain embodiments, n is 10. Another exemplary Gly-Ser polypeptide linker comprises the amino acid sequence (Gly4Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n-3. In certain embodiments, n-4. In certain embodiments, n-5. In certain embodiments, n is 6. Another exemplary Gly-Ser polypeptide linker comprises the amino acid sequence (Gly3Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n-3. In certain embodiments, n-4. In certain embodiments, n-5. In certain embodiments, n is 6.

As used herein, the term "human antibody" includes antibodies having the variable and constant regions (if present) of human germline immunoglobulin sequences. The human antibodies of the present disclosure may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo) (see, Lonberg, N.et al, (1994) Nature 368(6474): 856-; lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; lonberg, N.and Huszar, D. (1995) Intern.Rev.Immunol.Vol.13:65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y.Acad.Sci 764: 536-. However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences (i.e., humanized antibodies).

As used herein, the term "heterologous antibody" is defined with respect to a transgenic non-human organism that produces such an antibody. The term refers to an antibody having an amino acid sequence or coding nucleic acid sequence corresponding to that found in an organism not consisting of a transgenic non-human animal, typically from the species of the non-transgenic non-human animal.

As used herein, an "immune cell" is a cell of hematopoietic origin and plays a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

As used herein, "immune checkpoint" refers to the modulation of co-stimulatory and inhibitory signals of immune cells. In certain embodiments, the immune checkpoint inhibitor is an inhibitory signal. In certain embodiments, the inhibitory signal is an interaction between PD-1 and PD-L1. In certain embodiments, the inhibitory signal is an interaction between CTLA-4 and CD80 or CD86 in place of CD28 binding. In certain embodiments, the inhibitory signal is an interaction between LAG3 and an MHC class II molecule. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9.

As used herein, "immune checkpoint blocker" refers to a molecule that reduces, inhibits, interferes with, or modulates, in whole or in part, one or more checkpoint proteins. In certain embodiments, the checkpoint blockade agent blocks inhibitory signals associated with an immune checkpoint. In certain embodiments, the immune checkpoint blockade agent is an antibody or fragment thereof that disrupts inhibitory signaling associated with an immune checkpoint. In certain embodiments, the immune checkpoint blockade agent is a small molecule that disrupts inhibitory signaling. In certain embodiments, the immune checkpoint blocking agent is an antibody, fragment thereof, or antibody mimetic that prevents interaction between checkpoint blocking agent proteins, e.g., an antibody or fragment thereof that prevents interaction between PD-1 and PD-L1. In certain embodiments, the immune checkpoint blocking agent is an antibody or fragment thereof that prevents an interaction between CTLA-4 and CD80 or CD 86. In certain embodiments, the immune checkpoint blocking agent is an antibody or fragment thereof that prevents the interaction between LAG3 and its ligand or TIM-3 and its ligand.

As used herein, the term "immunomodulatory fusion protein" refers to a polypeptide comprising a collagen binding domain operably linked to at least one immunomodulatory domain. In some embodiments, the collagen binding domain is operably linked to the immunomodulatory domain by a linker.

As used herein, the term "immunomodulatory domain" refers to a polypeptide (e.g., a cytokine, agonist, or antagonist antibody) that confers activity that results in the activation or inhibition of an immune response (e.g., stimulation of CD8+ T cells). In some embodiments, the immunomodulatory domain refers to a polypeptide that binds to its cognate ligand or receptor resulting in activation or suppression of an immune response.

The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a particular antigen. The term "induction" as used in relation to the induction of CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.

As used herein, a subject "in need of prevention", "in need of treatment", or "in need thereof" refers to a subject as judged by an appropriate physician (e.g., a doctor, nurse, or nurse practitioner for humans; a veterinarian for non-human mammals) who would reasonably benefit from a given treatment (e.g., treatment with a composition comprising a fusion protein as described herein).

The term "in vivo" refers to a process that occurs in a living organism.

As used herein, "Interleukin (IL) -2" refers to a pleiotropic cytokine that activates and induces the proliferation of T cells and Natural Killer (NK) cells. IL-2 signals by binding to its receptor IL-2R, which consists of alpha, beta and gamma subunits. IL-2 signaling stimulates antigen-activated T cell proliferation.

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to an immune checkpoint blocker or co-stimulatory molecule) and is substantially free of antibodies that specifically bind to antigens other than the target of interest. However, an isolated antibody that specifically binds to an epitope may be cross-reactive with other targets from different species. Furthermore, the isolated antibody is typically substantially free of other cellular material and/or chemicals.

As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid encoding a fusion protein, polypeptide, antibody or antibody portion disclosed herein, which is intended to refer to a nucleic acid molecule wherein the nucleotide sequence encoding the fusion protein, polypeptide, antibody or antibody portion is free of other nucleotide sequences that may naturally flank the nucleic acid in human genomic DNA.

As used herein, "isotype" refers to the type of antibody (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. In some embodiments, the antibody of the present disclosure is an IgG1 isotype. In some embodiments, the antibody of the present disclosure is an IgG2 isotype. In some embodiments, the antibody of the present disclosure is an IgG3 isotype. In some embodiments, the antibody of the present disclosure is an IgG4 isotype.

As used herein, the term "KD" or "K_D"refers to the equilibrium dissociation constant of, for example, the binding reaction between a ligand and a receptor, an antigen and an antibody, or a collagen-binding protein and collagen. K_DIs a numerical representation of the ratio of the binding protein dissociation rate constant (kd) to the binding protein association rate constant (ka). K_DThe value of (a) is inversely proportional to the binding affinity of the binding protein to its binding partner. K_DThe smaller the value, the greater the affinity of the binding protein for its binding partner. Affinity is the strength with which a single molecule binds to its ligand, usually by balancing the dissociation constant (K)_D) Measurements and reports were performed and the equilibrium dissociation constants were used to evaluate and rank the strength of bimolecular interactions.

As used herein, the term "kd" or "k _d"(or" koff "or" k_off") is intended to refer to the off-rate constant for dissociation of the binding protein from the binding protein/partner complex. Kd is a numerical representation of the fraction of complex decayed or dissociated per second and is expressed in sec^-1The units are expressed.

As used herein, the term "ka" or "k_a"(or" kon "or" k_on") is intended to refer to the binding rate constant for binding of the binding protein to the binding partner. The value of ka is a numerical representation of the number of antibody/antigen complexes formed per second in a 1 molar (1M) binding partner solution, and is expressed as M^-1sec^-1The units are expressed.

As used herein, the terms "connected," "operably connected," "fused," or "fused" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains by any means including chemical conjugation, formation of non-covalent compositions, or recombinant means. Chemical conjugation methods (e.g., using heterobifunctional crosslinkers) are known in the art.

As used herein, "topical administration" or "local delivery" refers to delivery that does not rely on transport of a composition or agent through the vascular system to its intended target tissue or site. For example, an immunomodulatory fusion protein or a composition comprising a fusion protein can be delivered by injection or implantation of the fusion protein or composition, or injection or implantation of a device comprising the fusion protein or composition. After topical application near a target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site. In some embodiments, the immunomodulatory fusion protein is administered locally via a viral vector, electroporation, transplantation of cells expressing the immunomodulatory fusion protein, or replicon.

"lymphocyte activation gene-3 (LAG-3)" is an inhibitory receptor associated with the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits CD8+ effector T cell function. As used herein, the term "LAG 3" includes human LAG3(hLAG3), variants, subtypes, and species homologs of hLAG3, and analogs having at least one common epitope. The complete hLAG3 sequence can be found in GenBank accession number P18627(SEQ ID NO: 47).

As used herein, the term "mammal" or "subject" or "patient" includes humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

"nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081,1991; Ohtsuka et al, biol. chem.260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, mol. cell. probes 8:91-98,1994). For arginine and leucine, the modification at the second base may also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The polynucleotide used herein may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed of single-and double-stranded DNA, DNA being a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA being a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA in which the antigen is single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. In addition, a polynucleotide may be composed of a triple-stranded region comprising RNA or DNA or both RNA and DNA. Polynucleotides may also comprise one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms.

As used herein, "parenteral administration," "parenteral administration," and other grammatical equivalents refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intranasal, intraocular, intramuscular, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, submucosal, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracervical, intracoronary/intestinal, intracervical/intravaginal, and intrasternal injection and infusion.

The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to a specified percentage of two or more sequences or subsequences that have the same nucleotide or amino acid residue when compared and aligned for maximum correspondence, as measured using one of the sequence alignment algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art), or by visual inspection. Depending on the application, "percent identity" may be present over a region of the sequences being compared, e.g., over a functional domain, or over the entire length of two sequences being compared. For sequence comparison, one sequence is typically used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

Optimal sequence alignments for comparison can be made, for example, by the local homology algorithm of Smith & Waterman, adv.appl.Math.2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.mol.biol.48:443(1970), by the similarity algorithm study of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), by computerized execution of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group,575Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al, infra).

An example of an algorithm suitable for determining sequence identity and percent sequence similarity is the BLAST algorithm, which is described in Altschul et al, J.Mol.biol.215: 403-. Software for performing BLAST analysis is publicly available through the national center for Biotechnology information website.

As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The "programmed death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in vivo predominantly on previously activated T cells and binds to two ligands PD-Ll and PD-L2. As used herein, the term "PD-1" includes variants, subtypes, and species homologs of human PD-1(hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank accession AAC51773(SEQ ID NO: 44).

"programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that down-regulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1(hPD-Ll), variants, subtypes and species homologies of hPD-Ll, and analogs having at least one common epitope with hPD-Ll. The complete hPD-Ll sequence can be found in GenBank accession No. Q9NZQ7(SEQ ID NO: 45).

As used herein, the term "purified" or "isolated" as applied to any protein (fusion protein, antibody or fragment) described herein refers to a polypeptide that has been isolated or purified from a component (e.g., a protein or other naturally occurring biological or organic molecule) with which it is naturally associated, e.g., other proteins, lipids, and nucleic acids in a prokaryote expressing the protein. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99)% by mass of the total protein in the sample.

As used herein, the terms "specific binding" and "selective binding" refer to binding to collagen through a collagen binding domain, or to binding to an epitope on a predetermined antigen by an antibody. In some embodiments, based on K for collagen _DThe collagen binding domain specifically binds or selectively binds to collagen (i.e., to collagen-binding K)_DLower than a K for at least fibronectin, vitronectin, osteopontin, tenascin-C or fibrinogen_D)。

The term "effective amount" or "an amount sufficient to … …" refers to an amount sufficient to produce a desired effect, for example, an amount sufficient to reduce the diameter of a tumor.

The term "T cell" refers to one of the leukocytes, which is present through the cell surfaceT cell receptors can distinguish them from other leukocytes. There are several subsets of T cells, including but not limited to T helper cells (i.e., T)_HCells or CD4⁺T cells) and subtypes, including T_H1、T_H2、T_H3、T_H17、T_H9 and T_FHCells, cytotoxic T cells (i.e., T)_CCell, CD8⁺T cells, cytotoxic T lymphocytes, T killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (T cells)_CMCells), effector memory T cells (T)_EMAnd T_EMRACells) and resident memory T cells (T)_RMCells), regulatory T cells (i.e., T cells)_regCells or suppressor T cells) and subtypes, including CD4⁺FOXP3⁺T_regCell, CD4⁺FOXP3^-T_regCells, Tr1 cells, Th3 cells, and T_reg17 cells, natural killer T cells (i.e., NKT cells), mucosa-associated invariant T cells (MAIT), and γ δ T cells (γ δ T cells), which include V γ 9/V δ 2T cells. Any one or more of the T cells, previously described or not mentioned, can be the target cell type for use in the methods of use of the present invention.

The term "T cell cytotoxicity" includes any immune response mediated by CD8+ T cell activation. Exemplary immune responses include cytokine production, CD8+ T cell proliferation, production of granzymes or perforins, and clearance of infectious agents.

"T cell membrane protein-3 (TIM-3)" is obtained by inhibiting T_H1 are involved in inhibitory receptors that inhibit lymphocyte activity in response. Its ligand is galectin 9, which is upregulated in a variety of cancer types. As used herein, the term "TIM 3" includes human TIM3(hTIM3), variants, subtypes, and species homologs of hTIM3, and analogs having at least one common epitope. The complete hTIM3 sequence can be found in GenBank accession No. Q8TDQo (SEQ ID NO: 48).

A "therapeutic antibody" is an antibody, fragment of an antibody, or construct derived from an antibody, and can bind to a cell surface antigen on a target cell to elicit a therapeutic effect. Such antibodies may be chimeric, humanized or fully human. Methods for producing such antibodies are known in the art. Such antibodies include single chain Fc fragments of antibodies, miniantibodies and diabodies. Any therapeutic antibody known in the art to be useful in the treatment of cancer may be used in combination therapy suitable for use in the methods disclosed herein. The therapeutic antibody may be a monoclonal antibody or a polyclonal antibody. In preferred embodiments, the therapeutic antibody targets a cancer antigen.

The term "therapeutically effective amount" is an amount effective to ameliorate the symptoms of a disease. A therapeutically effective amount may be a "prophylactically effective amount" since prophylaxis may be considered treatment.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are typically in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

Immunomodulatory fusion proteins

In some aspects, the present disclosure provides an immunomodulatory fusion protein comprising a collagen binding domain operably linked to an immunomodulatory domain. In some embodiments, the immunomodulatory fusion protein further comprises a linker, such that the collagen binding domain is operably linked to the linker and the linker is operably linked to the immunomodulatory domain.

I. Collagen binding domains

In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising a collagen binding domain. In some embodiments, the collagen binding domain has a MW of about 5-100kDa, about 10-80kDa, about 20-60kDa, about 30-50kDa, or about 10kDa, about 20kDa, about 30kDa, about 40kDa, about 50kDa, about 60kDa, about 70kDa, about 80kDa, about 90kDa, or about 100 kDa. In some embodiments, the collagen binding domain is about 5kDa, about 10kDa, about 20kDa, about 30kDa, about 40kDa, about 50kDa, about 60kDa, about 70kDa, about 80kDa, about 90kDa, or about 100 kDa. In some embodiments, the collagen binding domain is about 30 kDa. In some embodiments, the collagen binding domain is about 40 kDa.

In some embodiments, the collagen binding domain is about 10-350, about 10-300, about 10-250, about 10-200, about 10-150, about 10-100, about 10-50, or about 10-20 amino acids in length. In some embodiments, the collagen binding domain is about 10 amino acids in length. In some embodiments, the collagen binding domain is about 15 amino acids in length. In some embodiments, the collagen binding domain is about 20 amino acids in length. In some embodiments, the collagen binding domain is about 30 amino acids in length. In some embodiments, the collagen binding domain is about 40 amino acids in length. In some embodiments, the collagen binding domain is about 50 amino acids in length. In some embodiments, the collagen binding domain is about 60 amino acids in length. In some embodiments, the collagen binding domain is about 70 amino acids in length. In some embodiments, the collagen binding domain is about 80 amino acids in length. In some embodiments, the collagen binding domain is about 90 amino acids in length. In some embodiments, the collagen binding domain is about 100 amino acids in length. In some embodiments, the collagen binding domain is about 120 amino acids in length. In some embodiments, the collagen binding domain is about 150 amino acids in length. In some embodiments, the collagen binding domain is about 200 amino acids in length. In some embodiments, the collagen binding domain is about 250 amino acids in length. In some embodiments, the collagen binding domain is about 300 amino acids in length. In some embodiments, the collagen binding domain is about 350 amino acids in length.

A. Isoelectric point

The isoelectric point (pI, pH (I), IEP) is the pH at which a particular molecule (e.g., collagen binding domain) has no net charge or is electrically neutral. Table 1 provides the calculated pI for the exemplary collagen binding domains described herein. The ExPASY tool from Switzerland bioinformatics institute (https:// web. ExPASy. org/computer _ pI /) was used to calculate the isoelectric point (pI) of the collagen binding domains shown in Table 1.

In some embodiments, the collagen binding domain has an isoelectric point less than (<) about 10, about 8, about 6, about 4, about 2, or about 1. In some embodiments, the collagen binding domain has an isoelectric point less than (<) 10. In some embodiments, the collagen binding domain has an isoelectric point less than (<)10 and a Molecular Weight (MW) greater than (>)5 kDa.

Table 1: pI calculated for exemplary collagen binding domains

Type I collagen

Collagen is the major structural protein located in the extracellular space, and type I collagen is the most abundant protein in mammals (Di Lullo et al, (2002) J Biol Chem 277(6): 4223-. The basic building block of type I collagen is a long (300-nm) and thin (1.5-nm diameter) protein, which is composed of three coiled subunits: two α 1(I) chains and one α 2 (I). Each chain comprises 1050 amino acids, which are intertwined with each other in a unique right-handed triple helix pattern. In humans, type I collagen is encoded by COL1a1 and COL1a2 genes. The COL1a1 gene encodes the pre- α 1 chain of collagen type I. The COL1a2 gene encodes the pre- α 2 chain of collagen type I, whose triple helix comprises two α 1 chains and one α 2 chain. Type I collagen is a fibrillogenic collagen present in most connective tissues, and is most abundant in bone, cornea, dermis, and tendon.

An exemplary amino acid sequence of the human alpha 1 chain precursor of type I collagen is shown in SEQ ID NO:90 (NCBI reference: NP-000079.2).

An exemplary amino acid sequence of the human alpha 2 chain precursor of type I collagen is shown in SEQ ID NO:91 (NCBI reference sequence: NP-000080.2).

Type IV collagen

Type IV collagen consists of a family of polypeptides that are the major component of mammalian basement membrane (Timpl (1989) Eur J Biochem 180: 487-502; Paulsson (1992) Crit Rev Biochem Mol Biol 27: 93-127). The α 1(IV) and α 2(IV) chains are products of different genes (COL 4A1 and COL4A2, respectively) which are located in pairs head-to-head on human chromosome 13 (Hudson et al, (1993) J Biol Chem 268: 26033-. The α 3(IV) and α 4(IV) chains (encoded by the COL4A3 and COL4A4 genes, respectively) are present in the same orientation on human chromosome 2, and the α 5(IV) and α 6(IV) chains (encoded by the COL4A5 and COL4A6 genes, respectively) are located on human chromosome X (Hudson et al, (1991) in Pathiobiochemistry, ed Kang A. (CRC Press, Boca Raton, FL), pp 17-30).

An exemplary amino acid sequence of the human α 1 chain of type IV collagen is shown in SEQ ID NO:92 (NCBI reference sequence: XP-011519350.1).

An exemplary amino acid sequence of the human α 2 chain of type IV collagen is shown in SEQ ID NO:93 (NCBI reference: NP-001837.2).

An exemplary amino acid sequence of the human α 3 chain of type IV collagen is shown in SEQ ID NO:94 (NCBI reference: NP-000082.2).

An exemplary amino acid sequence of the human α 4 chain of type IV collagen is shown in SEQ ID NO:95 (NCBI reference: NP-000083.3).

An exemplary amino acid sequence of the human α 5 chain of type IV collagen is shown in SEQ ID NO:96 (NCBI reference sequence: XP-011529151.2).

An exemplary amino acid sequence of the human α 6 chain of type IV collagen is shown in SEQ ID NO:97 (NCBI reference sequence: XP-006724680.1).

Thus, in some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising a collagen binding domain that specifically binds collagen. In some embodiments, the collagen binding domain specifically binds human type I collagen and/or human type IV collagen. In some embodiments, the collagen binding domain binds human type I collagen. In some embodiments, the collagen binding domain binds human collagen type IV. In some embodiments, the collagen binding domain specifically binds human type I collagen and human type IV collagen. In some embodiments, the collagen binding domain specifically binds human type I collagen or human type IV collagen.

D. Binding affinity to collagen

In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 0.5nM as determined by a collagen binding assay_D) A collagen binding domain that specifically binds collagen. In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 5nM as determined by a collagen binding assay_D) A collagen binding domain that specifically binds collagen. In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 50nM as determined by a collagen binding assay_D) A collagen binding domain that specifically binds collagen. In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 500nM as determined by a collagen binding assay_D) A collagen binding domain that specifically binds collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 0.5-5nM, 5-50nM, or 50-500nM, as determined by a collagen binding assay _D) Specifically binds to collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 50-100nM, 100-200nM, 200-300nM, 300-400nM, or 400-500nM as determined by the collagen binding assay_D) Specifically binds to collagen.

In some embodiments, the collagen binding assay determines the binding affinity for the collagen binding domain of collagen. In some embodiments, the collagen binding assay determines binding affinity for type I collagen. In some embodiments, the collagen binding assay determines binding affinity for type IV collagen.

In some embodiments, the collagen binding assay is an ELISA. Methods and techniques for performing collagen binding ELISA are well known in the art (see, e.g., Smith et al, (2000) J Biol Chem 275: 4205-4209). Thus, in some embodiments, the disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 0.5nM as determined by ELISA_D) A collagen binding domain that specifically binds collagen. Thus, in some embodiments, the disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 5nM as determined by ELISA _D) A collagen binding domain that specifically binds collagen. Thus, in some embodiments, the disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 50nM as determined by ELISA_D) A collagen binding domain that specifically binds collagen. Thus, in some embodiments, the present disclosure provides immunomodulatory fusionsA hybrid protein comprising an affinity (K) of less than about 500nM as determined by ELISA_D) A collagen binding domain that specifically binds collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 0.5-5nM, 4-40nM, or 50-500nM, as determined by ELISA_D) Specifically binds to collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 50-100nM, 100-200nM, 200-300nM, 300-400nM or 400-500nM, as determined by ELISA_D) Specifically binds to collagen.

In some embodiments, the collagen binding assay is a Surface Plasmon Resonance (SPR) assay. Methods and techniques for performing collagen binding SPR assays are well known in the art (see, e.g., Saenko et al, (2002) Anal Biochem 302(2): 252-. Thus, in some embodiments, the disclosure provides immunomodulatory fusion proteins comprising an affinity (K) at less than about 500nM as determined by SPR assay _D) A collagen binding domain that specifically binds collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 50-500nM, as determined by SPR assay_D) Specifically binds to collagen. In some embodiments, the collagen binding domain has an affinity (K) of about 50-100nM, 100-200nM, 200-300nM, 300-400nM or 400-500nM, as determined by the SPR assay_D) Specifically binds to collagen.

The phrase "surface plasmon resonance" includes an optical phenomenon whereby real-time biospecific interactions can be analyzed by detecting changes in protein concentration in a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description seeU.S., et al, (1993) Ann.biol.Clin.51: 19-26;U.S., et al, (1991) Biotechniques 11: 620-627; johnsson, B., et al, (199)5) J.mol.Recognit.8: 125-131; and Johnnson, B., et al, (1991) anal. biochem.198: 268. sup. 277.

E. Binding specificity to collagen

In some embodiments, the present disclosure provides immunomodulatory fusion proteins comprising a collagen binding domain that specifically binds collagen and does not specifically bind to one or more non-collagen extracellular matrix (ECM) components including, but not limited to, fibronectin, vitronectin, tenascin C, osteopontin, and fibrinogen. In some embodiments, the collagen binding domain has a lower K as compared to one or more non-collagen ECM components _DBinding with collagen. In some embodiments, the collagen binding domain is directed against type I collagen K_DA K lower than the collagen binding domain for a component of the extracellular matrix selected from fibronectin, vitronectin, osteopontin, tenascin C or fibrinogen_D. In some embodiments, the collagen-binding domain has a K that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99% lower than one or more non-collagen ECM components_DBinding with collagen. In some embodiments, the collagen-binding domain has a K that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold lower than one or more non-collagen ECM components_DBinding collagen.

In some embodiments, the collagen binding domain is not a promiscuous conjugate of an ECM component. In some embodiments, the collagen binding domain does not comprise a heparin binding domain. In some embodiments, the collagen binding domain is not a growth factor or portion thereof that binds extracellular matrix.

In some embodiments, the collagen binding domain has a lower K compared to type IV collagen _DBinding to type I collagen. In some embodiments, the collagen binding domain has a lower K compared to type I collagen_DWith type IV collagenWhite binding.

In some embodiments, the collagen binding domain competes with a reference collagen binding domain for binding to collagen. In some embodiments, the collagen binding domain competes for binding to type I collagen with a reference collagen binding domain. In some embodiments, the collagen binding domain competes with a reference collagen binding domain for binding to type IV collagen. In some embodiments, the collagen binding domain competes with a reference collagen binding domain for binding to type I collagen and type IV collagen. In some embodiments, the collagen binding domain competes with a reference collagen binding domain for binding to type I collagen, but not type IV collagen. In some embodiments, the collagen binding domain competes with a reference collagen binding domain for binding to type IV collagen, but not type I collagen.

In some embodiments, a reference collagen binding domain comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) leucine-rich repeats that bind collagen. In some embodiments, the reference collagen binding domain comprises a proteoglycan. In some embodiments, the reference collagen binding domain comprises a proteoglycan, wherein the proteoglycan is selected from the group consisting of: decorin, biglycan, fibromodulin, basement membrane glycan, cartilage protein, apocrin, PRELP, osteonectin/osteomodulin, optically active protein, osteochann/mimecan, podocan, basement membrane glycan, nestin. In some embodiments, the reference collagen binding domain is a basement membrane glycan. In some embodiments, the reference collagen binding domain comprises a class I leucine-rich proteoglycan (SLRP). SLRP is known to bind collagen (Chen and Birk (2013) FEBS Journal 2120-. In some embodiments, the reference collagen binding domain comprises a class II SLRP. In some embodiments, the reference collagen binding domain comprises a class III SLRP. In some embodiments, the reference collagen binding domain comprises a class IV SLRP. In some embodiments, the reference collagen binding domain comprises a class V SLRP. Further description of SLRP types is disclosed in Schaefer & Iozzo (2008) J Biol Chem 283(31): 21305-.

In some embodiments, the reference collagen binding domain comprises a leukocyte-associated immunoglobulin-like receptor 1(LAIR-1) protein. In some embodiments, the reference collagen binding domain comprises a leukocyte-associated immunoglobulin-like receptor 2(LAIR-2) protein. In some embodiments, the reference collagen binding domain comprises glycoprotein IV.

F. Exemplary collagen binding domains

In some embodiments, the collagen-binding domain comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) leucine-rich repeats that bind collagen. In some embodiments, the collagen binding domain comprises proteoglycans. In some embodiments, the collagen binding domain comprises a proteoglycan, wherein the proteoglycan is selected from the group consisting of: decorin, biglycan, testosterone, bikunin (bikunin), fibromodulin, basement membrane glycan, cartilage protein, keratin, ECM2, epiphysin, apocrin, PRELP, corneal protein, osteoadhesin glycan, optically active protein, osteocan, nyctan, Tsukushi, podocan-like protein 1 versican, basement membrane glycan, nidogen, neuroglycan, aggrecan, and brevican.

In some embodiments, the collagen binding domain comprises a class I leucine-rich proteoglycan (SLRP). In some embodiments, the collagen binding domain comprises a class II SLRP. In some embodiments, the collagen binding domain comprises a class III SLRP. In some embodiments, the collagen binding domain comprises a class IV SLRP. In some embodiments, the collagen binding domain comprises a class V SLRP. Further description of SLRP types is disclosed in Schaefer & Iozzo (2008) J Biol Chem 283(31): 21305-.

In some embodiments, the collagen binding domain comprises one or more leucine-rich repeats of a human class II proteoglycan member from the leucine-rich proteoglycan (SLRP) family. In some embodiments, the SLRP is selected from the group consisting of basisan, decorin, biglycan, fibromodulin, keratin, epiphyseal proteoglycan, ascherin, and osteochanan. In some embodiments, the SLRP is a basement membrane glycan. In some embodiments, the basement membrane glycan comprises the amino acid sequence set forth in SEQ ID NO: 107.

Basement membrane polysaccharide

The basement membrane glycan (also known as LUM) is an extracellular matrix protein encoded by the LUM gene on chromosome 12 in humans (Chakravarti et al, (1995) Genomics 27(3): 481-488). Basement membrane glycans are class II proteoglycan members of the leucine rich small proteoglycan (SLRP) family, which include decorin, biglycan, fibromodulin, keratoprotein, epiphysin, and osteochann (Iozzo & Schaefer (2015) Matrix Biology 42: 11-55).

Like other SLRPs, the molecular weight of the basement membrane glycan is about 40kDa and has four major intramolecular domains: 1) a signal peptide of 16 amino acid residues, 2) a negatively charged N-terminal domain containing sulfated tyrosine and one or more disulfide bonds, 3)10 leucine-rich tandem repeats to enable binding of the basement membrane glycan to collagen, and 4) a carboxy-terminal domain of 50 amino acid residues containing two conserved cysteines separated by 32 residues. Kao et al (2006) Experimental Eye Research 82(1): 3-4). There are four N attachment sites in the leucine-rich repeat domain of the protein core that can be replaced by keratan sulfate. The decorin of basement membrane glycans (like decorin and fibromodulin) is horseshoe-shaped. This enables it to bind to the collagen molecules in the collagen fibrils, thereby helping to keep the adjacent collagen fibrils apart (Scott (1996) Biochemistry 35(27): 8795-8799).

Leukocyte-associated immunoglobulin-like receptors (LAIR-1 and LAIR-2)

Leukocyte-associated Ig-like receptor (LAIR) -1 is a collagen receptor that inhibits immune cell function upon binding to collagen. Beside LAIR-1, the human genome encodes LAIR-2 (a soluble homolog). Human (h) LAIR-1 is expressed in most PBMC and thymocytes (Maasho et al, (2005) Mol Immunol 42: 1521-. hLAIR-1 cross-linking by mAb in vitro provides potent inhibitory signals that inhibit immune cell function (4, 10-15). Collagen is known to be a natural, high affinity ligand for LAIR molecules. The interaction of hlAIR-1 with collagen directly inhibits the activation of immune cells in vitro. (Meyaard et al (1997) Immunity 7: 283-.

In some embodiments, the collagen-binding domain comprises a human type I glycoprotein having an Ig-like domain or binding to the extracellular portion of collagen. In some embodiments, the type I glycoprotein competes with the basement membrane glycan for binding to type I collagen. In some embodiments, the human type I glycoprotein is selected from LAIR1, LAIR2, and glycoprotein IV. In some embodiments, the human type I glycoprotein is LAIR 1. In some embodiments, the human type I glycoprotein is LAIR1 and the collagen binding domain comprises amino acid residues 22-122 of the amino acid sequence set forth in SEQ ID NO: 98.

In some embodiments, the collagen binding domain is a variant of LAIR1, LAIR2, or glycoprotein IV. In some embodiments, the LAIR1 variant, LAIR2 variant, or glycoprotein IV variant comprises one or more amino acid substitutions, additions, or deletions (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) relative to the protein sequence of wild-type LAIR1, LAIR2, or glycoprotein IV. In some embodiments, the collagen binding domain is a LAIR1 variant comprising one or more amino acid substitutions, additions, or deletions (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) relative to the LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the collagen binding domain is a LAIR1 variant comprising one or more amino acid substitutions, additions, or deletions (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) at the LAIR1 binding pocket (e.g., the LAIR1 binding site comprises one or more residues E61, S66, Y68, I102, W109, Y115, R59, E63, R100, E111, and Q112, and combinations thereof) (Brondijk et al, (2010) Blood 115: 1364-. In some embodiments, the collagen-binding domain is a LAIR1 variant comprising one or more amino acid substitutions, additions, or deletions (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) outside of the LAIR1 binding pocket.

In some embodiments, the collagen binding domain is a LAIR1 variant having increased collagen binding affinity relative to the collagen binding affinity of wild-type LAIR1 protein. In some embodiments, the LAIR1 variant exhibits increased binding affinity to collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1 variant has a reduced collagen binding affinity relative to the collagen binding affinity of wild-type LAIR1 protein. In some embodiments, the LAIR1 variant exhibits reduced binding affinity to collagen relative to the collagen binding affinity of LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

Glycoprotein IV (CD36)

In some embodiments, the collagen binding domain comprises glycoprotein iv (gpiv). Glycoprotein IV binds many ligands including collagen (Tandon (1989) J Biol Chem 264(13): 7576-7583). The multifunctional glycoprotein GPIV can act as a receptor for a variety of ligands including thrombospondin, fibronectin, collagen or beta-amyloid as well as lipidic, such as oxidized low density lipoprotein (oxLDL), anionic phospholipids, long chain fatty acids and bacterial diacyllipopeptides. GPIV is a protein encoded by the CD36 gene in humans. The CD36 antigen is an integral membrane protein that is present on the surface of many cell types in vertebrates. It can introduce fatty acids into the interior of cells and is a member of the class B scavenger receptor family of cell surface proteins. In some embodiments, CD36 comprises the amino acid sequence set forth in SEQ ID NO 100.

Immunomodulatory domains

Immunomodulatory fusion proteins disclosed herein comprise at least one immunomodulatory domain operably linked to a collagen binding domain. In some embodiments, the immunomodulatory fusion protein comprises one, two, three, four, or five immunomodulatory domains. In some embodiments, when more than one immunomodulatory domain is present in the fusion protein, the immunomodulatory domains are the same. In some embodiments, when more than one immunomodulatory domain is present in the fusion protein, the immunomodulatory domains are different. In some embodiments, when more than one immunomodulatory domain is present in the fusion protein, each domain is N-terminal to the collagen binding domain. In some embodiments, when more than one immunomodulatory domain is present in the fusion protein, each domain is located C-terminal to the collagen binding domain. In some embodiments, when more than one immunomodulatory domain is present in the fusion protein, at least one domain is N-terminal to the collagen binding domain and at least one domain is C-terminal to the collagen binding domain.

In some embodiments, the immunomodulatory domain activates the activity of a cell of the immune system. For example, in some embodiments, the immunomodulatory domain is an immune response stimulus, such as, but not limited to, a cytokine, such as an interleukin, a chemokine, a TNF family member, an agonistic antibody, an immune checkpoint blocker, or a combination thereof. In some embodiments, the immunomodulatory domain enhances an immune response. In some embodiments, enhancing the immune response comprises stimulating T cells, stimulating B cells, stimulating a dendritic cell response, or a combination thereof. In some embodiments, the enhancement of the immune response results in cytokine production, antibody production, production of antigen-specific immune cells (e.g., CD8+ T cells or CD4+ T cells), stimulation of a type I interferon response, or a combination thereof.

In some embodiments, the immunomodulatory domain comprises a polypeptide that activates, enhances, or promotes a response of an immune cell. In some embodiments, the immunomodulatory domain comprises a polypeptide that inhibits, reduces, or suppresses the response of an immune cell. In some embodiments, the immune cells are lymphoid cells, including but not limited to T cells, B cells, NK cells, and innate lymphoid cells. In some embodiments, the immune cell is a myeloid lineage cell, including but not limited to monocytes, neutrophils, macrophages, dendritic cells, mast cells, and granulocytes.

In some embodiments, the immune cell response is cytokine production, antibody production, production of antigen-specific immune cells, or a combination thereof.

A. Interleukin

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an Interleukin (IL). Interleukins are secreted proteins that bind to their specific receptors and play a role in communication between leukocytes. Interleukins suitable for use as the immunomodulatory domain of the immunomodulatory fusion protein include, but are not limited to: IL-2, IL-12, IL-15 superagonists (IL-15SA), IL-21, IL-6, IL-5, IL-8, IL-7, IL-17, IL-23, IL-18, IL-1, IL-4, IL-3, IL-10, IL-13 and IL-9. In some embodiments, an interleukin suitable for use as an immunomodulatory domain comprises a sequence selected from the group consisting of SEQ ID NO: 1-5 and 9-24. In some embodiments, the immunomodulatory domain is an IL-2 polypeptide. In some embodiments, the immune modulatory domain is an IL-12 polypeptide. In some embodiments, the immunomodulatory domain is an IL-15 polypeptide. In some embodiments, the immunomodulatory domain is an IL-15SA polypeptide.

In some embodiments, the immunomodulatory domain is an interleukin polypeptide that binds a common gamma chain receptor. Interleukins that bind to common gamma chain receptors include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15/IL-15 Ra and IL-21.

In some embodiments, the immune regulatory domain is a polypeptide belonging to the IL-12 family. The IL-12 family comprises heterodimeric ligands consisting of an alpha subunit (e.g., IL-12p35, IL-23p19, IL-27p28) and a beta subunit (e.g., IL-12p40, IL-23p40 (which is the same as IL-12p 40), EBI3) with helical structures. Exemplary members include IL-12, IL-23, IL-27 and IL-35.

In some embodiments, the immune modulatory domain is a polypeptide belonging to the IL-1 superfamily. The interleukin-1 (IL-1) family consists of 11 structurally related family members (IL-1 α, IL-1 β, IL-1Ra, IL-18, IL-33, and IL-1F5 through IL-1F10), which are the most potent signaling molecules of the immune system, acting through a set of closely related receptors. All IL-1 receptors share a similar activation pattern: following ligand binding to the primary receptor subunit (i.e., IL-1R1 for IL-1 α and β, IL-18R for IL-18, and ST2 for IL-33), recruitment of the secondary receptor subunit (i.e., IL-1RAP for IL-1 α and β, IL-18RAP for IL-18, and IL-1RAP for IL-33) initiates signaling through the juxtaposition of the cytoplasmic Toll/IL-1 receptor (TIR) domains of the receptor subunits. The dimerized TIR domain provides a docking platform for MYD88 aptamer proteins, which leads to activation of the proinflammatory nuclear factor-kb (NF-kb) and mitogen-activated protein kinase (MAPK) pathways through recruitment of other intermediates. IL-1 family members are produced primarily by innate immune cells and act on a variety of cell types during the course of an immune response. Thus, in some embodiments, the immunomodulatory domain is an IL-18 polypeptide.

Interleukin-2 (IL-2)

In some embodiments, the immunomodulatory fusion protein comprises an IL-2 family member operably linked to a collagen binding domain, optionally through a linker. In some embodiments, the IL-2 family member is IL-2. Interleukin-2 (IL-2) is a cytokine that induces antigen-activated T cell proliferation and stimulates Natural Killer (NK) cells. The biological activity of IL-2 is through a multi-subunit IL-2 receptor complex (IL-2R) that spans three polypeptide subunits of the cell membrane: p55(IL-2R α, α subunit, also known as CD25 in humans), p75(IL-2RP, β subunit, also known as CD122 in humans) and p64(IL-2R γ, γ subunit, also known as CD132 in humans). The response of T cells to IL-2 depends on a number of factors, including: (1) the concentration of IL-2; (2) the number of IL-2R molecules on the cell surface; and (3) the number of IL-2 Rs occupied by IL-2 (i.e., the affinity of the binding interaction between IL-2 and IL-2R (Smith, "Cell Growth Signal Transduction is Quantal" In Receptor Activation by antibodies, Cytokines, Hormes, and Growth Factors 766:263-271, 1995)). IL-2: the IL-2R complex is internalized and the different components are differentially sorted. IL-2R α circulation to the cell indicates, whereas IL-2 associated with the IL-2: IL-2RP γ complex is delivered to lysosomes and degraded. IL-2 has rapid systemic clearance when administered as an intravenous (i.v.) bolus (initial clearance phase, half-life of 12.9 minutes, followed by a slow clearance phase, half-life of 85 minutes) (Konrad et al, Cancer Res.50: 2009-.

The outcome of systemic administration of IL-2 in cancer patients is far from ideal. Although between 15% and 20% of patients produce objective remission from high doses of IL-2, most patients do not respond and many suffer from serious, life-threatening side effects including nausea, confusion, hypotension and septic shock. The severe toxicity associated with high dose IL-2 treatment is due in large part to the activity of Natural Killer (NK) cells. NK cells expressing intermediate affinity receptor IL-2RP gamma_cIt is therefore stimulated when it actually results in nanomolar concentrations of IL-2 in the serum of a patient during treatment with high doses of IL-2. Attempts have been made to reduce serum concentrations by reducing the dose and adjusting the dosing regimen to selectively stimulate IL-2RaP γ carrier_cDespite the low toxicity, the effect of this treatment is also poor. In view of the toxicity problems associated with high dose IL-2 cancer therapy, many research groups have attempted to improve the anti-cancer efficacy of IL-2 by simultaneously administering therapeutic antibodies. However, this is not soSuch efforts have been largely unsuccessful, producing no additional clinical benefit or limited clinical benefit compared to IL-2 therapy alone. Therefore, new IL-2 therapies are needed to more effectively combat various cancers.

In some embodiments, the attachment of IL-2 to the collagen binding domain localizes the cytokine to the cell, thereby preventing its systemic toxicity. Furthermore, in some embodiments, when administered directly to a tumor or lesion, the collagen binding domain localizes the cytokine to the tumor or lesion microenvironment, thereby preventing systemic toxicity associated with IL-2 treatment.

In some embodiments, the IL-2 is a human recombinant IL-2, e.g.(aldesleukin).Is a human recombinant interleukin-2 product produced in E.Differs from natural interleukin-2 in the following respects: a) it is not glycosylated; b) it does not have an N-terminal alanine; and c) its serine at amino acid position 125 is replaced with cysteine.In the form of biologically active, non-covalently bonded microaggregates with an average size of 27 recombinant interleukin-2 molecules.(aldesleukin) is administered by intravenous infusion. In some embodiments, IL-2 is wild-type IL-2 (e.g., its precursor form of human IL-2 or mature IL-2). In some embodiments, IL-2 comprises the amino acid sequence set forth in SEQ ID NO 1.

In certain embodiments, IL-2 is mutated such that it has an altered affinity (e.g., higher affinity) for the IL-2R α receptor as compared to unmodified IL-2. Site-directed mutagenesis can be used to isolate IL-2 mutants that exhibit higher binding affinity for CD25 (i.e., IL-2R α) than wild-type IL-2. Increasing the affinity of IL-2 for IL-2R α at the cell surface will increase receptor occupancy within a limited range of IL-2 concentrations and increase the local concentration of IL-2 at the cell surface.

In some embodiments, the disclosure relates to IL-2 mutants that may, but need not, be substantially purified and may act as high affinity CD25 binders. IL-2 is a T cell growth factor that induces antigen-activated T cell proliferation and stimulates NK cells. Exemplary IL-2 mutants that are high affinity binders include those described in WO2013/177187a2 (the entire contents of which are incorporated herein by reference). Other exemplary IL-2 mutants with increased affinity for CD25 are disclosed in US7,569,215, the contents of which are incorporated herein by reference.

In some embodiments, the disclosure relates to IL-2 mutants having reduced binding affinity for CD25 relative to wild-type IL-2. In some embodiments, the IL-2 mutant does not bind to CD 25.

In some embodiments, the IL-2 mutant comprises an amino acid sequence having at least 80% identity to SEQ ID No. 1 that binds CD 25. For example, in some embodiments, the IL-2 mutant has at least one mutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) that has increased affinity for the alpha subunit of the IL-2 receptor as compared to wild-type IL-2. It will be appreciated that the mutations identified in mouse IL-2 can be made in full-length human IL-2 (nucleic acid sequence (accession number: NM 000586); amino acid sequence (accession number: P60568)) or the corresponding residues of human IL-2 without the signal peptide. Thus, in some embodiments, IL-2 is human IL-2. In other embodiments, IL-2 is a mutant human IL-2.

In some embodiments, the IL-2 mutant is at least or about 50%, at least or about 65%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 87%, at least or about 90%, at least or about 95%, at least or about 97%, at least or about 98%, or at least or about 99% identical in amino acid sequence to wild-type IL-2 (in its precursor form, or, preferably, mature form). Mutations may consist of changes in the number or content of amino acid residues. For example, IL-2 mutants may have a greater or lesser number of amino acid residues than wild-type IL-2. Alternatively, or in addition, the IL-2 mutant may contain a substitution of one or more amino acid residues present in wild-type IL-2.

For example, a polypeptide comprising an amino acid sequence that is at least 95% identical to the reference amino acid sequence of SEQ ID NO. 1 is a polypeptide comprising a sequence that is identical to the reference sequence except that up to 5 changes are introduced in the reference sequence of SEQ ID NO. 1. For example, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a plurality of amino acids not exceeding 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed either within residues of the reference sequence or within one or more contiguous groups within the reference sequence.

The substituted amino acid residue or residues may be, but need not be, conservative substitutions, which typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. These mutations may occur at amino acid residues that are in contact with IL-2R α.

Interleukin-12 (IL-12)

In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide operably linked to a collagen binding domain by a linker. Interleukin-12 (IL-12) is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al, Annu Rev Immunol.16:495-521 (1998). IL-12 mainly functions as a 70kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits. The precursor form of the IL-12P40 subunit (NM-002187; P29460; also known as IL-12B, Natural killer cell stimulating factor 2, cytotoxic lymphocyte mutational factor 2) is 328 amino acids in length, whereas its mature form is 306 amino acids in length. The precursor form of the IL-12P35 subunit (NM-000882; P29459; also known as IL-12A, Natural killer cell stimulating factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids in length, and the mature form is 197 amino acids in length. Genes for the IL-12p35 and p40 subunits are located on different chromosomes and are regulated independently of each other. Gately, MK et al, Annu Rev Immunol.16:495-521 (1998). Many different immune cells (e.g., dendritic cells, macrophages, monocytes, neutrophils, and B cells) produce IL-12 upon stimulation by an antigen. Active IL-12 heterodimers are formed after protein synthesis.

Due to its ability to activate NK cells and cytotoxic T cells, the IL-12 protein has been studied as a promising anti-cancer therapeutic since 1994. See, Nastala, C.L., et al, J Immunol 153:1697-1706 (1994). Despite the high expectations, early clinical studies have not achieved satisfactory results. Lasek W, et al, Cancer Immunol Immunother 63:419-435,424 (2014). In most patients, repeated administration of IL-12 will result in an adaptive response and a gradual decrease in IL-12-induced interferon gamma (IFN γ) levels in the blood. Furthermore, although it has been recognized that IL-2-induced anti-cancer activity is mediated primarily by secondary secretion of IFN γ, the concomitant induction of IFN γ and other cytokines (e.g., TNF- α) or chemokines (IP-10 or MIG) by IL-12 results in severe toxicity.

In addition to negative feedback and toxicity, the marginal effects of IL-12 therapy in a clinical setting may be caused by a strong immunosuppressive environment in humans. In order to minimize IFN gamma toxicity and improve IL-12 effectiveness, scientists have tried different methods, such as directed to IL-12 therapy of different dosage and time scheme. See, Sacco, S. et al, Blood 90:4473-4479 (1997); leonard, J.P., et al, Blood 90:2541-2548 (1997); coughlin, C.M., et al, Cancer Res.57:2460-2467 (1997); Asselin-Paturel, C. et al, Cancer 91:113-122 (2001); and Saudemont, A. et al, Leukemia 16: 1637-. Nevertheless, these methods do not significantly affect patient survival. Kang, W.K., et al, Human Gene Therapy 12: 671-.

Using retrovirus and adenovirus vectors in tumor cell expression, membrane anchoring form of IL-12 has been studied as a means of reducing and systemic drug delivery related toxicity. See Pan, W-Y, et al, mol. Ther.20(5):927 and 937 (2012). However, there are potential health risks associated with the use of viral vectors, as potential viruses may act as oncogenes, and viral vectors may be immunogenic.

Thus, in some embodiments, the immunomodulatory fusion proteins disclosed herein comprise an IL-12 polypeptide operably linked to a collagen binding domain. In some embodiments, the IL-12 polypeptide and collagen binding domain connection will cytokine localization to cells, thus preventing systemic toxicity. Furthermore, in some embodiments, when administered directly to a tumor or lesion, the collagen binding domain localizes the cytokine to the tumor or lesion microenvironment, thereby preventing systemic toxicity.

In some embodiments, the IL-12 polypeptide contains IL-12A (e.g., SEQ ID NO: 3). In some embodiments, the IL-12 polypeptide contains IL-12B (e.g., SEQ ID NO: 2). In some embodiments, IL-12 polypeptide contains IL-12A and IL-12B.

In some embodiments, IL-12B is located in the IL-12 polypeptide IL-12A N terminal. In some embodiments, IL-12A is located in the IL-12 polypeptide IL-12B N terminal. The phrase "at the N-terminus of … …" refers to a position in a polypeptide relative to the N-terminus of the polypeptide, relative to other sequences in the polypeptide. For example, IL-12B in IL-12A "N terminal" refers to IL-12B than IL-12A closer to the IL-12 polypeptide N terminal.

In some embodiments, IL-12 polypeptide contains IL-12B and IL-12A single polypeptide chain, which are directly fused to each other or through a linker (referred to herein as "subunit linker") connected to each other. Non-limiting examples of linkers are disclosed elsewhere herein.

In some embodiments, the IL-12 polypeptides of the disclosure comprise IL-12A and/or IL-12B that is a variant, is a functional fragment, or comprises substitutions, insertions, and/or additions, deletions, and/or covalent modifications as compared to the wild-type IL-12A or IL-12B sequence. In some embodiments, the IL-12 polypeptide carboxyl, amino terminal or internal region of the amino acid residues are deleted, thereby providing a fragment.

In some embodiments, IL-12 polypeptide contains IL-12A and/or IL-12B amino acid sequence of substitution variants, which can contain one, two, three or more than three substitutions. In some embodiments, a substituted variant may comprise one or more conservative amino acid substitutions. In other embodiments, the variant is an insertion variant. In other embodiments, the variant is a deletion variant.

As known to the technicians in this field, IL-12 protein fragments, functional protein domain, variants and homologous protein (orthologues) are also considered in the present disclosure of IL-12 polypeptide scope. Non-limiting examples of IL-12 polypeptides suitable for use in the immunomodulatory fusion proteins disclosed herein are set forth in SEQ ID NOs: 2-3.

In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the immunomodulatory fusion protein comprises an IL-12 polypeptide comprising the amino acid sequences set forth in SEQ ID NOs: 2 and 3.

Interleukin-15 (IL-15)

In some embodiments, the immunomodulatory fusion protein comprises an IL-15 polypeptide operably linked to a collagen binding domain, optionally through a linker. IL-15 is a member of the 4 α -helical bundle cytokine family and plays an important role in the development of a potent immune response. Waldmann, T.A., Cancer Immunol.Res.3:219-227 (2015). IL-15 is crucial for the normal development of NK cells and the long-term maintenance of memory CD8+ T cells. The IL-15 gene encodes a 162 amino acid preprotein with a 48 amino acid signal peptide, the mature protein of which is 114 amino acids in length. BamHord, R.N., et al, Proc.Natl.Acad.Sci.USA 93: 2897-. See also, e.g., GenBank accession nos. NM _000585 (homo sapiens IL-15 transcript variant 3mRNA sequence) and NP _000576 (the corresponding IL15 isoform 1 preprotein).

IL-15 shares some structural similarities with interleukin-2 (IL-2). Like IL-2, IL-15 signals through the IL-2 receptor beta chain (CD122) and the common gamma chain (CD 132). However, unlike IL-2, IL-15 by itself is not able to bind CD122 and CD132 efficiently. IL-15 must first bind to the IL-15 alpha receptor subunit (IL-15R alpha). The IL-15R α gene encodes a 267 amino acid preprotein with a 30 amino acid signal peptide, the mature protein of which is 237 amino acids in length. See, e.g., GenBank accession No. NM-002189 (homo sapiens IL-15R α transcript variant 1mRNA) and NP-002180 (homo sapiens IL-15R α isoform 1 precursor amino acid sequence).

Human IL-15R α is a mainly transmembrane protein, and binds to IL-15 on the cell surface of cells such as activated dendritic cells and monocytes. Waldmann, T.A., Cancer Immunol.Res.3:219-227 (2015). The membrane bound complex of IL-15/IL-15R α then presents the IL-15 subunit in trans to the CD122 and CD132 subunits. Thus, IL-15R α is an important component of IL-15 activity.

To overcome the problem of short half-life of systemically injected IL-15, it has been demonstrated that pre-complexing IL-15 with soluble recombinant IL-15Ra results in IL-15 superagonists (IL-15SA) enhancing the systemic potency of IL-15 by about 50-fold, and also extends the half-life of cytokines in serum to about 20hr after systemic injection (Stoklasek et al, J Immunol 177(9):6072,2006; Dubois et al, J Immunol 180(4):2099,2008; Rubinstein et al, Proc Natl Acad Sci U S A103 (24):9166,2006).

Thus, in some embodiments, the immunomodulatory fusion protein's immunomodulatory domain is an IL-15 polypeptide. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO 5. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO 4. In some embodiments, the IL-15 polypeptide is an IL-15 superagonist, comprising IL-15 and IL-15 Ra. In some embodiments, the IL-15 superagonists comprise the amino acid sequences set forth in SEQ ID NOs 4 and 5.

B. Interferon

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an Interferon (IFN). Interferons comprise a class of secreted proteins that are induced in response to specific extracellular stimuli by stimulating toll-like receptors (TLRs). In some embodiments, the interferon may potentiate the antiviral defenses (e.g., antigen presentation) of the immune system. Receptors are indicated by high affinity cells, and IFNs stimulate genes using signaling molecules. Interferons suitable for use as the immunomodulatory domain of an immunomodulatory fusion protein include, but are not limited to: IFN gamma and IFN alpha.

In some embodiments, the immunomodulatory fusion protein comprises an IFN γ polypeptide operably linked to a collagen binding domain. IFN γ is produced by a variety of immune cells, such as activated T cells and NK cells. IFN γ interacts with specific receptors on the cell surface and activates signaling pathways that produce immunomodulatory effects. Thus, in some embodiments, the immunomodulatory domain is an IFN γ polypeptide. In some embodiments, the IFN γ polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 7.

In some embodiments, the immunomodulatory fusion protein comprises an IFN α polypeptide operably linked to a collagen binding domain. IFN α is produced by B lymphocytes, surface marker-free lymphocytes and macrophages, and activates NK cells, while having antiviral and antitumor activities. Thus, in some embodiments, the immunomodulatory domain is an IFN α polypeptide. In some embodiments, the IFN alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO 6.

C. Stimulating factor for immune cell differentiation

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an immune cell differentiation stimulating factor. In some embodiments, the immune cell differentiation stimulating factor activates intracellular signaling pathways that drive differentiation, development and proliferation of hematopoietic progenitor cells into specific subtypes of immune cells. Immune cell differentiation stimulating factors suitable for use in the immunomodulatory fusion proteins disclosed herein include, but are not limited to: GM-CSF (granulocyte-macrophage colony stimulating factor), G-CSF (granulocyte colony stimulating factor), and FLT3L (FMS-like tyrosine kinase 3 ligand).

In some embodiments, the immunomodulatory domain is a GM-CSF polypeptide. GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts. In addition to having the function of stimulating growth and differentiation in hematopoietic progenitor cells, GM-CSF has a variety of roles on immune cells expressing the GM-CSF receptor. In some embodiments, the GM-CSF polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 27.

In some embodiments, the immunomodulatory domain is an FLT3L polypeptide. FLT3 is a Receptor Tyrosine Kinase (RTK) that is expressed by immature hematopoietic precursor cells. FLT3L is a transmembrane or soluble protein expressed by a large number of cells, including hematopoietic and stromal cells in the bone marrow. In combination with other growth factors, FLT3L stimulates proliferation and development of various cell types, including myeloid and lymphoid precursor cells, dendritic cells, and NK cells. In some embodiments, the FLT3L polypeptide comprises the amino acid sequence set forth in SEQ ID NO 28.

In some embodiments, the immunomodulatory domain is a G-CSF polypeptide. In some embodiments, G-CSF modulates proliferation, differentiation, and functional activation of neutrophils. In some embodiments, the G-CSF polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 29.

D. Chemotactic factor

In some embodiments, a immunomodulatory fusion protein of the disclosure is a fusion protein with a immunomodulatory domain. In some embodiments, the chemokine is a protein that induces directional chemotaxis of reactive cells (e.g., leukocytes). In general, chemokines are divided into four subfamilies: CXC, CC, (X) C and CX 3C. In CXC chemokines, one amino acid separates the first two cysteines ("CXC motif"). ELR + CXC chemokines are ligands for CXCR1 and/or CXCR2 chemokine receptors, and CXCR1 and/or CXCR2 chemokine receptors are G protein-coupled seven transmembrane domain type receptors that specifically bind ELR + CXC chemokines. Seven human ELR + CXC chemokines are human Gro α (also known as CXCL1), human Gro- β (also known as CXCL2), human Gro γ (also known as CXCL3), human ENA-78 (also known as CXCL5), human GCP-2 (also known as CXCL6), human NAP-2 (also known as CXCL7) and human IL-8 (also known as CXCL 8). All ELR + CXC chemokines bind to CXCR2 receptor; in addition, some ELR + CXC chemokines bind both CXCR1 and CXCR2 receptors (i.e., CXCL6 and CXCL8), all of which contribute to redundancy in the activation pathway. The five mouse ELR + CXC chemokines are Keratinocyte Chemokine (KC) (also known as CXCL1), macrophage inflammatory protein 2(MIP-2) (also known as CXCL2), dendritic cell inflammatory protein 1(DCIP-1) (also known as CXCL3), lipopolysaccharide-induced CXC chemokine (LIX) (also known as CXCL5), and neutrophil activating peptide 2(NAP-2) (also known as CXCL 7).

Chemokines suitable for use in the immunomodulatory fusion proteins disclosed herein include, but are not limited to: LIF, M-CSF, MIP-2, MIP-1 β, KP (CXLC1), MIG (CXCL9), IP-10(CXCL10), MCP-1, eotaxin, RANTES, LIX and MIP-1 α.

The amino acid sequences encoding exemplary chemokines suitable for use as the immunomodulatory domains of the immunomodulatory fusion proteins disclosed herein are shown below:

E. tumor Necrosis Factor (TNF) superfamily

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is the extracellular domain of a member of the Tumor Necrosis Factor (TNF) superfamily. The tumor necrosis factor ligand and receptor superfamily are a series of structurally homologous cell surface proteins that signal through the formation of a trimeric cluster of ligand-receptor complexes. The linkage of activated TNF superfamily receptors can lead to a wide range of pro-immune responses including proliferation, enhancement of effector functions, and production of chemokines and cytokines. Some ligands (e.g., Fas) can lead to induction of apoptosis, as well as expression on the surface of immune cells. In addition, other ligands act as inhibitory receptors, which attenuate the immune response. In some embodiments, the extracellular domain is derived from: TNF alpha, LIGHT, LT alpha, LT-beta, BTLA, CD160, CD40L, FasL, CD30L, 4-1BBL, CD27L, OX40L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1, EDA2, or GITRL. The extracellular domain is capable of binding to a receptor of a selected TNF superfamily member, thereby inducing or stimulating an immune response.

The following table shows the receptors corresponding to the derived extracellular domains:

CD28 family

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is the extracellular domain of a CD28 family member. The CD28 family is a family of inhibitory (PD1, CTLA-4) and activating (CD28, ICOS) receptors that bind to members of the B7 ligand family. CD28 is a co-stimulatory receptor that provides the secondary signal required to activate naive T cells (along with TCR ligation) and has two natural ligands, CD80 and CD 86. CD28 signaling can be used to increase proliferation, effector function, and anti-apoptotic signaling. Recently, it has been shown that CD28 signaling is required in an effective PD1/PDL1 blockade. ICOS (inducible T cell co-stimulator) is a closely related surface receptor that is expressed on activated T cells and shows similar function as CD 28.

Thus, in some embodiments, the immunomodulatory domain is the extracellular domain of CD80 (B7-1). In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO 71.

Thus, in some embodiments, the immunomodulatory domain is an extracellular domain of CD86(B7-2) that is capable of binding CD 28. In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 72.

Thus, in some embodiments, the immunomodulatory domain is the extracellular domain of ICOSLG. In some embodiments, the immunomodulatory domain comprises the amino acid sequence set forth in SEQ ID NO 73.

G. Agonistic antibodies

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof. In contrast to antagonistic antibodies that block their target function, agonistic antibodies activate their target. In some embodiments, the agonistic antibody, or antigen-binding fragment thereof, binds to an immune activating receptor. In some embodiments, immune activating receptors include, but are not limited to: tumor Necrosis Factor (TNF) receptors, CD28 family members, T Cell Receptors (TCRs), killer Ig-like receptors (KIRs), leukocyte Ig-like receptors (LIRs), CD94/NKG2 receptors, Fc receptors, Signaling Lymphocyte Activation Molecules (SLAMs), and activating Siglec receptors.

Tumor Necrosis Factor (TNF) superfamily

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a Tumor Necrosis Factor (TNF) superfamily member receptor. The TNF superfamily is as described above. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment thereof that binds TNFR1, thereby activating the receptor.

The following table provides a list of TNF superfamily member receptors that can generate agonistic antibodies or antigen-binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein:

in some embodiments, the immunomodulatory domain is an anti-4-1 BB agonist antibody. In some embodiments, the immunomodulatory domain is an anti-OX 40 agonist antibody. In some embodiments, the immunomodulatory domain is a CD40 agonist antibody.

Superfamily of CD28 receptors

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a CD28 superfamily receptor. The CD28 superfamily is as described above. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment that binds to CD28, thereby activating the receptor.

The following table provides a list of CD28 superfamily member receptors that can generate agonistic antibodies or antigen-binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein:

ligands	Receptors	Receptor Uniprot KB
			CD80(B7-1)	CD28	P10747
CD86(B7-2)	CD28	P10747
			ICOSLG	ICOS	Q9Y6W8

T Cell Receptor (TCR) complexes

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a T Cell Receptor (TCR) complex. The T Cell Receptor (TCR) is a cell surface receptor responsible for conferring specificity to a T cell antigen. Each TCR is specific for a particular peptide presented by MHC class I (for CD8+ T cells) or MHC class II (for CD4+ T cells). For naive T cells, ligation of the TCR provides the first of the two signals required to activate the T cell. TCR ligation of CD8+ T cells results in death of cells displaying homologous pmhcs (and potentially bystander cells) by release of soluble factors (such as perforin and granzyme B) and upregulation of apoptosis-inducing ligands (such as Fas ligand). For CD4+ helper T cells, ligation of the TCR to its cognate pMHC results in the release of cytokines.

Thus, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment thereof that binds to a TCR. For example, in some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment that binds to CD3 γ, thereby activating the receptor.

The following table provides a list of TCR complex members suitable for use in the immunomodulatory fusion proteins described herein that can generate agonistic antibodies, or antigen-binding fragments thereof, to target:

TCR conjugates	TCR Complex Member	Member Unit KB
			pMHC	CD3γ	P09693
pMHC	CD3δ	P04234
			pMHC	CD3ζ	P20963
pMHC	CD3ε	P07766

Killer cell Ig like receptor (KIR)

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a killer cell Ig-like receptor (KIR). Killer cell immunoglobulin-like receptors (KIRs) are a class of receptors that are expressed primarily on NK cells and on some subset of T cells. By binding to MHC class I (HLA-A, HLA-B and HLA-C) molecules, these receptors are primarily responsible for their recognition (and thus inhibitory function). These receptors may be either active or inhibitory, depending on the length of the cytoplasmic tail. Inhibitory receptors have a longer tail and contain an ITIM domain. Activated KIRs have shorter cytoplasmic domains and bind to DAP12 to mediate signaling.

The following table provides activated KIRs that can generate agonistic antibodies or antigen-binding fragments thereof to target suitable for use in the immunomodulatory fusion proteins described herein:

Ligands	Receptors	Receptor Uniprot KB
			HLA molecules	KIR 2DS1	Q14954
HLA molecules	KIR 2DS2	P43631
			HLA molecules	KIR 2DS3	Q14952
HLA molecules	KIR 2DS4	P43632
			HLA molecules	KIR 2DS5	Q14953
HLA molecules	KIR 3DS1	Q14943

Leukocyte Ig-like receptor (LIR)

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to leukocyte Ig-like receptor (LIR). LIR receptors are a class of immune receptors that are expressed primarily on innate immune cells. The main ligands are MHC class I molecules, some of which, although active as agonists, exhibit inhibitory functions to a large extent. For example, LIRA2 acts as a congenital sensor for immunoglobulin fragments that have been cleaved by microbial proteases.

In some embodiments, the immunomodulatory domain is an agonistic antibody or antigen-binding fragment thereof that binds to LIRA 2. In some embodiments, an antibody capable of binding to LIRA2 can be generated based on Uniprot ID Q8N 149.

Family of CD94/NKG2 receptors

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to the CD94/NKG2 receptor. CD94/NKG2 is a heterodimeric C-type lectin receptor expressed on the surface of NK cells and some subset of CD 8T cells. It binds to HLA-E molecules (non-classical MHC class I molecules) and can transmit inhibitory and activating signals to NK cells. Inhibitory receptors contain an ITIM domain in their cytoplasmic tail, while activating receptors bind DAP12 and DAP10 that contain ITAM domains.

The following table provides activated CD94/NKG2 receptors that can generate agonistic antibodies or antigen-binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein:

in some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is the extracellular domain of a CD94/NKG2 ligand. The following table shows the receptors corresponding to the derived extracellular domains.

Fc receptor

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to an Fc receptor. Fc receptors are immune cell receptors expressed primarily on innate immune cells, which bind to the constant region of antibodies and elicit a wide range of functions. The Fc receptor is almost fully activated (except for Fc γ RIIB, which transmits inhibitory signals). Fc receptor attachment can result in the transmission of ADCC, phagocytosis, degranulation, and activation signals, thereby increasing effector function.

The following table provides a list of Fc receptors suitable for use in the immunomodulatory fusion proteins described herein that can generate agonistic antibodies or antigen-binding fragments thereof to target:

Ligands	Receptors	Receptor Uniprot KB
			IgG	FcγRI	P12314
IgG	FcγRIIC	P31995
			IgG	FcγRIIIA	P12318
IgG	FcγRIIIB	P31994
			IgE	FcεRI	P30273
IgE	FcεRII	P06734
			IgA	FcαR	P24071
IgA/IgM	FcμR	Q8WWV6

Signaling Lymphocyte Activating Molecules (SLAM)

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a Signaling Lymphocyte Activating Molecule (SLAM) receptor. SLAM receptors are a series of molecules that act as both receptors and ligands. SLAM molecules interact with each other on neighboring cells to send activation or inhibition signals. SLAM molecules contain an immunoreceptor tyrosine-based switch motif at their cytoplasmic tail, allowing them to bind intracellularly to activating and inhibitory signaling molecules.

The following table provides a list of suitable SLAM receptors for use in the immunomodulatory fusion proteins described herein that can generate agonistic antibodies or antigen-binding fragments thereof to target:

in some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is the extracellular domain of a SLAM ligand. The following table shows the receptors corresponding to the derived extracellular domains.

Siglec family receptors

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an agonistic antibody or antigen-binding fragment thereof that binds to a Siglec family receptor. Siglecs are a family of surface receptors that are predominantly present on immune cells, which are part of the lectin family (carbohydrate binding proteins). These receptors bind to sialic acid containing ligands. These receptors are mainly used as inhibitory receptors for a variety of immune cell types, but some (siglecs 14, 15 and 16) contain an ITAM activation domain.

The following table provides activated Siglec receptors that can generate agonistic antibodies or antigen-binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein:

receptors	Receptor Uniprot KB
		Siglec 14	Q08ET2
Siglec 15	Q6ZMC9
		Siglec 16	A6NMB1

H. Antagonistic antibodies

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof. Antagonistic antibodies block the function of their target. In some embodiments, the antagonist antibody or antigen-binding fragment thereof binds to an immunosuppressive receptor such that an immune response is induced. In some embodiments, the antagonist antibody or antigen-binding fragment thereof binds to an immunosuppressive ligand such that an immune response is induced. In some embodiments, immunosuppressant receptors and ligands include, but are not limited to: CD28 receptor, Tumor Necrosis Factor (TNF) superfamily receptor, Siglec receptor, CD94/NKG2 receptor, leukocyte Ig-like receptor (LIR), killer Ig-like receptor (KIR), Fc receptor, adenosine pathway molecule, other checkpoint inhibitors, and LAIR 1.

CD28 molecule

In some embodiments, the immunomodulatory domains suitable for use in immunomodulatory fusion proteins of the disclosure bind an antagonist antibody or antigen-binding fragment thereof of the CD28 molecule. As described above, the CD28 family contains both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

The following table provides a list of CD28 molecules suitable for use in the immunomodulatory fusion proteins described herein that can generate antagonist antibodies or antigen-binding fragments thereof to target:

molecule	Molecule Unit KB
		PD1	Q15116
PDL1	Q9NZQ7
		PDL2	Q9BQ51
CTLA-4	P16410
		B7-H4	Q7Z7D3
B7-H3	Q5ZPR3

In some embodiments, the immunomodulatory domain is an antagonistic antibody or antigen-binding fragment thereof that binds to PD-1. In some embodiments, the immunomodulatory domain binds to an antagonistic antibody or antigen-binding fragment thereof of PD-L1. In some embodiments, the immunomodulatory domain is an antagonistic antibody or antigen-binding fragment thereof that binds to CTLA-4.

TNF superfamily molecules

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds a TNF superfamily member. As described above, the TNF superfamily comprises both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

The following table provides a list of TNF superfamily molecules that can generate antagonist antibodies or antigen binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein:

Molecule	Molecule Unit KB
		TIGIT	Q495A1
BTLA	Q7Z6A9

Siglec receptor

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds a Siglec receptor. As described above, the Siglec family contains both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

The following table provides a list of suitable Siglec receptors for use in the immunomodulatory fusion proteins described herein that can generate antagonist antibodies or antigen-binding fragments thereof to target:

CD94/NKG2 receptor

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds the CD94/NKG2 receptor. As described above, the CD94/NKG2 family comprises both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

Thus, in some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds CD94/NKG 2A. In some embodiments, such antibodies are generated based on UniProt ID P26715.

In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds CD94/NKG 2B. In some embodiments, such antibodies are generated based on UniProt ID Q13241.

Leukocyte Ig-like receptor (LIR)

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds leukocyte Ig-like receptor (LIR). As described above, the LIR family contains both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

The following table provides a list of LIRs that can be produced antagonistic antibodies or antigen binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein.

Receptors	Receptor Uniprot KB
		LIRB1	Q8NHL6
LIRB2	Q8N423
		LIRB3	O75022
LIRB4	Q8NHJ6

Killer cell Ig like receptor (KIR)

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds a killer cell Ig-like receptor (KIR). As described above, the KIR family contains both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

The following table provides a list of KIRs suitable for use in the immunomodulatory fusion proteins described herein that can generate antagonist antibodies or antigen-binding fragments thereof to target.

Receptors	Receptor Uniprot KB
		KIR 2DL1	P43626
KIR 2DL2	P43627
		KIR 2DL3	P43628
KIR 2DL4	Q99706
		KIR 2DL5A	Q8N109
KIR 2DL5B	Q8NHK3
		KIR 3DL1	P43629
KIR 3DL2	P43630
		KIR 3DL3	Q8N743

Fc receptor

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds an Fc receptor. As described above, the Fc receptor family contains both activating and inhibitory molecules. Thus, in some embodiments, antagonizing the inhibitory molecule results in the induction or stimulation of an immune response.

In some embodiments, the inhibitor Fc receptor is fcyriib. In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds Fc γ RIIB. In some embodiments, such antibodies are generated based on UniProt ID P31994.

Adenosine pathway molecules

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds an adenosine pathway member. For example, CD39 and CD73 are enzymes expressed on the surface of cells that catalyze the conversion of ATP to adenosine. Extracellular ATP is a dangerous molecule for eliciting an immune response, whereas adenosine has immunosuppressive effects. These molecules contribute to the local immunosuppressive environment by producing adenosine.

Thus, in some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds CD 39. In some embodiments, such antibodies are generated based on UniProt ID P49961.

In some embodiments, the immunomodulatory domain is an antagonist antibody or antigen-binding fragment thereof that binds CD 73. In some embodiments, such antibodies are generated based on UniProt ID P21589.

Other checkpoint inhibitors

In some embodiments, an immunomodulatory domain suitable for use in an immunomodulatory fusion protein of the disclosure is an antagonistic antibody or antigen-binding fragment thereof that binds an immune checkpoint inhibitor. In some embodiments, by antagonizing such immune checkpoint inhibitors, an immune response is induced or stimulated.

The following table provides a list of immune checkpoint inhibitors that can generate antagonist antibodies or antigen-binding fragments thereof to target that are suitable for use in the immunomodulatory fusion proteins described herein.

Molecule	Molecule Unit KB
		VISTA	Q9H7M9
TIM-3	Q8TDQ0
		LAG-3	P18627
CD47	Q08722
		SIRPα	P78324

III. Joint

In some embodiments, the immunomodulatory fusion protein comprises an immunomodulatory domain operably linked to a collagen binding protein by a linker. In some embodiments, the linker between the immunomodulatory domain and the collagen binding protein provides spatial separation such that the immunomodulatory domain retains its activity (e.g., promotes receptor/ligand engagement). In some embodiments, the linker between the immunomodulatory domain and the collagen binding protein is of sufficient length or mass to reduce adsorption of the immunomodulatory domain on the collagen fibers. Methods for measuring adsorption are well known to those skilled in the art. For example, adsorption can be measured by Ellipsometry (ELM), Surface Plasmon Resonance (SPR), optical waveguide mode spectroscopy (OWLS), attenuated total internal reflection infrared spectroscopy (ATR-IR), Circular Dichroism (CD), total internal reflection infrared spectroscopy (TIRF), and other high resolution microscopy techniques. In some embodiments, these methods demonstrate spatial arrangement between immunomodulatory fusion protein domains.

In some embodiments, the linker between the immunomodulatory domain and the collagen binding protein provides sufficient molecular weight to slow or reduce diffusion from the tissue. Methods for measuring diffusion from tissue are well known to those skilled in the art. For example, diffusion can be measured by in vivo imaging over time or by tissue section microscopy. Exemplary Methods are described at least in Schmidt & Wittrup, Mol Canc ther.2009' and Wittrup et al, Methods in Enzymol 2012, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the linker is a hydrophilic polypeptide comprising "N" amino acids in length, wherein N ═ 1-1000, 50-800, 100-.

A. Serum albumin

In some embodiments, the linker is serum albumin or a fragment thereof. Methods of fusing serum albumin to proteins are disclosed in, for example, US2010/0144599, US2007/0048282, and US2011/0020345, the entire contents of which are incorporated herein by reference. In some embodiments, the linker is Human Serum Albumin (HSA) or a variant or fragment thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

Suitable albumins for immunomodulating fusion proteins may be from human, primate, rodent, bovine, equine, donkey, rabbit, goat, sheep, dog, chicken or pig. In some embodiments, the albumin is a serum albumin, e.g., human serum albumin (SEQ ID NO:42), primate serum albumin (e.g., chimpanzee serum albumin, gorilla serum albumin), rodent serum albumin (e.g., hamster serum albumin, guinea pig serum albumin, mouse serum albumin, and rat serum albumin), bovine serum albumin, horse serum albumin, donkey serum albumin, rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin, and pig serum albumin.

In some embodiments, the albumin, or variant or fragment thereof, HAS at least 50%, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the sequence of wild-type HAS as set forth in SEQ ID No. 42.

In some embodiments, the number of alterations (e.g., substitutions, insertions, or deletions) in an albumin variant is 1-20, e.g., 1-10, and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations as compared to a corresponding wild-type albumin (e.g., HSA).

In some embodiments, fragments of albumin or variants thereof are suitable for use in immunomodulatory fusion proteins. Exemplary albumin fragments are disclosed in WO 2011/124718. In some embodiments, the albumin fragment (e.g., HAS fragment) is at least 20 amino acids in length, such as at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids in length.

In some embodiments, the albumin fragment may comprise at least one entire subdomain of albumin. The domain of HAS HAS been expressed as a recombinant protein (Dockal et al, JBC 1999; 274:9303-10), wherein domain I of HAS (SEQ ID NO:42) is defined as consisting of amino acids 1-197 (SEQ ID NO:116), domain II is defined as consisting of amino acids 189-385 (SEQ ID NO:117), and domain III is defined as consisting of amino acids 381-585 (SEQ ID NO: 118). Partial overlap of domains gives rise to a given extended a-helix structure (h10-h1), which exists between domains I and II, and between domains II and III (Peters,1996, op. cit, tables 2-4). HAS also comprises six sub-domains (sub-domains IA, IB, NA, NB, INA and NIB). Sub-domain IA comprises amino acids 6 to 105 of SEQ ID NO:42, sub-domain IB comprises amino acids 120-177 of SEQ ID NO:42, sub-domain NA comprises amino acids 200-291 of SEQ ID NO:42, sub-domain NB comprises amino acids 316-369 of SEQ ID NO:42, sub-domain INA comprises amino acids 392-491 of SEQ ID NO:42 and sub-domain NIB comprises amino acids 512-583 of SEQ ID NO: 42.

In some embodiments, a fragment comprises all or part of one or more domains or subdomains as defined above, or any combination of those domains and/or subdomains. In some embodiments, the albumin fragment comprises at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of albumin or albumin domain, or variant or fragment thereof.

Fc domains

In some embodiments, a linker suitable for use in the immunomodulatory fusion proteins disclosed herein is an Fc domain. In some embodiments, the Fc domain is a component of an agonist or antagonist antibody described above, and thus a separate Fc domain is not required.

In certain embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO 115. In some embodiments, the Fc domain does not comprise a variable region that binds to an antigen. In some embodiments, the Fc domain comprises a variable region that binds to an antigen. Suitable Fc domains for the immunomodulatory fusion proteins disclosed herein can be obtained from a variety of different sources. In certain embodiments, the Fc domain is from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgG1 constant region. The Fc domain of human IgG1 is shown in SEQ ID NO: 115. However, it is understood that the Fc domain may be from an immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the Fc domain or portion thereof may be from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG 4.

In some embodiments, the immunomodulatory fusion protein comprises a mutated Fc domain. In some embodiments, the immunomodulatory fusion protein comprises a mutated IgG1 Fc domain. In some embodiments, the mutant Fc domain comprises one or more mutations at the hinge, CH2, and/or CH3 domain. In some aspects, the mutant Fc domain comprises the D265A mutation.

Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) can be obtained from publicly accessible sources. Constant region domains comprising Fc domain sequences that lack particular effector functions and/or have particular modifications can be selected to reduce immunogenicity. A number of antibodies and sequences of antibody-encoding genes have been disclosed and suitable Fc domain sequences (e.g., hinge, CH2 and/or CH3 sequences or portions thereof) can be derived from these sequences using techniques well known in the art. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain polypeptides suitable for use in the methods disclosed herein. It will also be appreciated that the scope of the present disclosure encompasses alleles, variants and mutations of constant region DNA sequences.

The Fc domain sequence can be cloned, for example, using the polymerase chain reaction and primers selected for amplification of the target domain. To clone the Fc domain sequence from the antibody, mRNA can be isolated from hybridomas, spleen, or lymphocytes, reverse transcribed into DNA and the antibody gene amplified by PCR. In U.S. Pat. nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, for example, "PCR Protocols: A Guide to Methods and Applications" Innis et al, Academic Press, San Diego, Calif. (1990); ho et al, 1989, Gene 77: 51; PCR amplification Methods are described in detail in Horton et al, 1993.Methods Enzymol.217: 270. PCR can be primed by consensus constant region primers or more specific primers based on published heavy and light chain DNA and amino acid sequences. PCR may also be used to isolate DNA clones encoding the antibody light and heavy chains, as discussed above. In this case, the library can be screened by consensus primers or larger homologous probes (e.g., mouse constant region probes). A variety of primer pairs suitable for antibody gene amplification are well known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan.1994.protein Engineering 7: 1509); rapid amplification of cDNA ends (Ruberti, F. et al, 1994.J. Immunol. methods 173: 33); antibody leader sequence (Larrick et al, Biochem Biophys Res Commun 1989; 160: 1250)). Cloning of antibody sequences is further described in Newman et al, U.S. Pat. No. 5,658,570, filed on 25.01.1995, which is incorporated herein by reference.

In some embodiments, the disclosed immunomodulatory fusion proteins comprise one or more Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Fc domains). In certain embodiments, the Fc domains may be of different types. In certain embodiments, at least one Fc domain present in the immunomodulatory fusion protein comprises a hinge domain or a portion thereof. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH2 domain or portion thereof. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH3 domain or portion thereof. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH4 domain or portion thereof. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., oriented at hinge-CH 2). In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CH2-CH3 orientation). In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one hinge domain or portion thereof, at least one CH2 domain or portion thereof, and at least one CH3 domain or portion thereof, e.g., in the hinge-CH 2-CH3, hinge CH3-CH3, or CH2-CH3 hinge orientations.

In certain embodiments, the immunomodulatory fusion protein comprises at least one intact Fc domain derived from one or more immunoglobulin heavy chains (e.g., the Fc domain comprises a hinge, CH2, CH3 domain, but these need not be from the same antibody). In certain embodiments, the immunomodulatory fusion protein comprises at least two intact Fc domains derived from one or more immunoglobulin heavy chains. In certain embodiments, the complete Fc domain is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG 1).

In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising the entire CH3 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising the entire CH2 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising at least one CH3 domain, and at least one hinge region, and a CH2 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising a hinge and a CH3 domain. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain comprising a hinge, CH2, and CH3 domains. In certain embodiments, the Fc domain is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG 1).

The constant region domains or fragments thereof that make up the Fc domain of the immunomodulatory fusion protein may be derived from different immunoglobulin molecules. For example, a polypeptide suitable for use in an immunomodulatory fusion protein disclosed herein may comprise a CH2 domain derived from an IgG1 molecule or fragment thereof and a CH3 region derived from an IgG3 molecule or portion thereof. In some embodiments, the immunomodulatory fusion protein comprises an Fc domain comprising a hinge domain derived in part from an IgG1 molecule and in part from an IgG3 molecule. As described herein, one of ordinary skill in the art will appreciate that the Fc domain can be altered such that its amino acid sequence differs from that of a naturally occurring antibody molecule.

In certain embodiments, the immunomodulatory fusion protein lacks one or more constant regions of a complete Fc domain, i.e., it is partially or completely deleted. In certain embodiments, the immunomodulatory fusion protein lacks the entire CH2 domain. In certain embodiments, the immunomodulatory fusion protein comprises a CH2 domain deleted of the Fc region derived from a vector encoding an IgG1 human constant region domain (e.g., from IDEC Pharmaceuticals, San Diego) (e.g., WO02/060955a2 and WO02/096948a 2). This exemplary vector was engineered to delete the CH2 domain and provide a synthetic vector expressing the domain deleted IgG1 constant region. It should be noted that these exemplary constructs are preferably engineered to fuse the bound CH3 domain directly to the hinge region of the corresponding Fc domain.

In other constructs, it may be desirable to provide a peptide spacer between one or more of the constituent Fc domains. For example, peptide spacers may be located between the hinge region and the CH2 domain and/or between the CH2 and CH3 domains. For example, a compatible construct will be expressed in which the CH2 domain is deleted and the remaining CH3 domain (synthetic or non-synthetic) is linked to the hinge region with a 1-20, 1-10 or 1-5 amino acid peptide spacer. For example, such peptide spacers may be added to ensure that the regulatory elements of the constant region domains remain free and accessible, or that the hinge region remains flexible. Preferably, any linker peptide used in the present disclosure that is compatible will be relatively non-immunogenic and will not prevent proper folding of the Fc.

In certain embodiments, the Fc domain used in the immunomodulatory fusion protein is altered or modified, e.g., by amino acid mutation (e.g., addition, deletion, or substitution). As used herein, the term "Fc domain variant" refers to an Fc domain having at least one amino acid modification (e.g., amino acid substitution) as compared to the wild-type Fc from which the Fc domain is derived. For example, wherein the Fc domain is derived from a human IgG1 antibody, which is a variant comprising at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position in the Fc region of human IgG 1.

In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the hinge domain or portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH2 domain or portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH3 domain or portion thereof. In certain embodiments, the Fc variant comprises a substitution at an amino acid position located in the CH4 domain or portion thereof.

In certain embodiments, the immunomodulatory fusion protein comprises an Fc variant comprising more than one amino acid substitution. The immunomodulatory fusion proteins can comprise, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in the Fc domain. Preferably, the amino acid substitutions are spatially separated from each other by at least 1 or more amino acid positions, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. More preferably, the engineered amino acids are spatially separated from each other by at least 5, 10, 15, 20, or 25 or more amino acid positions.

In some embodiments, the Fc domain comprises an alteration in the region between amino acids 234 and 238, including the sequence LLGGP beginning at the CH2 domain. In some embodiments, the Fc variant alters Fc-mediated effector function, particularly ADCC, and/or reduces binding affinity for an Fc receptor. In some aspects, sequence alterations proximal to the CH2-CH3 linkage at positions such as K322 or P331 are capable of abolishing complement-mediated cytotoxicity and/or altering affinity for FcR binding. In some embodiments, the Fc domain introduces alterations at residues P238 and P331, e.g., changes the wild-type proline at these positions to serine. In some embodiments, one or more of the three hinge cysteines altered at the hinge region to encode CCC, SCC, SSC, SCS, or SSS at these residues may also affect FcR binding and molecular homogeneity, for example by eliminating unpaired cysteines that may destabilize the folded protein.

Other amino acid mutations in the Fc domain are expected to reduce binding to Fc γ receptors and Fc γ receptor subtypes. For example, mutations at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376, 378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 of the Fc region can alter binding, as described in U.S. patent No. 6,737,056 filed on 18/05 in 2004, which is incorporated herein by reference in its entirety. This patent reports that the change of Pro331 to Ser in IgG3 resulted in a 6-fold decrease in affinity compared to unmutated IgG3, indicating that Pro331 is involved in Fc γ RI binding. In addition, amino acid modifications in positions 234, 235, 236 and 237, 297, 318, 320 and 322 were disclosed as possible changes in receptor binding affinity in U.S.5,624,821, filed 29/04/1997, the entire content of which is incorporated herein by reference.

Other mutations contemplated for use include, for example, those described in U.S. patent application publication No. 2006/0235208, published 2006, 10/19, the entire contents of which are incorporated herein by reference. In addition, the use of mutations described in U.S. patent application publication No. 2006/0235208, the entire contents of which are incorporated herein by reference, is contemplated. The mutation L234A/L235A is described, for example, in U.S. patent application publication No. 2003/0108548, published 12/06/2003, the entire contents of which are incorporated herein by reference. In embodiments, the modifications described may be included alone or in combination. In certain embodiments, the mutation is D265A in human IgG 1.

In certain embodiments, the immunomodulatory fusion protein comprises an Fc variant, e.g., an Fc variant comprising an amino acid substitution that alters antigen-dependent effector function of the polypeptide, particularly ADCC or complement activation, as compared to a wild-type Fc region. Such immunomodulatory fusion proteins exhibit reduced binding to FcR γ when compared to the wild-type polypeptide, thereby mediating reduced effector function. Fc variants with reduced FcR γ binding affinity are expected to reduce effector function, and such molecules are also useful, for example, for treating diseases in which destruction of target cells is undesirable, e.g., where normal cells may express target cells, or where long term administration of the polypeptide may result in deleterious immune system activation.

In certain embodiments, the immunomodulatory fusion protein exhibits altered binding to an activating Fc γ R (e.g., Fc γ l, Fc γ lla, or Fc γ RIIIa). In certain embodiments, the immunomodulatory fusion protein exhibits altered binding affinity to an inhibitory Fc γ R (e.g., Fc γ RIIb). Exemplary amino acid substitutions that alter FcR or complement binding activity are disclosed in international PCT publication No. WO05/063815, which is incorporated herein by reference.

In some embodiments, the immunomodulatory fusion protein comprises an amino acid substitution that alters glycosylation of the fusion protein. For example, in some embodiments, the Fc domain comprises a mutation that results in reduced glycosylation (e.g., N-or O-linked glycosylation) or comprises a glycoform alteration of a wild-type Fc domain (e.g., a low fucose or an afucose glycan). In certain embodiments, the immunomodulatory fusion protein has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif, comprising the amino acid sequence NXT or NXS. Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in WO05/018572 and US2007/0111281, the contents of which are incorporated herein by reference. In certain embodiments, the immunomodulatory fusion protein comprises at least one Fc domain with an engineered cysteine residue or analog thereof located on a surface exposed to a solvent. In certain embodiments, the immunomodulatory fusion protein comprises an Fc domain comprising at least one engineered free cysteine residue or analog thereof that does not substantially form a disulfide bond with a second cysteine residue. Any of the above engineered cysteine residues or analogs thereof can then be conjugated to the functional domain using techniques well known in the art (e.g., conjugation with a thiol-reactive heterobifunctional linker).

In certain embodiments, the immunomodulatory fusion protein comprises a genetically fused Fc domain having two or more constituent Fc domains independently selected from the Fc domains described herein. In certain embodiments, the Fc domains are identical. In certain embodiments, at least two Fc domains are different. For example, Fc domains comprise the same number of amino acid residues, or may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In certain embodiments, the Fc domains differ in sequence at one or more amino acid positions. For example, the at least two Fc domains can differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions.

C. Other joints

In some embodiments, a linker suitable for use in the immunomodulatory fusion proteins described herein is a polyethylene glycol (PEG) domain. PEG is a well-known water-soluble Polymer, commercially available, or can be prepared by ring-opening polymerization of polyethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol.3, p. 138-. The term "PEG" is used broadly to encompass any polyethylene glycol molecule and may be represented by the formula: x-0 (CH) ₂CH₂O)_n-1CH₂CH₂OH, wherein n is 20 to 2300 and X is H or a terminal modification, e.g., C_1-4An alkyl group. In certain embodiments, one terminus of a PEG suitable for use in the methods disclosed herein terminates with a hydroxyl or methoxy group, i.e., X is H or CH₃("methoxy PEG"). PEG may contain other chemical groups required for conjugation reactions; this is due to the chemical synthesis of the molecule; or it is a spacer for optimal distance between parts of the molecule. Further, such PEG may be composed of one or more PEG side chains linked together. PEGs with more than one PEG chain are referred to as multi-arm or branched PEGs. For example, branched PEGs can be prepared by adding polyethylene oxide to various polyols, including glycerol, pentaerythritol, and sorbitol. For example, four-arm branched PEG can be prepared from pentaerythritol and ethylene oxide. Branched PEGs are described, for example, in EP-A0473084 and U.S. Pat. No. 5,932,462, both of which are incorporated herein by reference. One form of PEG includes two PEG side chains (PEG2) linked by a primary amino group of lysine (Monfardini et al, Bioconjugate Chem 1995; 6: 62-9).

In certain embodiments, the PEG is conjugated to the cysteine moiety at the N-or C-terminus of a domain of the immunomodulatory fusion protein (e.g., the immunomodulatory domain and the collagen binding domain). PEG moieties can also be attached by other chemical methods, including conjugation to amines. Conjugation of PEG to a peptide or protein typically involves activation of PEG and coupling of the activated PEG intermediate directly to the protein/peptide or linker of interest, which is then activated and coupled to the protein/peptide of interest (see, Abuchowski et al, JBC 1977; 252:3571 and JBC 1977; 252:3582, and Harris et al, in Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (J.M. Harris ed.) Plenum Press: New York, 1992; chapters 21 and 22). Various molecular weight forms of PEG may be selected, for example, from about 1,000 daltons (Da) to 100,000Da (n is 20 to 2300). The number of repeat units "n" in PEG approximates the molecular weight described in daltons.

The skilled person can select a molecular weight suitable for PEG, e.g. based at least on the molecular weight of the PEG-free immunomodulatory fusion protein.

In certain embodiments, PEG molecules can be activated to react with amino groups on domains, such as lysine (Benchamam C.O. et al, anal. biochem.,131,25 (1983); Veronese, F.M. et al, appl. biochem.,11,141 (1985); Zalipsky, S. et al, Polymeric Drugs and Drug Delivery Systems, ads 9-110ACS Symposium Series 469 (1999); Zalipsky, S. et al, Europ. Polymer.J., 19, 1177. supplement 1183 (1983); Delgado, C. et al, Biotechnology and Applied Biochemistry,12, 1990, 128 (1990)).

In certain embodiments, carbonates of PEG may be used to conjugate PEG. Ν, Ν' -disuccinimidyl carbonate (DSC) can be used to react with PEG to form an active mixed PEG-succinimidyl carbonate, which can then be reacted with the nucleophilic group of a linker or the amino group of IL-2 (see U.S. patent No. 5,281,698 and U.S. patent No. 5,932,462). In a similar type of reaction, 1' - (dibenzotriazolyl) carbonate and di- (2-pyridyl) carbonate may be reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed carbonates, respectively (U.S. Pat. No. 5,382,657). Pegylation can be carried out according to methods of the prior art, for example by reacting IL-2 with electrophilically active PEG (Shearwater Corp., USA, www.shearwatercorp.com). Preferred PEG reagents suitable for use in the methods disclosed herein are, for example, N-hydroxysuccinimidyl propionate (PEG-SPA), butyrate (PEG-SBA), PEG-succinimidyl propionate or branched N-hydroxysuccinimids such as mPEG2-NHS (Monfardii, C et al, Bioconjugate chem.6(1995) 62-69).

In some embodiments, a linker suitable for use in the immunomodulatory fusion proteins described herein is transferrin, as disclosed in US 7,176,278 and US 8,158,579, the entire contents of which are incorporated herein by reference.

In some embodiments, linkers suitable for use in the immunomodulatory fusion proteins described herein are serum immunoglobulin-binding proteins, such as those described in US2007/0178082, the entire contents of which are incorporated herein by reference.

In some embodiments, a linker suitable for use in the immunomodulatory fusion proteins described herein is a globulin, such as thyroxine-binding globulin, a2 macroglobulin, or a binding globulin.

In some embodiments, linkers suitable for use in the immunomodulatory fusion proteins described herein are fibronectin (Fn) -based scaffold domain proteins, such as those disclosed in US2012/0094909, the entire contents of which are incorporated herein by reference. Methods of preparing fibronectin based scaffold domain proteins are also disclosed in US 2012/0094909. A non-limiting example of an extended PK group based on Fn3 is Fn3 (HAS).

D. Other joints

In some embodiments, the immunomodulatory domain is operably linked to the collagen binding domain via a linker (e.g., gly-ser linker). In some embodiments, the immunomodulatory domain is operably linked to the collagen binding domain via a linker (e.g., serum albumin), wherein the linker is linked to the collagen binding domain and the immunomodulatory domain via an additional linker (e.g., gly-ser linker). Linkers suitable for fusing a collagen binding domain and an immunomodulatory domain, or a collagen binding domain, an immunomodulatory domain and a linker (e.g. serum albumin) are well known in the art and are disclosed in, for example, US2010/0210511, US2010/0179094 and US2012/0094909, the entire contents of which are incorporated herein by reference. Exemplary linkers include gly-ser polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a gly-ser polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

An exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser (Gly)₄Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n ═ 3, i.e., Ser (Gly)₄Ser) 3. In certain embodiments, n ═ 4, i.e., Ser (Gly)₄Ser) 4. In certain embodiments, n-5. In certain embodiments, n is 6. In certain embodiments, n ═ 7. In certain embodiments, n is 8. In certain embodiments, n is 9. In certain embodiments, n is 10. Another exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser (Gly)₄Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n-3. In certain embodiments, n-4. In certain embodiments, n-5. In certain embodiments, n is 6. Another exemplary Gly-ser polypeptide linker comprises (Gly)₄Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n-3. In certain embodiments, n-4. In certain embodiments, n-5. In certain embodiments, n is 6. Another exemplary Gly-ser polypeptide linker comprises (Gly) ₃Ser) n. In certain embodiments, n ═ l. In certain embodiments, n ═ 2. In certain embodiments, n-3. In certain embodiments, n-4. In certain embodiments, n-5. In certain embodiments, n is 6.

Other linkers suitable for use in immunomodulatory fusion proteins are well known in the art, e.g., serine-rich linkers disclosed in US 5,525,491, in Arai et al, Protein Eng 2001; helix-forming peptide linkers as disclosed in 14:529-32 (e.g. a (eaaak) nA (n ═ 2-5)), and in Chen et al, Mol Pharm2011; 8:457-65, namely, the dipeptide linker LE, the thrombin-sensitive dithiocyclic peptide linker, and the alpha helix-forming linker LEA (EAAAK)₄ALEA(EAAAK)₄ALE(SEQ ID NO:119)。

Other exemplary linkers include GS linkers (i.e., (GS) n), GGSG linkers (i.e., (GGSG) n), GSAT linkers, SEG linkers, and GGS linkers (i.e., (GGSGGS) n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for hybridising nuclease-albumin molecules can be found using publicly available databases, such as the linker database (ibi. vu. nl/programs/linkerdbwww). Linker databases are databases of interdomain linkers in multifunctional enzymes that serve as potential linkers in novel fusion proteins (see, e.g., George et al, Protein Engineering 2002; 15: 871-9).

It will be appreciated that variant forms of these exemplary polypeptide linkers may be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the polypeptide linker, thereby introducing one or more amino acid substitutions, additions or deletions into the polypeptide linker. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

The polypeptide linker of the present disclosure is at least one amino acid in length and may vary in length. In one embodiment, the polypeptide linker of the present disclosure is from about 1 to about 50 amino acids in length. As used herein, the term "about" means +/-two amino acid residues. Since the linker must be a positive integer in length, it is about 1 to about 50 amino acids in length, meaning 1 to 48-52 amino acids in length. In another embodiment, the polypeptide linker of the present disclosure is about 10-20 amino acids in length. In another embodiment, the polypeptide linker of the present disclosure is about 15 to about 50 amino acids in length.

In another embodiment, the polypeptide linker of the present disclosure is about 20 to about 45 amino acids in length. In another embodiment, the polypeptide linker of the present disclosure is about 15 to about 25 amino acids in length. In another embodiment, the polypeptide linker of the disclosure is 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, or 61 or more amino acids in length.

Polypeptide linkers can be introduced into the polypeptide sequence using techniques well known in the art. Modification can be confirmed by DNA sequence analysis. Plasmid DNA may be used to transform a host cell for stable production of the polypeptide produced.

Exemplary immunomodulatory fusion proteins

The present disclosure provides immunomodulatory fusion proteins comprising an immunomodulatory domain and a collagen binding domain, optionally a linker, wherein the immunomodulatory domain is operably linked to the collagen binding domain with or without a linker. The immunomodulatory fusion proteins of the disclosure are modular and can be configured to introduce a variety of separate domains.

IL-2 fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises IL-2 and a basement membrane glycan, wherein IL-2 is operably linked to the basement membrane glycan. In some embodiments, albumin IL-2 is used with the basement membrane glycans. In some embodiments, IL-2 is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, IL-2 is operably linked to the C-terminus of the basement membrane glycan.

In some embodiments, the immunomodulatory fusion protein comprises a human IL-2 sequence shown in SEQ ID NO:1 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises a human IL-2 sequence shown in SEQ ID NO:1 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO:107 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises IL-2 and LAIR-1, wherein IL-2 is operably linked to LAIR-1. In some embodiments, albumin IL-2 is used in operable linkage with LAIR-1. In some embodiments, IL-2 is operably linked to the N-terminus of LAIR-1. In some embodiments, IL-2 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises a human IL-2 sequence shown in SEQ ID NO:1 operably linked to a human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises a human IL-2 sequence shown in SEQ ID NO. 1 operably linked to a human LAIR-1 sequence shown in SEQ ID NO. 98 using a human serum albumin sequence selected from SEQ ID NO. 42 and SEQ ID NO. 43.

IL-12 fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises IL-12 and a basement membrane glycan, wherein IL-12 is operably linked to the basement membrane glycan. In some embodiments, albumin IL-12 is used in operable linkage with the basement membrane glycan. In some embodiments, IL-12 and basement membrane glycan N terminal operatively connected. In some embodiments, IL-12 and basement membrane glycan operably connected to the C terminal.

In some embodiments, the immunomodulatory fusion protein comprises the human IL-12 sequences shown in SEQ ID NO:2 and SEQ ID NO:3 operably linked to the human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises the human IL-12 sequences shown in SEQ ID NO:2 and SEQ ID NO:3 operably linked to the human basement membrane glycan sequence shown in SEQ ID NO:107 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises IL-12 and LAIR-1, wherein IL-12 is operably linked to LAIR-1. In some embodiments, albumin IL-12 is used in operable linkage with LAIR-1. In some embodiments, IL-12 and LAIR-1N terminal operatively connected. In some embodiments, IL-12 and LAIR-1C terminal operatively connected.

In some embodiments, the immunomodulatory fusion protein comprises the human IL-12 sequences shown in SEQ ID NO:3 and SEQ ID NO:2 operably linked to the human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises the human IL-12 sequence shown in SEQ ID NO:2 and SEQ ID NO:3 operably linked to the human LAIR-1 sequence shown in SEQ ID NO:98 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

CCL-3 fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-3 and basement membrane glycan, wherein CCL-3 is operably linked to basement membrane glycan. In some embodiments, albumin CCL-3 is used in operable linkage with the basement membrane glycan. In some embodiments, CCL-3 is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, CCL-3 is C-terminal to which the basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-3 sequence shown in SEQ ID NO:41 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-3 sequence shown in SEQ ID NO:41 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO:107 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-3 and LAIR-1, wherein CCL-3 is operably linked to LAIR-1. In some embodiments, albumin CCL-3 is used in operable linkage with LAIR-1. In some embodiments, CCL-3 is operably linked to the N-terminus of LAIR-1. In some embodiments, CCL-3 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-3 sequence shown in SEQ ID NO:41 operably linked to a human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-3 sequence shown in SEQ ID NO:41 operably linked to a human LAIR-1 sequence shown in SEQ ID NO:98 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

CCL-4 fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-4 and basement membrane glycan, wherein CCL-4 is operably linked to basement membrane glycan. In some embodiments, albumin CCL-4 is used in operable linkage with the basement membrane glycan. In some embodiments, CCL-4 is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, CCL-4 is C-terminal to which the basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-4 sequence shown in SEQ ID NO:33 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-4 sequence shown in SEQ ID NO:33 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO:107 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-4 and LAIR-1, wherein CCL-4 is operably linked to LAIR-1. In some embodiments, albumin CCL-4 is used in operable linkage with LAIR-1. In some embodiments, CCL-4 is operably linked to the N-terminus of LAIR-1. In some embodiments, CCL-4 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-4 sequence shown in SEQ ID NO:33 operably linked to a human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-4 sequence shown in SEQ ID NO:33 operably linked to a human LAIR-1 sequence shown in SEQ ID NO:98 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

CCL-5 fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-5 and basement membrane glycan, wherein CCL-5 is operably linked to basement membrane glycan. In some embodiments, albumin CCL-5 is used in operable linkage with the basement membrane glycan. In some embodiments, CCL-5 is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, CCL-5 is C-terminal operably linked to the basement membrane glycan.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-5 sequence shown in SEQ ID NO:39 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-5 sequence shown in SEQ ID No. 39 operably linked to a human basement membrane glycan sequence shown in SEQ ID No. 107 using a human serum albumin sequence selected from SEQ ID No. 42 and SEQ ID No. 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-5 and LAIR-1, wherein CCL-5 is operably linked to LAIR-1. In some embodiments, albumin CCL-5 is used in operable linkage with LAIR-1. In some embodiments, CCL-5 is operably linked to the N-terminus of LAIR-1. In some embodiments, CCL-5 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises a human CCL-5 sequence shown in SEQ ID NO:39 operably linked to a human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises a human CCL-5 sequence shown in SEQ ID NO:39 operably linked to a human LAIR-1 sequence shown in SEQ ID NO:98 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

F. Eotaxin fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises an eotaxin and a basement membrane glycan, wherein the eotaxin is operably linked to the basement membrane glycan. In some embodiments, albumin eotaxin is used in operable linkage with the basement membrane glycan. In some embodiments, the eotaxin is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, the eotaxin is C-terminally operably linked to the basement membrane glycan.

In some embodiments, the immunomodulatory fusion protein comprises a human eotaxin sequence shown in SEQ ID NO:38 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO: 107. In some embodiments, the immunomodulatory fusion protein comprises a human eotaxin sequence shown in SEQ ID NO:38 operably linked to a human basement membrane glycan sequence shown in SEQ ID NO:107 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises an eotaxin and LAIR-1, wherein the eotaxin is operably linked to LAIR-1. In some embodiments, albumin eotaxin is used in operable linkage with LAIR-1. In some embodiments, the eotaxin is operably linked to the N-terminus of LAIR-1. In some embodiments, the eotaxin is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises a human eotaxin sequence shown in SEQ ID NO:38 operably linked to a human LAIR-1 sequence shown in SEQ ID NO: 98. In some embodiments, the immunomodulatory fusion protein comprises a human eotaxin sequence shown in SEQ ID NO:38 operably linked to a human LAIR-1 sequence shown in SEQ ID NO:98 using a human serum albumin sequence selected from SEQ ID NO:42 and SEQ ID NO: 43.

G. Antibody fusion proteins

In some embodiments, the immunomodulatory fusion protein comprises an anti-CD 3 antibody and a basement membrane glycan, wherein the anti-CD 3 antibody is operably linked to the basement membrane glycan. In some embodiments, the anti-CD 3 antibody is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, the anti-CD 3 antibody is C-terminal to which a basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises an anti-CD 3 antibody and LAIR-1, wherein the anti-CD 3 antibody is operably linked to LAIR-1. In some embodiments, the anti-CD 3 antibody is operably linked to the N-terminus of LAIR-1. In some embodiments, the anti-CD 3 antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises an anti-4-1-BB antibody and a basement membrane glycan, wherein the anti-4-1 BB antibody is operably linked to the basement membrane glycan. In some embodiments, the anti-4-1 BB antibody is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, the anti-4-1-BB antibody is C-terminal to which the basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises an anti-4-1-BB antibody and LAIR-1, wherein the anti-4-1 BB antibody is operably linked to LAIR-1. In some embodiments, the anti-4-1 BB antibody is operably linked to the N-terminus of LAIR-1. In some embodiments, the anti-4-1 BB antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises an anti-CD 40 antibody and a basement membrane glycan, wherein the anti-CD 40 antibody is operably linked to the basement membrane glycan. In some embodiments, the anti-CD 40 antibody is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, the anti-CD 40 antibody is C-terminal to which a basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises an anti-CD 40 antibody and LAIR-1, wherein the anti-CD 40 antibody is operably linked to LAIR-1. In some embodiments, the anti-CD 40 antibody is operably linked to the N-terminus of LAIR-1. In some embodiments, the anti-CD 40 antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises an anti-OX 40 antibody and a basement membrane glycan, wherein the anti-OX 40 antibody is operably linked to the basement membrane glycan. In some embodiments, the anti-OX 40 antibody is operably linked to the N-terminus of the basement membrane glycan. In some embodiments, the anti-OX 40 antibody is C-terminal to which a basement membrane glycan is operably linked.

In some embodiments, the immunomodulatory fusion protein comprises an anti-OX 40 antibody and LAIR-1, wherein the anti-OX 40 antibody is operably linked to LAIR-1. In some embodiments, the anti-OX 40 antibody is operably linked to the N-terminus of LAIR-1. In some embodiments, the anti-OX 40 antibody is operably linked to the C-terminus of LAIR-1.

Method for producing immunomodulatory fusion proteins

In some aspects, the polypeptides described herein (e.g., collagen binding domains, cytokines, antibodies) are prepared in transformed host cells using recombinant DNA techniques. To this end, recombinant DNA molecules encoding the peptides were prepared. Methods for preparing such DNA molecules are well known in the art. For example, sequences encoding peptides can be excised from the DNA using suitable restriction enzymes. Alternatively, the antigen is synthesized as a DNA molecule using chemical synthesis techniques, such as the phosphoramidite method. Also, a combination of these techniques may be used.

Methods of producing the polypeptides also include vectors capable of expressing the peptides in a suitable host. The vector comprises a DNA molecule encoding a peptide operably linked to an appropriate expression control sequence. Methods of effecting such operative ligation, either before or after insertion of the DNA molecule into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, initiation signals, termination signals, cap signals, polyadenylation signals, and other signals involved in transcriptional or translational control.

The resulting vector with the DNA molecule thereon is used to transform a suitable host. The transformation can be performed using methods well known in the art.

Any of a number of available and well-known host cells are suitable for use in the methods of the present disclosure. The choice of a particular host depends on many factors recognized in the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of recovery of the peptide, expression characteristics, biosafety and cost. A balance must be struck between these factors, provided that it is understood that not all hosts may be equally effective for expression of a particular DNA sequence. Among these general guidelines, useful microbial hosts include bacteria (e.g., E.coli), yeast (e.g., Saccharomyces cerevisiae), and other fungi, insects, fabrics, cultured mammalian (e.g., human) cells, or other hosts known in the art.

Subsequently, the transformed host is cultured and purified. The host cell may be cultured under conventional fermentation conditions in order to express the desired compound. Such fermentation conditions are well known in the art. Finally, the peptide is purified from the culture by methods well known in the art.

The compounds may also be prepared by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art and include those described in Merrifield (1973), chem.polypeptides, pp.335-61(Katsoyannis and Panayotis); merrifield (1963), J.Am.chem.Soc.85: 2149; davis et al, (1985), biochem. Intl.10: 394-414; stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. patent nos. 3,941,763; finn et al, (1976), The Proteins (3 rd edition) 2: 105-253; and those of Erickson et al, (1976), The Proteins (3 rd edition) 2: 257-. Solid phase synthesis is the preferred technique for preparing individual peptides because it is the most cost-effective method for preparing small peptides. Compounds containing derivatized peptides or containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

Other methods of molecular expression/synthesis are generally known to those of ordinary skill in the art.

The nucleic acid molecule may be comprised in a vector, which is capable of directing its expression, for example in a cell which has been transduced by the vector. Thus, in addition to polypeptide mutants, certain embodiments include expression vectors comprising nucleic acid molecules encoding the mutants and cells transfected with these vectors.

Vectors suitable for use include T7-based vectors for bacteria (see, e.g., Rosenberg et al, Gene 56:125,1987), pMSXND expression vectors for mammalian cells (Lee and Nathans, J.biol.chem.263:3521,1988), and baculovirus-derived vectors for insect bacteria (e.g., pBacPAKS, an expression vector for Clontech, Palo Alto, Calif). The nucleic acid insert encoding the polypeptide of interest in such a vector may be operably linked to a promoter, which is selected, for example, based on the cell type in which expression is sought. For example, the T7 promoter that can be used in bacteria, the polyhedral protein promoter that can be used in insect cells, and the cytomegalovirus or metallothionein promoter that can be used in mammalian cells. Also, in the case of higher eukaryotes, specific and cell type specific promoters are prevented from being widely available. These promoters are known for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type in vivo. The skilled artisan is well aware of many promoters and other regulatory elements that can be used to direct expression of a nucleic acid.

In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, the vector may contain an origin of replication and other genes encoding selectable markers. For example, the neomycin resistance (neor) gene confers G418 resistance to cells expressing it and thus allows phenotypic selection of transfected cells. One skilled in the art can readily determine whether a given regulatory element or selectable marker is appropriate for use in a particular experimental environment.

Suitable Viral Vectors include, for example, retroviral, adenoviral and adeno-associated Viral Vectors, herpes virus, simian virus 40(SV40) and bovine papilloma virus Vectors (see, e.g., Gluzman (Ed.), European Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding the polypeptide mutants are also suitable. The cell is a transfected cell, i.e., a nucleic acid molecule, e.g., a nucleic acid molecule encoding a mutant polypeptide, has been introduced into the cell by recombinant DNA techniques. Progeny of such cells are also considered suitable for use in the methods of the present disclosure.

The precise composition of the expression system is not critical. For example, the polypeptide mutants may be in prokaryotic hosts (e.g., E.coli) or in eukaryotic hosts (e.g., insect cells (e.g., Sf21 cells)) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from a number of sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it is only important that the components are compatible with each other. The skilled or ordinary person can make such a decision. Furthermore, if guidance is required in selecting an expression system, the skilled worker can consult Ausubel et al (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y.,1993) and Pouwels et al (Cloning Vectors: A Laboratory Manual,1985 Suppl.1987).

The expressed polypeptide can be purified from the expression system using conventional biochemical procedures, and can be used, for example, as a therapeutic agent as described herein.

Pharmaceutical compositions and modes of administration

In certain embodiments, the present disclosure provides pharmaceutical compositions comprising an immunomodulatory fusion protein, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant.

In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material is for subcutaneous injection and/or intravenous injection. In certain embodiments, the formulation material is for local administration, e.g., intratumoral administration. In certain embodiments, the pharmaceutical composition may comprise components for modifying, maintaining or maintaining the composition, such as pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, aspartic acid, arginine, or lysine); an antibiotic; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; a disaccharide; and other carbohydrates (e.g., glucose, mannose, or dextrins); proteins (e.g., serum albumin, gelatin, or immunoglobulins); coloring, flavoring and diluting agents; an emulsifier; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tirofloxacin); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancing agents (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); a vehicle; a diluent; excipients and/or pharmaceutical adjuvants. Remington's Pharmaceutical Sciences, 18 th edition, a.r. gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20mM NaOAC, pH 5.2, 50mM NaCl; and/or 10mM NAOAC, pH 5.2, 9% sucrose. In certain embodiments, the optimal pharmaceutical composition is determined by one of skill in the art based on, for example, the intended route of administration, the form of delivery, and the desired dosage. See, e.g., Remington's Pharmaceutical Sciences, as described above. In certain embodiments, such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the immunomodulatory fusion protein.

In some embodiments, a formulation comprising an immunomodulatory fusion protein described herein is 4 ℃ to 37 ℃ when administered to a subject.

In some embodiments, the primary carrier or vehicle in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier is water for injection, a physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other substances commonly found in parenterally administered compositions. The saline comprises isotonic phosphate buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin is a further exemplary carrier. In some embodiments, the pharmaceutical composition comprises a Tris buffer at about pH 7.0-8.5, or an acetate buffer at about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In some embodiments, a composition comprising an immunomodulatory fusion protein can be prepared for storage by mixing a selected composition of the desired purity with an optional formulation agent (Remington's Pharmaceutical Sciences, as described above) lyophilized and or in aqueous solution. In addition, in some embodiments, the composition comprising the immunomodulatory fusion protein may be formulated as a lyophilizate using a suitable excipient, such as sucrose.

In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. In some embodiments, the composition may be selected for inhalation or for delivery through the alimentary canal, e.g., orally. The preparation of such pharmaceutically acceptable compositions is within the ability of those skilled in the art.

In some embodiments, the formulation components are present at concentrations acceptable to the site of administration. In some embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8.

In some embodiments, when parenteral administration is contemplated, the therapeutic composition may be in a pyrogen-free, parenterally acceptable water-soluble form comprising the immunomodulatory fusion protein in a pharmaceutically acceptable carrier. In some embodiments, the carrier for parenteral injection is sterile distilled water, wherein the immunomodulatory fusion protein is formulated as a sterile isotonic solution for proper storage. In some embodiments, preparation may include formulating the desired molecule with an agent, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, which may provide controlled or sustained release of the product, which may then be delivered by depot injection. In some embodiments, hyaluronic acid may also be used and may have the effect of promoting duration in circulation. In some embodiments, the implantable drug delivery device can be used to introduce a desired molecule.

In some embodiments, the pharmaceutical composition may be formulated for inhalation. In some embodiments, the immunomodulatory fusion protein can be formulated as a dry powder for inhalation. In some embodiments, an inhalation solution comprising an immunomodulatory fusion protein can be formulated with a propellant for aerosol delivery. In some embodiments, the solution may be atomized. Pulmonary administration is further described in PCT application PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

In some embodiments, it is contemplated that the formulation may be administered orally. In some embodiments, immunomodulatory fusion proteins administered in this manner may be formulated with or without those carriers typically used in complex solid dosage forms such as tablets and capsules. In some embodiments, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. In some embodiments, at least one additional agent may be included to facilitate uptake of the immunomodulatory fusion protein. In certain embodiments, diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.

In some embodiments, the pharmaceutical composition may comprise an effective amount of the immunomodulatory fusion protein in admixture with non-toxic excipients suitable for tablet manufacture. In some embodiments, the solution may be prepared in unit dosage form by dissolving the tablet in sterile water or another suitable carrier. In some embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that include the immunomodulatory fusion protein in a sustained or controlled delivery formulation. In some embodiments, techniques for preparing a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art. See, e.g., PCT application PCT/US93/00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. In some embodiments, the sustained release formulation may include a semipermeable polymer matrix in the form of a cross-sectional shape, such as a film or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. Nos. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-L-glutamic acid (Sidman et al, Biopolymers,22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (Langer et al, J.biomed.Mater.Res.,15:167-277(1981) and Langer, chem.Tech.,12:98-105(1982)), ethylene vinyl acetate (Langer et al, supra), or poly-D (-) -3-hydroxybutyric acid (EP 133,988). In some embodiments, the sustained release composition may further comprise liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc.Natl.Acad.Sci.USA,82: 3688-; EP 036,676; EP 088,046 and EP 143,949.

Pharmaceutical compositions for in vivo administration are sterile. In some embodiments, sterility is achieved by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization is performed by administering such a method prior to or after lyophilization and reconstitution. In some embodiments, the composition for parenteral administration is stored in lyophilized form or in solution form. In some embodiments, the parenteral composition is placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

In certain embodiments, once the pharmaceutical composition is formulated, it is stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. In some embodiments, such formulations are stored as ready-to-use formulations or in a form that is reconstituted prior to administration (e.g., lyophilized).

In some embodiments, kits for producing a single dose administration unit are provided. In some embodiments, a kit may comprise a first container for drying a protein and a second container having an aqueous formulation. In some embodiments, kits (e.g., liquid syringes and hemolysis syringes) comprising single-chamber and multi-chamber pre-filled syringes are included.

In some embodiments, an effective amount of a pharmaceutical composition comprising an immunomodulatory fusion protein will depend, for example, on the therapeutic context and purpose. Those skilled in the art will appreciate that, according to certain embodiments, the appropriate dosage level for treatment will thus depend, in part, on the molecule delivered, the indication of the use of the immunomodulatory fusion protein, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. In some embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect.

In some embodiments, in the formulation used, the frequency of administration will take into account the pharmacokinetic parameters of the immunomodulatory fusion protein. In some embodiments, the clinician will administer the composition up to a dosage to achieve the desired effect. In some embodiments, the composition may thus be administered over time in a single dose, or in two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter. Those of ordinary skill in the art routinely further refine appropriate dosages and are within the scope of tasks they routinely perform. In some embodiments, the appropriate dose may be determined by using appropriate dose response data.

In some embodiments, the route of administration of the pharmaceutical composition is consistent with known methods, e.g., oral, injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intralesional routes; by a sustained release system or by an implanted device. In certain embodiments, the composition may be administered by bolus injection or continuously by infusion or by an implanted device. In certain embodiments, the individual components of the combination therapy may be administered by different routes.

In some embodiments, the composition may be administered topically by implantation of a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. In some embodiments, where an implanted device is administered, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be by infusion, timed-release bolus injection, or continuous administration. In some embodiments, it may be desirable to use a pharmaceutical composition comprising an immunomodulatory fusion protein ex vivo. In this case, the cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an immunomodulatory fusion protein, and then the cells, tissues and/or organs are subsequently implanted back into the patient.

In some embodiments, the immunomodulatory fusion proteins can be delivered by implanting certain cells that have been genetically engineered to express and polypeptide using those methods described herein. In some embodiments, such cells may be animal or human cells, and may be autologous, allogeneic or xenogeneic. In some embodiments, the cell may be immortalized. In some embodiments, to reduce the chance of an immune response, cells are encapsulated to avoid infiltration of surrounding tissues. In some embodiments, the encapsulating material is generally a biocompatible, semi-permeable polymeric shell or membrane that allows the release of the protein product but prevents the cells from being destroyed by other deleterious factors of the patient's immune system or surrounding tissues.

Method of treatment

The immunomodulatory fusion proteins and/or nucleic acids expressing the same described herein are useful for treating disorders associated with aberrant apoptosis or differentiation processes (e.g., cell proliferative disorders (e.g., hyperproliferative disorders) or cell differentiation disorders, such as cancer). Non-limiting examples of cancers suitable for treatment with the methods of the present disclosure are described below.

Examples of cell proliferative and/or differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic tumor disorders, such as leukemia). Metastatic tumors can originate from a variety of primary tumor types, including but not limited to prostate, colon, lung, breast and liver cancers. Thus, a composition comprising, for example, an immunomodulatory fusion protein, as used herein, can be administered to a patient having cancer.

As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," and "tumorous" refer to cells that have the ability to grow autonomously (i.e., an abnormal state or condition characterized by rapidly proliferating cells). Hyperproliferative and neoplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state), or they can be classified as non-pathological (i.e., deviating from normal but not associated with a disease state). The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. "pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include proliferation of cells associated with wound repair.

The term "cancer" or "tumor" is used to refer to malignancies of various organ systems, including those affecting the lungs, breast, thyroid, lymphoid tissues, gastrointestinal organs and genitourinary tract, and adenocarcinomas which are generally considered to include malignancies such as most colon, renal cell, prostate and/or testicular tumors, non-small cell carcinoma of the lungs, small bowel and esophageal cancers.

The term "cancer" is art-recognized and refers to malignancies of epithelial or endocrine tissue, including respiratory system cancers, gastrointestinal system cancers, genitourinary system cancers, testicular cancers, breast cancers, prostate cancers, endocrine system cancers and melanomas. The immunomodulatory fusion proteins can be used to treat patients with, suspected of having, or likely at high risk of developing any type of cancer including renal cancer or melanoma or any viral disease. Exemplary cancers include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of cancerous and sarcoma tissues. "adenocarcinoma" refers to a cancer derived from glandular tissue or in which tumor cells form recognizable glandular structures.

Other examples of proliferative diseases include hematopoietic tumor diseases. As used herein, the term "hematopoietic tumor disease" includes diseases involving proliferating/tumor cells of hematopoietic origin, e.g., diseases derived from myeloid, lymphoid or erythroid lineages or their precursor cells. Preferably, the disease is derived from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryocytic leukemia). Other exemplary bone marrow diseases include, but are not limited to, acute promyelocytic leukemia (APML), Acute Myelogenous Leukemia (AML), and Chronic Myelogenous Leukemia (CML) (reviewed in Vaickus, L. (1991) crit. Rev. in Oncol./Hemotol.11: 267-97); lymphoid malignancies include, but are not limited to, Acute Lymphocytic Leukemia (ALL) including B-lineage ALL and T-lineage ALL, Chronic Lymphocytic Leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's Macroglobulinemia (WM). Other forms of malignant lymphoma include, but are not limited to, non-hodgkin's lymphoma and variants thereof, peripheral T cell lymphoma, adult T cell leukemia/lymphoma (ATL), Cutaneous T Cell Lymphoma (CTCL), large granular lymphocytic Leukemia (LGF), hodgkin's disease, and reed-schenberg disease.

One skilled in the art will recognize that the amount of immunomodulatory fusion protein or therapeutically useful amount sufficient to reduce tumor growth and size will vary not only by the particular compound or composition selected, but will also depend on the route of administration, the nature of the disease being treated, and the age and condition of the patient, and will ultimately be at the discretion of the physician or pharmacist of the patient. The length of time given by the compound used in the method will vary from person to person.

Those skilled in the art will appreciate that the B16 melanoma model used herein is a general model for solid tumors. That is, the effectiveness of the treatment in this model is also predictive of the effectiveness of the treatment in other non-melanoma solid tumors. For example, the usefulness of cps (a parasitic strain that induces an adaptive immune response) to mediate anti-tumor immunity against B16F10 tumors was found to be generalizable to other solid tumor models, including lung and ovarian cancer models, as described in Baird et al (J Immunology 2013; 190: 469-78; Epub Dec 7,2012). In another example, results from a series of studies directed against VEGF-targeted lymphocytes also suggested that the results of B16F10 tumors could be generalized to other studied tumor types (Chinnanamy et al, JCI 2010; 120: 3953-68; Chinnamy et al, Clin Cancer Res 2012; 18: 1672-83). In yet another example, immunotherapy involving LAG-3 and PD-1 may reduce tumor burden and produce a generalized outcome in fibrosarcoma and colon adenocarcinoma cell lines (Woo et al, Cancer Res 2012; 72: 917-27).

In certain embodiments, the immunomodulatory fusion proteins disclosed herein are used to treat cancer. In certain embodiments, the immunomodulatory fusion proteins disclosed herein are used to treat melanoma, leukemia, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, and brain cancer.

In certain embodiments, the immunomodulatory fusion proteins disclosed herein inhibit the growth and/or proliferation of tumor cells. In certain embodiments, the immunomodulatory fusion proteins disclosed herein reduce tumor size. In certain embodiments, the immunomodulatory fusion proteins disclosed herein inhibit metastasis of a primary tumor.

It will be appreciated by those skilled in the art that the treatment referred to herein extends to the prevention as well as treatment of the indicated cancers and symptoms.

Combination therapy

In some embodiments, the immunomodulatory fusion protein is used in combination with other therapies. For example, in some embodiments, the immunomodulatory fusion proteins are used in combination with other immunotherapies. Exemplary immunotherapy includes, but is not limited to, Chimeric Antigen Receptor (CAR) T cell therapy, tumor-associated antigen-targeting antibodies, immune checkpoint inhibitors, and cancer vaccines.

I. Chimeric Antigen Receptor (CAR) effector cells

In some aspects, the disclosure provides immunomodulatory fusion proteins for use in combination with or in conjunction with Chimeric Antigen Receptor (CAR) effector cell therapy (e.g., CAR T cells).

Chimeric Antigen Receptors (CARs) are genetically engineered artificial transmembrane receptors that confer arbitrary specificity to an immune effector cell (e.g., T cell, natural killer cell, or other immune cell) ligand and which cause it to recognize and bind ligand prior to activating the effector cell. Typically these receptors are used to confer antigen specificity of monoclonal antibodies to T cells.

In some embodiments, the CAR comprises three domains: 1) an extracellular domain, which typically comprises a signal peptide, a ligand or antigen recognition region (e.g., scFv), and a flexible spacer; 2) a Transmembrane (TM) domain; 3) an intracellular domain (also referred to as an "activation domain") that typically comprises one or more intracellular signaling domains. The extracellular domain of the CAR is located outside the cell and is exposed to the extracellular space, so it is susceptible to interaction with its cognate ligand. The TM domain allows the CAR to anchor in the cell membrane of effector cells. The third intracellular domain (also referred to as the "activation domain") contributes to the responsive cell activation upon binding of the CAR to its specific ligand. In some embodiments, effector cell activation includes induction of cytokines and production of chemokines, as well as activation of cytolytic activity of the cell. In some embodiments, the CAR redirects cytotoxicity to the tumor cell.

In some embodiments, the CAR comprises a ligand or antigen-specific recognition domain (also referred to as a binding domain) that binds to a specific target ligand or antigen. In some embodiments, the binding domain is a single chain antibody variable fragment (scFv), an extracellular domain of a tethered ligand or co-receptor, fused to a transmembrane domain, which in turn is linked to a signaling domain. In some embodiments, the signaling domain is derived from CD3 ζ or FcR γ. In some embodiments, the CAR comprises one or more costimulatory domains derived from proteins such as CD28, CD137 (also known as 4-IBB), CD134 (also known as OX40), and CD278 (also known as ICOS).

Binding of the antigen binding domain of the CAR to its target antigen on the surface of the target cell results in aggregation of the CAR and delivers an activation stimulus to the CAR-containing cell. In some embodiments, the primary feature of a CAR is its ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or production of molecules that can mediate apoptosis of cells expressing a target antigen in a Major Histocompatibility (MHC) independent manner, thereby exploiting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors. Although scFv-based CARs comprising signaling domains from CD3 ζ or FcR γ have been shown to be capable of delivering effective signals for T cell activation and effector function, they are insufficient to elicit signals that promote T cell survival and expansion in the absence of concomitant costimulatory signals. A new generation of CARs comprising a binding domain, a hinge, a transmembrane and a signaling domain derived from CD3 ζ or FcR γ together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD 278) has been shown to more effectively direct antitumor activity and enhance cytokine secretion, lytic activity, survival and proliferation in vitro of CAR-expressing T cells in animal models and cancer patients (Milone et al, Molecular Therapy, 2009; 17: 1453-.

In some embodiments, an effector cell (e.g., a CAR-T cell) expressing a chimeric antigen receptor is a cell derived from a patient having a disease or disorder and typically modified in vitro to express at least one CAR specific for a ligand. The cell performs at least one effector function (e.g., induction of a cytokine) that is stimulated or induced by specific binding of the ligand to the CAR and which is useful for treatment of a disease or disorder in the same patient. The effector cell is a T cell (e.g., a cytotoxic T cell or a helper T cell). One skilled in the art will appreciate that other cell types (e.g., natural killer cells or stem cells) may express the CAR, and that the chimeric antigen receptor effector cells may comprise effector cells other than T cells. In some embodiments, the effector cell is a T cell (e.g., a cytotoxic T cell) that, when contacted or brought into proximity with a target or target cell (e.g., a cancer cell), exerts its effector function (e.g., a cytotoxic T cell response) on the target cell (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5): 430-.

Prolonged exposure of T cells to their cognate antigen can lead to effector failure, thereby perpetuating infected or transformed cells. Recently developed strategies to stimulate or restore host effector function using immune checkpoint blockade agents have been successful in treating a variety of cancers. Emerging evidence suggests that T cell failure may also represent a significant obstacle for T cells expressing chimeric antigen receptors (CAR-T cells) to maintain long-lived anti-tumor activity. In some embodiments, the differentiation state of patient-harvested T cells prior to CAR transduction, as well as the opsonization protocol (e.g., addition or exclusion of alkylating agents, fludarabine, systemic irradiation) that the patient undergoes prior to reintroduction of CAR-T cells, can profoundly affect the persistence and cytotoxic potential of CAR-T cells. The differentiation status and effector function of CAR-T cells can also be altered by in vitro culture conditions that stimulate (anti-CD 3/CD28 or stimulator cells) and expand (by cytokines such as IL-2) the T cell population (Ghoneim et al, (2016) Trends in Molecular Medicine 22(12): 1000-.

In some embodiments, particularly for the treatment of ALL and/or NHL, a suitable CAR targets CD19 or CD 20. Non-limiting examples include CARs comprising the following structure: (i) an anti-CD 19 scFv, a CD 8H/TM domain, a 4-1BB CS domain, and a CD3 ζ TCR signaling domain; (ii) anti-CD 19 scFv, CD28 hinge and transmembrane domain, CD28 costimulatory domain, and CD3 TCR signaling domain; and (iii) an anti-CD 20 scFv, an IgG hinge and transmembrane domain, a CD28/4-1BB co-stimulatory domain, and a CD3 ζ TCR signaling domain. In some embodiments, CAR effector cells suitable for use in combination with the compositions and methods disclosed herein target CD19 or CD20, including but not limited to kymeriah^TM(tisagenlecucel; Novartis; formerly CTL019) and Yescatta^TM(axicabtagene ciloleucel；Kite Pharma)。

A. Retargeted CAR T cells

In some embodiments, a CAR-T therapy suitable for use in combination with an immunomodulatory fusion protein is a retargeted CAR-T cell. In some embodiments, modifying effector cells (e.g., T cells) to express a CAR that binds to a universal immune receptor, tag, switch, or Fc region on an immunoglobulin is suitable for use in the methods described herein.

In some embodiments, effector cells (e.g., T cells) are modified to express universal immunoreceptors or UnivIR. One type of UnivIR is biotin-binding immunoreceptor (BBIR) (see, e.g., U.S. patent publication No. US20140234348a1, incorporated herein by reference in its entirety). Further examples of methods and compositions related to universal chimeric receptors and/or effector cells expressing universal chimeric receptors are described in international patent application nos. WO2016123122a1, WO2017143094a1, WO2013074916a1, U.S. patent application No. US20160348073a1, all of which are incorporated herein by reference in their entirety.

In some embodiments, effector cells (e.g., T cells) are modified to express universal, modular, anti-tag chimeric antigen receptors (unicars). This system allows for the re-targeting of UniCAR implanted immune cells against a variety of antigens (see, e.g., U.S. patent publication No. US20170240612a1, incorporated by reference in its entirety; Cartellieri et al, (2016) Blood Cancer Journal 6, e458), incorporated by reference in its entirety).

In some embodiments, an effector cell (e.g., a T cell) is modified to express a switchable chimeric antigen receptor and chimeric antigen receptor effector cell (CAR-ES) switch. In this system, the CAR-ES switch has a first region that is bound by a chimeric antigen receptor on the CAR-EC and a second region that binds to a cell surface molecule on the target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In some embodiments, the CAR-EC is a T cell, wherein the CAR-EC switch can act as a "molecular switch" for CAR-EC activity. Activity can be "turned off" by reducing or stopping the management of the switch. These CAR-EC switches can be used with the CAR-ECs of the present disclosure as well as existing CAR T cells for the treatment of a disease or disorder, such as cancer, where the target cell is a malignant cell. Such treatment may be referred to herein as switchable immunotherapy (U.S. patent publication US9624276B2, which is incorporated herein by reference in its entirety).

In some embodiments, effector cells (e.g., T cells) are modified to express a receptor (e.g., CD 16V-BB-zeta) (Kudo et al, (2014) Cancer Res 74(1): 93-103) that binds the Fc portion of human immunoglobulins, which is incorporated herein by reference in its entirety.

In some embodiments, effector cells (e.g., T cells) are modified to express a universal immunoreceptor (e.g., switchable CAR, sscar) that binds to a peptide neo-epitope (PNE). In some embodiments, a peptide neo-epitope (PNE) has been introduced into a defined different position within an antibody (antibody switch) targeting an antigen. Thus, the sscar-T cell specificity is only redirected towards PNE, not occurring in the human proteome, thus allowing orthogonal interaction between the sscar-T cells and the antibody switch. In this way, sscar-T cells are strictly dependent on the presence of an antibody switch to be fully activated, thus excluding CAR T cell off-target recognition of endogenous tissues or antigens without an antibody switch (Arcangeli et al, (2016) trans Cancer Res 5(Suppl 2): S174-S177, the entire contents of which are incorporated herein by reference). Other examples of switchable CARs are provided by U.S. patent application US20160272718a1, which is incorporated by reference herein in its entirety.

As used herein, the term "tag" encompasses the universal immune receptor, tag, switch or Fc region of an immunoglobulin as described above. In some embodiments, the effector cell is modified to express a CAR comprising a tag binding domain. In some embodiments, the CAR binds Fluorescein Isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, a polymethacrylic chlorophyll protein complex, green fluorescent protein, Phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, or maltose binding protein.

B. anti-TAG chimeric antigen receptor (AT-CAR)

In some embodiments, the CAR-T therapy suitable for use in combination with an immunomodulatory fusion protein is an anti-tag CAR T cell. There are several limitations to the general clinical application of CAR T cells. For example, since there is no single tumor antigen that is ubiquitously expressed by all cancer types, specific engineering of each scFv in a CAR against a desired tumor antigen is required. Furthermore, tumor antigens targeted by CARs may be down-regulated or mutated in response to a therapy that results in tumor escape.

Alternatively, universal anti-tag chimeric antigen receptors (AT-CAR) and CAR-T cells have been developed. For example, human T cells have been engineered to express an anti-Fluorescein Isothiocyanate (FITC) CAR (referred to as anti-FITC-CAR). This platform takes advantage of the high affinity interaction between anti-FITC scFv (at the cell surface) and FITC and the ability of FITC molecules (or other tags) to bind to any anti-cancer monoclonal antibody, such as cetuximab (anti-EGFR), rituximab (anti-CD 20) and herceptin (anti-Her 2).

Thus, in some embodiments, effector cells (e.g., T cells) are modified to express universal anti-tag chimeric antigen receptors (AT-CARs), as described AT least in WO 2012082841 and US20160129109a1, which are incorporated herein by reference in their entirety. In this AT-CAR system, T cells recognize and bind to a marker protein, such as an antibody. For example, in some embodiments, AT-CAR T cells recognize a tag-labeled antibody, e.g., a FITC-labeled antibody. In some embodiments, the anti-tumor antigen antibody is conjugated to a tag (e.g., FITC) and administered prior to, concurrently with, or after AT-CAR therapy. Anti-tumor antigen antibodies are known to those skilled in the art.

As noted, the binding specificity of the tag binding domain depends on the identity of the protein conjugated tag used to bind the target cell. For example, in some aspects of the disclosure, the tag is FITC and the tag binding domain is anti-FITC scFv. Alternatively, in some aspects of the disclosure, the tag is biotin or PE (phycoerythrin) and the tag binding domain is an avidin scFv or an anti-PE scFv.

In some embodiments, the protein of each preparation of marker protein is the same or different, and the protein is an antibody or antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof is cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), rituximab (anti-CD 20), omalizumab (anti-CD 20), tositumomab (anti-CD 20), trastuzumab (anti-Her 2), gemtuzumab (anti-CD 33), alemtuzumab (anti-CD 52), and bevacizumab (anti-VEGF).

Thus, in some embodiments, the marker protein comprises a FITC-conjugated antibody, a biotin-conjugated antibody, a PE-conjugated antibody, a histidine-conjugated antibody, and a streptavidin-conjugated antibody, wherein the antibody binds to TAA or TSA expressed by the target cell. For example, marker proteins include, but are not limited to, FITC-conjugated cetuximab, FITC-conjugated rituximab, FITC-conjugated herceptin, biotin-conjugated cetuximab, biotin-conjugated rituximab, biotin-conjugated herceptin, PE-conjugated cetuximab, PE-conjugated rituximab, PE-conjugated herceptin, histidine-conjugated cetuximab, histidine-conjugated rituximab, histidine-conjugated herceptin, streptavidin-conjugated cetuximab, streptavidin-conjugated rituximab, and streptavidin-conjugated herceptin.

In some embodiments, the AT-CAR of each population of AT-CAR expressing T cells is the same or different, and the AT-CAR comprises a tag binding domain, a transmembrane structure, and an activation domain. In some embodiments, the tag binding domain is an antibody or antigen binding fragment thereof. In some aspects, the tag binding domain specifically binds to FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the tag binding domain is an antigen binding fragment and the antigen binding fragment is a single chain variable fragment (scFv), such as an scFv that specifically binds FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the transmembrane structure is associated with a hinge and transmembrane region that is the human CD8 a chain. In some embodiments, the activation domain comprises one or more of the cytoplasmic region of CD28, the cytoplasmic region of CD137(41BB), OX40, HVEM, CD3 ζ, and FcR epsilon.

In some embodiments, the tag of each preparation of labeled protein is the same or different, and the tag is selected from the group consisting of Fluorescein Isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, the polymethacrylic chlorophyll protein complex, green fluorescent protein, Phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.

The tag may be conjugated to the protein using techniques such as chemical conjugation and chemical cross-linking agents. Alternatively, polynucleotide vectors can be prepared that use the marker protein as a fusion protein. The cell line can then be engineered to express the marker protein, and the marker protein can be isolated from the culture medium, purified, and used in the methods disclosed herein.

In some embodiments, the marker protein is administered to the subject prior to, concurrently with, or subsequent to the administration of the T cells expressing the AT-CAR. In some embodiments, the present disclosure provides a method of treating cancer in a subject, comprising: (a) administering a preparation of a marker protein to a subject in need of treatment, wherein the marker protein binds to cancer cells in the subject; and (b) administering therapeutically effective T cells expressing an anti-tag chimeric antigen receptor (AT-CAR) to the subject, wherein the T cells expressing the AT-CAR bind to the marker protein and induce cancer cell death, thereby treating the cancer in the subject.

C. Tandem car (tancar) effector cells

In some embodiments, the CAR-T therapy suitable for use in combination with an immunomodulatory fusion protein is a tandem CAR effector cell. It has been observed that tumor heterogeneity and immune editing can lead to escape from CAR therapy when using the CAR approach for cancer therapy (Grupp et al, New eng.j. med (2013)368: 1509-. As an alternative approach, bispecific CARs, also known as tandem CARs or tancars, have been developed to attempt to target multiple cancer-specific markers simultaneously. In TanCAR, the extracellular domain comprises two antigen binding specificities in tandem, connected by a linker. Thus both binding specificities (scFv) are linked to a single transmembrane portion: one scFv is juxtaposed to the membrane, the other at a distal position. Exemplary TanCAR, Grada et al (Mol Ther Nucleic Acids (2013)2, e105) describe TanCAR, which includes a CD 19-specific scFv followed by a Gly-to-Ser linker and a HER 2-specific scFv. HER2-scFv was located at the membrane proximal position and CD19-scFv was located at the distal position. TanCAR was demonstrated to induce T cell reactivity that was different against both tumor-restricted antigens.

Accordingly, some aspects of the present disclosure relate to tandem chimeric antigen receptors that mediate bispecific activation and targeting of T cells. Although the present disclosure relates to the bispecific of CARs, in some aspects, the CARs are capable of targeting three, four, or more tumor antigens. Targeting multiple antigens using CAR T cells can enhance T cell activation and/or counteract tumor escape through antigen loss. TanCAR can also target multiple expressed antigens, target multiple tumors using the same cellular product with broad specificity, and/or provide better toxicity study progress and less intense signaling CAR, as multiple specificities achieve the same result.

In some embodiments, the present disclosure provides a TanCAR that induces two targeting domains. In some embodiments, the present disclosure provides a multispecific TanCAR that induces three or more targeting domains. In another embodiment, the disclosure provides a first CAR and a second CAR at the surface of a cell, each CAR comprising an antigen binding domain, wherein the antigen binding domain of the first CAR binds to a first tumor antigen (e.g., CD19, CD20, CD22, HER2) and the antigen binding domain of the second CAR binds to another (different) tumor antigen. TanCAR is described in US20160303230a1 and US20170340705a1, which are incorporated herein by reference.

In some embodiments, the TanCAR of the present disclosure targets two or more tumor antigens. Exemplary tumor antigens include one or more of CD19, CD20, CD22, kappa light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor alpha 2, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CALX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-alpha, CD44v7/8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, 1 and/TEM 8.

In some embodiments, the present disclosure provides bispecific tancars targeting CD19 and another tumor antigen. In some embodiments, the present disclosure provides bispecific tancars targeting CD22 and another tumor antigen. In some embodiments, the present disclosure provides bispecific tancars targeting HER2 and another tumor antigen. In some embodiments, the disclosure provides a bispecific TanCAR targeting IL 13R-a 2 and another tumor antigen. In some embodiments, the present disclosure provides bispecific tancars targeting VEGF-a and another tumor antigen. In some embodiments, the present disclosure provides bispecific tancars targeting Tem8 and another tumor antigen. In some embodiments, the disclosure provides a bispecific TanCAR that targets FAP and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR targeting EphA2 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets one or more, two or more, three or more, or four or more of the following tumor antigens: CD19, CD22, HER2, IL13R- α 2, VEGF-A, Tem8, FAP or EphA, and any combination thereof. In some embodiments, the disclosure provides bispecific tancars targeting HER2 and IL 13R-a 2. In some embodiments, the present disclosure provides bispecific tancars targeting CD19 and CD 22.

D. Methods of generating chimeric antigen receptors and CAR effector cells

In some embodiments, effector cells (e.g., T cells) of a subject are genetically modified with a chimeric antigen receptor (Sadelain et al, Cancer Discov.3:388-398, 2013). For example, effector cells (e.g., T cells) are provided, and a recombinant nucleic acid encoding a chimeric antigen receptor is introduced into patient-derived effector cells (e.g., T cells) to generate CAR cells. In some embodiments, effector cells (e.g., T cells) that are not the source subject are genetically modified with a chimeric antigen receptor. For example, in some embodiments, the effector cells (e.g., T cells) are allogeneic cells that have been engineered for use as "off-the-shelf" adoptive cell therapies, such as universal chimeric antigen receptor T cells (UCART), developed by celllectis. UCART is an allogeneic CAR T cell that has been engineered for the treatment of patients with the most specific cancer types. Non-limiting examples of UCART under development by celectis include those targeting the following tumor antigens: CD19, CD123, CD22, CS1, and CD 38.

Any of the nucleic acids or expression vectors of the present disclosure can be introduced into effector cells (e.g., T cells) using a variety of different methods known in the art. Non-limiting examples of methods for introducing nucleic acids into effector cells (e.g., T cells) include: lipofection, transfection (calcium phosphate transfection, transfection using hyperbranched organic compounds, transfection using cationic polymers, dendron molecular transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microscopic injection, electroporation, cell extrusion, sonoporation, protoplast fusion, imperfections, hydrodynamic delivery, gene gun, magnetic transfection, viral transfection, and nuclear transfection. Furthermore, CRISPR/Cas9 genome editing techniques known in the art can be used to introduce CAR nucleic acids into effector cells (e.g., T cells) and/or to introduce other genetic modifications (e.g., as described below) into effector cells (e.g., T cells) to enhance CAR cell activity (using CRISPR/Cas9 techniques in conjunction with CAR T cells, see, e.g., us 9,890,393; us 9,855,297; us 2017/0175128; us 2016/0184362; us 2016/0272999; WO 2015/161276; WO 2014/191128; CN 106755088; CN 106591363; CN 106480097; CN 106399375; CN 104894068).

Provided herein are methods useful for producing any of the cells or compositions described herein, wherein each cell can express a CAR (e.g., any of the CARs described herein).

A Chimeric Antigen Receptor (CAR) includes an antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain that includes a cytoplasmic sequence of a CD3 zeta sequence sufficient to stimulate T cells when the antigen binding domain binds to an antigen, and optionally one or more (e.g., two, three, or four) cytoplasmic sequences of a costimulatory protein (e.g., cytoplasmic sequences of one or more of B7-H3, BTLA, CD2, CD7, CD27, CD8, CD30, CD40, CD40L, CD80, CD160, CD244, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX40, PD-1, PD-L1, TIM3, and a ligand that specifically binds to CD 83) that provide costimulation of T cells when the antigen binding domain binds to an antigen. In some embodiments, the CAR can further comprise a linker. Non-limiting aspects and features of the CAR are described below. CARs and other aspects of CAR cells, including exemplary antigen binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described, for example, in Kakarla et al, Cancer j.20: 151-; srivastava et al, Trends Immunol.36:494-502, 2015; nishio et al, Oncoimmunology 4(2) e988098,2015; ghorashian et al, Br.J.Haematol.169:463-478, 2015; levine, Cancer Gene ther.22:79-84,2015; jensen et al, curr. Opin. Immunol.33:9-15,2015; singh et al, Cancer Gene ther.22:95-100,2015; li et al, Zhongguo Shi Yan Xue Ye Xue Za Zhi 22: 1753-; gill et al, Immunol.Rev.263:68-89,2015; magee et al, Discov.Med.18:265-271, 2014; gargett et al, front. Pharmacol.5:235,2014; yuan et al, Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1137-1141, 2014; pedgram et al, Cancer J.20: 127-; eshhar et al, Cancer J.20: 123-; ramos et al, Cancer J.20: 112-; maus et al, Blood 123: 2625-; jena et al, curr.Hematol.Malig.Rep.9:50-56,2014; maher et al, curr. Gene ther.14:35-43,2014; riches et al, Discov. Med.16:295-302, 2013; cheadle et al, Immunol.Rev.257:83-90,2014; davila et al, int.J.Hematol.99:361-371, 2014; xu et al, Cancer Lett.343:172-178, 2014; kochenderfer et al, nat. Rev. Clin. Oncol.10:267-276, 2013; hosing et al, curr. Hematol. Malig. Rep.8:60-70,2013; hombach et al, curr. mol. Med.13:1079-1088, 2013; xu et al, Leuk.Lymphoma 54: 255-; gilham et al, Trends mol. Med.18:377-384,2012; Lipowska-Bhalla et al, Cancer Immunol.Immunother.61:953-962,2012; chmielewski et al, Cancer Immunol.Immunother.61:1269-1277, 2013; jena et al, Blood 116:1035-1044, 2010; dotti et al, Immunology Reviews 257(1):107-126, 2013; dai et al, Journal of the National Cancer Institute 108(7): djv439,2016; wang and Riviere, Molecular Therapy-Oncomytics 3:16015,2016; U.S. patent application publication numbers 2018/0057609; 2018/0037625, respectively; 2017/0362295, respectively; 2017/0137783, respectively; 2016/0152723, respectively; 2016/0206656, respectively; 2016/0199412, respectively; 2016/0208018, respectively; 2015/0232880, respectively; 2015/0225480, respectively; 2015/0224143, respectively; 2015/0224142, respectively; 2015/0190428, respectively; 2015/0196599, respectively; 2015/0152181, respectively; 2015/0140023, respectively; 2015/0118202, respectively; 2015/0110760, respectively; 2015/0099299, respectively; 2015/0093822, respectively; 2015/0093401, respectively; 2015/0051266, respectively; 2015/0050729, respectively; 2015/0024482, respectively; 2015/0023937, respectively; 2015/0017141, respectively; 2015/0017136, respectively; 2015/0017120, respectively; 2014/0370045, respectively; 2014/0370017, respectively; 2014/0369977, respectively; 2014/0349402, respectively; 2014/0328812, respectively; 2014/0322275, respectively; 2014/0322216, respectively; 2014/0322212, respectively; 2014/0322183, respectively; 2014/0314795, respectively; 2014/0308259, respectively; 2014/0301993, respectively; 2014/0296492, respectively; 2014/0294784, respectively; 2014/0286973, respectively; 2014/0274909, respectively; 2014/0274801, respectively; 2014/0271635, respectively; 2014/0271582, respectively; 2014/0271581, respectively; 2014/0271579, respectively; 2014/0255363, respectively; 2014/0242701, respectively; 2014/0242049, respectively; 2014/0227272, respectively; 2014/0219975, respectively; 2014/0170114, respectively; 2014/0134720, respectively; 2014/0134142, respectively; 2014/0120622, respectively; 2014/0120136, respectively; 2014/0106449, respectively; 2014/0106449, respectively; 2014/0099340, respectively; 2014/0086828, respectively; 2014/0065629, respectively; 2014/0050708, respectively; 2014/0024809, respectively; 2013/0344039, respectively; 2013/0323214, respectively; 2013/0315884, respectively; 2013/0309258, respectively; 2013/0288368, respectively; 2013/0287752, respectively; 2013/0287748, respectively; 2013/0280221, respectively; 2013/0280220, respectively; 2013/0266551, respectively; 2013/0216528, respectively; 2013/0202622, respectively; 2013/0071414, respectively; 2012/0321667, respectively; 2012/0302466, respectively; 2012/0301448, respectively; 2012/0301447, respectively; 2012/0060230, respectively; 2011/0213288, respectively; 2011/0158957, respectively; 2011/0104128, respectively; 2011/0038836, respectively; 2007/0036773, respectively; and 2004/0043401. CARs and other aspects of CAR cells, including exemplary antigen binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in WO 2016/168595; WO 12/079000; 2015/0141347, respectively; 2015/0031624, respectively; 2015/0030597, respectively; 2014/0378389, respectively; 2014/0219978, respectively; 2014/0206620, respectively; 2014/0037628, respectively; 2013/0274203, respectively; 2013/0225668, respectively; 2013/0116167, respectively; 2012/0230962, respectively; 2012/0213783, respectively; 2012/0093842, respectively; 2012/0071420, respectively; 2012/0015888, respectively; 2011/0268754, respectively; 2010/0297093, respectively; 2010/0158881, respectively; 2010/0034834, respectively; 2010/0015113, respectively; 2009/0304657, respectively; 2004/0043401, respectively; 2014/0322253, respectively; 2015/0118208, respectively; 2015/0038684, respectively; 2014/0024601, respectively; 2012/0148552, respectively; 2011/0223129, respectively; 2009/0257994, respectively; 2008/0160607, respectively; 2008/0003683, respectively; 2013/0121960, respectively; 2011/0052554, respectively; and 2010/0178276.

Antigen binding domains

The antigen binding domain comprised in the Chimeric Antigen Receptor (CAR) may specifically bind to an antigen (e.g. a Tumor Associated Antigen (TAA) or an antigen expressed on non-cancer cells) or a universal receptor (e.g. a tag). Non-limiting examples of antigen binding domains include: monoclonal antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) (e.g., fully human or chimeric (e.g., humanized) antibodies), antigen-binding fragments of antibodies (e.g., Fab ', or F (ab')₂Fragments) (e.g.fragments of fully human or chimeric (e.g.humanized) antibodies), diabodies, triabodies, tetrabodies, minibodies, scFv-Fc, (scFv)₂scFab, bis-scFv, hc-IgG, BiTE, single domain antibodies (e.g. V-NAR domain orVhH domain), IgNAR and multispecific (e.g., bispecific antibody) antibodies. Methods for preparing these antigen binding domains are known in the art.

In some embodiments, the antigen binding domain comprises at least one (e.g., one, two, three, four, five, or six) CDR (e.g., any one from a single CDR of an immunoglobulin light chain variable region, or any one from three CDRs of an immunoglobulin heavy chain variable region) of an antibody capable of specifically binding to a target antigen, e.g., an immunoglobulin molecule (e.g., a light chain or heavy chain immunoglobulin molecule) and an immunologically active (antigen binding) fragment of an immunoglobulin molecule.

In some embodiments, the antigen binding domain is a single chain antibody (e.g., V-NAR domain or V)_HH domain, or any single chain antibody described herein). In some embodiments, the antigen binding domain is a whole antibody molecule (e.g., a human, humanized or chimeric antibody) or a multimeric antibody (e.g., a bispecific antibody).

In some embodiments, the antigen binding domain includes an antibody fragment and a multispecific (e.g., bispecific) antibody or antibody fragment. Examples of antibodies and antigen-binding fragments thereof include, but are not limited to: single chain fv (scFv), Fab fragment, F (ab')₂Fv with disulfide linkages (sdFv), Fv and fragments comprising a VL or VH domain.

Other antigen binding domains provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single chain antibodies, intrabodies (e.g., intrabodies), and antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subtype. In some embodiments, the antigen binding domain is an IgG1 antibody or antigen binding fragment thereof. In some embodiments, the antigen binding domain is an IgG4 antibody or antigen binding fragment thereof. In some embodiments, the antigen binding domain is an immunoglobulin comprising a heavy chain and a light chain.

Other examples of antigen binding domains are an antigen binding fragment of an IgG (e.g., an antigen binding fragment of an IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), an antigen binding fragment of an IgA (e.g., an antigen binding fragment of IgA1 or IgA2) (e.g., an antigen binding fragment of human or humanized IgA, e.g., human or humanized IgA1 or IgA2), an antigen binding fragment of an IgD (e.g., an antigen binding fragment of a human or humanized IgD), an antigen binding fragment of an IgE (e.g., an antigen binding fragment of a human or humanized IgE), or an antigen binding fragment of an IgM (e.g., an antigen binding fragment of a human or humanized IgM).

In some embodiments, the antigen binding domain may be at about or less than 1 × 10^-7M (e.g. about or less than 1X 10^-8M, about or less than 5X 10^-9M, about or less than 2X 10^-9M, or about or less than 1X 10^-9M) affinity (K)_D) Binding to a specific antigen (e.g., a tumor associated antigen) in, for example, saline or phosphate buffered saline.

In some embodiments, the CAR effector cell (e.g., CAR T cell) comprises a CAR molecule that binds to a tumor antigen (e.g., comprises a tumor antigen binding domain). In some embodiments, the CAR molecule comprises an antigen binding domain of a tumor antibody that recognizes a solid tumor (e.g., breast cancer, colon cancer, etc.). In some embodiments, the CAR molecule is a tandem CAR molecule as described above, comprising at least two antigen binding domains. In some embodiments, the CAR molecule comprises an antigen binding domain that recognizes a tumor antigen of a hematologic malignancy (e.g., leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic myelogenous (myelocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, burkitt's lymphoma, and marginal zone B cell lymphoma, polycythemia vera, lymphogranulomatosis, non-hodgkin's disease, multiple myeloma, etc.).

In some embodiments, the tumor antigen is a Tumor Specific Antigen (TSA). TSA is unique to tumor cells and does not occur on other cells in the body. In some embodiments, the tumor antigen is a Tumor Associated Antigen (TAA). TAAs are not unique to tumor cells, but are expressed on normal cells under conditions that do not induce an immune-tolerant state against the antigen. Expression of antigen on the tumor antigen occurs under conditions that enable the immune system to respond to the antigen. In some embodiments, when the immune system is immature and unable to respond, TAAs are expressed on normal cells during fetal development, or are typically present at very low levels on normal cells, but are expressed at much higher levels on tumor cells.

In certain embodiments, the tumor-associated antigen is determined by sequencing patient tumor cells and identifying muteins found only in the tumor. These antigens are also referred to as "neoantigens". Once a neoantigen is identified, therapeutic antibodies thereto can be generated and used in the methods described herein.

In some embodiments, the tumor antigen is an epithelial cancer antigen (e.g., breast, gastrointestinal tract, lung), prostate-specific cancer antigen (PSA), or prostate-specific membrane antigen (PSMA), bladder cancer antigen, lung (e.g., small cell lung) cancer antigen, colon cancer antigen, ovarian cancer antigen, brain cancer antigen, stomach cancer antigen, renal cell carcinoma antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen, or colorectal cancer antigen. In certain embodiments, the tumor antigen is a lymphoma antigen (e.g., non-hodgkin's lymphoma or hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma (e.g., multiple myeloma or plasma cell myeloma) antigen, an acute lymphocytic leukemia antigen, a chronic myelogenous leukemia antigen, or an acute myelogenous leukemia antigen.

Tumor antigens (e.g., Tumor Associated Antigens (TAA) and Tumor Specific Antigens (TSA) that can be targeted by CAR effector cells (e.g., CAR T cells) include, but are not limited to, 1GH-IGK, 43-9F, 5T4, 791Tgp72, cyclophilin C-associated protein, alpha-fetoprotein (AFP), alpha-actin 4, A3, A33 antibody-specific antigen, ART-4, B7, Ba 733, BAGE, BCR-ABL, beta-catenin, beta-HCG, BrE3 antigen, BCA225, BTAA, CA125, CA 15-3, CA 27.29, BCAA, CA195, CA242, CA-50, CAM 8, CAMEL, CAP-1, carbonic anhydrase IX, C-Met, CA19-9, CA72-4, CAM 17.1, CASP-8/m, CCCL19, CCCL21, CD 43 4, CD 4642, CD 4624, CD 598, CD 465, CD 598, CD 359, CD 598, CD 465, CD 598, CD19, CD20, CD21, CD22, CD23, CD25, CD32 25, CD40 25, CD66 25-E, CD25, CD70 25, CD79 25, CD132, CD133, CD138, CD147, CD154, CDC 25, CDK4 25, CDKN 225, CO-029, CTLA 25, CXCR 25, CXCL 25, EGFL-EGFA, specific antigen-p, ACAGC 4, ACAGN 225, CGN-25, CGOP 3-GCE 3, GCOV-GCE 3, GCE-25, GCE-3, GCE-GCE 3, GCE-3, CGE-3, CGE 3, and its antigen (CGE-3-III), CGE-3, CGE-III, CGE 3, CGE-3, CG, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, HTgp-175, Ia, IGF-1R, IFN-gamma, IFN-alpha, IFN-beta, IFN-lambda, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC 4-antigen, KSA, KS-1-antigen, KS1-4, LAGE-1a, Le-Y, LDR/FUT, M344, MA-50, macrophage Migration Inhibitory Factor (MIF), MAGE, MAGE-1, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MART-1, MART-2, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MG7-Ag, MOV18, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, MYL-RAR, NB/70K, Nm H1, NuMA, NCA 1, PSMA 1, NY-ESO-1, p1, p185erbB 1, p180erbB 1, PAM 1 antigen, pancreatic mucin, GF 1 receptor (PD-1), PD-1 receptor ligand (PD-1), receptor ligand (PI-1-PD-P2-P), receptor ligand (P-72), P1, P72, RCAS1, RS5, RAGE, RANTES, Ras, T101, SAGE, S100, survivin-2B, SDDCAG16, TA-90\ Mac2 binding protein, TAAL6, TAC, TAG-72, TLP, tenascin, TRAIL receptor, TRP-1, TRP-2, TSP-180, TNF- α, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, tyrosinase, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factor C3, C3a, C3B, C5a, C5, angiogenic markers, bc1-2, bc1-6, and K-Ras, oncogenic markers and oncogenic products (see, e.g., Sensi et al, Clin Cancer Res, 12: 5023-32; Parmiani et al, Parmii, 24-187: 197207; Cancer 19: 79; Cancer 19: 3: 197207).

In some embodiments, the tumor antigen is a viral antigen derived from a virus associated with a human chronic disease or cancer (e.g., cervical cancer). For example, in some embodiments, the viral antigen is derived from epstein-barr virus (EBV), HPV antigens E6 and/or E7, Hepatitis C Virus (HCV), hepatitis a virus (HBV), or Cytomegalovirus (CMV).

Exemplary cancers or tumors and specific tumor antigens associated with such tumors, but not limited thereto, include acute lymphocytic leukemia (etv6, aml1, cyclophilin B), B-cell lymphoma (Ig-inherited), glioma (E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn), bladder cancer (p21ras), biliary tract cancer (p21ras), breast cancer (MUC family, HER2/neu, c-erbB-2), cervical cancer (p53, p21ras), colon cancer (p21ras, HER2/neu, c-erbB-2, MUC family), colorectal cancer (large intestine-associated antigen (CRC) -CO17-1A/GA733, APC), Choriocarcinoma (CEA), epithelial cell carcinoma (cyclophilin B), gastric cancer (HER2/neu, c-glycoprotein B-2, 733B-2, and, Hepatocellular carcinoma (alpha-fetoprotein), Hodgkin lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoblastic leukemia (cyclophilin b), melanoma (p5 protein, gp75, carcinoembryonic antigen, GM2 and GD2 ganglioside, Melan-A/MART-1, cdc27, MAGE-3, p21ras, gp100), myxoma (MUC family, p21ras), non-small cell lung cancer (HER2/neu, c-erbB-2), nasopharyngeal carcinoma (Imp-1, EBNA-1), ovarian cancer (HER2/neu, c-erbB-2), prostate cancer (prostate specific antigen (PSA) and its epitopes PSA-1, PSA-2 and PSA-3, PSMA, HER2/neu, HER-35b-2, ga733 glycoprotein), renal cancer (HER2/neu, c-erbB-2), cervical and esophageal squamous cell carcinoma, testicular cancer (NY-ESO-1) and T-cell leukemia (HTLV-1 epitope), and viral products or proteins.

In some embodiments, an immune effector cell comprising a CAR molecule (e.g., a CAR T cell) for use in the methods of the present disclosure expresses a CAR comprising a binding domain (i.e., the CAR T cell specifically recognizes mesothelin). Mesothelin is a tumor antigen that is overexpressed in a variety of cancers, including ovarian, lung, and pancreatic cancers.

In some embodiments, an immune effector cell comprising a CAR molecule (e.g., a CAR T cell) for use in the methods of the present disclosure expresses a CAR comprising a CD19 binding domain. In some embodiments, an immune effector cell comprising a CAR molecule (e.g., a CAR T cell) for use in the methods of the present disclosure expresses a CAR comprising a HER2 binding domain. In some embodiments, an immune effector cell comprising a CAR molecule (e.g., a CAR T cell) for use in the methods of the present disclosure expresses a CAR comprising an EGFR-binding domain.

In some embodiments, the CAR effector cell expressing a CAR comprising a CD19 targeting or binding domain is kymeriah^TM(tisagenlecucel; Novartis; see WO2016109410, incorporated herein by reference in its entirety) or Yescatta^TM(axicabtagene ciloleucel; Kite; see US 20160346326, incorporated herein by reference in its entirety).

Joint

The CARs provided herein can optionally include a linker between the antigen binding domain and the transmembrane domain (1), and/or a linker between the transmembrane domain and the cytoplasmic signaling domain (2). In some embodiments, the linker may be a polypeptide linker. For example, a linker can have from about 1 amino acid to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 amino acids, or about 2 amino acids; about 2 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, or about 4 amino acids; about 4 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, or about 6 amino acids; about 6 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, or about 8 amino acids; about 8 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, or about 10 amino acids; about 10 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, or about 12 amino acids; about 12 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, or about 14 amino acids; about 14 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, or about 16 amino acids; from about 16 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, or about 18 amino acids; about 18 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids; about 20 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids; about 25 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, or about 30 amino acids; about 30 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, or about 35 amino acids; about 35 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, or about 40 amino acids; about 40 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, or about 50 amino acids; about 50 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, or about 60 amino acids; about 60 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 150 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, or about 70 amino acids; about 70 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, or about 80 amino acids; about 80 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, or about 90 amino acids; about 90 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, or about 100 amino acids; from about 100 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, or about 200 amino acids; from about 200 amino acids to about 500 amino acids, about 400 amino acids, or about 300 amino acids; a length of between about 300 amino acids to about 500 amino acids or about 400 amino acids to about 500 amino acids.

Additional examples and aspects of linkers are described in the references cited herein, and are therefore incorporated herein in their entirety.

Transmembrane domain

In some embodiments, the CAR described herein also includes a transmembrane domain. In some embodiments, the transmembrane domain is naturally associated with a sequence in the cytoplasmic domain. In some embodiments, the transmembrane domain may be modified with one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions to avoid binding of the domain to other transmembrane domains (e.g., transmembrane domains of the same or different surface membrane proteins) to minimize interaction with other members of the receptor complex.

In some embodiments, the transmembrane domain may be derived from a natural source. In some embodiments, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains useful herein can be derived from (e.g., comprise at least the following transmembrane sequences or partial transmembrane sequences): an α, β, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

In some embodiments, the transmembrane domain may be synthetic. For example, in some embodiments, if the transmembrane domain is from a synthetic source, the transmembrane domain may include (e.g., predominantly include) hydrophobic residues (e.g., leucine and valine). In some embodiments, a synthetic transmembrane domain will include at least one (e.g., at least two, at least three, at least four, at least five, or at least six) triad of alanine glutamate, tryptophan, and valine at the end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain of the CAR may comprise the CD8 hinge domain.

Other specific examples of transmembrane domains are described in the references cited herein.

Cytoplasmic Domain

Also provided herein are CAR molecules comprising, e.g., a cytoplasmic signaling domain that includes a cytoplasmic sequence sufficient to stimulate CD3 ζ of a T cell when the antigen binding domain binds an antigen, and optionally a cytoplasmic sequence of one or more costimulatory proteins (e.g., a cytoplasmic sequence of one or more of CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, B7-H3, a ligand that specifically binds to CD83, and a cytoplasmic sequence of one or more of any of the ITAM sequences described herein or known in the art) that provide costimulation of T cells. Stimulation of the CAR immune effector cell may result in activation of one or more anti-cancer activities of the CAR immune effector cell. For example, in some embodiments, stimulation of the CAR immune effector cell can result in an increase in cytolytic or accessory activity of the CAR immune effector cell, which includes secretion of cytokines. In some embodiments, the entire intracellular signaling domain of the costimulatory protein is included in the cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain comprises a truncated portion of an intracellular signaling domain of the co-stimulatory protein (e.g., a truncated portion of an intracellular signaling domain that can transduce an effector function signal in a CAR immune effector cell). Non-limiting examples of intracellular signaling domains that may be included in a cytoplasmic signaling domain include T Cell Receptors (TCRs) and co-receptors that co-initiate signal transduction following participation of an antigen receptor, as well as any variant of these sequences that includes at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) substituent, and that have the same or about the same functional capacity.

In some embodiments, the cytoplasmic signaling domain includes two different types of cytoplasmic signaling sequences: a signaling sequence that initiates antigen-dependent activation by a TCR (primary cytoplasmic signaling sequence), such as the CD3 ζ cytoplasmic signaling sequence, and cytoplasmic sequences of one or more costimulatory proteins that function in an antigen-dependent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequence).

In some embodiments, the cytoplasmic domain of the CAR is designed to include the CD3 zeta signaling domain by itself or in combination with any other desired cytoplasmic signaling sequence useful in the context of the CAR. In some examples, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory cytoplasmic signaling sequence. Costimulatory cytoplasmic signaling sequences refer to portions of CARs that include cytoplasmic signaling sequences of costimulatory proteins (e.g., CD27, CD28, 40IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind CD 83).

In some embodiments, the cytoplasmic signaling sequences within the cytoplasmic signaling domain of the CAR are located in random order. In some embodiments, the cytoplasmic signaling sequences within the cytoplasmic signaling domain of the CAR are linked to each other in a specific order. In some embodiments, a linker (e.g., any of the linkers described herein) can be used to form a pre-junction of different cytoplasmic signaling sequences.

In some embodiments, the cytoplasmic signaling domain is designed to include the CD3 zeta signaling sequence and the cytoplasmic signaling sequence of costimulatory protein CD 28. In some embodiments, the cytoplasmic signaling domain is designed to include the CD3 zeta signaling sequence and the cytoplasmic signaling sequence of costimulatory protein 4-IBB. In some embodiments, the cytoplasmic signaling domain is designed to include the CD3 zeta signaling sequence and the cytoplasmic signaling sequences of the costimulatory proteins CD28 and 4-IBB. In some embodiments, the cytoplasmic signaling domain does not include the cytoplasmic signaling sequence of 4-IBB.

Additional modification of CAR T cells

In another embodiment, the therapeutic efficacy of a CAR effector cell (e.g., a CAR T cell) is enhanced by disruption of methylcytosine dioxygenase genes (e.g., Tet1, Tet2, Tet3), as described in PCT publication WO 2017/049166, which results in a decrease in the total level of 5-hydroxymethylcytosine associated with enhanced proliferation, modulation of effector cytokine production and degranulation, and thereby increased proliferation and/or function of the CAR effector cell (e.g., a CAR T cell). Thus, effector cells (e.g., T cells) may be engineered to express a CAR, and wherein expression and/or function of Tet1, Tet2, and/or Tet3 in the smile cell (e.g., T cell) has been reduced or eliminated.

In another embodiment, the therapeutic efficacy of CAR effector cells (e.g., CAR T cells) is enhanced by the use of effector cells (e.g., T cells) that constitutively express a CAR (referred to as an unconditional CAR) and conditionally express another drug useful for the treatment of cancer, as described in PCT publication WO 2016/126608 and U.S. patent 2018/0044424. In such embodiments, the conditionally expressed drug is expressed upon activation of an effector cell (e.g., a T cell), e.g., binding of the unconditional CAR to its target. In one embodiment, the conditionally expressed drug is a CAR (referred to herein as a conditional CAR). In another embodiment, the conditionally expressed drug inhibits a checkpoint inhibitor of the immune response. In another embodiment, the conditionally expressed drug improves or enhances the efficacy of the CAR, and may include a cytokine.

In another embodiment, the therapeutic efficacy of the CAR T cells is enhanced by modifying the CAR T cells with a nucleic acid that is capable of or alters (e.g., down-regulates) the expression of an endogenous gene selected from the group consisting of TCR a chain, TCR β chain, β -2 microglobulin, HLA molecule, CTLA-4, PD1, and FAS, as described in PCT publication WO 2016/069282 and U.S. patent No. 2017/0335331.

In another embodiment, the therapeutic effectiveness of the CAR T cell is enhanced by co-expressing the CAR and one or more T cell priming ("ETP") enhancers in the T cell, as described in PCT publication WO 2015/112626 and U.S. publication No. 2016/0340406. Addition of ETP components to CAR T cells can enhance "professional" Antigen Presenting Cell (APC) function. In one embodiment, the CAR and one or more EPTs are transiently co-expressed in a T cell. Thus, engineered T cells are safe (given the transient nature of CAR/ETP expression) and induce prolonged immunity through APC function.

In another embodiment, the therapeutic efficacy of the CAR T cell is enhanced by co-expressing the CAR and an Integral Membrane Protein (IMP) comprising a binding (or dimerization) domain in the T cell, as described in PCT publication WO 2016/055551 and U.S. publication No. 2017/0292118. Both CAR and IMP are made reactive to soluble compounds, in particular by the second binding domain comprised in the CAR, allowing co-localization of the IMP-carried inhibitory signaling domain and the CAR-carried signaling domain by dimerization or ligand recognition, which has the effect of reducing CAR activation. The inhibitory signaling domain is preferably programmed death 1(PD-1), which attenuates T cell receptor (TCP) -mediated activation of IL-2 production and T cell proliferation.

In another embodiment, the therapeutic effectiveness of CAR T cells is enhanced using a system that achieves controlled changes in the conformation of the extracellular portion of the CAR containing the antigen binding domain by the addition of small molecules, as described in PCR publication WO 2017/032777. The integrated system switches the interaction between the antigen and the antigen binding domain between on/off states. By being able to control the conformation of the extracellular portion of the CAR, the antigen directly modulates downstream functions of the CAR T cell, such as cytotoxicity. Thus, a CAR is characterized in that it comprises: a) at least one extracellular domain comprising: i) an extracellular antigen-binding domain; and ii) a switch domain comprising at least a first and a second polyligand binding domain that is capable of binding a predetermined multivalent ligand to form a multimer comprising the second binding domain and the multivalent ligand to which they are capable of binding; b) at least one transmembrane domain; and c) at least one endodomain comprising a signaling domain and optionally a costimulatory domain; wherein the switch domain is located between the extracellular antigen-binding domain and the transmembrane domain.

Tumor associated antigen targeting antibodies

In some aspects, the disclosure provides immunomodulatory fusion proteins for use in combination with or performed on antibodies that target tumor antigens.

Therapeutic monoclonal antibodies have been envisaged as a class of pharmaceutically active agents which should be tumor-selective treated by targeting tumor-selective antigens or epitopes.

Methods of producing antibodies and antigen binding fragments thereof are well known in the art and are disclosed, for example, in U.S. patent nos. 7,247,301, 7,923,221 and U.S. patent application 2008/0138336, the entire contents of which are incorporated herein by reference.

Therapeutic antibodies that may be used in the methods of the present disclosure include, but are not limited to, any art-recognized anti-cancer antibody approved for use in clinical trials or for development of clinical use. In certain embodiments, more than one anti-cancer antibody may be included in the combination therapies of the present disclosure.

Non-limiting examples of anti-cancer antibodies include the following but are not limited to: trastuzumab (herceptin)^TMGenentech, South San Francisco, Calif.), for use in the treatment of HER-2/neu positive breast cancer or metastatic breast cancer; bevacizumab (AVASTIN)^TMGenentech) for the treatment of colorectal cancer, metastatic colorectal cancer, breast cancer, metastatic breast cancer, non-small cell lung cancer or renal cell carcinoma; rituximab (RITUXAN) ^TMGenentech) for use in the treatment of non-hodgkin's lymphoma or chronic lymphocytic leukemia; pertuzumab (OMNITARG)^TMGenentech) for the treatment of breast, prostate, non-small cell lung or ovarian cancer; cetuximab (ERBITUX)^TMImClone Systems Incorporated, New York, n.y.) for use in the treatment of colorectal cancer, metastatic colorectal cancer, lung cancer, head and neck cancer, colon cancer, breast cancer, prostate cancer, gastric cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, renal cell carcinoma, prostate cancer, cervical cancer, or bladder cancer; IMC-1C11(Imclone Systems Incorporated) for use in the treatment of colorectal cancer, head and neck cancer, and other potential cancer targets; tositumomab and iodine I131 (BEXXAR XM, Corixa Corporation, Seattle, Wash.) for the treatment of non-hodgkin's lymphoma, which may be CD20 positive, follicular non-hodgkin is lymphoma, with or without transformation, the disease rituximab refractory and chemotherapyThe disease has relapsed; in¹¹¹Ibritumomab tiuxetan (ibirtumomab tiuxetan); y is⁹⁰Ibritumomab tiuxetan; in¹¹¹Ibritumomab tiuxetan and Y⁹⁰Ibritumomab tiuxetan (ZEVALIN)^TMBiogen Idee, Cambridge, Mass.) for use in the treatment of lymphoma or non-hodgkin's lymphoma, which may include recurrent follicular lymphoma; recurrent or refractory, low grade or follicular non-hodgkin's lymphoma; or transformed B cell non-hodgkin's lymphoma; EMD 7200(EMD Pharmaceuticals, Durham, N.C.), for use in the treatment of non-small cell lung cancer or cervical cancer; SGN-30 (genetically engineered monoclonal antibodies targeting the CD30 antigen, Seattle Genetics, Bothell, Wash.) for use in the treatment of Hodgkin's lymphoma or non-Hodgkin's lymphoma; SGN-15 (genetically engineered monoclonal antibody targeting a Lewis-associated antigen conjugated to doxorubicin, Seattle Genetics) for use in the treatment of non-small cell lung cancer; SGN-33 (humanized antibody targeting CD33 antigen, Seattle Genetics) for use in the treatment of Acute Myeloid Leukemia (AML) and myelodysplastic syndrome (MDS); SGN-40 (a humanized monoclonal antibody targeting CD40, Seattle Genetics) for use in the treatment of multiple myeloma or non-Hodgkin's lymphoma; SGN-35 (a genetically engineered monoclonal antibody targeting CD30 antigen conjugated with auristatin E, Seattle Genetics) for use in the treatment of non-hodgkin's lymphoma; SGN-70 (humanized antibody targeting CD70, Seattle Genetics) for the treatment of renal and nasopharyngeal carcinoma; SGN-75 (conjugates consisting of SGN70 antibody and auristatin derivatives, Seattle Genetics); and SGN-17/19 (fusion protein comprising an antibody and an enzyme conjugated to a melphalan prodrug, Seattle Genetics) for the treatment of melanoma and metastatic melanoma.

It is to be understood that therapeutic antibodies used in the methods of the present disclosure are not limited to those described above. For example, the following approved therapeutic antibodies may also be used in the methods of the present disclosure: bentuximab (ADCETRIS) for anaplastic large cell lymphoma and Hodgkin's lymphoma^TM) Ipilimumab for melanoma (MDX-101; YERVOY^TM) Australian for chronic lymphocytic leukemiaAnti (ARZERRA)^TM) Panitumumab (VECTIBIX) for colorectal cancer^TM) Alemtuzumab (CAMPATH) for chronic lymphocytic leukemia^TM) Aframucimumab (ARZERRA) for chronic lymphocytic leukemia^TM) Gemtuzumab ozogamicin (MYLOTARG) for acute myelogenous leukemia^TM)。

Antibodies suitable for use in the methods disclosed herein can also be target molecules expressed by immune cells, such as, but not limited to OX86, which target OX40 and increase antigen-specific CD8+ T cells at the tumor site and enhance tumor rejection; BMS-663513, which targets CD137 and causes established tumor regression, as well as expansion and maintenance of CD8+ T cells, and daclizumab (ZENAPAAX)^TM) It targets CD25 and results in transient depletion of CD4+ CD25+ FOXP3+ tregs and enhances tumor regression and increases the number of effector T cells. A more detailed discussion of these antibodies can be found, for example, in Weiner et al, Nature rev. immunol 2010; 10:317-27.

Other therapeutic antibodies can be identified that target tumor antigens (e.g., tumor antigens associated with different types of cancers, such as carcinomas, sarcomas, myelomas, leukemias, lymphomas, and combinations thereof). For example, in the methods disclosed herein, the tumor antigens described below can be targeted by therapeutic antibodies.

The tumor antigen can be an epithelial cancer antigen (e.g., breast, gastrointestinal tract, lung), prostate-specific cancer antigen (PSA), or prostate-specific membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen. In certain embodiments, the tumor antigen is a lymphoma antigen (e.g., non-hodgkin's lymphoma or hodgkin's lymphoma), a B cell lymphoma cancer antigen, a leukemia antigen, a myeloma (e.g., multiple myeloma or plasma cell lymphoma) antigen, an acute lymphocytic leukemia antigen, a chronic myelogenous leukemia antigen, or an acute myelogenous leukemia antigen. It is to be understood that the tumor antigens described are merely exemplary, and that any tumor antigen used in the methods disclosed herein can be targeted.

In certain embodiments, the tumor antigen is a mucin-1 protein or peptide (MUC-1), which is present in most or all human adenocarcinoma: pancreas, colon, breast, ovary, lung, prostate, head and neck, including multiple myeloma and some B cell lymphomas. Patients with inflammatory bowel disease (crohn's disease or ulcerative colitis) are at increased risk of colorectal cancer. MUC-1 is a type I transmembrane glycoprotein. The major extracellular portion of MUC-1 has a large number of tandem repeats consisting of 20 amino acids, which contain immunogenic epitopes. In some cancers, it is exposed in a non-glycosylated form that can be recognized by the immune system (Gendler et al, J Biol Chem 1990; 265: 15286-.

In certain embodiments, the tumor antigen is a mutated B-Raf antigen, which is associated with melanoma and colon cancer. Most of these mutations represent a single nucleotide change of T-A at nucleotide 1796, resulting in a change of valine at residue 599 of the B-Raf activating fragment to glutamic acid. As an effector of the activation of Ras protein, Raf protein is also indirectly associated with cancer, the oncogenic form of which is present in about one third of all human cancers. Normal non-mutated B-Raf participates in cell banking transduction, conducting signals from the cell membrane to the nucleus. The protein is typically only active when a relay signal is required. In contrast, mutant B-Raf was reported to be active all the time, disrupting signal transduction. (Merce and Pritchad, Biochim Biophys Acta (2003)1653(1): 25-40; Sharkey et al, Cancer Res. (2004)64(5): 1595-.

In certain embodiments, the tumor antigen is a human epidermal growth factor receptor-2 (HER-2/neu) antigen. Cancers with cells that overexpress HER-2/neu are referred to as HER-2/neu + cancers. Exemplary HER-2/neu + cancers include prostate, lung, breast, ovarian, pancreatic, skin, liver (e.g., hepatocellular), intestinal, and bladder cancers.

HER-2/neu has an extracellular binding domain (ECD) of about 645aa, a highly hydrophobic transmembrane anchoring domain (TMD) with 40% homology to Epidermal Growth Factor Receptor (EGFR), and EGFR carboxy terminal intracellular domain (ICD) of about 580 aa with 80% homology. The nucleotide sequence of HER-2/neu can be obtained from GENBANK^TMAnd (4) obtaining. Accession number AH002823 (human HER-2 gene, promoter region and exon 1); m16792 (human HER-2 gene, exon 4); m16791 (human HER-2 gene, exon 3); m16790 (human HER-2 gene, exon 2); and M16789 (human HER-2 gene, promoter region and exon 1). The amino acid sequence of the HER-2/neu protein can be obtained from GENBANK^TMAnd (4) obtaining. Accession number AAA 58637. Based on these sequences, the skilled person is able to develop the HER-2/neu antigen using known assays to know the appropriate epitope for generating an effective immune response. Exemplary HER-2/neu antigens include p369-377 (HER-2/neu-derived HLA-A2 peptide); dHER2(Corixa Corporation); li-Key class II MHC epitope hybridization (Generex Biotechnology Corporation); peptide P4 (amino acids 378-398); peptide P7 (amino acids 610-623); a mixture of peptide P6 (amino acid 544-; a mixture of peptides P4, P6 and P7; HER2[9754 ] ]And the like.

In certain embodiments, the tumor antigen is an Epidermal Growth Factor Receptor (EGFR) antigen. The EGFR antigen can be an EGFR variant 1 antigen, an EGFR variant 2 antigen, an EGFR variant 3 antigen, and/or an EGFR variant 4 antigen. Cancers with cells overexpressing EGFR are referred to as EGFR⁺Cancer. Exemplary EGFR⁺The cancer includes lung cancer, head and neck cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, stomach cancer, ovarian cancer, brain cancer, and bladder cancer.

In certain embodiments, the tumor antigen is a Vascular Endothelial Growth Factor Receptor (VEGFR) antigen. VEGFR is believed to be a modulator of cancer-induced angiogenesis. VEGFR-overexpressing cancers with cells termed VEGFR⁺Cancer. Exemplary VEGFR⁺The cancer includes breast cancer, lung cancer, small cell lung cancer, colon cancer, colorectal cancer, kidney cancer, leukemia and lymphocytic leukemia.

In certain embodiments, the tumor antigen is Prostate Specific Antigen (PSA) and/or Prostate Specific Membrane Antigen (PSMA), which is ubiquitously expressed in androgen-independent prostate cancer.

In certain embodiments, the tumor antigen is glycoprotein 100(gp 100), a tumor specific antigen associated with melanoma.

In certain embodiments, the tumor associated antigen is a Carcinoembryonic (CEA) antigen. Cancers with CEA overexpressing cells are referred to as CEA⁺Cancer. Exemplary CEA⁺Cancers include colorectal, gastric, and pancreatic cancers. Exemplary CEA antigens include CAP-1 (i.e., CEA aa 571-579), CAP1-6D, CAP-2 (i.e., CEA aa 555-.

In certain embodiments, the tumor antigen is the carbohydrate antigen 10.9(CA 19.9). CA 19.9 is an oligosaccharide associated with Lewis A blood group substances and is associated with colorectal cancer.

In certain embodiments, the tumor antigen is a melanoma cancer antigen. Melanoma cancer antigen is used for treating melanoma. Exemplary melanoma cancer antigens include MART-1 (e.g., MART-126-35 peptide, MART-127-35 peptide); MART-1/Melan A; pMel 17; pMel17/gp 100; gp100 (e.g., gp100 peptide 280-288, gp100 peptide 154-162, gp100 peptide 457-467); TRP-1; TRP-2; NY-ESO-1; p 16; beta-catenin; mum-1, and the like.

In certain embodiments, the tumor antigen is a mutant or wild-type ras peptide. The mutant ras peptide may be a mutant K-ras peptide, a mutant N-ras peptide, and/or a mutant H-ras peptide. Mutations in ras proteins typically occur at positions 12 (e.g., arginine or valine to glycine), 13 (e.g., asparagine to glycine), 61 (e.g., glutamine to leucine), and/or 59. The mutant ras peptides may be used as lung cancer antigens, gastrointestinal cancer antigens, liver cancer antigens, myeloid cancer antigens (e.g., acute leukemia, myelodysplasia), skin cancer antigens (e.g., melanoma, basal cells, squamous cells), bladder cancer antigens, colon cancer antigens, colorectal cancer antigens, and renal cell carcinoma antigens.

In certain embodiments, the tumor antigen is a mutant and/or wild-type p53 peptide. The p53 peptide can be used as colon cancer antigen, lung cancer antigen, breast cancer antigen, hepatocellular carcinoma antigen, lymphoma cancer antigen, prostate cancer antigen, thyroid cancer antigen, bladder cancer antigen, pancreatic cancer antigen, and ovarian cancer antigen.

Other tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxyesterase, mut hsp70-2, M-CSF, protease, Prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, tyrosinase, protein, PSMA, ras, Her2/neu, TRP-1, TRP-2, TAG-72, KSA, CA-125, PSA, BRCI, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1, survivin and telomerase, prostate cancer antigen-1 (PCTA-1), MAGE, GAGE, GP-100, MUC-1, MUC-2, ELF2M, neutral elastase, ephrin B2, CD22, Insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor, and mesothelin.

In certain embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignancy. Malignant tumors express a variety of proteins that can be targeted antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma, and Prostate Acid Phosphatase (PAP) and Prostate Specific Antigen (PSA) in prostate cancer. Other target molecules belong to the class of molecules involved in transformation, such as the oncogene ene HER-2/Neu ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B cell lymphomas, tumor-specific idiotypic immunoglobulins constitute a true tumor-specific immunoglobulin antigen that is unique to a single tumor. B cell differentiation antigens (e.g., CD19, CD20, and CD37) are other candidates as target antigens in B cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for monoclonal antibody dynamic immunotherapy, but with limited success.

The tumor antigen may also be a Tumor Specific Antigen (TSA) or a Tumor Associated Antigen (TAA). TSA is unique among tumor cells and does not appear in other cells of the body. TAA-associated antigens are not specific for tumor cells, but are expressed on normal cells under conditions that do not induce an immune-tolerant state to the antigen. Expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells when the immune system is immature and unable to respond during fetal development, and may also be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l/Melana (MART-1), Pmel 17, tyrosinase, TRP-1, TRP-2 and tumor specific multispectral antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as epstein-barr virus antigen EBVA and Human Papilloma Virus (HPV) antigens E6 and E7. Other protein-based macroantigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p l80erbB-3, c-met, nm-23H l, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β -catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4(791Tgp72), alpha fetoprotein, β -HCG, BCA225, BTAA, CA 125, CA 15-3\ CA 27.29\ BCAA, CA 195, CA 242, CA-50, CAM43, CD68\ I, CO-029, FGF-5, G250, Ga733VEpCAM, HTMA-175, M344, MG-50, MOVAV 24-7, MOV 18-NB 70, Ag-1/32, RCAS 18, RCAS 70K, NY, RC1/K, NY, RC1 Tgp72, RCA, and RCA, SDCCAG16, TA-90\ Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

In certain embodiments, the tumor-associated antigen is determined by sequencing tumor cells of the patient and identifying muteins found only in the tumor. These antigens are referred to as "neoantigens". Once a neoantigen is identified, therapeutic antibodies thereto can be generated and used in the methods described herein.

The therapeutic antibody may be an antibody fragment; a complex comprising an antibody; or a conjugate comprising an antibody. The antibody may optionally be chimeric or humanized or fully human.

Immune checkpoint blockade

In some aspects, the disclosure provides immunomodulatory fusion proteins for use in combination with or performed by an immune checkpoint inhibitor or an immune checkpoint blocker.

T cell activation and effector functions are balanced by costimulatory and inhibitory signals called "immune checkpoints". Inhibitory ligands and receptors that regulate T cell effector function are overexpressed on tumor cells. Subsequently, agonists of co-stimulatory receptors or antagonists of inhibitory signals result in the expansion of antigen-specific T cell responses. Unlike therapeutic antibodies that directly target tumor cells, immune checkpoint blockers enhance endogenous anti-tumor activity. In certain embodiments, an immune checkpoint blocker suitable for use in the methods disclosed herein is an antagonist of inhibitory signaling, e.g., an antibody that targets, e.g., PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM 3. These ligands and receptors are reviewed in Pardol, D., Nature.12: 252-one 264,2012.

In certain embodiments, the immune checkpoint blockade agent is an antibody or antigen-binding portion thereof that disrupts or inhibits signaling from an inhibitory immunomodulator. In certain embodiments, the immune checkpoint blockade agent is a small molecule that destroys or inhibits an inhibitory immune modulator.

In certain embodiments, the inhibitory immunomodulator (immune checkpoint blocker) is part of the PD-1/PD-L1 signaling pathway. Accordingly, certain embodiments of the present disclosure provide methods for immunotherapy of a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that disrupts the interaction between the PD-1 receptor and its ligand PD-L1. Antibodies known in the art that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1, as well as stimulate an anti-tumor immune response, are suitable for use in the methods disclosed herein. In certain embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. For example, antibodies that target PD-1 and are in clinical trials include, for example, nivolumab (BMS-936558, Bristol-Myers Squibb) and pembrolizumab (lambrolizumab, MK03475, Merck). Other suitable antibodies for use in the methods disclosed herein are anti-PD-1 antibodies disclosed in U.S. patent No. 8,008,449, which is incorporated herein by reference. In certain embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Antibodies known in the art that bind to PD-L1 and disrupt the interaction between PD-1 and PD-L1, as well as stimulate anti-tumor immune responses, are suitable for use in the methods disclosed herein. For example, antibodies that target PD-L1 and are in clinical trials include BMS-936559(Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. patent No. 7,943,743. One of ordinary skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunomodulatory agent is part of a CTLA-4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide methods for immunotherapy of a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that targets CTLA-4 and disrupts its interaction with CD80 and CD 86. Exemplary antibodies targeting CTLA-4 include ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb), which is FDA approved, and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently in human testing. Other suitable antibodies targeting CTLA-4 are disclosed in WO 2012/120125, U.S. patent nos. 6,984720, 6,682,7368, and U.S. patent applications 2002/0039581, 2002/0086014, and 2005/0201994, which are incorporated herein by reference. One of ordinary skill in the art will appreciate that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunomodulator is part of the LAG3 (lymphocyte activator gene 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide methods for immunotherapy of a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that targets LAG3 and disrupts its interaction with MHC class II molecules. An exemplary antibody targeting LAG3 is IMP321(Immutep), currently undergoing human testing. Other suitable antibodies targeting LAG3 are disclosed in U.S. patent application 2011/0150892, which is incorporated herein by reference. One of ordinary skill in the art will appreciate that any antibody that binds to LAG3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunomodulatory agent is part of a B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Accordingly, certain embodiments of the present disclosure provide methods for immunotherapy of a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that targets B7-H3 or H4. The B7 family does not have any defined receptors, but these ligands are upregulated on tumor cells or tumor infiltrating cells. Preclinical mouse models have demonstrated that blocking these ligands can enhance anti-tumor immunity. An exemplary antibody targeting B7-H3 is MGA271 (macrogenetics), which is currently undergoing human trials. Other suitable antibodies targeting LAG3 are disclosed in U.S. patent application 2013/0149236, which is incorporated herein by reference. One of ordinary skill in the art will appreciate that any antibody that binds to B7-H3 or H4 and stimulates an anti-tumor immune response is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunomodulatory agent is part of the TIM3(T cell membrane protein 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide methods for immunotherapy of a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that targets LAG3 and disrupts its interaction with galectin 9. Suitable antibodies targeting TIM3 are disclosed in U.S. patent application 2013/0022623, which is incorporated herein by reference. One of ordinary skill in the art will appreciate that any antibody that binds to TIM3, disrupts its interaction with galectin 9, and stimulates an anti-tumor immune response is suitable for use in the methods disclosed herein.

It is to be understood that antibody-targeted immune checkpoints suitable for use in the methods disclosed herein are not limited to those described above. Furthermore, one of ordinary skill in the art will appreciate that other immune checkpoint targets can also be targeted by the antagonists or antibodies in the methods described herein, provided that the targeting stimulates an anti-tumor immune response, which is reflected in, for example, an increase in T cell proliferation, an increase in T cell activation, and/or an increase in cytokine production (e.g., IFN- γ, IL-2).

Cancer vaccine

In some aspects, the disclosure provides immunomodulatory fusion proteins for use in combination with or for administration to a cancer vaccine. In certain embodiments, the cancer vaccine stimulates a specific immune response against a specific target (e.g., a tumor-associated antigen).

In certain embodiments, the cancer vaccine comprises a viral, bacterial, or yeast vector to deliver a recombinant gene to an Antigen Presenting Cell (APC).

In certain embodiments, the cancer vaccine uses autologous or allogeneic tumor cells. In certain embodiments, these tumor cells can be modified to express MHC, co-stimulatory molecules, or cytokines.

In certain embodiments, the tumor-associated antigen is determined by sequencing tumor cells of the patient and identifying muteins found only in the tumor. These antigens are referred to as "neoantigens". Once a neoantigen is identified, therapeutic antibodies thereto can be generated and used in the methods described herein. It can be used as an antigen for vaccines or to develop monoclonal antibodies specifically reactive with a novel antigen.

In certain embodiments, the vaccine comprises irradiated tumor cells transduced with a cytokine (e.g., GM-CSF) or loaded with an adjuvant compound, such as GM-CSF secreting tumor cell vaccine GVAX (Immunological Reviews,222(1): 287-298, 2008). In certain embodiments, the vaccine comprises one or more tumor-associated antigens traveling with the immunomer composition, optionally in combination with an adjuvant. For example, vaccination against HPV-16 oncoprotein resulted in a positive clinical outcome of vulvar intraepithelial neoplasms (The New England Journal of Medicine,361(19), 1838-1847, 2012). Similarly, the polypeptide immune response to the cancer vaccine IMA901 following a single dose administration of cyclophosphamide can prolong the survival of patients (Nature Medicine,18(8):1254-61, 2012). Alternatively, DNA-based methods can be used to immunize a patient with one or more tumor-associated antigens. Improved tumor immunity was observed in mice melanoma using a DNA vaccine in combination with an anti-tyrosinase related protein-1 monoclonal antibody (Cancer Research,68 (23)), 9884-.

Other vaccine approaches utilize patient immune cells, such as dendritic cells, which can be cultured with tumor associated antigens to produce antigen-antigen presenting cells that will stimulate the immune system and target the antigen of interest. The current FDA-approved cancer therapy vaccine using this method is (Dendreon), approved for use in some men with metastatic prostate cancer. The vaccine stimulates an immune response to prostate phosphonate (PAP), an antigen present on most prostate cancer cells. The vaccine is prepared by isolating and culturing PAP-bearing dendritic cells from immune cells of a particular patient to produce antigen presenting cells that will stimulate the immune system and target PAP. These and other cancer vaccines can be used in combination with other therapies described herein.

A. Amphiphilic vaccines

In some embodiments, a cancer vaccine suitable for use with the immunomodulatory fusion proteins described herein is an amphiphilic vaccine, as described in US 2013/0295129, which is incorporated herein by reference. Amphiphilic vaccines combine albumin-binding lipids with peptide antigens or molecular adjuvants to effectively target the peptide or adjuvant to lymph nodes in vivo. The lipid conjugate binds to endogenous albumin, targeting lymphatic and draining lymph nodes and accumulating there as a result of the filtration of albumin by antigen presenting cells. When the lipid conjugate includes an antigenic peptide or a molecular adjuvant, the conjugate induces or enhances a strong immune response.

Lymph node targeting conjugates typically include three domains: a highly lipophilic albumin binding domain (e.g., albumin-binding lipid), a cargo (e.g., a molecular adjuvant or a peptide antigen), and a polar block linker, which promotes solubility of the conjugate and reduces the ability of the lipid to insert into the plasma membrane of the cell. Thus, in certain embodiments, the generic structure of the conjugate is L-P-C, wherein "L" is a lipid that binds albumin, "P" is a polar block, and "C" is a cargo such as a molecular adjuvant or polypeptide. In some embodiments, the cargo itself may also serve as a polar block domain, and a separate polar block domain is not required. Thus, in certain embodiments, the conjugate has only two domains: albumin-binding lipids and cargo.

The cargo of conjugates suitable for use in the methods disclosed herein are typically molecular adjuvants, such as immunomodulatory oligonucleotides, or peptide antigens. However, the cargo may also be other oligonucleotides, peptides, Toll-like receptor agonists or other immunomodulatory compounds, dyes, MRI contrast agents, fluorophores, or small molecule drugs that require efficient transport to lymph nodes.

In certain embodiments, the lipid-oligonucleotide conjugate comprises an immunostimulatory oligonucleotide linked directly to a lipid-conjugated linker. Other embodiments relate to lipid-peptide conjugates comprising an antigenic peptide directly conjugated to a lipid or linked to a linker conjugated to a lipid.

Lipid

Lipid conjugates typically include a hydrophobic lipid. Lipids may be linear, branched or cyclic. The length of the lipid is preferably at least 17 to 18 carbons, but may be shorter if it shows good albumin binding and sufficient targeting to lymph nodes. Lymph node targeting conjugates include lipid-oligonucleotide conjugates and lipid peptide conjugates, which can translocate lymph to lymph nodes from the site of delivery. In certain embodiments, the activity is dependent in part on the ability of the conjugate to bind to albumin in the blood of the subject. Thus, lymph node targeting conjugates typically include lipids that can bind albumin under physiological conditions. Lipids suitable for targeting lymph nodes can be selected based on the ability of the lipid or lipid conjugate comprising the lipid to bind albumin. Suitable methods for detecting the ability of a lipid or lipid conjugate to bind to albumin are well known in the art.

For example, in certain embodiments, multiple lipid conjugates are allowed to spontaneously form micelles in aqueous solution. The micelles are incubated with albumin or an albumin-containing solution, such as Fetal Bovine Serum (FBS). Samples can be analyzed, e.g., by ELISA, size exclusion chromatography, or other methods to determine whether binding has occurred. If albumin or an albumin-containing solution (e.g., Fetal Bovine Serum (FBS)) is present, the micelles dissociate and the lipid conjugate binds to albumin as described above, the lipid conjugate can be selected as a lymph node targeting conjugate.

Examples of preferred lipids for use in the lipid conjugates targeting lymph nodes include, but are not limited to, fatty acids having an aliphatic tail of 8-30 carbon atoms including, but not limited to, linear unsaturated and saturated fatty acids, branched chain saturated and unsaturated fatty acids, and fatty acid derivatives such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, cholesterol derivatives, and steroid acids such as bile acids, lipid a, or combinations thereof.

In certain embodiments, the lipid is a diacyl lipid or a two-tailed lipid. In some embodiments, the tail in the diacyl lipid comprises from about 8 to 30 carbon atoms and can be saturated, unsaturated, or a combination thereof. The tail may be coupled to the head group via an ester bond, an amide bond, a thioester bond, or a combination thereof. In a particular embodiment, the diacyl lipid is a phospholipid, a glycolipid, a sphingolipid, or a combination thereof.

Preferably, the lymph node targeting conjugate comprises a lipid of 8 or more carbon units in length. It is believed that the number of myogenic lipid units can reduce the insertion of lipids into the plasma membrane of the cell, thereby leaving the lipid conjugate free to bind albumin and transport to lymph nodes.

For example, the lipid may be a diacyl lipid consisting of two C18 hydrocarbon tails.

In certain embodiments, the lipid used to prepare the lymph node targeting lipid conjugate is not a single chain hydrocarbon (e.g., C18) or cholesterol. Cholesterol conjugates have been studied to enhance the immunomodulation of molecular adjuvants (such as CPG) and the immunogenicity of peptides, but cholesterol conjugates that bind in combination with lipoproteins but poorly bind albumin show poorer lymph node targeting and lower immunogenicity in vaccines compared to optimal target protein binding conjugates.

Molecular adjuvant

In certain embodiments, the lipid oligonucleotide conjugates are used in vaccines. Oligonucleotide conjugates typically contain an immunostimulatory oligonucleotide.

In certain embodiments, the immunostimulatory oligonucleotides may serve as ligands for Pattern Recognition Receptors (PRRs). Examples of PRRs include Toll-like family signal molecules that play a role in the innate immune response and also affect later and more antigen-specific adaptive immune responses. Thus, the oligonucleotides may be used as ligands for Toll-like family signalling molecules, such as Toll-like receptor 9(TLR 9).

For example, unmethylated CpG sites can be detected on human plasmacytoid dendritic cells and B cells by TLR9 (Zaida et al, Infection and Immunity,76(5):2123-2129, (2008)). Thus, the sequence of the oligonucleotide may include one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) but also a nucleotide motif. "p" refers to the phosphodiester backbone of DNA, and as discussed in more detail below, some CG-containing oligonucleotides may have modified backbones, such as Phosphorothioate (PS) backbones.

In certain embodiments, the immunostimulatory oligonucleotide may comprise more than one CG dinucleotide, either arranged consecutively or separated by intervening nucleotides. The CpG motif may be internal to the oligonucleotide sequence. Many nucleotide sequences stimulate TLR9 with the number and location of CG dinucleotides and the precise base sequence flanking the CG dinucleotides.

Generally, CG ODNs are classified according to their sequence, secondary structure, and impact on human Peripheral Blood Mononuclear Cells (PBMCs). These five classes are class A (type D), class B (type K), class C, class P and class S (Vollmer, J & Krieg, A M, Advanced drug delivery reviews 61(3): 195-. CG ODNs can stimulate the production of type I interferons (e.g., IFN α) and induce Dendritic Cell (DC) maturation. Some classes of ODNs are also strong activators of Natural Killer (NK) cells through indirect cytokine signaling. Some classes are strong stimulators of human B-cell and mononuclear maturation (Weiner, G L, PNAS USA 94(20):10833-7 (1997); Dalpke, A H, Immunology 106(1):102-12 (2002); Hartmann, G, J of Immun.164(3):1617-2(2000), each of which is incorporated herein by reference).

According to some embodiments, lipophilic CpG oligonucleotide conjugates may be used to enhance immune responses to antigens. lipophilic-CpG oligonucleotides are described by, wherein "L" is a lipophilic compound, e.g. a diacyl lipid, "Gn" is a guanine repeat linker and "n" represents 1, 2, 3, 4 or 5.

5'-L-G_nTCCATGACGTTCCTGACGTT-3'

Other PRR Toll-like receptors include TLR3 and TLR7, which recognize double-stranded RNA, single-stranded and short double-stranded RNA, respectively, and retinoic acid-inducible gene I (RIG-1) -like receptors, i.e., RIG-I and melanoma differentiation-associated gene 5(MDA5), the most well known of which are RNA-inducible receptors in the cytoplasm. Thus, in certain embodiments, the oligonucleotide comprises a functional ligand for TLR3, TLR7, or a RIG-I like receptor, or a combination thereof.

Examples of immunostimulatory oligonucleotides, as well as methods of preparation, are known in the art, see, e.g., Bodera, p.recent Pat infllam Allergy Drug discov.5(1):87-93(2011), which is incorporated herein by reference.

In certain embodiments, the oligonucleotide cargo comprises two or more immunostimulatory sequences.

The length of the oligonucleotide can be between 2-100 nucleotide bases, which includes, for example, 5 nucleotide bases in length, 10 nucleotide bases in length, 15 nucleotide bases in length, 20 nucleotide bases in length, 25 nucleotide bases in length, 30 nucleotide bases in length, 35 nucleotide bases in length, 40 nucleotide bases in length, 45 nucleotide bases in length, 50 nucleotide bases in length, 60 nucleotide bases in length, 70 nucleotide bases in length, 80 nucleotide bases in length, 90 nucleotide bases in length, 95 nucleotide bases in length, 98 nucleotide bases in length, 100 nucleotide bases in length, or more.

The 3 'end or 5' end of the oligonucleotide may be conjugated to a polar block or lipid. In certain embodiments, the 5' end of the oligonucleotide is linked to a polar block or a lipid.

The oligonucleotide may be a DNA or RNA nucleotide, which typically includes a heterocyclic base (nucleobase), a sugar moiety linked to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl functionality of the down moiety. The main naturally occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as heterocyclic bases, as well as ribose or deoxyribose through phosphodiester linkages. In certain embodiments, the oligonucleotide consists of a nucleotide analog that has been chemically modified to improve stability, half-life or specificity or affinity for a target receptor relative to a DNA or RNA counterpart. Chemical modifications include chemical modifications of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide having one or more chemical modifications to the composition of a heterocyclic base, sugar moiety, or phosphate moiety. In certain embodiments, the modified nucleotide has a reduced charge as compared to a DNA or RNA oligonucleotide of the same nucleobase sequence. For example, the oligonucleotide may have a low negative charge, no charge, or a positive charge.

Typically, nucleoside analogs support bases that are capable of hydrogen bonding with a base in a standard polynucleotide by Watson-Crick base pairing, wherein the analog backbone provides the bases (e.g., single-stranded RNA or single-stranded DNA) in a manner that allows hydrogen bonding between the oligonucleotide analog molecule and the bases in the standard polynucleotide in a sequence-specific manner. In certain embodiments, the analogs have a substantially uncharged phosphorus-containing backbone.

Peptide antigens

Peptide conjugates suitable for use in the methods disclosed herein generally include antigenic proteins or polypeptides, such as tumor-associated antigens or portions thereof.

The peptide can be 2-100 amino acids, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide may be greater than 50 amino acids. In some embodiments, the peptide may be greater than 100 amino acids.

The protein/peptide is linear, branched or cyclic. The peptide may include D amino acids, L amino acids, or a combination thereof. The peptide or protein may be conjugated to the polar block or lipid at the N-terminus or C-terminus of the peptide or protein.

The protein or polypeptide may be any protein or peptide that can induce or increase the immune system to produce antibodies and T cell responses to the protein or peptide. Cancer antigens are antigens that are generally preferentially expressed by cancer cells (i.e., they are expressed at a higher level in cancer cells than on non-cancer cells), and in some cases, by cancer cells alone. The cancer antigen may be expressed within or on the surface of a cancer cell. The cancer antigen can be, but is not limited to, TRP-1, TRP-2, MART-1/Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC) -C017-1A/GA 733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, Prostate Specific Antigen (PSA), PSA-1, PSA-2, PSA-3, Prostate Specific Membrane Antigen (PSMA), T cell receptor/CD 3 zeta chain, and CD 20. The cancer antigen may be selected from the following: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-05, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, MAGE-1, MAGE-72, MAGE-1, MAGE, Beta-catenin, gamma-catenin, P120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, P15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus protein, Smad tumor antigen family, lmp-1, P1A, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 or c-erbB-2. Other cancer antigens include tumor antigens as described herein.

Suitable antigens are known in the art and can be obtained from commercial government and scientific sources. In certain embodiments, the antigen is an intact inactivated or irradiated tumor cell. The antigen may be a purified or partially purified polypeptide derived from a tumor. The antigen may be a recombinant polypeptide produced by expressing the DAN encoding the polypeptide antigen in a heterologous expression system. The antigen may be DNA encoding all or part of the antigenic protein. The DNA may be in the form of vector DNA, for example plasmid DNA.

In certain embodiments, the antigens may be provided as a single antigen or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids.

Polar block/linker

In order to effectively transport the conjugate to the lymph nodes, the conjugate should remain soluble. Thus, a polar block linker is included between the cargo and the lipid to increase the solubility of the conjugate. The polar block reduces or prevents the ability of the lipid to insert into the plasma membrane of a cell, such as a cell in a tissue adjacent to the injection site. The polar block may also reduce or prevent the ability of the cargo, e.g., synthetic oligonucleotides containing a PS backbone to non-specifically bind extracellular matrix proteins at the site of administration. The polar block increases the solubility of the conjugate without preventing its ability to bind to albumin. It is believed that the composition of this character allows the conjugate to bind to albumin present in serum or interstitial fluid and maintain circulation until albumin is transported to or retained in the lymph nodes.

The length and composition of the polar block can be adjusted depending on the lipid and cargo selected. For example, for oligonucleotide conjugates, the oligonucleotide itself may be sufficiently polar to ensure solubility of the conjugate, e.g., an oligonucleotide of 10, 15, 20, or more nucleotides in length. Thus, in certain embodiments, no additional dosage form block linker is required. However, depending on the amino acid sequence, some lipidated peptides may be substantially insoluble. In these cases, it may be desirable to include a polar block that mimics the action of the polar oligonucleotide.

The polar block is used as part of any lipid conjugate suitable for use in the methods of the present disclosure, e.g., lipid oligonucleotide conjugates and lipid peptide conjugates, which reduce cell membrane insertion/preferential partitioning on albumin. Suitable polar blocks include, but are not limited to, oligonucleotides such as those discussed above, hydrophilic polymers including, but not limited to, polyethylene glycol (MW: 500Da to 20,000Da), polyacrylamide (MW: 500Da to 20,000Da), polyacrylic acid; a string of hydrophilic amino acids, such as serine, acid, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or combinations thereof including, but not limited to, dextran (MW: 1,000Da to 2,000,000Da), or combinations thereof.

The hydrophobic lipid and the linker/cargo are covalently linked. The covalent bond is a non-cleavable bond or a cleavable bond. Non-cleavable bonds include amide or phosphate bonds, and cleavable bonds include disulfide bonds, acid-cleavable bonds, ester bonds, anhydride bonds, biodegradable bonds, or enzymatically cleavable bonds.

a. Ethylene glycol linker

In certain embodiments, the polar block is one or more Ethylene Glycol (EG) units, preferably two or more EG units (e.g., polyethylene glycol (PEG)). For example, in certain embodiments, a peptide conjugate comprises a protein or peptide (e.g., a peptide antigen) and an amphiphilic lipid linked by a polyethylene glycol (PEG) molecule or derivative or analog thereof.

In certain embodiments, protein conjugates suitable for use in the methods of the present disclosure comprise a protein conoton linked to PEG, which in turn is linked to a hydrophobic lipid or lipid-Gn-ON conjugate, either covalently or through formation of a protein-oligonucleotide conjugate that is hybridized to an oligomicelle. The exact number of EG units depends on the lipid and cargo, however, typically the polar block may have from about 1 to about 100, from about 20 to about 80, from about 30 to about 70, or from about 40 and about 60 EG units. In certain embodiments, the polar block has from about 45 to about 55 EG units. For example, in certain embodiments, the polar block has 48 EG units.

b. Oligonucleotide linker

As described above, in certain embodiments, the polar block is an oligonucleotide. The polar block linker may have any sequence, for example the oligonucleotide sequence may be a random sequence, or a sequence specifically selected for its molecular or biochemical properties (e.g. high polarity). In certain embodiments, the polar block linker comprises one or more series of consecutive adenines (a), cytosines (C), guanines (G), thymines (T), uracils (U), or analogs thereof. In certain embodiments, the polar block linker consists of a series of consecutive adenines (a), cytosines (C), guanines (G), thymines (T), uracils (U), or analogs thereof.

In certain embodiments, the linker is one or more guanines, for example between 1-10 guanines. It has been found that altering the number of guanines between cargo, such as CpG oligonucleotides, and the lipid tail in the presence of serum proteins controls micelle stability. Thus, the amount of guanine in the linker can be selected based on the desired affinity of the conjugate for a serum protein, such as albumin. When the cargo is a CpG immunostimulatory oligonucleotide and the lipid tail is a diacyl lipid, the amount of guanine affects the ability of micelles formed in aqueous solution in the presence of serum to dissociate: 2 0% of unstable micelles (lipo-G)₀T₁₀CG) was intact, while the remaining 80% of the micelles were destroyed and bound to the FBS component. The percentage of intact micelles in the presence of guanine was from 36% (lipo-G)₂T⁸-CG) increased to 73% (lipo-G)₄T₆-CG) and finally 90% (lipo-G)₆T₄-CG). The number of guanines increased to eight (lipo-G)₈T₂-CG) and ten (lipo-G)₁₀T₀-CG) does not further improve the micelle stability.

Thus, in certain embodiments, the linker in a targeted lymph node conjugate suitable for use in the methods of the present disclosure may include 0, 1, or 2 guanines. As discussed in more detail below, linkers comprising 3 or more consecutive guanines can be used to form micelle-stable conjugates having properties suitable for use in the methods of the present disclosure.

B. Immunogenic compositions

Conjugates suitable for use in the methods of the present disclosure may be used in immunogenic compositions or as components in vaccines. Generally, the immunogenic compositions disclosed herein include an adjuvant, an antigen, or a combination thereof. The combination of adjuvant and antigen may be referred to as a vaccine. When administered in combination to a subject, the adjuvant and antigen may be administered in separate pharmaceutical compositions, or they may be administered together in the same pharmaceutical composition. When administered in combination, the adjuvant may be a lipid conjugate, the antigen may be a lipid conjugate, or both the adjuvant and the antigen may be lipid conjugates.

Immunogenic compositions suitable for use in the methods of the present disclosure include lipid conjugates, which are antigens, such as antigenic polypeptide-lipid conjugates, administered alone or in combination with an adjuvant. Adjuvants may be, but are not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from bark of the saponaria tree, such as QS21 (glycolipids eluting in the 21 st peak by HPLC fractionation; antibiotics, inc., Worcester, Mass.); poly (bis (carboxyphenoxy) phosphazene) (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulatory complex comprising mixed saponins, lipids and forming Virus-sized particles with pores that can accommodate antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4(SmithKline Beecham adjuvant System #4 comprising alum and MPL; SBB, Belgium), micelle-forming nonionic block copolymers such AS CRL 1005 (these comprise linear chains of hydrophobic polyoxypropylene, pendant chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide 1312 (e.g., IMS, binding of aqueous nanoparticles to soluble immunostimulants, Seppic).

The adjuvant may be a TLR ligand, such as those discussed above. Adjuvants that act through TLR3 include, but are not limited to, double stranded RNA. Adjuvants that act through TLR4 include, but are not limited to, derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont) and pyrimidine dipeptides (MDP; Ribi) and threonyl-pyrimidine dipeptides (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, OM Pharma SA, Switzerland). Adjuvants that act through TLR5 include, but are not limited to, flagellin. Adjuvants that act through TLR7 and/or TLR8 include single stranded RNA, Oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazolinamines (e.g., imiquimod (R-837), rasimod (R-848)). Adjuvants that act through TLR9 include DNA of viral or bacterial origin, or synthetic Oligodeoxynucleotides (ODNs), such as CpG ODNs. Another class of adjuvants are phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbones.

The adjuvant is selected from oil emulsions (e.g., Freund's adjuvant); a saponin preparation; virosomes and virus-like particles; bacterial and microbial derivatives; an immunostimulatory oligonucleotide; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing ingredients (e.g., mineral salts such as aluminum and calcium salts, hydroxides, phosphates, sulfates, and the like); a bioadhesive and/or mucoadhesive; microparticles; a liposome; polyoxyethylene ethers and polyoxyethylene ester formulations; polyphosphazene; a murine amido peptide; an imidazoquinolone compound; and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

Adjuvants may also include immunomodulators, such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor, and tumor necrosis factor.

Reagent kit

In some aspects, the disclosure provides kits comprising at least an immunomodulatory fusion protein described herein and instructions for use. In some embodiments, the kit comprises, in a suitable container, the immunomodulatory fusion protein, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art. In some embodiments, the kit further comprises instructions for use in combination with immunotherapy.

In some embodiments, the container is at least one vial, well, test tube, flask, bottle, syringe, or other container device into which the amphiphilic ligand conjugate is placed, and in some cases, suitably aliquoted. When other components are provided, the kit may comprise a container for the other antigen to place the compound. The kit may also include a device for containing the amphiphilic ligand conjugate, as well as any other reagent containers that are hermetically sealed for commercial sale. Such containers may include injection or blow molded plastic containers that retain the desired vials therein. The container and/or kit may include a label with instructions and/or warnings for use.

In some embodiments, the disclosure provides a kit comprising a container comprising an immunomodulatory fusion protein described herein, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of a composition for treating or delaying progression of cancer in an individual receiving immunotherapy (e.g., CAR-T cells, cancer vaccines, anti-tumor associated antigen antibodies, and/or immune checkpoint blockade).

In some embodiments, the disclosure provides a kit comprising a medicament comprising an immunomodulatory fusion protein described herein, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament alone or in combination with an immunotherapy (e.g., CAR-T cells, cancer vaccines, anti-tumor associated antigen antibodies, and/or immune checkpoint blockade) to treat or delay progression of cancer in an individual receiving CAR-T cell therapy.

Other embodiments-collagen binding IgG binding fusion proteins

In another aspect, the present disclosure provides a collagen-binding IgG-binding fusion protein comprising an Ig-binding domain and a collagen-binding domain. The collagen-binding IgG-binding fusion proteins provided by the present disclosure bind to IgG (e.g., immunomodulatory IgG) and collagen, thereby localizing and sequestering the IgG in the tumor when administered.

In some embodiments, the collagen binding domain is a collagen binding domain as described above. Exemplary IgG binding domains include the dimerization Z domain (one of the five IgG binding domains of protein A, referred to herein as "ZZ") (Jendeberg et al, (1995) J Mol Recognit 8: 270-942), the dimerization IgG binding domain of protein G (referred to herein as "SpG 2") (Jung et al, (2009) Anal Chem 81: 936-942), IgG binders isolated from Sso7d yeast display libraries (Gera et al, (2011) J Mol Biol 409: 601-616), IgG binders isolated from a fibronectin type III domain (Fn3) yeast display library (Hackel et al, (2010) J Mol Biol 401: 84-96) and two small peptides designed to bind to the IgG Fc region (referred to herein as "Fc-III-4C" and "RRGW") (Gong et al, (2015) bioconjugateg Chem 27: 1569-15720143; Tsai et al, (Anal Chem 86: 2931-2938).

In some embodiments, the collagen-binding IgG-binding fusion protein is suitable for use in any of the methods described herein. In some embodiments, the collagen-binding IgG-binding fusion protein is used in combination with a therapeutic antibody (e.g., an immunomodulatory antibody). In some embodiments, the collagen-binding IgG-binding fusion protein is administered in combination with a therapeutic antibody for the treatment of cancer.

Examples

While the disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to fall within the scope of this disclosure.

Example 1: recombinant expression of collagen binding fusion proteins in mammalian cells

To evaluate the ability to express collagen-binding immunomodulatory molecules in mammalian cells, five His-tagged collagen-binding polypeptides fused to gaussian luciferase (Gluc) (collagen imaging probe (CNA35-Gluc)), bacterial collagenase ColG domain s3a/s3b (ColG s3a/s3b-Gluc), heparin-binding domain of mouse placental growth factor-2 (PLGF2HBD-Gluc), bacterial collagenase ColH domain s3(ColH s3-Gluc), and mouse protein-based membrane glycan (basement membrane glycan-Gluc) were transiently expressed in human embryonic kidney 293(HEK293) cells. The amino acid sequences of these constructs are shown in SEQ ID NO: 128-132. Briefly, HEK293 cells (density 100 ten thousand cells/mL) were transfected with polyethyleneimine (2 mg per liter of cell culture) in OptiPro serum-free medium (20 mL per liter of cell culture) (Thermo Fisher) using sterile-filtered plasmid DNA (1 mg per liter of cell culture). TA99 was purified as described previously using rProtein A Sepharose Fast Flow resin (GE Healthcare) (Zhu et al, 2015). His-tagged proteins were isolated from HEK293 supernatants using TALON Metal affinity resin (Takara Bio Inc.). Then, by size exclusion chromatography using a HiLoad 16/600Superdex 200pg column The cytokine fusion protein was further purified on a FPLC system (GE Healthcare) that had been pretreated with 1M NaOH for 4 hours to remove endotoxins and subsequently equilibrated in sterile pbs (corning). After purification, all proteins were buffer-crossedChange to sterile PBS (Corning), sterile filter 0.2 microns (Pall Corporation) and confirm that it contained minimal endotoxin levels (per injection) using the chromogenic LAL assay (Lonza)<0.1 EU). To confirm their molecular weight, the Protein Novex Prestained Sharp Protein Ladder was run together on 4-12% NuPAGE Bis-Tris Protein gel (Life Technologies) with 1% MES running buffer. All proteins were stored at 4 ℃, but prior to therapeutic injection, cytokine fusion proteins were rewarmed to room temperature to salvage basement membrane glycans that showed reversible cold denaturation. After size exclusion chromatography of the talen-purified eluate, the relative expression level of His-tagged collagen-binding fusion protein in the resulting eluate was evaluated by SDS-PAGE and absorbance spectrophotometry.

Transient expression of the separate Gluc (19.8kDa) and Gluc-fused collagen-binding polypeptides ColG s3a/s3b-Gluc (46.1kDa), ColH s3-Gluc (32.8kDa) and basement membrane glycan-Gluc (56.6kDa) was achieved in HEK293 cells as determined by SDS-PAGE analysis of purified His-tagged proteins from HEK cell lysates. Protein staining at or near the respective expected molecular weight of each fusion protein was observed (data not shown). However, no expression of CNA35-Gluc (54.4kDa) or PLGF2HBD-Gluc (22.8kDa) was observed as measured by SDS-PAGE of purified proteins from HEK cell lysates (data not shown).

Transient expression of Gluc, ColG s3a/s3b-Gluc, ColH s3-Gluc, and basement membrane glycan-Gluc was achieved in HEK293 cells as measured by absorption spectrophotometry during size exclusion chromatography of recombinantly expressed and purified His-tagged protein. For each fusion protein, a monomeric peak in UV radiation absorption was observed at 280nm (a280) (data not shown). No absorption peaks were detected for CNA35-Gluc or PLGF2 HBD-Gluc.

In addition to the basement membrane glycans, other mammalian collagen binding polypeptides can be expressed as collagen binding fusion proteins. Extracellular domains of mammalian leukocyte-associated immunoglobulin-like receptor 1(LAIR-1) proteins are known to bind collagen (Lebbink et al, (2006) J Exp Med 203(6): 1419-. Transient expression of the His-tagged mouse LAIR-1 extracellular domain (amino acid sequence as shown in SEQ ID NO: 181) in HEK293 as determined by absorption spectrophotometry during size exclusion chromatography of the recombinantly expressed protein has been achieved as described above. LAIR-1 eluted as a single peak as measured by UV radiation absorption at 280nm (a280) (data not shown).

These results indicate that collagen binding fusion proteins comprising prokaryotic or mammalian collagen binding polypeptides are expressed in mammalian cells. The expression of His-tagged collagen-binding fusion proteins, ColG s3a/s3b-Gluc, ColH s3-Gluc, and basement membrane glycan-Gluc, as well as Gluc alone, was achieved in HEK293 cells, but no expression of CNA35-Gluc or PLGF2HBD-Gluc was observed. In addition, the extracellular domain of His-tagged LAIR-1 was expressed in HEK293 cells and purified. These results indicate that collagen-binding immunomodulatory molecules comprising prokaryotic or mammalian collagen-binding polypeptides (e.g., ColG s3a/s3b, ColH s3, basement membrane glycans, or LAIRs) will be expressed in mammalian cells.

Example 2: recombinant collagen binding fusion proteins bind collagen in vitro

To evaluate the ability of collagen to bind to immunomodulatory molecules to collagen, collagen-binding fusion proteins expressed and purified as described in example 1 were tested for their ability to bind to type I collagen and collagen IV coated plates by ELISA. Briefly, type I collagen (Gibco) and type IV collagen (Corning) coated 96-well plates were blocked for 1 hour at room temperature using PBS + 0.1% wt/vol Bovine Serum Albumin (BSA) + 0.05% wt/vol tween 20(PBSTA), and then incubated for 3 hours at room temperature using PBSTA containing various concentrations of basement membrane glycans. The basement membrane glycans were preheated at 37C for 10 minutes to reverse their low temperature mediated denaturation. The wells were washed with PBSTA and then incubated with horseradish peroxidase conjugated polyclonal anti-6 xHis (ab1187, Abcam) diluted 1:4000 in PBSTA for 1 hour at room temperature. The wells were washed again with PBSTA and then treated for 10min with 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) followed by 1M sulfuric acid to stop the color reaction. The absorbance at 450nm was measured using a M1000 microplate reader (Tecan) (corrected using the reference absorbance at 570 nm). Wells titrating MSA served as negative controls.

Purified His-tagged collagen-binding fusion proteins, basement membrane glycan-Gluc, ColG s3a/s3b-Gluc and ColH s3-Gluc, were evaluated on collagen type I coated plates by ELISA. As shown in FIG. 1A, only the basal lamina polysaccharides-Gluc and ColG s3a/s3b-Gluc bind to type I collagen with KD's of 130nM and 139nM, respectively. ColH s3-Gluc did not have specific binding to type I collagen beyond the Gluc background binding.

Purified His-tagged collagen-binding fusion proteins, basement membrane glycan-Gluc, ColG s3a/s3b-Gluc and ColH s3-Gluc, were evaluated on collagen IV coated plates by ELISA. As shown in fig. 1B, the basal lamina glycan-Gluc binds to collagen type IV with KD 600 nM. ColG s3a/s3b-Gluc or ColH s3-Gluc did not have specific binding to type IV collagen beyond the Gluc background binding.

Purified His-tagged mouse LAIR-1 (denoted mLAIR1-His) and His-tagged biotinylated basement membrane glycan (denoted Lwt-His-b) were evaluated for binding to type I collagen as a function of concentration by ELISA. Binding was determined by ELISA using an anti-His antibody conjugated to horseradish peroxidase (HRP). As shown in fig. 1C, LAIR-1 and basement membrane glycans have similar binding affinities to type I collagen. Binding of plates blocked with bovine serum albumin was also assessed by ELISA. No binding was observed for either protein, indicating specific binding to type I collagen.

To further confirm the collagen binding activity of the mammalian collagen-binding polypeptides described above, the ability of the purified His-tagged LAIR-1 and the basement membrane glycan collagen-binding polypeptides described in example 1 to compete for binding to collagen was examined. Briefly, type I collagen (Gibco) coated 96-well plates were blocked for 1 hour at room temperature using PBS + 0.1% wt/vol Bovine Serum Albumin (BSA) + 0.05% wt/vol tween 20(PBSTA), and then incubated for 3 hours at room temperature using PBSTA containing various concentrations of LAIR in the presence of 50nM of biotinylated basement membrane glycan. The basement membrane glycans and LAIR were preheated at 37C for 10 minutes to reverse their denaturation mediated by low temperature. The wells were washed with PBSTA and then incubated with horseradish peroxidase conjugated polyclonal streptavidin-HRP diluted 1:400 in PBSTA for 1 hour at room temperature. The wells were washed again with PBSTA and then treated for 10min with 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) followed by 1M sulfuric acid to stop the color reaction. The absorbance at 450nm was measured using a M1000 microplate reader (Tecan) (corrected using the reference absorbance at 570 nm). Wells titrating MSA served as negative controls. Results of the competition ELISA assay for basement membrane glycan binding in the presence of different concentrations of LAIR on collagen coated plates are shown in figure 1D.

To evaluate the stability of the purified collagen-binding polypeptide, the collagen-binding activity of the basement membrane glycan was examined after thawing from the solution frozen state. Briefly, type I collagen affinity (K) of the basement membrane glycans incubated with various excipients (trehalose (T), bsa (b), collagen (C), protein (P) only) under different conditions after thawing (K)_D): 3 weeks at 37C (blue), 3 weeks at 4C (grey), 2 weeks at 4C followed by 1 week at 37C (red). As described previously, the binding affinity of the basement membrane glycans to type I collagen in the presence of excipients (trehalose, bovine serum albumin and collagen) after freeze thawing was determined by ELISA. Binding affinity (K) despite the use of dressing_D) Still similar. However, if the basement membrane glycans were not rewarming to 37 ℃ prior to measurement, the binding affinity decreased by about 50-fold (data not shown).

These results show the binding of collagen binding fusion proteins comprising a prokaryotic (e.g., ColG s3a/s3b) or mammalian (e.g., basement membrane glycan) collagen binding polypeptide to type I collagen (fig. 1A). These results also indicate that collagen-binding fusion proteins comprising mammalian collagen-binding polypeptides (e.g., basement membrane glycans) bind to both type I collagen and type IV collagen (fig. 1B). Furthermore, these results indicate that the extracellular domain of LAIR-1 competes with basement membrane glycans for binding to type I collagen (fig. 1C, 1D). These results suggest that collagen-binding immunomodulatory molecules comprising prokaryotic or eukaryotic collagen-binding polypeptides (e.g., ColG s3a/s3b, basement membrane glycans, and LAIR-1) will bind to collagen.

Example 3: the recombinant collagen-binding fusion protein is retained after intratumoral injection

Type I and type IV collagen are components of thick fibrotic sacs surrounding tumor and perivascular basement membranes, respectively. To evaluate the expression of type I and type IV collagen in mice, mice were inoculated with 1 million 4T1, MC38, or B16F10 tumor cells on day 0. Tumors were excised on day 10 and fixed in 10% neutral buffered formalin overnight, then embedded in paraffin and cut into 5 micron thick sections (CryoStar NX 70). Immunohistochemistry was used to assess the presence of type I and type IV collagen in tumor sections. Briefly, Tris buffer salt containing 0.1% vol/vol tween-20 was used as described in 1: slices were stained with 500 dilutions of rabbit antibodies against collagen type I (ab34710, Abcam) and collagen type IV (ab6586, Abcam) followed by secondary staining using goat HRP conjugated anti-rabbit antibodies (ab6721, Abcam). Positive staining was observed for collagen type I (upper) and collagen type IV (lower) in sections of 4T1 (left), MC38 (middle) and B16F10 (right) tumors (data not shown). Therefore, type I collagen and type IV collagen are abundantly expressed in many syngeneic mouse tumor models.

To evaluate the intratumoral retention of collagen-binding immunomodulatory molecules, the ability of collagen-binding fusion proteins (basement membrane glycan-Gluc and ColG s3a/s3b-Gluc) bound to type I collagen in example 2 to maintain binding at the intratumoral injection site was examined by in vivo fluorescence imaging. Briefly, 5x10⁵Individual 4T1 cells (mouse mammary tumor cells) were injected into the mammary fat pad of BALB/c mice followed by intratumoral injection of Gluc alone, GolG s3a/s3b-Gluc or basisan-Gluc on day 7 after tumor cell injection, each mouse being monitored by in vivo bioluminescence imaging (epi-illumination, auto exposure setup).

Bioluminescent signals from mice injected with Gluc only dropped to background levels approximately 36hr after injection (data not shown). Signals from mice injected with ColG s3a/s3b-Gluc dropped to background levels approximately 8.5 days after injection (data not shown). During the experiment, the signal from mice injected with the basement membrane glycan-Gluc did not decrease below background levels (about 16.5 days after injection, data not shown).

These results indicate that, over time, the collagen-binding fusion proteins, ColG s3a/s3b-Gluc and the basement membrane glycan-Gluc, are physically retained at the intratumoral injection site. These results suggest that collagen-binding immunomodulatory molecules comprising prokaryotic or mammalian collagen-binding polypeptides (e.g., ColG s3a/s3b or basement membrane glycans) will exhibit intratumoral retention and limited systemic dissemination.

Example 4: the intratumoral retention of collagen-binding fusion proteins depends on molecular weight and collagen-binding activity

Several factors may determine the intratumoral retention of collagen-binding fusion proteins: affinity for collagen, collagen concentration, size-dependent escape by diffusion or convection, and protein turnover. As a protein below 60kDa, the basement membrane glycan (37kDa) has high permeability across the vascular endothelium and is readily absorbed into the circulatory system, which may help in distribution from the injection site and reduce intratumoral retention (McLennan et al, (2005) Drug Discov Today Technol 2: 89-96; Egawa et al, (2013) Sci Rep 3: 1932).

To evaluate the effect of molecular weight on the intratumoral retention and systemic distribution of collagen-binding immunomodulatory molecules, the retention of collagen-binding polypeptide-based peptidoglycan fused to 67kDa Mouse Serum Albumin (MSA) protein after intratumoral injection was determined by in vivo fluorescence imaging. Briefly, to inoculate B16F10-Trp2KO tumors, 10 suspended in 50uL sterile PBS^{6 are}Cells were injected subcutaneously into the right flank of C57BL/6 female mice. Fusion proteins were labeled with a 5 molar excess of Alexa Fluor 647NHS ester (Life Technologies) in PBS adjusted to pH8 for 30 min. Excess dye was removed using a PD10 desalting column (GE Healthcare) and the degree of labelling (DOL) of each protein was calculated. The proteins compared in the retention study contained equimolar amounts of dye. For the basement membrane glycan-MSA (SEQ ID NO:126) and MSA (SEQ ID NO:183), 0.11nmol of each construct (110 ug of basement membrane glycan was administered) was added 5 days after inoculation MSA and 71.7ug MSA) were injected intratumorally into B16F10-Trp2KO tumor-bearing mice.

To assess the retention of the fusion protein, mice were imaged with a Xenogen IVIS imaging system 100(Xenogen) under an auto-exposure epi-illumination fluorescence setting at the indicated time points. During this period, the mice were kept on casein feed without alfalfa (test diet) to minimize gastrointestinal background fluorescence. The total radiation efficiency was determined using a Living Image (Caliper Life Sciences) Image analyzer. B16F10 cells lacking tyrosinase-related protein 2, B16F10-Trp2KO, were used to inoculate non-pigmented tumors to maximize fluorescence signal-to-noise ratio.

Fluorescently labeled basement membrane glycan, basement membrane glycan-MSA or MSA is injected intratumorally and the total radiation efficiency of the mice is monitored over time. Although the basement membrane glycans had a smaller molecular weight, the fluorescence signal from the basement membrane glycans (37kDa) was retained to a greater extent than the fluorescence signal from MSA (67kDa) during the first 5 hours after injection (data not shown). These data suggest that intratumoral retention of basement membrane glycans depends at least in part on their collagen binding activity. The fluorescence signal from the basement membrane glycan-MSA also had a greater retention than the fluorescence signal from MSA (fig. 2A). The basement membrane glycan-MSA (104kDa) is significantly larger than the basement membrane glycan (37kDa), and although each has the same collagen binding site, the degree of retention of the basement membrane glycan-MSA is higher than the basement membrane glycan alone (fig. 2A), probably due to the faster clearance rate of smaller basement membrane glycan constructs.

To further evaluate the effect of systemic distribution on molecular weight, the amount of fluorescence over time from sera from mice injected with fluorescently labeled basement membrane glycans fused to MSA was determined. Fluorescently labeled MSA was used as a positive control. To assess serum fluorescence after injection, small amounts of blood (<10uL) were drawn by capillary action from the tail tip into glass microplasminoplasty heparin-coated tubes (VWR) at the indicated time points. The tube was sealed at one end using a parafilm and placed upright at 4C protected from light to allow the serum to separate from the clotted blood under the force of gravity. Tubes were scanned using a flat panel Typhoon Trio variable mode imager (GE Healthcare) (excitation laser: 633 nm; emission filter: 670 BP; PMT: 450-.

As shown in figure 2B, serum from mice injected with basement membrane glycan-MSA produced a lower fluorescence signal over time compared to serum from mice injected with MSA (as a percentage of the injected dose). These results indicate that the basement membrane glycan-MSA shows a lower systemic distribution than MSA alone.

Co-localization of basement membrane glycans with collagen types I and IV was assessed following intratumoral administration by immunofluorescence imaging. Briefly, B16F10 tumors were excised after intratumoral injection of fluorescently labeled basement membrane glycans. The tumors were stored overnight in periodate-lysine-paraformaldehyde at 4 ℃, then cryo-protected with 30% sucrose in PBS for 8 hours at 4 ℃, and then slowly frozen in a cryogenic mold containing 100% OCT compound (Sakura Finetek USA Inc.) on dry ice. Frozen tumor molds were cut into 50 micron thick sections (CryStar NX70), the sections were dried at room temperature for one hour, and then antigen repaired. For antigen retrieval, sections were heated in 10mM sodium citrate with 0.05% tween 20 for 1 hour at 60 ℃, washed in PBS, and then treated with 2mg/mL hyaluronidase for 30 minutes at room temperature. Sections were washed in immune mixture (immix) (PBS with 0.2% wt/vol bovine serum albumin, 0.05% wt/vol sodium azide, 0.3% vol/vol Triton X-10, 10% vol/vol donkey serum), then permeabilized with cold acetone at-20 ℃ for 10 minutes, then blocked in immix at room temperature for 1 hour. Rabbit antibodies against collagen type I (ab34710, Abcam) and collagen type IV (ab6586, Abcam) diluted 1:200 in immix were used to stain overnight in a humidified chamber at room temperature. After several PBS washes, sections were stained in immix with AF 488-conjugated goat anti-rabbit secondary antibody (diluted 1: 500) overnight in a moisture box at room temperature. After several washes, the sections were fixed in PBS containing 1% neutral formalin buffer and in VECTASHIELD anti-fade fixing medium. The sections were imaged by confocal microscopy. Superposition of the fluorescent signals of type I or type IV collagen confirmed that the degree of co-localization of the basement membrane glycans with type I and type IV collagen in B16F10 tumors was high (data not shown).

These results indicate that both affinity for collagen and increased molecular weight contribute to intratumoral retention and systemic distribution of collagen-binding fusion proteins. These results suggest that increased affinity for collagen and increased collagen binding immunomodulatory molecular weight will increase intratumoral retention and decrease systemic distribution, thereby increasing therapeutic effect.

Example 5: proximity of bound collagen decreases the payload activity of collagen-binding fusion proteins

To evaluate the effect of collagen binding on payload activity, intratumoral bioluminescence was compared in vivo between the basal lamina glycan-Gluc and ColG-Gluc. Briefly, equimolar (0.3nmol) basement membrane glycan-Gluc or GolG-Gluc tumors were injected intratumorally into B16F10 melanoma-bearing mice. Despite equimolar (0.3nmol) injection, lower intratumoral bioluminescence was observed with the basileman-Gluc compared to ColG-Gluc. Specifically, 4/5 mice injected with ColG-Gluc had detectable bioluminescence above background, while only 1/5 mice injected with basilar glycan-Gluc had detectable bioluminescence above background (data not shown). Interestingly, bioluminescent signals were only detected outside the tumor, presumably from constructs that leaked out of the tumor. The lack of bioluminescent signals in tumors from collagen binding constructs suggests that the enzyme does not function optimally when forced against collagen. This result indicates that a spacer protein (e.g., MSA) is required to help better separate collagen from future payloads.

Since collagen is an insoluble protein and thus susceptible to solid phase behavior (e.g., surface adsorption of proximal soluble proteins), MSA acts as a large hydrophilic spacer between the payload and the basement membrane glycans to protect the payload from adsorption and functional disruption. For our localization method, any soluble payload is forced by the basement membrane glycans into the solid-liquid interface and adsorbed onto the collagen fibers, possibly rendering them functionally inert (as seen with Gluc). To prevent adsorption of the payload to collagen, we operably linked Mouse Serum Albumin (MSA) to the basement membrane glycans as a large spacer between the payload and collagen.

Example 6: synergistic effect of immunomodulatory collagen binding molecules and anti-tumor antigen antibodies in mouse melanoma tumor model

The synergistic effect between anti-tumor antigen antibodies and interleukin-2 (IL-2) has been well characterized (Zhu et al, (2015) Cancer Cell 27: 489-501). The effect of IL-2 fusion to collagen binding molecules was evaluated by making IL-2 fused to the C-terminus of the basement membrane glycan-MSA. MSA was introduced to ensure steric entry of the receptor for IL-2 when bound to collagen fibers, and may also increase the molecular weight of the construct, thereby reducing the tumor's diffusional flux. As a positive control with equivalent biological activity, IL-2 was expressed as a fusion with MSA (MSA-IL2), thereby greatly separating the collagen-binding effect from the size-dependent improvement effect of small cytokines such as wild-type IL-2 on tumor retention. Thus, using the method described in example 1, IL-2 is expressed as fused to MSA alone or to the C-terminus of the basal-lamina glycan-MSA. The His-tagged cytokine fusion protein was purified by talen metal affinity data as described in example 1. The cytokine-fusion protein was then further purified by FPLC (AKTA, GE Healthcare) using a size exclusion chromatography column (HiLoad 16/600Superdex 200pg) that had been pretreated with 1M NaOH for 4 hours to remove endotoxins, followed by equilibration in sterile PBS. After purification, the protein buffer was exchanged to sterile PBS, sterile filtered on a 0.2 micron membrane filter (Pall Corporation), and confirmed to contain minimal endotoxin using chromogenic LAL assay (Lonza) (< 0.1EU per dose). The protein showed > 90% monomer expression when evaluated by size exclusion chromatography at 280nm by absorbance (data not shown). In addition, the ability of the protein to induce cell proliferation was evaluated. CTLL-2 cells were seeded at a density of 5000 cells/well on tissue culture plates coated with or without type IV collagen. Cells were stimulated for 48 hours with various concentrations of MSA-IL2, basement membrane glycan-MSA-IL 2, or basement membrane glycan. Cell proliferation was determined by WST-1 based colorimetric assay (Roche) according to the manufacturer's instructions. Although basement membrane glycans failed to cause proliferation, treatment with MSA-IL2 and basement membrane glycan-MSA-IL 2 resulted in similar levels of cell proliferation regardless of the presence of collagen type IV (data not shown). Thus, MSA-IL2 and the basement membrane glycan-MSA-IL 2 have equivalent biological activity.

In addition, serum levels of IL-2 fusion were quantified after intratumoral injection as described in example 4. Fluorescently labeled basement membrane glycan-MSA-IL 2 and MSA-IL2 were injected into B16F10-Trp2KO tumors and serum fluorescence levels were quantified at various time points post injection. The fluorescence signal from serum from mice injected with basement membrane glycan-MSA-IL 2 was lower over time when measured as a percentage of injected dose compared to the fluorescence signal from serum from mice injected with MSA-IL2 (data not shown). These results indicate that the basement membrane glycan-MSA-IL 2 binds to intratumoral collagen in vivo and shows a lower systemic distribution compared to MSA-IL2 alone.

To characterize the anti-tumor effectiveness of the combination of immunomodulatory collagen binding molecules and tumor antigen targeting antibodies, the synergistic effect of a fusion protein comprising a collagen binding polypeptide fused to cytokine IL-2 with a mouse monoclonal anti-TYRP 1 antibody (TY99) was evaluated in a B16-F10 melanoma model.

Briefly, 1 × 10 in 50 μ L sterile PBS⁶A B16-F10 mouse melanoma cell (ATCC) was injected subcutaneously into the right flank of a 6-8 week old C57BL/6 mouse. Mice with established B16-F10 melanoma tumors were treated by intraperitoneal (i.p.) injection of 100 μ g/dose systemically of anti-TYRP-1 antibody (TA99) and by intratumoral (i.tu.) injection of 13 μ g/dose of collagen-binding IL-2 fusion protein-based glycosaminoglycan-MSA-IL 2. Mice injected with MSA-IL2(SEQ ID NO:121) (9. mu.g/dose) or basement membrane glycan (SEQ ID NO:182) (4. mu.g/dose) served as positive controls. Mice injected with IL-2 fusion protein (either basement membrane glycan-MSA-IL 2(SEQ ID NO:120) or MSA-IL2) received IL-2 equivalent to 0.11 nmol/dose. At the end of euthanasia to pair Animals were euthanized, i.e. reduced by 20% of total body weight or tumor area over 100mm²(length x width).

The percent survival of tumor-bearing mice treated with MSA-IL2 alone, basement membrane glycan-MSA-IL 2, basement membrane glycan, or in combination with TA99 is shown in FIGS. 3A-3B. Mice given either basement membrane glycan-MSA-IL 2 or MSA-IL2 alone had limited survival benefit with single drug treatment (fig. 3A). The combination of TA99 with basement membrane glycans did not provide a survival benefit (fig. 3B), however, administration of a combination of TA99 with MSA-IL-2 or basement membrane glycan-MSA-IL 2 showed a synergistic survival benefit to mice (fig. 3B). The combination with the basal lamina glycan-MSA-IL 2 had a greater survival benefit compared to the combination with MSA-IL2 (fig. 3B).

After discontinuation of the treatment, several mice developed local skin discoloration or vitiligo, indicating a melanocyte-specific T cell response. Almost all mice injected with the basement membrane glycans MSA-IL2 and TA99 showed vitiligo plaques at the site of injection (16 out of 17 mice), whereas only one of the 17 mice treated with MSA-IL2 and TA99 showed this side effect. Taken together, these observations suggest that the basement membrane glycan-MSA-IL 2 anchors IL-2 within the tumor and enhances IL-2 synergy with TA99 by increasing anti-tumor T cell responses and overall survival.

To determine whether intratumoral injection of the basement membrane glycan-MSA-IL-2 was necessary to improve outcome when used in combination with TA99, we evaluated the effectiveness of this combination when the basement membrane glycan-MSA-IL 2 was injected to other contralateral sites. When the basal lamina glycan-MSA-IL 2 was administered paratumorally (peri. tu) (i.e., adjacent to the lesion) (fig. 3C) or intratubercularly (i.e., to the tumor drain inguinal lymph node) (fig. 3D), the effectiveness was diminished. When the basement membrane glycan-MSA-IL 2 was administered subcutaneously at the tail root (2 cm from the tumor site), all survival benefits of the combination were reduced (fig. 3C).

These results indicate that tumor-bearing mice treated with the combination of basement membrane glycan-MSA-IL 2 and TA99 provide synergistic antitumor effects, whereas the intratumoral localization of IL-2 is required for maximum effectiveness of this combination therapy.

Example 7: the synergistic effect of immunomodulating collagen binding molecules and anti-tumor antigen antibodies is dependent on CD8+ T cells, dendritic cells and IFN γ

High doses of IL-2 support proliferation and effector function in T cells and NK cells, but also promote neutrophilia and eosinophilia (Macdonald et al, (1990) Br J Haematol 76(2): 168-. Given the diverse known effects of IL-2 on immune cells, the contribution of different types of leukocytes to the therapeutic effectiveness of the basement membrane glycan-MSA-IL 2 was determined by antibody-mediated cell depletion. The subset of immune cells or IFN γ is cleared by intraperitoneal (i.p.) administration of depleting antibody the day before the first treatment to one week after the last treatment. TA99 and basement membrane glycan-MSA-IL 2 were administered as described in FIG. 3B. CD8+ T cells, NK cells or neutrophils were depleted every four days with 400 μ g of anti-CD 8 α (2.43, BioXCell), anti-NK 1.1(PK136, BioXCell) or anti Ly6G (1a8, BioXCell) antibody, respectively. Macrophage or soluble IFN γ were depleted every other day using 300 μ g of anti-CSF 1R (AFS98, BioXCell) or 200 μ g of anti-IFN γ (XMG1.2, BioXCell), respectively. Eosinophils were depleted using 1mg of anti-IL-5 (TRFK5, BioXCell).

To assess the contribution of the immune cell subsets, 1 × 10 in 50 μ L sterile PBS⁶A B16-F10 mouse melanoma cell (ATCC) was injected subcutaneously into 6-8 weeks old C57BL/6 Wild Type (WT) or BatF3^-/-Mouse (B6.129S (C) -Batf3^tm1KmmJ; jackson Laboratory) which is cross-presenting Dendritic Cell (DC) deficient. Depletion of Natural Killer (NK) cells in WT mice did not alter the effectiveness of the combination of basement membrane glycan-MSA-IL 2 with TA99 (figure 4). Depletion of neutrophils, eosinophils or macrophages in wild type mice did not alter the effectiveness of the combination of the basement membrane glycan-MSA-IL 2 with TA99 (data not shown), suggesting that no single innate cell population was solely responsible for tumor control. However, CD8+ T cells (anti-CD 8 α), cross-presenting DCs (BatF 3)^-/-) And depletion of IFN γ (anti-IFN γ) did alter the effectiveness of the treatment, suggesting that it is essential for tumor rejection (fig. 4). Survival statistics were determined by log rank Mantel-Cox test.

These results indicate that treatment of tumor-bearing mice with basement membrane glycan-MSA-IL 2 in combination with TA99 provides a synergistic anti-tumor effect dependent on CD8+ T cells, dendritic cells and IFN γ.

Example 8: the combination of immunomodulatory collagen binding molecules with anti-tumor antigen antibodies establishes protective memory and induces systemic tumor-specific cellular immunity

As shown above, the persistent disease-free survival and dependence on the adaptive immune component observed after treatment with the basal lamina glycan-MSA-IL 2 in combination with TA99 suggests that cured tumor-free mice may be resistant to tumor re-challenge. To determine whether mice treated with basement membrane glycan-MSA-IL 2 in combination with TA99 were cured tumor-free mice, 1x10 was administered on day 100⁶A cured C57BL/6 mouse from the experiment shown in FIG. 3B was re-challenged with a single B16-F10 cell seeded in the contralateral flank. Most of the treatment with basement membrane glycan-MSA-IL 2 in combination with TA ((treated long-term survivors excluded a re-challenge of B16-F10 inoculated in the contralateral flank (9/15 mice).

Although tumor eradication by Cell-mediated immunity requires systemic immune activation (Spitzer et al, (2017) Cell 168:487-502), a strict localization of vitiligo plaques was observed after treatment with the basement-membrane glycan-MSA-IL 2 in combination with TA99 (see example 6), suggesting an assessment of tumor-specific T Cell responses outside of the treated tumor lesions. To determine whether treatment with basement membrane glycan-MSA-IL 2 in combination with TA99 induced a synergistic anti-tumor T cell response, IFN γ ELISPOT was performed as in fig. 3B using splenocytes collected 4 days after treatment with mice. Treatment with TA99 in combination with MSA-IL2 or basement membrane glycans was used as a positive control. The number of IFN γ Spot Forming Units (SFU) in response to stimulation with B16F10 target cells was quantified. Treatment with TA99 in combination with basement membrane glycan-MSA-IL 2 produced more IFN γ -expressing peripheral splenocytes than the combination with MSA-IL 2. Specifically, treatment with TA99+ basement-membrane glycan-MSA-IL 2 resulted in the production of about 20SFU/100 ten thousand splenocytes, whereas treatment with TA99+ MSA-IL2, with or without TA99+ basement-membrane glycan resulted in the production of less than about 7SFU/100 ten thousand splenocytes.

To confirm that intracellular IFN γ staining on splenocytes was generated by tumor specific CD8+ T cells, splenocytes were collected 4 days after treatment of mice as shown in fig. 3B. Harvested splenocytes were stimulated with irradiated B16-F10 or 4T1 for 12 hours in the presence of brefeldin a, followed by staining for surface markers (CD4, CD3 and CD8) and intracellular IFN γ (n-5 mice/group). Figure 5A shows the quantification of IFN γ + cells in live CD45+ CD3+ CD8+ T cells as determined by flow cytometry. Data were analyzed by one-way ANOVA combined with Tukey multiple comparison test.

The results shown in figure 5A demonstrate that the use of irradiated B16-F10 tumor cells, but not 4T1 tumor cells, stimulated the induction of IFN γ expression in spleen cells, confirming that peripheral B16F 10-specific CD8+ T cell responses in the spleen were induced by treatment of tumor-bearing mice with the basal lamina polysaccharide-MSA-IL 2 in combination with TA 99. These results also indicate that treatment of tumor-bearing mice with TA99 in combination with tumor collagen-anchored basement membrane glycan-MSA-IL 2 induces a greater tumor-specific systemic response than in combination with unanchored MSA-IL 2.

To determine the ability of peripheral effectors (e.g., peripheral tumor-specific T cells) induced by treatment with TA99 in combination with basement membrane glycan-MSA-IL 2 to control distal untreated tumor lesions, support of the lesions by exogenous cytokines was limited, mice were inoculated subcutaneously with 1x10 ⁶B16-F10 cells were plated to establish tumors in bilateral flank regions and administered TA99 systemically (i.p) and IL-2 (in the form of basal lamina glycan-MSA-IL 2 or MSA-IL 2) only in right or ipsilateral tumor tumors (i.tu). Figure 5B shows the average tumor area over time for non-injected contralateral and intratumorally injected ipsilateral tumors. The results in fig. 5B show that the combination of TA99 and MSA-IL2 can leak out of the ipsilateral tumor and leak to the contralateral lesion after injection, giving some contralateral tumor control, but most mice succumb to tumor burden. In contrast, the combination of TA99 with the basement membrane glycan-MSA-IL 2 (which is isolated from ipsilateral tumors) organized the growth of ipsilateral and contralateral tumors, resulting in a durable cure in several mice. These results indicate that in the control and eradication of disseminated disease, the systemic anti-tumor response elicited by tumor collagen binding to anchored IL-2 is stimulated by disseminated IL-2.

Example 9: treatment of tumor-bearing mice with collagen-binding IL-12 fusion protein did not induce IL-12-associated weight loss

As shown in the above examples, after observing the improvement of the collagen-anchored antitumor therapeutic effect by IL-2, the effect of anchoring IL-12, another dose-limiting cytokine, to collagen, was evaluated. IL-12 plays a key regulatory factor in type 1 cell-mediated immunity, and this pathway is known to be critical for effective anti-tumor responses (Green et al, (2017) J Biol Chem 292: 13925-13933). Although preclinical work is promising, severe toxicity and lethality have terminated early clinical trials with systemic administration of IL-12 (Lasek et al, (2014) Cancer Immunol Immunother 63(5): 419-. IL-12 stimulation of NK and T cell-induced IFN γ is implicated in toxicity, however, IL-12 is also poorly suited to its effectiveness (Leonard et al, (1997) Blood 90: 2541-2548).

Thus, IL-12 was expressed and purified as a fusion with MSA alone or as a fusion with the N-terminus of the basement membrane glycan-MSA (IL 12-MSA-basement membrane glycan) using the methods described in examples 1 and 6. Briefly, mouse IL-12 is expressed in a single chain form with a 15 amino acid glycine-serine linker between the p40 and p35 subunits (scIL 12). To produce the collagen-anchored form of IL-2, scIL12 was fused to the N-terminus of the basement membrane glycan using an MSA spacer, hereafter referred to as IL 12-MSA-basement membrane glycan (SEQ ID NO: 123). The non-anchored form of IL-12, IL12-MSA (SEQ ID NO:122) was used as a positive control. IL12-MSA and IL 12-MSA-based proteoglycan proteins showed > 90% monomer expression when evaluated by absorbance at 280nm during size exclusion chromatography (data not shown).

In addition, as described in example 4, the tumor injection after IL-12 fusion serum levels were quantified. Fluorescently labeled IL 12-MSA-basement membrane glycans and IL12-MSA were injected into B16F10-Trp2KO tumors, and serum fluorescence levels were quantified at various time points post injection. The fluorescence signal from serum from mice injected with IL 12-MSA-based xylan was lower over time when measured as a percentage of injected dose compared to the fluorescence signal from serum from mice injected with IL12-MSA (data not shown). These results indicate that IL 12-MSA-based basement membrane glycans bind to intratumoral collagen in vivo and as a result show a lower systemic distribution compared to IL12-MSA alone.

To assess whether retention of IL-12 in tumors by collagen binding would mitigate IL-12-mediated toxicity, IL-12 fused to basal lamina glycan-MSA was assessed in a mouse melanoma tumor model. On day 0, 1X10⁶A single B16-F10 melanoma cell was implanted into 6-8 week old C57BL/6 mice. On days 6 and 12, these mice were tested for weight change from baseline following intratumoral (i.tu.) injection of PBS (n-6), 17.8 μ g/dose of IL12-MSA (n-7) or 23.1 μ g/dose of IL 12-MSA-basement membrane glycan (n-7), or intraperitoneal (i.p.) injection of 17.8 μ g/dose of IL12-MSA (n-7). Mice injected with IL-12 fusion protein (IL 12-MSA-based basement membrane glycan or IL12-MSA) received an equivalent of 140 pmol/dose of IL-12.

As shown in figure 6A, intratumoral administration of IL12-MSA in B16-F10 tumor-bearing mice resulted in significant weight loss, which is a non-invasive reading of the toxicity of systemic cytokines (e.g., IFN γ). Systemic administration of IL12-MSA by intraperitoneal injection resulted in the same weight loss profile. In contrast, intratumoral injection of equimolar IL 12-MSA-based peptidoglycan did not result in weight loss. Collectively, these results indicate that local administration is insufficient to mitigate IL-2 mediated toxicity in the absence of efforts to achieve intratumoral retention through collagen binding, and that collagen anchoring provides sufficient intratumoral inhibition to inhibit significant systemic toxicity of IL-12, thereby improving the therapeutic index.

The survival of the treated animals was also evaluated as shown in figure 6A. Mice were euthanized according to the criteria described in example 6. As shown in figure 6B, treatment with IL-12 fusion improved survival compared to untreated control mice or mice treated with basement membrane glycans. Treatment with IL 12-MSA-based basement membrane glycans resulted in modest survival improvement compared to IL 12-MSA.

The IL12 dose for treatment of B16F10 tumors was titrated to determine the dose-dependent effects on toxicity and antitumor efficacy. Mice vaccinated with the B16F10 tumor on day 0 as described above were treated with different doses of IL12-MSA or IL 12-MSA-based mannan intratumoral injection on day 5. Untreated control mice received an intratumoral injection of PBS. The effect of the dose on tumor area (e.g., efficacy index) and body weight change (e.g., toxicity index) was evaluated. The doses of IL12-MSA and IL 12-MSA-based glycans evaluated included a mass equivalent of 140pmol IL12, 14pmol IL12, 1.4pmol IL12, or 0.14pmol IL 12. IL 12-MSA-based mannan showed no toxicity at any dose tested, but reduced efficacy at IL12 doses of 1.4 or 0.4pmol (data not shown). While IL12-MSA showed reduced toxicity and reduced efficacy with decreasing dose (data not shown). Thus, to evaluate IL12 fusions in combination with other therapeutic agents, a 14pmol dose of IL12 was identified as having a tolerable toxicity index while maintaining therapeutic effectiveness.

Example 10: synergistic antitumor effects of collagen binding to IL-2 and IL-12 fusion proteins in a mouse tumor model

Theoretical studies have been conducted on combinations that enhance the antitumor effect of IL-12, but the possibility of its realization has been largely excluded from safety considerations (Lasek et al, (2014) Cancer Immunol 63(5): 419-. As indicated by the absence of treatment-related weight loss in example 9, the improved therapeutic index of IL 12-MSA-based mannan prompted the evaluation of this cytokine in combination with other therapeutic agents. IL-2 and IL-12 are known to be involved in the complement signaling pathway to stimulate NK and T cells (Wigginton & Wiltrout (2002) Expert Opin Biol Ther 2: 513-. In addition, IL-2 upregulates the expression of the IL-12 receptor subunit β 2 (Wang et al, (2000) Blood 95:3183) and IL-12 persistently surface expresses the high affinity IL-2 receptor CD25(Starbeck-Miller et al, (2013) J Exp Med 211: 105-120). IL-2 and IL-12 increase and prolong the effect of each other by reciprocal positive feedback (Wigginton et al, (1996) J Natl Cancer Inst 88: 38-43). Although encouraging in efficacy, some clinical trials surrounding this combination have been terminated (Wigginton & Wiltrout (2002) Expert Opin Biol Ther 2: 513-. It is noteworthy that the combination of IL-2 and IL-12 also significantly enhanced IFN-y production by T cells and NK cells (Wigginton & Wiltrout (2002) Expert Opin Biol Ther 2: 513-524).

To evaluate the therapeutic effect of the combination of immunomodulatory collagen-binding molecules comprising IL-2 or IL-12, the basement membrane glycans-MSA-IL 2 and IL 12-MSA-basement membrane glycans were tested in combination in a mouse melanoma tumor model essentially as described in example 9. However, given the known toxicity associated with the above non-collagen anchored cytokine co-administration, the doses of IL 12-MSA-basement membrane glycan and IL12-MSA-IL-12 were reduced to 1/10, to 14 pmol/dose, previously administered in example 9. Mice administered with basement membrane glycan-MSA-IL 2 received 13 μ g/dose as in example 6. Mice administered MSA-IL2 received 9 μ g/dose. Mice injected with IL-2 fusion protein (either basement membrane glycan-MSA-IL 2 or MSA-IL2) received IL-2 equivalent to 0.11 nmol/dose.

Intratumoral injection of IL12-MSA alone or MSA-IL2 alone at reduced doses (14 pmol/dose) did not result in weight loss or significant tumor growth delay in B16-F10 tumor-bearing mice (data not shown). In contrast, co-administration of IL12-MSA with MSA-IL2 resulted in weight loss and increased survival (FIG. 7). In contrast, treatment with a combination of collagen-anchored forms of IL 12-MSA-basement-membrane glycan and basement membrane glycan-MSA-IL 2 resulted in a greatly increased survival compared to the combination of IL12-MSA and MSA-IL2 without concomitant weight loss (figure 7).

These results indicate that treatment of tumor-bearing mice with a combination of collagen-bound IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 greatly increases the survival of the mice compared to treatment with a combination of non-collagen-bound IL12-MSA and MSA-IL 2. Furthermore, these results indicate that treatment of mice with a combination of collagen-bound IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 prevents treatment-related toxicity associated with co-administration of IL-12 and IL-2, thereby providing a therapeutic profile for this cytokine combination.

Example 11: the collagen binding IL-2 and IL-12 fusion protein synergy depends on CD8+ T cells and dendritic cells

The immune cell type responsible for the effectiveness of the intratumoral IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 combination therapy was determined by antibody-mediated cell depletion, essentially as described in example 7. As shown in figure 8A, CD8+ T cells and cross-presenting dendritic cells are essential for efficacy, as depletion of these cell types will reduce survival of tumor-bearing mice treated intratumorally with a combination of IL 12-MSA-basement membrane glycans and basement membrane glycans-MSA-IL 2. In contrast, depletion of NK cells, neutrophils, eosinophils or macrophages did not significantly affect survival outcome (fig. 8B). Antibody-mediated depletion of IFN γ, a cytokine known to be expanded by concomitant stimulation of IL-2 and IL-12 (Gollob et al, (1999) J Immunol 162(8):4472 and 4481), also did not significantly alter survival (FIG. 8A), however, the lack of therapeutic efficacy may be attributed to insufficient depletion of IFN γ.

In order to evaluate the contribution of immune cell types to the antitumor efficacy provided by the combination of IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 in tumor-bearing mice, immunophenotypic analysis of tumor-infiltrating immune cells was performed. Mice were inoculated with 1 million B16F10 tumor cells on day 0 and treated with PBS, IL12-MSA and MSA-IL2, or IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 intratumoral injection on day 5. Tumors were excised on day 11. Tumors were analyzed for immune cell infiltration as described previously. (Moynihan et al, (2016) Nat Med 22: 1402-1410); zhu et al, (2015) Cancer Cell 27: 489-501). Briefly, excised tumors were weighed, cut into small pieces, and incubated at 37C for 30 minutes in RPMI-1640 containing 1mg/mL collagenase and dispase (Roche) and 25. mu.g/mL DNase I (Roche). Further mechanical dissociation was used to generate single cell suspensions for staining. In BD FACS LSRFortessa^TMThe cells were analyzed, and(FlowJo, Inc) the data were analyzed. Cells were stained for surface and intracellular markers to describe cell types as follows:

NK thinCell (Living CD 45)⁺CD3^-NK1.1⁺)

Treg cells (Living CD 45)⁺CD3⁺NK1.1^-CD4⁺CD8^-CD25⁺FoxP3⁺)

CD4 cell (Living CD 45) ⁺CD3⁺NK1.1^-CD4⁺CD8^-CD25^+/-FoxP3^-)

CD 8T cells (Living CD 45)⁺CD3⁺NK1.1^-CD4^-CD8⁺)

Monocyte/macrophage (live CD 45)⁺CD11b⁺Ly6G^-CD11c^-F4/80⁺)

CD11b⁺DC (Living CD 45)⁺CD11b⁺MHCII⁺CD11c⁺)

CD11b^-DC (Living CD 45)⁺CD11b^-MHCII⁺CD11c⁺)

Neutrophils (live CD 45)⁺CD11b⁺Ly6G⁺)

As shown in figure 8C, fold change in CD8+ T cells in tumor infiltration from mice treated with IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 (basement membrane glycan form) versus PBS was higher than fold change in IL12-MSA + MSA-IL2(MSA form) treatment versus PBS treated mice. Furthermore, tumors treated with the basement membrane glycan-MSA cytokine fusion had more infiltrating CD8+ T cells than tumors treated with MSA cytokine fusion 6 days after initial treatment (fig. 8D), and higher surface PD-1 expression (fig. 8E).

The generation of tumor-specific T cells in response to treatment was further evaluated in splenocytes isolated 6 days post-treatment by IFN γ ELISPOT. The number of IFN γ Spot Forming Units (SFU) in response to B16F10 target cell stimulation was quantified. Treatment with the basement membrane glycan-MSA cytokine fusion produced about 20SFU/1 million splenocytes, compared to about 15SFU/1 million splenocytes for MSA fusion or about 2SFU/1 million splenocytes for untreated animals. Thus, treatment with a basement membrane glycan-MSA cytokine fusion results in an increase in the number of peripheral tumor-specific T cells compared to untreated or treated with MSA cytokine fusions.

These results indicate that the antitumor efficacy (e.g. increased survival) provided by the combination of IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 is dependent on CD8+ T cells and dendritic cells. Furthermore, these results demonstrate that intratumoral treatment of tumor-bearing mice with a combination of IL 12-MSA-basement-membrane glycan and basement-membrane glycan-MSA-IL 2 provides anti-tumor efficacy, at least in part, by inducing activated CD8+ T cells into the tumor and inducing the generation of peripheral tumor-specific T cells.

Example 12: synergistic effect of collagen binding to a combination of IL-2 and IL-12 fusion proteins and anti-PD-1 antibodies in a mouse melanoma tumor model

As described in example 11, upregulation of surface PD-1 on tumor-infiltrating CD 8T cells in response to treatment with a basement membrane glycan-cytokine prompted evaluation of the anti-tumor efficacy of a combination of anti-PD-1 antibody (clone 29f.1a12, BioXCell), IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 in a mouse melanoma tumor model. Briefly, 1x10 on day 0⁶A single B16-F10 tumor cell was inoculated subcutaneously into the right flank of a 6-8 week old C57BL/6 mouse. On days 5 and 11, tumor-bearing mice were treated intratumorally with a combination of basement membrane glycan-MSA-IL 2 and IL 12-MSA-basement membrane glycan or a combination of MSA-IL2 and IL12-MSA at the doses described in example 10, and with anti-PD-1 antibody (200 μ g/dose). Percent body weight change and percent survival from baseline were monitored and shown in figure 9.

As shown in figure 9, introduction of anti-PD-1 antibodies in combination with IL12-MSA and MSA-IL2 or IL 12-MSA-basement-membrane glycan and basement-membrane glycan-MSA-IL 2 did not alter the previously observed trend of weight loss (figure 7). However, the addition of anti-PD-1 antibodies improved survival outcome for mice treated with the combination of IL12-MSA + MSA-IL2, but did not further improve survival for mice treated with the combination of IL 12-MSA-basement-membrane glycan + basement-membrane glycan-MSA-IL 2.

The incidence of local vitiligo was higher in mice treated with anti-PD-1 antibody, IL12-MSA and MSA-IL2(1/5 mice had vitiligo at the injection site) compared to mice treated with anti-PD-1 antibody, IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2(4/5 mice had vitiligo at the injection site). Furthermore, survivors from cytokine (IL12-MSA + MSA-IL2 or IL 12-MSA-basilar glycan + basilar glycan-MSA-IL 2) and anti-PD-1 treatment were more than survivors from IL 12-MSA-basilar glycan + basilar glycan-MSA-IL 2 treatment but in the absence of anti-PD-1 treatment against subsequent tumor re-challenge with B16-F10 tumors. Although 4/4 mice treated with basement membrane glycan-MSA cytokine + anti-PD-1 and 2/2 mice treated with MSA cytokine + anti-PD-1 resisted re-challenge tumors, 1/3 mice treated with basement membrane glycan-MSA cytokine alone resisted re-challenge.

These results indicate that treatment of tumor-bearing mice with a combination of anti-PD-1, IL 12-MSA-basement membrane glycan and basement membrane glycan-MSA-IL 2 increases the incidence of treatment-induced vitiligo and effective immunological memory, thus showing a synergistic effect in enhancing the T cell response resulting from local IL-2 and IL-12 treatment.

Example 13: synergistic effect of collagen-binding IL-2 and IL-12 fusion proteins in combination with anti-PD-1 antibodies in mouse breast and colon cancer tumor models

The anti-tumor effect of collagen-binding IL-2 and IL-12 fusion proteins in combination with anti-PD-1 antibodies was further evaluated in an EMT6 breast cancer model. Briefly, on day 0, 1 × 10 suspended in 50 μ L sterile PBS⁶Individual EMT6 mouse breast cancer cells (ATCC) or 1x10⁶An individual MC38 mouse colon Cancer cell (National Cancer Institute, Bethesda, Md.) was injected subcutaneously into the right flank of a C57BL/6 female mouse. EMT6 tumor-bearing mice were treated with either the basement membrane glycan-MSA-IL 2+ IL 12-MSA-basement membrane glycan (i.tu.), anti-PD-1 antibody alone (i.p.,200 μ g/dose) or a combination of anti-PD-1 antibody with basement membrane glycan-MSA-IL 2 and IL 12-MSA-basement membrane glycan (i.tu), at doses as described in example 10 on days 5, 11 and 17. On days 5, 11 and 17, the base was used at the doses as described in example 10 MC38 tumor-bearing mice were treated with a combination of either the membranoglycan-MSA-IL 2+ IL 12-MSA-basement-membrane glycan (i.tu.), anti-PD-1 antibody alone (i.p.,200 μ g/dose) or the combination of the membranoglycan-MSA-IL 2+ IL 12-MSA-basement-membrane glycan (i.tu.). Tumor area (mm) was monitored over time for each tumor model²) And percent survival, and as shown in figures 10A-10B. Survival statistics were determined by log rank Mantel-Cox test.

As shown in figure 10A, treatment of EMT6 breast cancer tumor-bearing mice with anti-PD-1 antibody in combination with basement membrane glycans-MSA-IL 2 and IL 12-MSA-basement membrane glycans resulted in elimination of tumor lesions (as indicated by the absence of detectable tumor area) and a greatly increased survival compared to tumor-bearing mice treated with anti-PD-1 antibody alone.

As shown in figure 10B, treatment of MC38 colon cancer tumor-bearing mice with anti-PD-1 antibody in combination with basement-membrane glycans-MSA-IL 2 and IL 12-MSA-basement membrane glycans resulted in a reduction in tumor area and a greatly increased survival compared to tumor-bearing mice treated with either anti-PD-1 antibody alone or IL 12-MSA-basement membrane glycan + basement-MSA-IL 2 alone.

These results indicate that treatment of tumor-bearing mice with anti-PD-1 antibodies in combination with IL 12-MSA-basement-membrane glycan + basement-MSA-IL 2 can produce a synergistic anti-tumor effect in both EMT6 breast and MC38 colon cancer models.

Example 14: synergistic effect of collagen-binding IL-12 fusion proteins and cancer vaccines in mouse melanoma tumor model

To further evaluate the ability of collagen binding to the IL-12 fusion protein to synergistically enhance antitumor therapy, the antitumor effect of a combination of collagen binding to the IL-12 fusion protein IL 12-MSA-based basement membrane glycans with a cancer vaccine was evaluated in a B16-F10 mouse melanoma model. Briefly, on day 0, 1 × 10 in 50 μ L sterile PBS suspension was added⁶A single B16-F10 cell (ATCC) was inoculated subcutaneously into the right flank of a 6-8 week old C57BL/6 female mouse. Subcutaneous administration of a cancer vaccine comprising 90 μ g of a peptide derived from the B16-F10-related antigen TYRP-1 at the caudal root with a priming dose on day 5 and priming doses on days 11 and 17And a modified gp100 Peptide (PEG) and 50 μ g of cyclic dinucleotide adjuvant (Invivogen). Subcutaneous administration of the cyclic dinucleotide adjuvant and cancer vaccine allows for the assessment of the initiation of antigen-specific CD 8T cell responses. Briefly, peripheral blood was collected on day 16 and stimulated with the peptide antigens Trp1 and EGP for 6 hours. Brefeldin a was included during the last 4 hours of incubation. Peripheral blood cells were then stained for surface markers and intracellular IFN γ and analyzed by flow cytometry. The percentage of IFN γ + cells among live CD45+ CD3+ CD8+ T cells was evaluated. Vaccines alone or in combination with IL 12-MSA-based basement membrane glycans improved priming of antigen-specific CD 8T cells (data not shown).

The weight change from baseline, tumor area and survival (fig. 11, left to right) of each mouse treated with PBS (n-12) or IL-12 (n-10 for IL 12-MSA; n-10 for IL 12-MSA-basement-membrane glycan), or vaccine alone (n-7) or vaccine and IL12 (n-7 for IL 12-MSA; n-7 for IL 12-MSA-basement-membrane glycan) intratumoral (i.tu.) injection of each mouse treated mice was monitored over time on days 5, 7 and 17.

As shown in figure 11, vaccination alone did not affect body weight (left panel), moderately delayed B16F10 tumor growth (middle panel) and moderately increased survival of mice (right panel) relative to mice treated with PBS. In contrast, co-administration of cancer vaccine and IL-12 (using IL12-MSA or IL 12-MSA-based glycans) resulted in a synergistic decrease in tumor growth (middle panel) and an increase in survival (right panel). Administration of a cancer vaccine in combination with IL 12-MSA-based basement membrane glycans has a longer survival compared to that of IL12-MSA combination. Furthermore, the combination of the vaccine with IL12-MSA resulted in treatment-induced weight loss, while the combination with IL 12-MSA-based mannan was not detected (right panel).

These results indicate that administration of a cancer vaccine in combination with collagen-anchored IL-12(IL 12-MSA-basement-membrane glycan) to tumor-bearing mice results in a synergistic anti-tumor effect leading to a reduction in tumor growth and a greatly increased percentage of survival compared to administration of the cancer vaccine or IL 12-MSA-basement-membrane glycan alone. These results indicate that intratumoral collagen anchoring of cytokines (e.g., IL-12) synergistically improves tumor control of cancer vaccines.

Example 15: synergistic effect of collagen binding to IL12 fusion protein and CAR-T cells in mouse melanoma tumor model

To further evaluate the ability of collagen binding to IL-12 fusion protein to synergistically enhance antitumor therapy, the antitumor effect of a combination of collagen binding to IL-12 fusion protein IL 12-MSA-based basement membrane glycans with CAR-T cells was evaluated in a B16-F10 mouse melanoma model. Briefly, on day 0, 0.5 × 10 suspended in 50 μ L sterile PBS⁶A single B16-F10 cell (ATCC) was inoculated subcutaneously into the right flank of a 6-8 week old C57BL/6 female mouse. B16F10 specific CAR-T cells were generated by transduction of CD3+ splenocytes to express a CAR consisting of a single chain variable fragment (scFv) of TA99 fused to both CD28 and CD3 zeta costimulatory signaling domain. To ensure CAR-T cell engraftment, all mice were pre-treated with whole body irradiation the day before an intravenous (i.v.) bolus injection of 1000 ten thousand CAR-T cells. Treated mice were monitored over time for intratumoral (i.tu.) changes in weight from baseline, tumor area and survival (fig. 12, left to right) using PBS (n-9) or IL-12 (n-6 for IL 12-MSA; n-5 for IL 12-MSA-basement membrane glycan), or CAR-T alone (n-12), or CAR-T and IL12 (n-7 for IL 12-MSA; n-5 for IL 12-MSA-basement membrane glycan) on days 5 and 11.

As shown in figure 12, treatment with CAR-T cells or IL 12-MSA-based mannan (2.3 ug/dose) alone reduced tumor area (middle panel) and increased survival (right panel). However, administration of the combination of CAR-T cells and IL 12-MSA-basement-membrane glycans to tumor-bearing mice resulted in durable tumor regression, leading to a reduction in tumor area and a greatly increased survival compared to CAR-T cell or IL 12-MSA-basement-membrane glycan therapy alone. Treatment with CAR-T cells in combination with IL12-MSA also resulted in tumor regression and modest improvement in survival. However, the combination treatment showed significant inertia as indicated by a greater loss of body weight in the animals after treatment. Such toxicity was not observed in the combination of CAR-T cells and IL-12-MSA-based basement membrane glycans.

These results indicate that administration of tumor antigen specific CAR-T cells in combination with collagen anchored IL-12(IL 12-MSA-basement-membrane glycan) to tumor-bearing mice results in a synergistic anti-tumor effect leading to a reduction in tumor growth and a greatly increased percentage of survival compared to CAR-T cells or IL 12-MSA-basement-membrane glycan treatment alone. These results indicate that the intratumoral collagen anchoring of cytokines (e.g., IL-12) synergistically improves tumor control of tumor antigen specific CAR-T cells.

Example 16: novel adjuvant administration of collagen-binding IL-12 fusion proteins in combination with PD-1 checkpoint blockade in mouse breast tumor resection model to prevent metastatic relapse

To further evaluate the ability of collagen-binding IL-12 fusion proteins to synergistically enhance anti-tumor therapy, the anti-tumor effect of a combination of collagen-binding IL-12 fusion protein IL 12-MSA-based basement membrane glycans with anti-PD-1 antibodies (clone 29f.1a12, BioXCell) was evaluated in a 4T1 mouse breast tumor resection model and compared to a combination of scIL12 with anti-PD-1. Briefly, on day 0, 0.5 × 10 suspended in 100 μ L sterile PBS⁶4T1-Luc cells (MIT, Cambridge, MA) expressing luciferase were injected into mammary fat pads of 6-8 week old BALB/c female mice. Prior to surgical resection of the primary foci, mice were treated with both intratumoral administration of IL 12-MSA-basement membrane glycan or scIL12 and systemic administration of anti-PD-1 (neoadjuvant therapy). New co-therapy (anti-PD-1, 200 μ g/dose, i.p. administration + IL 12-MSA-basement membrane glycan, 4.6 μ g/dose (30pmol IL 12/dose) or scIL12, 1.9 μ g/dose (30pmol IL 12/dose)) was administered on day 7 and day 13 and the primary tumor was surgically excised on day 16. Mice were monitored for metastasis by In Vivo Imaging (IVIS) after surgery. Figure 13 shows the body weight change (left panel), primary tumor growth and weight (middle panel) and percent survival (right panel) during neoadjuvant treatment of mice treated with IL-12 (n-5 for scIL 12; n-5 for IL 12-MSA-based xylan) intratumoral (i.tu.) injection and anti-PD-1 intraperitoneal (i.p.) injection on days 7 and 13. The arrows indicate treatment time and the crosses indicate surgical time.

As shown in figure 13, the combination of both forms of IL-12(scIL12 or IL 12-MSA-based glycans) with anti-PD-1 antibodies was not significantly toxic based on the absence of weight loss (left panel). However, the new co-therapy with IL 12-MSA-based peptidoglycan resulted in more shrinkage of the primary tumor compared to the non-anchored form of scIL-12 (middle panel). After surgery, mice were monitored for metastasis by in vivo bioluminescence imaging. IL 12-MSA-based basement membrane glycans completely protected mice from metastatic growth, whereas several mice treated with scIL-12 relapsed.

These results indicate that administration of anti-PD-1 antibodies in combination with collagen-anchored IL-12(IL 12-MSA-based glycosaminoglycan) to tumor-bearing mice results in a synergistic anti-tumor effect, leading to a reduction in primary tumor growth and a greatly increased percentage of survival after surgical resection of the tumor, compared to administration of a combination of non-collagen-bound IL-12(scIL12) and anti-PD-1 antibodies. These results indicate that collagen-anchored IL-12 improves post-operative outcome in the context of new co-therapy.

Example 17: collagen-binding chemokine fusion proteins induce immune cell migration and tumor infiltration

T cell infiltration is critical for durable anti-tumor immunity. There was a correlation between T cell infiltration and tumor cell-derived CCL3, CCL4, and CCL5 expression. Spranger et al, (2015) Nature 523: 231-235 (2015); spranger et al, (2016) Proc Natl Acad Sci USA 113, E7759-E7768 (2016). CCL3(MIP-1a) has a high affinity for CCR1 and a low affinity for CCR5, mediating the recruitment of T cells, B cells, and monocytes. CCL4(MIP-1b) binds to CCR5 and mediates general lymphocyte recruitment. CCL5(RANTES) binds to a variety of chemokine receptors (CCR1, CCR3, CCR4, CCR5) and thereby attracts monocytes, T cells, eosinophils, and other immune cells. T cell recruitment chemokines are also more prevalent in tumors undergoing productive immune-mediated regression. Liang et al, (2016) Proc Natl Acad Sci USA 113: 5000-; schlecker et al, (2012) J Immunol 189: 5602-; brewitz et al, (2017) Immunity 46: 205-219; kanegasaki et al, (2014) Cancer Res 74: 5070-; wittrup (2017) Trends Cancer Res 3: 615-.

To evaluate the ability to express collagen-binding chemokines in mammalian cells, human embryonic kidney 293(HEK293) cellsTransiently expressing three His-tagged collagen-bound cytokines comprising a basement membrane glycan fused to CCL3, CCL4, or CCL 5. Briefly, HEK293 cells (density 100 ten thousand cells/mL) were transfected with polyethyleneimine (2 mg per liter of cell culture) in OptiPro serum-free medium (20 mL per liter of cell culture) (Thermo Fisher) using sterile-filtered plasmid DNA (1 mg per liter of cell culture). TA99 was purified as described previously using rProtein A Sepharose Fast Flow resin (GE Healthcare) (Zhu et al, 2015). His-tagged proteins were isolated from HEK293 supernatants using TALON Metal affinity resin (Takara Bio Inc.). Then, by size exclusion chromatography using a HiLoad 16/600Superdex 200pg columnThe cytokine fusion protein was further purified on a FPLC system (GE Healthcare) that had been pretreated with 1M NaOH for 4 hours to remove endotoxins and subsequently equilibrated in sterile pbs (corning). After purification, all protein buffers were exchanged to sterile pbs (corning), sterile filtered at 0.2 micron (Pall Corporation), and confirmed to contain minimal endotoxin levels using the chromogenic LAL assay (Lonza) (per injection) <0.1 EU). To confirm their molecular weight, the Protein Novex Prestained Sharp Protein Ladder was run together on 4-12% NuPAGE Bis-Tris Protein gel (Life Technologies) with 1% MES running buffer. The resulting eluate was evaluated by SDS-PAGE for the relative expression level of His-tagged collagen-binding fusion protein (data not shown).

Transient expression of basement membrane glycan, basement membrane glycan D213A (SEQ ID NO:125), basement membrane glycan-Gluc, basement membrane glycan-CCL 3(SEQ ID NO:153), basement membrane glycan-CCL 4(SEQ ID NO:156), and basement membrane glycan-CCL 5(SEQ ID NO:160) was achieved in HEK293 cells, as determined by SDS-PAGE analysis, and protein staining was observed at or near the respective expected molecular weight of each fusion protein.

These results indicate that collagen binding fusion proteins comprising chemokines can be expressed and purified from mammalian cells.

To evaluate the ability of the basement membrane glycan-chemokine fusion proteins produced as described above to induce inflammation (e.g., immune cell migration), an in vivo inflammatory peritonitis assay was performed as described above (Proudfoot et al, (2003) Proc Natl Acad Sci USA 100: 1885-. Briefly, the peritoneal cavity is lined with a collagen-rich and vasculature-rich mesothelium. When injected intraperitoneally, a matrix-binding construct (e.g., a collagen-binding chemokine fusion protein) adheres to the lining. In the case of the basal lamina glycan-chemokines, mesothelial localization can create a concentration gradient that mediates the extravasation of immune cells from nearby blood vessels and into the peritoneal cavity. These infiltrates were recovered by peritoneal lavage for ex vivo immunophenotypic analysis. Therefore, BALB/c mice were injected intraperitoneally with 1nmol equivalent of either basilar glycan-Gluc, basilar glycan-CCL 3, basilar glycan-CCL 4, or basilar glycan-CCL 5 in 200uL sterile PBS and the mice were sacrificed 18 hours post injection. The peritoneal cavity was washed 3 times by gently massaging 5mL of ice-cold PBS in the cavity, and the lavage fluid was combined. The collected cells were counted using an Accuri flow cytometer.

The basement membrane glycan-chemokine fusion proteins comprising CCL3, CCL4, or CCL5 were able to mediate overall cellular infiltration compared to injection of basement membrane glycan-GLuc (data not shown). When the infiltrate was immunophenotyped by surface marker staining, mice treated with either basement membrane glycan-CCL 3, basement membrane glycan-CCL 4, or basement membrane glycan-CCL 5 contained a large number of macrophages, followed by neutrophils, NK cells, DC, B cells, and T cells, compared to lavage of mice treated with PBS or basement membrane glycan-Gluc (data not shown).

These results indicate that intraperitoneal administration of collagen-binding chemokines (e.g., basement membrane glycan-CCL 3, basement membrane glycan-CCL 4, or basement membrane glycan-CCL 5) induces migration of immune cells, including T cells, into the abdominal cavity of mice. These results suggest that intratumoral administration of a basement membrane glycan-chemokine fusion protein will induce inflammation (e.g., immune cell infiltration) and thus mimic an immune-responsive tumor.

To assess the therapeutic effect of using a basement membrane glycan-chemokine to induce inflammation in a tumor background, basement membrane glycan-CCL 3(Lum CCL3) and basement membrane glycan-CCL 5(Lum CCL5) were tested in a 4T1 breast cancer model and a B16F10 melanoma model, as described previously. Intratumoral treatment of tumor-bearing mice was performed on day 7 and day 13 using either basement membrane glycan-GLuc (20 ug/dose), or a combination of basement membrane glycan-CCL 3(5.5 ug/dose), basement membrane glycan-CCL 4(5.4 ug/dose) and basement membrane glycan-CCL 5(5.5 ug/dose) in the presence or absence of IFN α. IFN alpha (50 ug/dose) was administered intraperitoneally on days 9 and 15.

As shown in figure 14A, intratumoral injection of the combination of basement membrane glycan-CCL 3, basement membrane glycan-CCL 4, and basement membrane glycan-CCL 5 slightly delayed the growth of the 4T1 tumor in the presence of systemic IFN α. Finally, the same treatment in B16F10 tumor-bearing mice showed that IFNa and basement membrane glycan-chemokine, administered alone or in combination, delayed growth equivalently (fig. 14B). To determine whether the basement membrane glycan-chemokines directly affected the survival of tumor cells, increasing concentrations of the fusion proteins basement membrane glycan-gluc (Lum gluc), basement membrane glycan-CCL 3(Lum CCL3), basement membrane glycan-CCL 5(Lum CCL5) were cultured with 4T1 cells or B16F10 in cell culture medium for 48 hours. After an additional 4 hours incubation with 10uL of cell proliferation assay reagent WST-1(Sigma Aldrich), proliferation was measured by absorbance at 450nm (reference 700 nm). As shown in fig. 14C and 14D, the presence of the membrane glycan cytokine had no effect on the proliferation of 4T1 cells or B16F10 cells, indicating that any tumor growth delay observed in vivo was caused by immune-mediated rather than direct tumoricidal effects.

Example 18: synergistic effect of collagen-binding CCL11 chemokine fusion protein and cancer vaccine in mouse melanoma tumor model

Eosinophils are known to secrete T cell chemokines and normalize tumor blood vessels, thereby reducing intratumoral infiltration. Carretero et al, (2015) Nat Immunol 16: 609-617. The chemokine CCL11(eoxtaxin) is known to recruit eosinophils (Menzies-Gow et al, (2002) J Immunol 169(5): 2712-2718). Thus, the combination of CCL 11-basement membrane glycan fusion protein (SEQ ID NO:172) described in example 14 with a cancer vaccine was used to detect its recruitment of eosinophils into tumors mediating the recruitment of eosinophils in the presence of systemic TNF α and IFN γAbility to follow tumor control. Briefly, on day 0, 3x10 was used⁵A single B16F10 cell was inoculated into the right flank of a C57BL/6 mouse. Vaccination was performed subcutaneously (s.c.) in the tail roots, with priming on day 5 and boosting on days 11 and 17. Vaccination consisted of 90ug TTR-Trp1-EGP and 50ug cyclic dinucleotide to elicit tumor specific CD8+ T cell responses. At day 11, day 17, day 23 and day 29, CCL 11-based peptidoglycan (5 ug/dose/mouse), TNF α (5.8 pmol/dose/mouse) and IFN γ (6.3 pmol/dose/mouse) were administered for intratumoral treatment. Tumor area over time was measured every other day (mean + SD).

As shown in figure 15, tumor-bearing mice treated with CCL 11-based peptidoglycan in combination with B16F10 specific cancer vaccine, TNF α and IFN γ had smaller tumor areas than cancer vaccine alone or in combination with systemic TNF α and IFN γ.

Example 19: synergistic effect of collagen-binding CCL11 chemokine fusion protein and cancer vaccine in mouse melanoma tumor model

To further evaluate the ability of collagen-binding chemokine fusion proteins to synergistically enhance antitumor therapy, the antitumor effect of the collagen-binding chemokine fusion protein CCL 11-basement membrane glycan in combination with MSA-IL2(30 μ g/dose) and a tumor targeting antibody targeting an A-V type integrin (2.5F-Fc; see Kwan et al, (2017) J Exp 20160215 (9): 10.1084/jem.831) was evaluated in the B16-F10 mouse melanoma model. Briefly, on day 0, 0.5 × 10 suspended in 50 μ L sterile PBS was used⁶A single B16-F10 cell (ATCC) was inoculated subcutaneously into the right flank of a 6-8 week old C57BL/6 female mouse. Tumor targeting antibodies 2.5F-Fc and MSA-IL2 were administered intraperitoneally (i.p.) on days 5, 11, and 17. Intratumoral treatment with CCL 11-basement membrane glycan (5 ug/dose/mouse) was performed on day 5 and day 11. Tumor growth was monitored every other day over time and individual tumor areas (mm) ²) As shown in fig. 16A.

As shown in figure 16B, tumor-bearing mice treated with CCL 11-basement membrane glycan and tumor-targeting antibodies 2.5F-Fc and MSA-IL-2 had a greatly reduced tumor area compared to treatment without CCL11 (basement membrane glycan only).

Overall, the results in example 17, example 18 and example 19 indicate that the basement membrane glycan-chemokines induce local inflammation, recruit immune cells to the local site of administration, and can be used in combination with other anti-tumor therapies to enhance their effects and provide synergistic tumor control.

Example 20: recombinant expression of collagen-binding antibody fusion proteins

To evaluate the ability of expressing collagen-binding antibody fusion proteins in mammalian cells, basement membrane glycans (4420-basement membrane glycans (SEQ ID NOS: 142 and 143; anti-fluorescein), LOB 12.3-basement membrane glycans (SEQ ID NOS: 146 and 147; anti-4-1 BB), 3/23-basement membrane glycans (SEQ ID NOS: 184 and 185; anti-CD 40), 2C 11-basement membrane glycans (SEQ ID NOS: 150 and 151; anti-CD 3), and OX 86-basement membrane glycans (SEQ ID NOS: 148 and 149; anti-OX 40)) fused to five different antibodies were generated and transiently expressed in human embryonic kidney 293(HEK293F) cells. All IgG-basement membrane glycan fusions were encoded on a single plasmid (data not shown). All IgG-based peptidoglycan constructs were expressed as a light chain (VL) with first a mouse kappa constant region (mK), followed by a T2A peptide (SEQ ID NO: 152; T2A) and finally a heavy chain (VH) with a mouse IgG2c constant region (mIgG2c) fused to a short linker ((G4S) ₃) Of (2) a basement membrane glycan (LUM). The T2A peptide allows ribosomes to jump the bond formed between the last two residues of the peptide, allowing the expression of two different proteins in one open reading frame. The furin cleavage site (F) was included upstream of the T2A peptide, allowing removal of the T2A peptide from the light chain end. Furthermore, inclusion of a GSG linker (GSG) upstream of the T2A peptide has been shown to increase the cleavage efficiency of the T2A peptide (Chng et al, (2015) mAbs 7(2): 403-. Both the light and heavy chains contain leader sequences to ensure proper transport of the protein to the secretory pathway. All constructs had silent LALA-PG effector functions that abrogated binding to Fc γ receptor and binding to C1q (Lo et al, (2017) J Biol Chem 292: 3900-3908).

Expression and purification of anti-luciferase (4420) antibody alone or fused to the basement membrane glycans was achieved as shown by SDS-PAGE analysis, which showed protein bands at predicted molecular weights under both reducing (R) and non-reducing (NR) conditions (data not shown). Similarly, expression of agonist IgG-based basement membrane glycan fusion proteins LOB 12.3-based membrane glycan (anti-4-1 BB), 3/23-based membrane glycan (anti-CD 40), 2C 11-based membrane glycan (anti-CD 3), and OX 86-based membrane glycan (anti-OX 40) was achieved as shown by SDS-PAGE analysis, which showed protein bands at predicted molecular weights under both reducing (R) and non-reducing (NR) conditions (data not shown). All IgG-based peptidoglycan fusion proteins were purified using recombinant protein a resin (rProtein a Sepharose, Fast Flow resin (GE Healthcare)) according to the manufacturer's recommendations.

These results indicate that collagen-binding antibody fusion proteins (e.g., IgG-based peptidoglycan) are expressed in mammalian cells and that collagen-binding polypeptide fusion does not affect purification.

Example 21: recombinant collagen-binding antibody fusion proteins bind collagen in vitro and are retained in vivo within tumors

To evaluate the ability of the collagen binding antibody fusion protein to bind collagen, IgG-based basement membrane glycan fusion proteins expressed and purified as described in example 20 were tested for their ability to bind to collagen type I coated plates by ELISA. Briefly, type I collagen (Gibco) coated 96-well plates were blocked for 1 hour at room temperature using PBS + 0.1% wt/vol Bovine Serum Albumin (BSA) + 0.05% wt/vol tween 20(PBSTA), and then incubated for 2 hours at room temperature using PBSTA containing various concentrations of basement membrane glycans. The wells were washed with PBSTA and then incubated with horseradish peroxidase conjugated goat anti-mouse IgG2c heavy chain (ab98722, Abcam) (final concentration. 5. mu.g/ml) diluted 1:1000 in PBSTA for 1 hour at room temperature. The wells were washed again with PBSTA and then treated for 10min with 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) followed by 1M sulfuric acid to stop the color reaction. The absorbance at 450nm was measured using a M1000 microplate reader (Tecan) (corrected using the reference absorbance at 570 nm). Purified collagen binding antibody fusion proteins LOB 12.3-basement membrane glycans (anti-4-1 BB), 3/23-basement membrane glycans (anti-CD 40), 2C 11-basement membrane glycans (anti-CD 3), and OX 86-basement membrane glycans (anti-OX 40) were evaluated on collagen I coated plates by ELISA. As shown in fig. 17A, all IgG-based peptidoglycan fusion proteins retained the ability to bind collagen with similar affinity. These results indicate that the basement membrane glycans fused to the heavy chain of IgG retain the ability to bind type I collagen.

The ability of the 4420-basement membrane glycan fusion protein and the 4420 antibody to be retained in vivo within tumors was also evaluated. Both proteins were purified using size exclusion chromatography and then labeled with NHS-AlexaFluor 647(Thermo Fisher) according to the manufacturer's instructions. On day 0, 1X10 was used⁶4T1 breast cancer cells were injected subcutaneously into 6 to 8 week old female BALB/c mice. On day 7, equimolar amounts of fluorescently labeled 4420 antibody and 4420-basement membrane glycan were injected intratumorally into mice, three mice each, setting up PBS control mice. Retention of fluorescence in the tumor was assessed by measuring fluorescence on an IVIS Spectrum instrument (Perkin Elmer) at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, 72, 96, 100, 124 and 148 hours (fig. 17B).

As shown in fig. 17B, the fluorescence signal from mice injected with the fluorescently labeled 4420 antibody (4420LALA-PG) decreased faster and was greater in magnitude than the fluorescence signal from the 4420-basement-membrane glycan fusion protein (4420-LUM LALA-PG).

These results indicate that over time, collagen-binding antibody fusion proteins (e.g., 4420-basement membrane glycan fusion proteins) are physically retained at the intratumoral injection site. These results suggest that collagen-binding immunomodulatory molecules comprising therapeutic antibodies or antigen-binding fragments will exhibit intratumoral retention and limited systemic dissemination.

Example 22: recombinant expression of collagen-binding IgG-binding fusion proteins

As a strategy to localize all IgG within the tumor without regenerating the antibody directly as a basement membrane glycan fusion, several different IgG-binding proteins can be fused to the basement membrane glycan. As with other basement membrane glycan fusion proteins described herein, a Mouse Serum Albumin (MSA) spacer is used to ensure that basement membrane glycan-collagen binding does not interfere with the function of the IgG binding domain. Several different IgG binders were selected for screening, including the dimerization Z domain (one of the five IgG binding domains of protein A, referred to herein as "ZZ") (Jendeberg et al, (1995) J Mol Recognit 8: 270-278), the dimerization IgG binding domain of protein G (referred to herein as "SpG 2") (Jung et al, (2009) Anal Chem 81: 936-942), IgG binders isolated from Sso7d yeast display libraries (Gera et al, (2011) J Mol Biol 409: 601-616), IgG binders isolated from a fibronectin type III domain (Fn3) yeast display library (Hackel et al, (2010) J Mol Biol 401: 84-96) and two small peptides designed to bind to the IgG Fc region (referred to herein as "Fc-III-4C" and "RRGW") (Gong et al, (2015) bioconjugateg Chem 27: 1569-15720143; Tsai et al, (Anal Chem 86: 2931-2938). In addition, a basement membrane glycan-MSA fused to 4m5.3 was also cloned and expressed. 4m5.3 is an scFv with femtomolar binding affinity for fluorescein (Midelfort et al, (2004) J Mol Biol 343: 685-701). Fluorescein can be conjugated to antibodies by a wide range of conjugation strategies in the field of Antibody Drug Conjugates (ADC) (Carter & Lazar (2017) Nat Rev Drug Discov 17: 197-223). 4m 5.3-MSA-based glycans with fluorescein labeled antibodies were used as an alternative strategy to localize IgG to tumors. This construct also serves as a universal platform that securely binds and localizes any fluorescein (or FITC) -labeled protein or small molecule.

To evaluate the ability of expressing collagen binding IgG binding fusion proteins in mammalian cells, a basement-membrane glycan (basement membrane glycan-MSA-Fc-III-4C (SEQ ID NO: 136; 105.7kDa), basement membrane glycan-MSA-Fn 3(SEQ ID NO: 137; 113.7kDa), basement membrane glycan-MSA-SpG 2(SEQ ID NO: 138; 117.7kDa), ZZ-MSA-basement membrane glycan (SEQ ID NO: 135; 117.5kDa), WGRR-MSA-basement membrane glycan (SEQ ID NO: 140; 104.6kDa), RRGW-MSA-basement membrane glycan (SEQ ID NO: 139; 104.6kDa), Sso7 d-MSA-basement membrane glycan (SEQ ID NO: 134; 111.5kDa) and 4m 5.3-MSA-basement membrane glycan (SEQ ID NO: 133; 132.2kDa) fused to 8 different IgG binding polypeptides were generated, and transiently expressed in human embryonic kidney 293(HEK293) cells. All basement membrane glycan-IgG binding fusion proteins were His-tagged according to the manufacturer's instructions to facilitate purification from HEK293 lysates using talen metal affinity resin (Takara Bio Inc.).

Expression and purification of all 8 basement membrane glycan-IgG binding fusion proteins was achieved as shown by SDS-PAFE analysis, which showed protein bands at predicted molecular weights under both reducing and non-reducing conditions (data not shown).

These results indicate that His-tagged collagen is expressed in mammalian cells to bind to and enable purification of IgG-binding fusion proteins (e.g., IgG-based peptidoglycan).

Example 23: recombinant collagen binding IgG binding fusion proteins bind collagen and IgG in vitro

To evaluate the ability of collagen to bind IgG-binding fusion protein to collagen, the expressed and purified basement membrane glycan-IgG-binding fusion protein as described in example 22 was tested for its ability to bind to plates coated with type I collagen and type IV collagen by ELISA. Briefly, Nunc MaxiSorp flat bottom 96-well plates (ThermoFisher) coated with mouse IgG2a isotype control antibody (100. mu.L, 2.5. mu.g/mL, BioXCell C1.18.4) were coated overnight at 4C. Then, at room temperature, PBS + 0.1% wt/vol Bovine Serum Albumin (BSA) + 0.05% wt/vol Tween 20(PBSTA) blocking for 1 hour, followed by incubation at room temperature for 2 hours with PBSTA containing various concentrations of basement membrane glycans. The wells were washed with PBSTA and then incubated with horseradish peroxidase conjugated polyclonal anti-6 xHis (ab1187, Abcam) (final concentration. 5 μ g/ml) diluted 1:2000 in PBSTA for 1 hour at room temperature. The wells were washed again with PBSTA and then treated for 10min with 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) followed by 1M sulfuric acid to stop the color reaction. The absorbance at 450nm was measured using a M1000 microplate reader (Tecan) (corrected using the reference absorbance at 570 nm).

Purified basement-membrane glycan-IgG-binding fusion proteins, basement-membrane glycan-MSA-Fn 3, basement-membrane glycan-MSA-SpG 2, ZZ-MSA-basement-membrane glycan and 4m 5.3-MSA-basement-membrane glycan were evaluated by ELISA on collagen type I coated plates and collagen type IV coated plates. Basement membrane glycans were used as positive controls. As shown in fig. 18A, all of the basement membrane glycan-IgG-binding fusion proteins retained the ability to bind collagen with similar affinity. These results indicate that the basement membrane glycans fused to various IgG-binding polypeptides retain the ability to bind to collagen type I and collagen type IV.

Purified basement membrane glycan-IgG-binding fusion protein from example 22 was tested for its ability to bind to the mouse IgG2a isotype control (clone C1.18.4) as measured by ELISA. Briefly, an anti-His secondary antibody conjugated with HRP (Abcam, ab1187), and a 1-step Ultra TMB-ELISA substrate (Thermo Fischer) were used to detect IgG conjugate-basement-xylan fusions.

As shown in FIG. 18B, all of the basement membrane glycan-IgG-binding fusion proteins tested had a range of affinities (K)_D) Binding to mouse IgG2 a.

Together, these results indicate that the basement membrane glycan-IgG-binding fusion protein binds to both collagen type I and collagen type IV as well as to IgGs. These results suggest that the use of a basement membrane glycan-IgG-binding fusion protein in combination with IgG (e.g., a therapeutic antibody) will bind both collagen and IgG and retain IgG at the local site of administration.

Example 24: the collagen combined with the basement membrane polysaccharide is retained in the abdominal cavity after the intraperitoneal injection

The foregoing examples have demonstrated intratumoral retention after intratumoral (i.tu.) administration using basement membrane glycans. The abdominal cavity is also lined with collagen-rich mesothelium. To evaluate the ability of the basement membrane glycans to be retained in the peritoneal cavity after intraperitoneal (i.p) injection, BALB/c mice were intraperitoneally injected with Gauss luciferase alone (GLuc; 20. mu.g/dose) or fused to the basement membrane glycans (basement membrane glycan-GLuc; 40. mu.g/dose). Immediately after injection, mice were imaged by in vivo fluorescence (epi-illumination, auto-exposure setup). Mice were re-imaged 24 hours after injection.

The basal lamina glycan-GLuc was retained in the cavity, while GLuc alone diffused rapidly in the abdominal cavity after injection, resulting in a low initial signal (data not shown). Basement membrane glycans were observed to reside in the lumen 24 hours after injection.

Tumors embedded in the intima or omentum majorana are also rich in collagen. To determine whether intraperitoneal administration of the basement membrane glycans would result in biased accumulation of tumors on the omentum majus, the basement membrane glycans fluorescently labeled with Alexa Fluor 647 were administered to mice with ovarian tumor microcolonies in the omentum tissue of the mice. Briefly, mice were intraperitoneally injected with OVCA433 cells (human ovarian tumor cells) and allowed to form micro-colonies on the omentum majus for 3 weeks. 20ug of labeled basement membrane glycan was injected intraperitoneally. The excised membranous tissue was imaged by fluorescence microscopy. The labeled basement membrane glycans were injected intraperitoneally into tumor-bearing mice. Mice were sectioned for imaging of omental tissue at 1 hour, 6 hours, and 24 hours post-injection.

As shown in figure 19, when the basement membrane glycans were injected intraperitoneally into mice lined with ovarian tumors within the large omentum, the basement membrane glycans were biased towards accumulation in these tumor microcolonies. 1 hour after injection (left panel), the fluorescence signal from the basement membrane glycans was evenly distributed (in yellow). At 6 hours post injection (middle), it remained only around the tumor microcolonies of the omentum majus (shown in red) (middle panel). At 24 hours (right panel), it was observed that the basement membrane glycans remained around the large tumor. These results indicate that intraperitoneally injected basement membrane glycans can accumulate at collagen-rich sites, and that basement membrane glycans are suitable for administration in several ways, including intraperitoneal injection.

Example 25: expression of collagen-binding IL-12 fusion proteins from self-replicating RNA in mammalian cells

To evaluate the expression of collagen-binding immunomodulatory molecules using RNA, their ability to be expressed in B16F10 mouse melanoma cells was examined using self-replicating RNA molecules (replicons) encoding IL12-MSA and IL 12-MSA-based peptidoglycan, either alone or fused to fluorescent protein (mCherry). The replicon used in this example was from alphavirus. The replicon does capsid structural proteins, but retains non-structural proteins. The non-structural proteins correspond to RNA-based RNA replication and RNA transcription. Ab1c1 is a mutant replicon that shows greater expression in vitro and in vivo compared to the wild-type replicon. The Ab1c1 replicon contained four mutations in the nsP2 and nsP3 genes, which prolonged the presence of the replicon in both cellular and subgenomic transcripts.

Briefly, 0.5x10 cultured in DMEM + 10% FBS was transfected with a NEOnN transfection reagent (electroporation) and Ab1c1 replicon⁶And B16F10 cells. Transfection efficiency was measured after 24 hours by FACS analyzer using an optical setup for mCherry. 24 hours after transfection, cell supernatants and commercial IL-12ELISA for IL-12 expression was quantified (according to the manufacturer's instructions). Replicon expression was assessed in B16F10 cells by flow cytometry by determining mCherry (fig. 20A). A commercial IL-12ELISA was used to quantitate IL-12 in B16F10 cells (FIG. 20B).

As shown in fig. 20A, mCherry + B16F10 cells transfected with IL12-MSA-mCherry or IL 12-MSA-basilar glycan-mCherry were observed by flow cytometry. In these replicates, mCherry fluorescent protein is C-terminally encoded, therefore, detection of mCherry expression indicates full-length expression of the encoded protein.

As shown in fig. 20B, the cell supernatants showed expression of these constructs in B16F10 cells transfected with IL-12 encoding replication, but Ab1c1-GIM control was not shown.

These results indicate that delivery of the basement membrane glycan-fusion protein can be achieved using means other than injection including replicons as indicated.

Example 26: in Braf^v600e/Pten^fl/flSynergistic effects of collagen-binding cytokine fusion protein, anti-PD-1 antibody and TA99 in mouse model

To evaluate the effect of the combination of basement membrane glycan-cytokine and anti-PD-1 blockade in a refractory mouse melanoma model, Braf was used^V600E/Pten^fl/flA genetically modified mouse model (GEMM) (Spranger et al, (2015) Nature 523: 231. sup. 235; Momin et al, (2019) Sci. Transl. Med.11, eeaw 2614). Melanoma is induced in the GEMN by Cre expression in melanocytes regulated by tamoxifen, which drives oncogenic Braf^V600EAnd a biallelic deletion of the tumor suppressor Pten. Braf^V600E/Pten^fl/flMelanoma has fewer neoantigens and greater heterogeneity than B16F10 tumors. This model has moderate T cell infiltration, but tumor growth is only slightly slowed by dual checkpoint blockade of PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

To determine whether the use of collagen binding to IL-2 and IL-12 and anti-PD-1 checkpoint blockade could restart ongoing responses and provoke new T cell responses, as observed in B16F10 tumors (fig. 9, 10 and 13), and whether de novo T cell priming and any distant effects could be enhanced by the introduction of an immunogenic cell death-inducing agent (such as the tumor-targeting antibody TA99), on day 0, by the Braf ^V600E/Pten^fl/flThe right flank of the mouse was treated with 4-hydroxyttamoxifen to induce melanoma. After induction, flat black lesions formed. Without treatment, the lesion would progress to large masses (data not shown). Mice were treated starting on day 25 with PBS control (i.tu), with basement-membrane glycan-MSA-IL 2(i.tu) + IL 12-MSA-basement-membrane glycan (i.tu) + TA99(i.p.) + anti-PD-1 (i.p.), with basement-membrane glycan-MSA-IL 2(i.tu) + IL 12-MSA-basement-membrane glycan (i.tu) + anti-PD-1 (i.p.), with MSA-IL2(i.tu) + IL12-MSA (i.tu) + TA99(i.p.) + anti-PD-1 (i.p.), or with MSA-IL2(i.tu) + IL12-MSA (i.tu) + anti-PD-1 (i.p.).

As shown in FIG. 21A, Braf treated with MSA-IL2(i.tu) + IL12-MSA (i.tu) + TA99(i.p.) + anti-PD-1 (i.p.)^V600E/Pten^fl/flTumor-bearing mice showed inhibition of lesion progression.

As shown in FIG. 21B, Braf treated with anti-PD-1 and collagen-bound IL-2 and IL-12 with or without TA99^V600E/Pten^fl/flThe overall survival of tumor-bearing mice was comparable. Therefore, TA99 is not required for effectiveness. These results demonstrate that IL-2, IL-12 and checkpoint blockade can be effective tumor agonistic combination therapies (fig. 9, fig. 10 and fig. 13).

Tumor control in this model can also be achieved using unanchored cytokines instead of collagen-anchored cytokines, but at the expense of significant and potentially lethal toxicity. One third of the mice treated with IL12-MSA and MSA-IL2 were euthanized due to > 20% weight loss, while none of the mice treated with IL 12-MSA-basement membrane glycans and basement membrane glycan-MSA-IL 2 exhibited treatment-related toxicity (fig. 21B). These results indicate that collagen-binding fusion proteins can safely improve overall survival in this effective tumor agonistic combination therapy (fig. 21B).

Example 27: LAIR ability to bind tumors in vivo

In this example, the ability of LAIR to bind to resected B16F10 tumors was measured. B16F10 tumors had little detectable collagen, and therefore, this was a low estimate for the tumor binding capacity of LAIR. Briefly, 1x10⁶Individual B16F10 cells were inoculated subcutaneously into the left flank of C57/mouse. Seven days later, the tumor was carefully initiated and separated from all residual skin and subcutaneous fat. The excised tumors were then incubated with a mild detergent (PBS + 0.1% v/v tween 20) for 2 hours at 37 ℃ and then dissociated/crushed through a 70 micron filter (fig. 22A). As shown in fig. 22B, stroma accounted for one third of the tumor weight.

The filtered fraction contained no extracellular matrix (i.e., cellular fraction), while the fraction that did not pass through the filter was enriched in matrix (i.e., matrix fraction), as confirmed by hydroxyproline assay (MAK008-1KT, Millipore Sigma) according to the manufacturer's instructions (fig. 22C). Hydroxyproline (4-hydroxyproline) is a non-protein amino acid formed by post-translational hydroxylation of proline. Hydroxyproline is a main component of collagen, and plays a role in stabilizing a helical structure in collagen. Since hydroxyproline is largely limited to collagen, measurement of hydroxyproline levels is used as an indicator of collagen content. In this assay, hydroxyproline concentration was detected by reaction of oxidized hydroxyproline with 4- (dimethylamino) benzaldehyde (DMAB), which resulted in the formation of a colorimetric (560nm) product proportional to the hydroxyproline present.

Each tumor stromal component was then combined with either antigen overdose (10. mu.M) or antigen depletionAF647 labeled LAIR was incubated at a concentration (10 μ M) to quantify the LAIR binding sites in the matrix composition. The solution fluorescence was monitored over time until a steady state was reached. As shown in fig. 22D, a decrease in fluorescence was observed with 1 μ M labeled LAIR, which corresponds to the removal of LAIR from the wash after binding to collagen in the matrix component. A 20% decrease in solution concentration indicates 0.2nmol uptake into the tumor stroma. Thus, from 1x10 ⁶Day 7 of the individual cell inoculum B16F10 tumors had 0.2nmol LAIR binding sites (fig. 22D). This amount correlated with the collagen content of the tumor as determined by hydroxyproline content (fig. 22E). This experiment indicates that LAIR is able to bind to B1610 because the B1610 matrix is collagen-rich.

Example 28: similar benefits were obtained with collagen-binding fusion proteins using LAIR compared to using basement membrane glycans

Like the basement membrane glycans, LAIR is able to bind to type I collagen. LAIR cytokine fusion proteins were used to determine whether collagen binding fusion proteins could enhance the effectiveness of the fusion cytokines. LAIR-cytokine fusion protein LAIR-MSA-IL2(SEQ ID NO:186) was expressed and purified as described for the basement membrane glycan-MSA-IL 2 (example 1). Using the B16F10 melanoma mouse model as described in example 6, LAIR-MSA-IL2 was at least as effective in reducing tumor size as the basement membrane glycan-MSA-IL 2 (compare fig. 3B to fig. 23A). LAIR-MSA IL-2 was also able to increase survival of mice at levels comparable to those of mice treated with a combination of basement membrane glycan-MSA-IL 2 and intraperitoneally administered TA99 (compare fig. 3B and fig. 3C to fig. 23B). These results indicate that collagen binding strategies can be performed using basement membrane glycans, LAIRs, and other collagen binding proteins in this class.

Example 29: LAIR engineering to produce higher and lower affinity collagen conjugates

In this example, a yeast display platform was used to engineer higher and lower affinity variants of LAIR. Briefly, the mouse LAIR gene was amplified using error-prone PCR to generate a library of LAI mutants. RJY200 yeast (EBY100 internal modified form, ATCC) was transformed with linearized pCTCON2 vector (41843, Addgene) and error-prone PCR products were collected and subjected to in vivo homologous recombination events to generate the final display plasmid. The pCTCON2 plasmid was formatted to fuse the LAIR mutant to the Aga2 protein, which binds to the yeast surface through disulfide bonds to the membrane bound Aga1 protein. The LAIR gene is followed by a c-myc tag that can be used to detect the complete expression of the mutant LAIR protein. Once expressed on the surface, the yeast can be stained with a marker antigen and an antibody against the c-myc tag (ACMYC, Ex. alpha.). Clones expressing LAIR, as determined by c-myc staining intensity, can then be sorted using FACS for lower or higher affinity. (Chao et al, (2006) nat. Protoc.1(2): 755-768-).

Soluble collagen peptide mimetics (CRP, collagen-related peptide) were used as antigens for FACS assays because collagen I, the natural ligand for LAIR, is insoluble. The protein sequence of the mimetic is GCO- (GPO) ₁₀-GCOG-NH2, wherein O represents a hydroxyproline amino acid. Like collagen I, these peptides spontaneously form helical structures in solution. These helical structures are held in place using a cross-linking agent (Cat #, inc.). After purification, the cross-linked peptide was used as our antigen (CRP-XL). Notably, the peptides were used in biotinylated (CRP-XL-biotin) and non-biotinylated (CRP-XL) forms. (details on these peptides and their functionalization into triple helix form can be obtained from CambCollab Inc.).

Two different strategies were used to isolate high and low affinity collagen binding mutants. To select low affinity mutants, equilibrium sorting was used. In this strategy, LAIR expression was induced on the surface of the yeast library. The library was incubated with CPP-XL-biotin and chicken anti-c-myc (ACMYC, Ex. alpha.) in sequence, followed by secondary antibody (streptavidin-AF 647(S21374, Thermo Fisher)) and goat anti-chicken AF488(A-1139, Invitrogen) until equilibrium was reached. Yeasts that showed weak or no AF647 signal but positive for AF488, indicating that they express LAIR but do not bind the collagen mimetic, were sorted on the BD FACS Aria machine. After several rounds of sorting, the yeast was micro-prepared to isolate display plasmids, transformed into bacterial colonies, and then sequenced. Popular clones (clones that appear in the sequence at a higher frequency (at least twice) than other clones) and/or clones containing mutations in the collagen binding pocket were selected for downstream analysis. (Brondijk et al, (2010) Blood 115: 1364-. These mutants were cloned into mammalian expression vectors, expressed solubly as a fusion with Mouse Serum Albumin (MSA), and tested for their ability to bind collagen I in an ELISA assay. As shown in fig. 24A-E and fig. 25A-C, the weak binding clones (fig. 24F, fig. 24D) contained mutations in the LAIR binding pocket. 187-192 SEQ ID NOS.

To isolate higher affinity mutations, a kinetic sorting strategy was used. After induction of expression, the CRP-XL-biotin tag library was used as described above. After equilibration, the yeast clones were washed and then incubated for 3-5 days in a 300:1 excess of CRP-XL (not biotinylated). Unlabeled CRP-XL will replace dissociated CRP-XL-biotin for LAIR binding (fig. 26C-D). Only the clones with the lowest shut-down rate, i.e. the highest affinity, will be labeled. The yeast clones with the highest AF647 signal were then sorted using FACS. After several rounds of sorting, the selected high affinity clones were subjected to downstream analysis in a competition assay. The isolated yeast clones were labeled with CRP-XL-biotin and then incubated with unlabeled CRP-XL. Samples were taken over time and analyzed for AF647 signals (fig. 26E-F). As shown in figure 26G, the high affinity mutant LAIR (LAIR30.2.k1.b) had a lower off-rate compared to the WT LAIR. The mutation seen in this clone was located outside the LAIR binding pocket (FIG. 26B, SEQ ID NO: 193). These results indicate that mutant LAIRs with a range of binding affinities to collagen can be isolated. These LAIR1 variants provide the opportunity to engineer immunomodulatory fusion proteins comprising therapeutic agents with different binding affinities for collagen-rich tumors.

Summary of the sequences