Fibroblast activation protein binding agents and uses thereof

文档序号：1966477 发布日期：2021-12-14 浏览：8次中文

阅读说明：本技术 成纤维细胞激活蛋白结合剂及其用途 (Fibroblast activation protein binding agents and uses thereof ) 是由 N·克雷 E·德普拉 L·扎比奥于 2020-03-27 设计创作，主要内容包括：本发明部分地涉及结合成纤维细胞激活蛋白(FAP)的剂、嵌合蛋白和蛋白复合物以及它们作为诊断剂和治疗剂的用途。本发明还涉及包含所述FAP结合剂、嵌合蛋白和蛋白复合物的药物组合物以及所述药物组合物在治疗各种疾病中的用途。(The present invention relates in part to agents that bind Fibroblast Activation Protein (FAP), chimeric proteins and protein complexes and their use as diagnostic and therapeutic agents. The invention also relates to pharmaceutical compositions comprising said FAP-binding agents, chimeric proteins and protein complexes and the use of said pharmaceutical compositions in the treatment of various diseases.)

1. A Fibroblast Activation Protein (FAP) binding agent comprising a targeting moiety, wherein the targeting moiety comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iii) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (ii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) any one of SEQ ID NOs 877 to 879;

(iii) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (ii).

2. The FAP-binding agent of claim 1, wherein the FAP-binding agent is a full-length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a miniprotein, a dappin, an anticalin, an adnectin, an aptamer, an Fv, a Fab ', a F (ab')₂A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

3. The FAP-binding agent of claim 2, wherein the targeting moiety is a single domain antibody.

4. The FAP-binding agent of claim 2, wherein the targeting moiety comprises a VHH, a humanized VHH or a camelized VHH.

5. The FAP-binding agent of any one of claims 1 to 4, wherein the FAP-binding agent comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) Any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NO 837-850; or

(e) Any one of SEQ ID NO 1045-1085.

6. The FAP-binding agent of any of claims 1 to 5, wherein the FAP-binding agent comprises one or more signaling agents.

7. The FAP-binding agent of claim 6, wherein the signaling agent is selected from one or more of a wild-type interferon, a wild-type interleukin, a wild-type tumor necrosis factor, or a modified form thereof.

8. The FAP-binding agent of claim 7, wherein the targeting moiety and the signaling agent are optionally linked to one or more linkers.

9. The FAP-binding agent of claim 6 or 7, wherein the signaling agent is a modified interferon alpha 2(IFN alpha 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the modified human IFN alpha 2 has one or more mutations conferring increased safety compared to a wild-type IFN alpha 2 having the amino acid sequence SEQ ID NO:176 or 177.

10. The FAP-binding agent of claim 9, wherein the IFN α 2 has:

(a) One or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

(b) 176 or 177, at positions L15, a19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, T106, D114, L117, R120, R125, K133, K134, R144, a145, M148, R149, S152, L153 and N156.

11. The FAP-binding agent of claim 9, wherein the modified IFN α 2 has one or more mutations at positions R149, M148, or L153, relative to SEQ ID NOs 176 or 177.

12. The FAP-binding agent of claim 10, wherein the mutation is:

(a) 176 or 177, optionally M15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q3661, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R144A, a 145A, M148A, R149A, S152A, L A, and N36156; or

(b) 176 or 177 relative to the amino acid sequence SEQ ID NO, one or more selected from R33A, T106X₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

13. The FAP-binding agent of claim 6 or 7, wherein the signaling agent is a modified interferon that is IFN α 1 having a mutation at one or more of amino acids at positions L, A, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154 and N157 relative to SEQ ID NO 1042, said mutation optionally being selected from L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, K126, R133, K134, K135, R145, R149, R145, R146A 146, M146, A146, M146, R146, A146, M146, R146, and S146, R146, A146, and S146, L154A and N157A.

14. The FAP-binding agent of claim 6 or 7, wherein the signaling agent is a modified interferon-beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the modified human IFN- β has one or more mutations conferring increased safety compared to a wild-type IFN- β having the amino acid sequence SEQ ID NO: 178.

15. The FAP-binding agent of claim 14, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178.

16. The FAP-binding agent of claim 15, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, relative to SEQ ID NO: 178.

17. The FAP-binding agent of claim 6 or 7, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:182, and wherein the modified TNF α has one or more mutations conferring reduced receptor binding affinity as compared to a wild-type TNF α having the amino acid sequence SEQ ID NO: 182.

18. The FAP-binding agent of claim 17, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, relative to SEQ ID No. 182.

19. The FAP-binding agent of claim 17, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, a109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, a145G, E146G, and S147G, relative to SEQ ID No. 182.

20. The FAP-binding agent of claim 19, wherein the modified TNF α has a mutation at position Y87, optionally selected from Y87Q, Y87L, Y87A, and Y87F.

21. The FAP-binding agent of claim 19, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

22. The FAP-binding agent of claim 19, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

23. The FAP-binding agent of any one of claims 1 to 22, wherein the FAP-binding agent comprises one or more additional targeting moieties.

24. The FAP-binding agent of claim 23, wherein the one or more additional targeting moieties recognizes or functionally modulates a tumor antigen.

25. The FAP-binding agent of claim 23, wherein the one or more additional targeting moieties recognizes or functionally modulates an antigen on an immune cell.

26. The FAP-binding agent of claim 25, wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

27. The FAP-binding agent of any one of claims 1 to 26, wherein the FAP-binding agent recruits cytotoxic T cells to a tumor cell or tumor environment.

28. The FAP-binding agent of claim 27, wherein the one or more additional targeting moieties recognizes or functionally modulates PD-1, PD-L1, PD-L2, CTLA4, OX40L, OX40, CD20, XCR1, Flt3, or Clec 9A.

29. The FAP-binding agent of any of claims 1 to 28, wherein the FAP-binding agent recognizes or binds FAP without substantially functionally modulating the activity of the FAP.

30. The FAP-binding agent of any one of claims 1 to 29, wherein the FAP-binding agent is suitable for use in a patient suffering from one or more of the following: cancer, infection, immune disorders, fibrotic diseases, and autoimmune diseases.

31. A recombinant nucleic acid encoding the FAP-binding agent of any one of claims 1 to 30.

32. A host cell comprising the nucleic acid of claim 31.

33. A method for treating or preventing cancer, comprising administering to a patient in need thereof an effective amount of the FAP-binding agent of any one of claims 1 to 30.

34. The method of claim 33, wherein the cancer is selected from one or more of: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; liver cancer; hepatoma; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHLs); small Lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; higher-grade immunocytogenic NHL; higher lymphoblastic NHL; high-grade small non-nucleated cell NHL; giant-mass NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nevus maculatus hamartoma; edema (e.g., edema associated with brain tumors); and megs syndrome.

35. The method of any one of claims 33 or 34, further comprising administering a DNA intercalating agent chemotherapeutic agent, such as doxorubicin, cisplatin, daunorubicin, or epirubicin.

36. The method of any one of claims 33 to 35, wherein the FAP-binding agent recruits immune cells, directly or indirectly, to a tumor or tumor microenvironment.

37. A method for treating or preventing an autoimmune disease and/or a neurodegenerative disease, the method comprising administering to a patient in need thereof an effective amount of the FAP-binding agent of any one of claims 1 to 30.

38. The method of claim 37, wherein the autoimmune disease and/or neurodegenerative disease is selected from multiple sclerosis, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, guillain-barre syndrome, scleroderma, goodpasture's syndrome, wegener's granulomatosis, autoimmune epilepsy, lasmassen encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, addison's disease, hashimoto's thyroiditis, fibromyalgia, meniere's syndrome; transplant rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, lupus erythematosus, myasthenia gravis, reiter's syndrome, and graves ' disease.

39. The method of claim 38, wherein the autoimmune disease and/or neurodegenerative disease is multiple sclerosis.

40. The method of any one of claims 37-39, wherein the chimeric protein causes immune suppression in the patient.

41. A method for treating or preventing a fibrotic disease, the method comprising administering to a patient in need thereof an effective amount of the FAP-binding agent of any one of claims 1 to 30.

42. The method of claim 41, wherein the fibrotic disease is selected from liver fibrosis, lung fibrosis, Primary Sclerosing Cholangitis (PSC), chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver injury, cirrhosis, and myelodysplastic syndrome.

43. A chimeric protein, comprising:

at least one targeting moiety that targets or binds to Fibroblast Activation Protein (FAP) and a wild-type or modified signaling agent, wherein the targeting moiety comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) Any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) any one of SEQ ID NOs 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, 1035; or

(v) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iv);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iii) any one of SEQ ID NOs 883, 916, 921, 924, 929, 932, 937, 940, 945, 948, 953, 956, 961, 964, 969, 972, 977, 980, 985, 988, 993, 996, 1001, 1004, 1009, 1012, 1017, 1020, 1025, 1028, 1033, 1036; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) Any one of SEQ ID NOs 877 to 879;

(iii) any of SEQ ID NOs 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii).

44. The chimeric protein of claim 43, wherein the chimeric protein directly or indirectly alters the microenvironment of disease-associated fibroblasts.

45. The chimeric protein of claim 43, wherein the chimeric protein polarizes disease-associated fibroblasts directly or indirectly.

46. The chimeric protein of any one of claims 44 or 45, wherein the fibroblast is associated with a disease selected from: cancer, infection, immune disorders, autoimmune diseases, fibrotic diseases and cardiovascular diseases.

47. The chimeric protein of any one of claims 43 to 46, wherein the targeting moiety targets F2 fibroblasts.

48. The chimeric protein of any one of claims 43 to 47, wherein the targeting moiety is a full-length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a miniprotein, a dappin, an anticalin, an adnectin, an aptamer, an Fv, a Fab ', a F (ab') ₂A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

49. The chimeric protein of claim 48, wherein the targeting moiety is a single domain antibody.

50. The chimeric protein of claim 49, wherein the targeting moiety comprises a VHH, a humanized VHH, or a camelized VHH.

51. The chimeric protein of any one of claims 43 to 50, wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NOs 837 to 850;

(e) any one of SEQ ID NOs 1045 to 1085;

(f) any of the VH chains of SEQ ID NOs 880, 918, 926, 934, 942, 950, 958, 966, 974, 982, 990, 998, 1006, 1014, 1022, 1030; or

(g) Any of VL chains of SEQ ID NOs 881, 919, 927, 935, 943, 951, 959, 967, 975, 983, 991, 999, 1007, 1015, 1023, 1031.

52. The chimeric protein of claim 43, wherein the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, and modified forms thereof.

53. The chimeric protein of claim 43, wherein the targeting moiety and the signaling agent are optionally linked to one or more linkers.

54. The chimeric protein of claim 52 or 53, wherein the signaling agent is a mutant interferon alpha 2(IFN alpha 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the mutant human IFN alpha 2 has one or more mutations conferring increased safety compared to a wild-type IFN alpha 2 having the amino acid sequence SEQ ID NO:176 or 177.

55. The chimeric protein of claim 54, wherein the IFN α 2 has:

(a) one or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

56. The chimeric protein according to claim 55, wherein the mutation is one of L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q61A, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R144A, A145A, M148, R A, S152A, L A, and N156, or more of SEQ ID NO:176 or 177, optionally one of L153, L148, L33, or L A.

57. The chimeric protein of claim 55, wherein the mutant human IFN α 2 has one or more amino acid sequences selected from the group consisting of R33A, T106X, relative to amino acid sequence SEQ ID NO 176 or 177₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

58. The chimeric protein of claim 52 or 53, wherein the signaling agent is a modified interferon that is IFNa 1 having a mutation at one or more of amino acids at positions L, A, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 relative to SEQ ID NO 1042, optionally selected from L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, R149, R145, R146A 146, M146, A146, M146, R146, A146, M146, and S146A 146, M146, R30, D32, D153, D146, and N157, L154A and N157A.

59. The chimeric protein of claim 52 or 53, wherein the signaling agent is a mutant interferon-beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the mutant human IFN- β has one or more mutations conferring increased safety compared to a wild-type IFN- β having the amino acid sequence SEQ ID NO: 178.

60. The chimeric protein of claim 59, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178.

61. The chimeric protein of claim 60, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G relative to SEQ ID NO 178.

62. The chimeric protein of claim 52 or 53, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:182, and wherein the modified TNF α has one or more mutations that confer reduced receptor binding affinity as compared to a wild-type TNF α having the amino acid sequence SEQ ID NO: 182.

63. The chimeric protein of claim 62, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, relative to SEQ ID NO 182.

64. The chimeric protein of claim 62, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, E146G, and S147G, relative to SEQ ID NO 182.

65. The chimeric protein of claim 63, wherein the modified TNF α has a mutation at position Y87, optionally selected from Y87Q, Y87L, Y87A, and Y87F.

66. The chimeric protein of claim 63, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

67. The chimeric protein of claim 63, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

68. The chimeric protein of any one of claims 43 to 67, wherein the chimeric protein comprises one or more additional targeting moieties.

69. The chimeric protein of claim 68, wherein the one or more additional targeting moieties recognizes or functionally modulates a tumor antigen.

70. The chimeric protein of claim 68, wherein the one or more additional targeting moieties recognizes or functionally modulates an antigen on an immune cell.

71. The chimeric protein of claim 70, wherein the immune cell is selected from a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

72. The chimeric protein of any one of claims 43 to 71, wherein the chimeric protein recruits cytotoxic T cells to a tumor cell or tumor environment.

73. The chimeric protein of claim 72, wherein the one or more additional targeting moieties recognize or functionally modulate PD-1, PD-L1, PD-L2, CTLA4, OX40L, OX40, CD20, XCR1, Flt3, or Clec 9A.

74. The chimeric protein of any one of claims 43 to 73, wherein the chimeric protein recognizes or binds to FAP without substantially functionally modulating the activity of FAP.

75. The chimeric protein of any one of claims 43 to 74, wherein the chimeric protein is suitable for use in a patient suffering from one or more of the following: cancer, infection, immune disorder, fibrotic disease and/or autoimmune disease.

76. A recombinant nucleic acid encoding the chimeric protein of any one of claims 43 to 75.

77. A host cell comprising the nucleic acid of claim 76.

78. A method for treating or preventing cancer, the method comprising administering to a patient in need thereof an effective amount of a chimeric protein comprising:

at least one targeting moiety that targets or binds Fibroblast Activation Protein (FAP) and a modified signaling agent, wherein the targeting moiety comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) any one of SEQ ID NOs 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, 1035; or

(v) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iv);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) any one of SEQ ID NOs 877 to 879;

(iii) any of SEQ ID NOs 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii).

79. The method of claim 78, wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NOs 837 to 850;

(e) any one of SEQ ID NOs 1045 to 1085;

(f) any of the VH chains of SEQ ID NOs 880, 918, 926, 934, 942, 950, 958, 966, 974, 982, 990, 998, 1006, 1014, 1022, 1030; or

(g) Any of VL chains of SEQ ID NOs 881, 919, 927, 935, 943, 951, 959, 967, 975, 983, 991, 999, 1007, 1015, 1023, 1031.

80. The method of claim 78 or 79, wherein the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, and modified forms thereof.

81. The method of claim 80, wherein the targeting moiety and the signaling agent are optionally linked to one or more linkers.

82. The method of claim 80, wherein the signaling agent is a mutant interferon alpha 2(IFN α 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the mutant human IFN α 2 has one or more mutations conferring increased safety compared to a wild-type IFN α 2 having the amino acid sequence of SEQ ID NO:176 or 177.

83. The method of claim 82, wherein the IFN alpha 2 has:

(a) one or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

(b) 176 or 177, at positions L15, a19, R22, R23, L26, F27, L30, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, D114, L117, R120, R125, K133, K134, R144, a145, M148, R149, S152, L153 and N156.

84. The method according to claim 83, wherein the mutation is one of L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q61A, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R144A, a 145A, M148, R A, S36149, S A, L153, and N156, optionally L153, or L A, or N36153, or L36148, with respect to SEQ ID No. 176 or 177.

85. The method of claim 82, wherein the mutant human IFN alpha 2 has one or more selected from R33A, T106X relative to the amino acid sequence of SEQ ID NO 176 or 177₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

86. The method of claim 80, wherein the modified interferon is IFN α 1 having a mutation at one or more amino acids at positions L, A, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 relative to SEQ ID NO 1042, said mutation optionally being selected from L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, 149, R145, A146, M146, A, and N157 relative to SEQ ID NO 1042.

87. The method of claim 80, wherein the signaling agent is a mutant interferon beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the mutant human IFN- β has one or more mutations conferring increased safety compared to a wild-type IFN- β having the amino acid sequence of SEQ ID NO: 178.

88. The method of claim 87, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO 178.

89. The method of claim 88, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G of SEQ ID NO 178.

90. The method of claim 80, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:182, and wherein the modified TNF α has one or more mutations conferring reduced receptor binding affinity as compared to wild-type TNF α having the amino acid sequence SEQ ID NO: 182.

91. The method of claim 90, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 relative to SEQ ID NO 182.

92. The method of claim 90, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D relative to SEQ ID NO 182.

93. The method of claim 90, wherein the modified TNF α has a mutation at position Y87, optionally selected from Y87Q, Y87L, Y87A, and Y87F.

94. The method of claim 90, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

95. The method of claim 90, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

96. The method of any one of claims 78 to 95, wherein the cancer is selected from one or more of: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; liver cancer; hepatoma; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHLs); small Lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; higher-grade immunocytogenic NHL; higher lymphoblastic NHL; high-grade small non-nucleated cell NHL; giant-mass NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nevus maculatus hamartoma; edema (e.g., edema associated with brain tumors); and megs syndrome.

97. The method of any one of claims 78-96, wherein the chimeric protein recruits an immune cell to a tumor or tumor microenvironment, directly or indirectly.

98. A method for treating or preventing an autoimmune disease and/or a neurodegenerative disease, the method comprising administering to a patient in need thereof an effective amount of a chimeric protein comprising:

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) any one of SEQ ID NOs 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, 1035; or

(v) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iv);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) any one of SEQ ID NOs 877 to 879;

(iii) any of SEQ ID NOs 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii).

99. The method of claim 98, wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NOs 837 to 850;

(e) any one of SEQ ID NOs 1045 to 1085;

(f) any of the VH chains of SEQ ID NOs 880, 918, 926, 934, 942, 950, 958, 966, 974, 982, 990, 998, 1006, 1014, 1022, 1030; or

(g) Any of VL chains of SEQ ID NOs 881, 919, 927, 935, 943, 951, 959, 967, 975, 983, 991, 999, 1007, 1015, 1023, 1031.

100. The method of claim 98 or 99, wherein the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, and modified forms thereof.

101. The method of claim 100, wherein the targeting moiety and the signaling agent are optionally linked to one or more linkers.

102. The method of claim 100, wherein the signaling agent is a mutant interferon alpha 2(IFN α 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the mutant human IFN α 2 has one or more mutations conferring increased safety compared to a wild-type IFN α 2 having the amino acid sequence of SEQ ID NO:176 or 177.

103. The method of claim 102, wherein the IFN α 2 has:

(a) one or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

104. The method according to claim 103, wherein the mutation is one of L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q3661, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R144A, a 36145, a A, M36148, R A, S149, S A, L153, and N36156, optionally L153, or L A, or N36153, with respect to SEQ ID No. 176 or 177, optionally one of L153, R148, A, or N36153.

105. The method of claim 102, wherein the mutant human IFN α 2 has one or more amino acid sequences selected from the group consisting of R33A, T106X, relative to amino acid sequence SEQ ID NOs 176 or 177₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

106. The method of claim 100, wherein the modified interferon is IFN α 1 having a mutation at one or more amino acids at positions L, a, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157 relative to SEQ ID NO:1042, said mutation being optionally selected from L15, a19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, 149, R145, 146 a, a146, N146 a, a146, N146, a, N146, and N146 a, M146, a, N157, to SEQ ID NO.

107. The method of claim 100, wherein the signaling agent is a mutant interferon-beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the mutant human IFN- β has one or more mutations conferring increased safety compared to a wild-type IFN- β having the amino acid sequence of SEQ ID NO: 178.

108. The method of claim 107, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178.

109. The method of claim 108, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G of SEQ ID NO: 178.

110. The method of claim 100, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID No. 182, and wherein the modified TNF α has one or more mutations conferring reduced receptor binding affinity as compared to wild-type TNF α having the amino acid sequence of SEQ ID No. 182.

111. The method of claim 110, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 relative to SEQ ID NO 182.

112. The method of claim 110, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, a109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, a145G, a145R, a145T, E146D, E146K, and S147D relative to SEQ ID No. 182.

113. The method of claim 112, wherein the modified TNF α has a mutation at position Y87, optionally selected from the group consisting of Y87Q, Y87L, Y87A, and Y87F.

114. The method of claim 112, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

115. The method of claim 112, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

116. The method of any one of claims 98-115, wherein the autoimmune disease and/or neurodegenerative disease is selected from multiple sclerosis, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, guillain-barre syndrome, scleroderma, goodpasture's syndrome, wegener's granulomatosis, autoimmune epilepsy, lasimason encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, addison's disease, hashimoto's thyroiditis, fibromyalgia, meniere's syndrome; transplant rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, lupus erythematosus, myasthenia gravis, reiter's syndrome, and graves ' disease.

117. The method of claim 116, wherein the autoimmune disease and/or neurodegenerative disease is multiple sclerosis.

118. The method of any one of claims 98-117, wherein the chimeric protein causes immune suppression in the patient.

119. A method for treating or preventing a fibrotic disease, the method comprising administering to a patient in need thereof an effective amount of a chimeric protein comprising:

At least one targeting moiety that targets or binds Fibroblast Activation Protein (FAP) and a modified signaling agent, wherein the targeting moiety comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) any one of SEQ ID NOs 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, 1035; or

(v) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iv);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) any one of SEQ ID NOs 877 to 879;

(iii) any of SEQ ID NOs 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii).

120. The method of claim 119, wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NOs 837 to 850;

(e) Any one of SEQ ID NOs 1045 to 1085;

(f) any of the VH chains of SEQ ID NOs 880, 918, 926, 934, 942, 950, 958, 966, 974, 982, 990, 998, 1006, 1014, 1022, 1030; or

(g) Any of VL chains of SEQ ID NOs 881, 919, 927, 935, 943, 951, 959, 967, 975, 983, 991, 999, 1007, 1015, 1023, 1031.

121. The method of claim 119 or 120, wherein the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, and modified forms thereof.

122. The method of claim 121, wherein the targeting moiety and the signaling agent are optionally linked to one or more linkers.

123. The method of claim 121, wherein the signaling agent is a mutant interferon alpha 2(IFN α 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the mutant human IFN α 2 has one or more mutations conferring increased safety compared to a wild-type IFN α 2 having the amino acid sequence of SEQ ID NO:176 or 177.

124. The method of claim 123, wherein the IFN α 2 has:

(a) One or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

125. The method according to claim 123, wherein the mutation is one of L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q3661, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R144A, a 36145, a A, M36148, R A, S149, S A, L153, and N36156, optionally L153, or L A, or N36153, relative to SEQ ID No. 176 or 177, optionally one of L149, L A, or N36153.

126. The method of claim 123, wherein the mutant human IFN α 2 has one or more amino acid sequences selected from the group consisting of R33A, T106X, relative to amino acid sequence SEQ ID NOs 176 or 177₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X ₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

127. The method of claim 121, wherein the modified interferon is IFN α 1 having a mutation at one or more amino acids at positions L, a, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157 relative to SEQ ID NO:1042, said mutation being optionally selected from L15, a19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, 149, R145, 146 a, a146, N146 a, N146, a146, N146, a, N146, and N146 a, M146, N157, a, to SEQ ID NO.

128. The method of claim 121, wherein the signaling agent is a mutant interferon beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the mutant human IFN- β has one or more mutations conferring increased safety compared to a wild-type IFN- β having the amino acid sequence of SEQ ID NO: 178.

129. The method of claim 128, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178.

130. The method of claim 129, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G of SEQ ID NO: 178.

131. The method of claim 121, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID No. 182, and wherein the modified TNF α has one or more mutations conferring reduced receptor binding affinity as compared to wild-type TNF α having the amino acid sequence of SEQ ID No. 182.

132. The method of claim 131, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 relative to SEQ ID No. 182.

133. The method of claim 131, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, a109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, a145G, a145R, a145T, E146D, E146K, and S147D relative to SEQ ID No. 182.

134. The method of claim 131, wherein the modified TNF α has a mutation at position Y87, optionally selected from the group consisting of Y87Q, Y87L, Y87A, and Y87F.

135. The method of claim 131, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

136. The method of claim 131, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

137. The method of claim 119, wherein the fibrotic disease is selected from liver fibrosis, lung fibrosis, Primary Sclerosing Cholangitis (PSC), chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis c infection, alcoholic liver disease, liver injury, cirrhosis, and myelodysplastic syndrome.

138. An Fc-based chimeric protein complex, comprising:

at least one targeting moiety that targets or binds to Fibroblast Activation Protein (FAP); and

an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and/or stabilize a hinge region in the Fc domain;

And a wild-type or modified signaling agent;

wherein the targeting moiety comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein

(a) CDR1 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 1108, 1129, 1093 to 1107 and 1109-1128;

(ii) any one of SEQ ID NOs 87 to 115;

(iii) any one of SEQ ID NOs 851 to 861;

(iv) any one of SEQ ID NOs 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032, 1035; or

(v) (iii) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iv);

(b) CDR2 comprises the following amino acid sequence:

(i) any one of SEQ ID NOs 126, 128, 116 to 125, 127, and 129 to 144;

(ii) any one of SEQ ID NOs 862 to 876;

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii); and is

(i) any one of SEQ ID NOs 157, 159, 145 to 156, 158, 160 to 175;

(ii) any one of SEQ ID NOs 877 to 879;

(iii) any of SEQ ID NOs 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037; or

(iv) (iv) an amino acid sequence having one to five amino acid substitutions, deletions or insertions in any of the amino acid sequences of (i) to (iii).

139. The Fc-based chimeric protein complex of claim 138, wherein the complex directly or indirectly alters the microenvironment of a disease-associated fibroblast.

140. The Fc-based chimeric protein complex of claim 138, wherein the complex directly or indirectly polarizes disease-associated fibroblasts.

141. The Fc-based chimeric protein complex of any one of claims 139 or 140, wherein the fibroblast is associated with a disease selected from: cancer, infection, immune disorders, autoimmune diseases, fibrotic diseases and cardiovascular diseases.

142. The Fc-based chimeric protein complex of any one of claims 138 to 141, wherein the targeting moiety targets F2 fibroblasts.

143. The Fc-based chimeric protein complex of any one of claims 138 to 142, wherein the targeting moiety is a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a miniprotein, a dappin, an anticalin, an adnectin, an aptamer, an Fv, a Fab ', a F (ab')₂A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.

144. The Fc-based chimeric protein complex of claim 143, wherein the targeting moiety is a single domain antibody.

145. The Fc-based chimeric protein complex of claim 144, wherein the targeting moiety comprises a VHH, a humanized VHH, or a camelized VHH.

146. The Fc-based chimeric protein complex of any one of claims 138 to 145, wherein the targeting moiety comprises an amino acid sequence having at least 90% sequence similarity to:

(a) any one of SEQ ID NOs 1087, 1092, 1086, 1088-1091;

(b) any one of SEQ ID NOs 2 to 42;

(d) any one of SEQ ID NOs 837 to 850;

(e) any one of SEQ ID NOs 1045 to 1085;

(g) any of the VH chains of SEQ ID NOs 880, 918, 926, 934, 942, 950, 958, 966, 974, 982, 990, 998, 1006, 1014, 1022, 1030; or

(h) Any of VL chains of SEQ ID NOs 881, 919, 927, 935, 943, 951, 959, 967, 975, 983, 991, 999, 1007, 1015, 1023, 1031.

147. The Fc-based chimeric protein complex of any one of claims 138-146, wherein the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, and modified forms thereof.

148. The Fc-based chimeric protein complex of claim 147, wherein the signaling agent is a mutant interferon alpha 2(IFN α 2) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:176 or 177, and wherein the mutant human IFN α 2 has one or more mutations conferring increased safety compared to a wild-type IFN α 2 having the amino acid sequence of SEQ ID NO:176 or 177.

149. The Fc-based chimeric protein complex of claim 148, wherein IFN α 2 has:

(a) One or more mutations at positions 144 to 154 relative to SEQ ID NO:176 or 177, relative to SEQ ID NO:176 or 177; or

(b) 176 or 177, at positions L15, a19, R22, R23, L26, F27, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61, F64, N65, T69, L80, Y85, Y89, D114, L117, R120, R125, K133, K134, R144, a145, M148, R149, S152, L153 and N156.

150. The Fc-based chimeric protein complex of claim 148, wherein the mutation is one of L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, H57A, E58A, Q61A, F64A, N65A, T69A, L80A, Y85A, Y89A, D114A, L117A, R120A, R125A, K133A, K134A, R36144A, a145 a A, a 145A, M148A, R A, S149, S A, L153, N156, L153, or L A, optionally L153, or N153, R A, relative to SEQ ID No. 176 or 177.

151. The Fc-based chimeric protein complex of claim 148, wherein the mutant human IFN α 2 has one or more amino acids selected from the group consisting of R33A, T106X, relative to the amino acid sequence of SEQ ID NO 176 or 177 ₃、R120E、R144X₁ A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

152. The Fc-based chimeric protein complex of claim 148, wherein the modified interferon is IFN α 1 having a mutation at one or more amino acids at positions L, a, R, S, L, D, R, H, Q, C, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157 relative to SEQ ID NO:1042, said mutation optionally selected from the group consisting of L15, a19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, 149, R145, R146 a146, M146, a, M146, N157, a, and N157 relative to SEQ ID NO.

153. The Fc-based chimeric protein complex of claim 147, wherein the signaling agent is a mutant interferon beta (IFN- β) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:178, and wherein the mutant human IFN- β has one or more mutations conferring increased safety as compared to a wild-type IFN- β having the amino acid sequence of SEQ ID NO: 178.

154. The Fc-based chimeric protein complex of claim 153, wherein the modified human IFN- β comprises one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155 of SEQ ID NO: 178.

155. The Fc-based chimeric protein complex of claim 154, wherein the modified human IFN- β comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G of SEQ ID NO: 178.

156. The Fc-based chimeric protein complex of claim 147, wherein the signaling agent is a modified tumor necrosis factor alpha (TNF α) comprising an amino acid sequence having at least 95% identity to SEQ ID No. 182, and wherein the modified TNF α has one or more mutations conferring reduced receptor binding affinity as compared to wild-type TNF α having the amino acid sequence of SEQ ID No. 182.

157. The Fc-based chimeric protein complex of claim 156, wherein the modified TNF α has one or more mutations at positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, relative to SEQ ID No. 182.

158. The Fc-based chimeric protein complex of claim 156, wherein the mutation is one or more of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, a109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, a145G, a145R, a145T, E146D, E146K, and S147 39 147D relative to SEQ ID No. 182.

159. The Fc-based chimeric protein complex of claim 156, wherein the modified TNF α has a mutation at position Y87, optionally selected from the group consisting of Y87Q, Y87L, Y87A, and Y87F.

160. The Fc-based chimeric protein complex of claim 156, wherein the modified TNF α has a mutation at position I97, optionally selected from I97A, I97Q, and I97S.

161. The Fc-based chimeric protein complex of claim 156, wherein the modified TNF α has a mutation at position Y115, optionally selected from Y115A and Y115G.

162. The Fc-based chimeric protein complex of claim 138, further comprising one or more linkers.

163. The Fc-based chimeric protein complex of claim 138, wherein the Fc domain is from IgG, IgA, IgD, IgM, or IgE.

164. The Fc-based chimeric protein complex of claim 163, wherein the IgG is selected from IgG1, IgG2, IgG3, or IgG 4.

165. The Fc-based chimeric protein complex of any one of claims 138-164, wherein the signaling agent is a modified signaling agent and has reduced affinity or activity for a receptor of the signaling agent relative to a wild-type signaling agent.

166. The Fc-based chimeric protein complex of any one of claims 138-165, wherein the signaling agent is a modified signaling agent and the targeting moiety restores the affinity or activity of the modified signaling agent for a receptor of the signaling agent.

167. The Fc-based chimeric protein complex of any one of claims 138-166, wherein the Fc chain pairing is facilitated by ionic pairing and/or knob-into-hole pairing.

168. The Fc-based chimeric protein complex of any one of claims 138-167, wherein the one or more mutations of the Fc domain result in ionic pairing between Fc chains in the Fc domain.

169. The Fc-based chimeric protein complex of any one of claims 138-168, wherein the one or more mutations of the Fc domain result in knob-in-hole pairing in the Fc domain.

170. The Fc-based chimeric protein complex of any one of claims 138-169, wherein the one or more mutations of the Fc domain result in a reduction or elimination of the effector function of the Fc domain.

171. The Fc-based chimeric protein complex of any one of claims 138-170, wherein the targeting moiety comprises a recognition domain that recognizes and binds an antigen or receptor on an immune cell.

172. The Fc-based chimeric protein complex of claim 171, wherein the immune cell is selected from the group consisting of a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

173. The Fc-based chimeric protein complex of any one of claims 138-172, wherein the Fc domain is homodimeric.

174. The Fc-based chimeric protein complex of any one of claims 138-162 and 165-173, wherein the Fc domain is heterodimeric.

175. The Fc-based chimeric protein complex of any one of claims 138 to 174, wherein the complex comprises one or more additional targeting moieties.

176. The Fc-based chimeric protein complex of claim 175, wherein the one or more additional targeting moieties recognizes or functionally modulates a tumor antigen.

177. The Fc-based chimeric protein complex of claim 175, wherein the one or more additional targeting moieties recognizes or functionally modulates an antigen on an immune cell.

178. The Fc-based chimeric protein complex of claim 177, wherein the immune cell is selected from the group consisting of a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

179. The Fc-based chimeric protein complex of any one of claims 138 to 178, wherein the chimeric protein recruits cytotoxic T cells to a tumor cell or tumor environment.

180. The Fc-based chimeric protein complex of claim 179, wherein the one or more additional targeting moieties recognizes or functionally modulates PD-1, PD-L1, PD-L2, CTLA4, OX40L, OX40, CD20, Flt3, XCR1, and Clec 9A.

181. The Fc-based chimeric protein complex of any one of claims 138 to 180, wherein the complex recognizes or binds FAP without substantially functionally modulating the activity of FAP.

182. The Fc-based chimeric protein complex of any one of claims 138 to 181, wherein the complex is suitable for use in a patient having one or more of the following: cancer, infection, immune disorder, fibrotic disease and/or autoimmune disease.

183. The Fc-based chimeric protein complex of any one of claims 138 to 182, wherein the Fc-based chimeric protein complex has the orientation/configuration of any one of: fig. 5A to 5F, fig. 6A to 6H, fig. 7A to 7H, fig. 8A to 8D, fig. 9A to 9F, fig. 10A to 10J, fig. 11A to 11D, fig. 12A to 12F, fig. 13A to 13J, fig. 14A to 14F, fig. 15A to 15L, fig. 16A to 16L, fig. 17A to 17F, fig. 18A to 18L, fig. 19A to 19L, fig. 20A to 20J, fig. 21A to 21J, fig. 22A to 22F, and fig. 23A to 23F.

184. The Fc-based chimeric protein complex of any one of claims 138 to 183, wherein the Fc is human IgG1, and optionally contains one or more of L234, L235, K322, D265, P329, and P331 (according to EU numbering).

185. The Fc-based chimeric protein complex of any one of claims 138-184, wherein the Fc-based chimeric protein complex has a trans-orientation/configuration as to any targeting moiety and signaling agent relative to each other, or any targeting moiety relative to each other, or any signaling agent relative to each other.

186. A recombinant nucleic acid encoding the Fc-based chimeric protein complex of any one of claims 138 to 185 or a component thereof.

187. A host cell comprising the nucleic acid of claim 186.

188. A method for treating or preventing cancer, the method comprising administering to a patient in need thereof an effective amount of the Fc-based chimeric protein complex of any one of claims 138 to 185.

189. A method for treating or preventing an autoimmune disease and/or a neurodegenerative disease, the method comprising administering to a patient in need thereof an effective amount of the Fc-based chimeric protein complex of any one of claims 138 to 185.

190. A method for treating or preventing a fibrotic disease, the method comprising administering to a patient in need thereof an effective amount of the Fc-based chimeric protein complex of any one of claims 138 to 185.

Technical Field

The present technology relates, in part, to binding agents that bind Fibroblast Activation Protein (FAP), chimeric proteins and Fc-based chimeric protein complexes and their use as therapeutic and diagnostic agents.

Description of electronically submitted text files

The contents of a text file electronically submitted with the present document are incorporated by reference herein in their entirety. A computer-readable format copy of the sequence table (filename: ORN-059PC _ ST25, recording date: 26 months and 3 years 2020; file size: 770,048 bytes).

Background

Fibroblasts regulate the structure and function of healthy tissue, participate in tissue repair shortly after acute inflammation, and exert abnormal stimulatory effects in chronic inflammatory states including cancer. Cancer-associated fibroblasts (CAF) regulate the tumor microenvironment and influence the behavior of neoplastic cells in a manner that promotes or inhibits tumors. Understanding the microenvironment of a tumor is important for cancer treatment. Fibroblasts express a variety of immunomodulatory factors, such as cytokines, lipid mediators, and growth factors. In addition, fibroblasts display many surface and intracellular receptors, as well as the molecular mechanisms necessary for responding to external signals. Fibroblasts can be considered as an extension of the "professional" immune system, given that they can trigger inflammation. Fibroblasts are involved in many normal and pathological processes. Illustrative diseases known to be associated with abnormal fibroblasts include cancer, cardiovascular disease and autoimmune disease. CAF is an important matrix component and plays an important role in regulating the tumor microenvironment and influencing tumor cell behavior, primarily through the release of proteolytic enzymes, growth factors and cytokines. Studies have shown that the pro-cancerous and anti-therapeutic properties of the matrix can be attributed to the activity of fibroblasts.

Human fibroblast activation protein (FAP; GenBank accession AAC 51668; NCBI reference sequence: NM 004460.3), also known as Seprase, is an integrated membrane serine peptidase of 170kDa (EC 3.4.21. B28). FAP belongs to the dipeptidyl peptidase IV family, is a homodimer containing two N-glycosylated subunits and having a large C-terminal extracellular domain in which the catalytic domain of the enzyme is located (Scanlan et al, Proc. Natl. Acad. Sci. USA 91(1994), 5657. SP. 5661). The glycosylated form of FAP has both post-prolyl dipeptidyl peptidase and gelatinase activities (Sun et al, Protein Expr. Purif.24(2002), 274-281). Thus, FAP is a serine protease that has both dipeptidyl peptidase activity and endopeptidase activity that cleaves both gelatin and type I collagen.

FAP has a unique organizational profile: its expression was found to be highly upregulated in more than 90% of reactive stromal fibroblasts of all primary and metastatic epithelial tumors, including lung, colorectal, bladder, ovarian and breast cancers, but is universally absent in normal adult tissues (Rettig et al, Proc. Natl. Acad. Sci. USA 85(1988), 3110-. Subsequent reports indicate that FAP is expressed not only in stromal fibroblasts but also in certain types of malignant cells of epithelial origin, and that FAP expression is directly associated with the malignant phenotype (Jin et al, Anticancer Res.23(2003), 3195-.

Since FAP is expressed in many common cancers and is restricted in expression in normal tissues, it is a promising antigenic target in the imaging, diagnosis and treatment of various carcinomas. There remains a need for improved therapies for treating diseases associated with abnormal fibroblasts, such as treating fibrotic diseases or treating cancer by altering CAF function.

Disclosure of Invention

In one aspect, the present technology relates to Fibroblast Activation Protein (FAP) binding agents that target or bind to FAP. In some embodiments, the FAP-binding agent comprises a moiety that targets FAP. The FAP-binding agent or the FAP-targeting moiety may, for example, be a full-length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a miniprotein, a dappin, an anticalin, an adnectin, an aptamer, an Fv, a Fab ', a F (ab')₂A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule. In some embodiments, the FAP targeting moiety is a single domain antibody (VHH). In some embodiments, the FAP-binding agent directly or indirectly alters a disease microenvironment comprising disease-associated F2 fibroblasts (e.g., a tumor microenvironment comprising disease-associated F2 fibroblasts). In some embodiments, the FAP-binding agent directly or indirectly polarizes F2 fibroblasts associated with a disease. In some embodiments, the FAP-binding agents further comprise signaling agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified to reduce their activity. In some embodiments, the FAP-binding agent comprises binding to other target of interest (e.g., anti-FAP) A pro or receptor). In another embodiment, the other target of interest (e.g., antigen or receptor) is present on a fibroblast. In some embodiments, the additional target of interest (e.g., an antigen or receptor) is present on fibroblasts in the cancer stroma. In some embodiments, the fibroblast-binding agents of the invention may recruit, directly or indirectly, immune cells (e.g., dendritic cells) to a site of action (such as a tumor microenvironment, as a non-limiting example). In some embodiments, the FAP-binding agents of the invention promote antigen (e.g., antigen or receptor) presentation by immune cells (e.g., dendritic cells, macrophages) in the tumor stroma or direct antigen presentation by fibroblasts.

In some embodiments, these FAP-binding agents bind to FAP, but do not functionally modulate (e.g., partially or fully neutralize) FAP. Thus, in some embodiments, FAP-binding agents of the invention are used, e.g., to directly or indirectly recruit FAP-expressing cells to a target site, while still allowing FAP-expressing cells to signal via FAP (i.e., binding of the FAP-binding agent does not reduce or eliminate FAP signaling at the target site). Conversely, in some embodiments, FAP-binding agents of the invention are used, e.g., to directly or indirectly recruit FAP-expressing cells to a target site, but do not allow FAP-expressing cells to signal via FAP (i.e., binding of the FAP-binding agent reduces or eliminates FAP signaling at the target site). In some embodiments, the FAP targeting moiety is a single domain antibody (VHH).

In another aspect, the present technology relates to a chimeric protein or Fc-based chimeric protein complex with a FAP-binding agent disclosed herein or with at least one targeting moiety that targets or binds to FAP. In another aspect, the FAP-binding agents or chimeric proteins or Fc-based chimeric protein complexes disclosed herein can be used in methods of treating various diseases or disorders (such as cancer, infections, inflammatory diseases or disorders, immune disorders, fibrotic diseases, and other diseases and disorders).

In another aspect, one or more targeting moieties (i.e., FAP binding agents) and one or more signaling agents are conjugated to one or more Fc domains to form an Fc-based chimeric protein complex. In some embodiments, the one or more targeting moieties (i.e., FAP-binding agents) and the one or more signaling agents are linked directly to the one or more Fc domains or via a linker. Such Fc-based chimeric protein complexes surprisingly have a significantly improved half-life in vivo, particularly when in the heterodimer configuration as described herein, as compared to chimeras lacking Fc, and are particularly suitable for production and purification. Thus, the Fc-based chimeric protein method of the invention results in agents that are particularly suitable for use as therapeutic agents.

In another aspect, the present technology relates to FAP-binding agents, chimeric proteins, or Fc-based chimeric protein complexes that are cross-reactive between humans, mice, and cynomolgus monkeys.

Drawings

Fig. 1 shows FAP VHH-bound fluorescence-activated cell sorting (FACS) data. FAP VHH periplasmic extracts were applied to HEK293T cells transiently transfected with human FAP, mouse FAP or empty vector (MOCK). Binding was measured and plotted as the Mean Fluorescence Intensity (MFI) difference between FAP and MOCK transfected cells.

Fig. 2A-2H show binding selection of VHH with human, mouse and cynomolgus FAP in FACS. HEK293T cells transiently transfected with human FAP, mouse FAP or empty vector (MOCK) were incubated with serial dilutions of purified FAP VHH. Binding was measured and plotted as the Mean Fluorescence Intensity (MFI) difference between FAP and MOCK transfected cells. Fig. 2A shows data of 2PE14, fig. 2B shows data of 2PE17, fig. 2C shows data of 2PE36, fig. 2D shows data of 2PE40, fig. 2E shows data of 2PE42, fig. 2F shows data of 2PE44, fig. 2G shows data of 3PE12, fig. 2H shows data of 3PE42, fig. 2I shows data of 3PE57, fig. 2J shows data of 3PE93, and fig. 2K shows data of 3PE 94.

Fig. 3A-3D show wild-type IFN α 2 and FAP VHH ActaFeron (AFN) signaling in HL116 and HL 116-hfp cells after targeting. Parental HL116 or derived HL 116-hfp cells were stimulated with wild-type IFNa2 or FAP VHH AFN for 6 hours as indicated. The mean luciferase values (. + -. STDEV) of three measurements were plotted. Figure 3A shows IFN alpha 2 data, figure 3B shows 2PE14-AFN data, figure 3C shows 3PE12-AFN data, and figure 3D shows 3PE42-AFN data.

Figure 4 shows a protein sequence alignment of some embodiments of VHHs that bind human FAP. The CDRs 1, CDR2, CDR3 of these VHH sequences are boxed and their alignment is shown.

Fig. 5A-5F, 6A-6H, 7A-7H, 8A-8D, 9A-9F, 10A-10J, 11A-11D, 12A-12F, 13A-13J, 14A-14F, 15A-15L, 16A-16L, 17A-17F, 18A-18L, 19A-19L, 20A-20J, 21A-21J, 22A-22F, and 23A-23F show various non-limiting illustrative schematics of Fc-based chimeric protein complexes of the present invention. In various embodiments, each schematic is a composition of the invention. Where applicable in the figures, "TM" refers to a "targeting moiety" as described herein. "SA" refers to a "signaling agent" as described herein. Is an optional "linker" as described herein. The two long parallel rectangles are human Fc domains as described herein, e.g., from IgG1, from IgG2, or from IgG4, and optionally have effector knockout and/or stabilizing mutations also as described herein. Two long parallel rectangles (one with a protrusion and the other with a depression) are human Fc domains as described herein, e.g., from IgG1, from IgG2, or from IgG4, with knob-in holes and/or ion pair (aka charged pair, ionic bond, or charged residue pair) mutations as described herein, and optionally with effector knockout and/or stabilization mutations also as described herein.

Fig. 5A-5F show illustrative homodimer 2-strand complexes. These figures show illustrative configurations of homodimer 2-strand complexes.

Fig. 6A-6H show illustrative homodimer 2-chain complexes with two Targeting Moieties (TM) (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, the constructs shown in boxes (i.e., fig. 6G and 6H) have Signaling Agents (SA) between TM1 and TM2 or between TM1 and Fc.

Fig. 7A-7H show illustrative homodimer 2-chain complexes with two signaling agents (more signaling agents may be present in some embodiments, as described herein). In various embodiments, the positions of SA1 and SA2 are interchangeable. In various embodiments, the constructs shown in boxes (i.e., fig. 7G and 7H) have a TM between SA1 and SA2, or a TM at the N-terminus or C-terminus.

Fig. 8A-8D show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the pore chain of Fc.

Fig. 9A-9F show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the pore chain of Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.

Fig. 10A-10J show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the pore chain of Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations/configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the positions of SA1 and SA2 are interchangeable.

Fig. 11A-11D show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e. SA on the knob chain of Fc and TM on the pore chain of Fc.

Fig. 12A-12F show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc, and both TM on the pore chain of Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.

Fig. 13A-13J show illustrative heterodimer 2 chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc, and TM on the pore chain of Fc, with two signaling agents (as described herein, more signaling agents may be present in some embodiments). In these orientations/configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the positions of SA1 and SA2 are interchangeable.

Fig. 14A-14F show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of Fc.

Fig. 15A-15L show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.

Fig. 16A-16L show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of Fc, with two signaling agents (as described herein, in some embodiments more signaling agents may be present). In various embodiments, the positions of SA1 and SA2 are interchangeable.

Fig. 17A-17F show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the pore chain of Fc.

Fig. 18A-18L show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the pore chain of Fc, with two targeting moieties (in some embodiments, more targeting moieties are present, as described herein). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, TM1 and TM2 may be the same.

Fig. 19A-19L show illustrative heterodimer 2 chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the pore chain of Fc, with two signaling agents (as described herein, in some embodiments more signaling agents may be present). In various embodiments, the positions of SA1 and SA2 are interchangeable.

Fig. 20A-20J show illustrative heterodimer 2 chain complexes with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments), and where SA is on knob Fc and TM is on each chain. In various embodiments, TM1 and TM2 may be the same.

Fig. 21A-21J show illustrative heterodimer 2 chain complexes with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments), and where SA is on the pore Fc and TM is on each chain. In various embodiments, TM1 and TM2 may be the same.

Fig. 22A-22F show illustrative heterodimer 2 chain complexes with two signaling agents (more signaling agents may be present in some embodiments as described herein) and with split SA and TM chains: SA on knob and TM on well Fc.

Fig. 23A-23F show illustrative heterodimer 2 chain complexes with two signaling agents (more signaling agents may be present in some embodiments as described herein) and with split SA and TM chains: TM on knob and SA on well Fc.

Fig. 24A-24H show sensorgrams of FAP VHH variants bound to biotinylated FAP in biolayer interferometry (BLI).

Detailed Description

The present technology is based, in part, on the discovery of fibroblast-binding agents or fibroblast activation protein-binding agents (e.g., antibodies, such as VHH, as non-limiting examples) that recognize, target, or bind to fibroblasts or fibroblast activation proteins. In some embodiments, the fibroblast-binding agent of the present invention is part of a chimeric or fusion protein or Fc-based chimeric protein complex having one or more targeting moieties and/or one or more signaling agents.

In some embodiments, the fibroblast-binding agent or fibroblast activation protein-binding agent targets F2 fibroblasts. In some embodiments, the fibroblast-binding agent or FAP-binding agent directly or indirectly alters a disease microenvironment comprising disease-associated F2 fibroblasts (e.g., a tumor microenvironment comprising the F2 fibroblasts). In some embodiments, the fibroblast-binding agent or FAP-binding agent directly or indirectly polarizes F2 fibroblasts into F1 fibroblasts, wherein in some cases, the fibroblasts are associated with a disease.

F2 fibroblasts refer to the tumorigenic (or tumor-promoting) cancer-associated fibroblasts (CAF) (also known as type II-CAF). F1 fibroblasts are known as oncostatic CAF (also known as type I-CAF). Polarization refers to changing the phenotype of the cell, for example, converting tumorigenic F2 fibroblasts into tumorigenic F1 fibroblasts.

In some embodiments, the fibroblast-binding agent or FAP-binding agent targets the FAP marker. In some embodiments, the fibroblast-binding agent or FAP-binding agent comprises a moiety that targets FAP. In some embodiments, the fibroblast-binding agent or FAP-binding agent portion targeting FAP is any FAP-targeting portion disclosed herein.

In some embodiments, the fibroblast-binding agent or FAP-binding agent comprises an amino acid sequence having at least 90% sequence similarity to any one of SEQ ID NOs 2-42 or 46-86.

In some embodiments, the fibroblast-binding agent or FAP-binding agent further comprises one or more signaling agents. In some embodiments, the signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor, any of which is optionally modified.

In some embodiments, a fibroblast-binding agent or FAP-binding agent further comprising one or more signaling agents directly or indirectly alters a disease microenvironment comprising disease-associated F2 fibroblasts (e.g., a tumor microenvironment comprising the F2 fibroblasts). In some embodiments, a fibroblast-binding agent or FAP-binding agent further comprising one or more signaling agents directly or indirectly polarizes F2 fibroblasts into F1 fibroblasts.

In some embodiments, the fibroblast-binding agent or FAP-binding agent further comprises one or more additional targeting moieties. In some embodiments, the one or more additional targeting moieties recognize and optionally functionally modulate a tumor antigen. In some embodiments, the one or more additional targeting moieties recognize and optionally functionally modulate an antigen on an immune cell.

In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, and an NK cell.

In some embodiments, the fibroblast-binding agent or FAP-binding agent recruits cytotoxic T cells to tumor cells or the tumor environment.

In some embodiments, the fibroblast-binding agent or FAP-binding agent recognizes and binds FAP without substantially functionally modulating the activity of the FAP.

In another aspect, the present technology is based, in part, on the discovery of agents (e.g., antibodies, such as VHH as non-limiting examples) that recognize and bind to Fibroblast Activation Protein (FAP). In some embodiments, the FAP-binding agents of the invention are part of a chimeric or fusion protein or Fc-based chimeric protein complex having one or more targeting moieties and/or one or more signaling agents. In some embodiments, these FAP-binding agents bind to FAP, but do not functionally modulate FAP. In some embodiments, these FAP-binding agents can bind to and recruit, directly or indirectly, immune cells to a site in need of a therapeutic effect (e.g., a tumor or tumor microenvironment). In some embodiments, FAP-binding agents enhance tumor antigen presentation to elicit an effective anti-tumor immune response.

In some embodiments, the FAP-binding agent modulates antigen presentation. In some embodiments, the FAP-binding agents mediate the immune response to avoid or reduce autoimmunity. In some embodiments, the FAP-binding agent provides immune suppression. In some embodiments, the FAP-binding agent increases the ratio of tregs to CD8+ T cells and/or CD4+ T cells in the patient. In some embodiments, the methods of the invention involve reducing autoreactive T cells in the patient.

In some embodiments, the present technology provides pharmaceutical compositions comprising the FAP-binding agents and uses of the pharmaceutical compositions in the treatment of various diseases, including fibrotic diseases. In some embodiments, the present technology provides pharmaceutical compositions comprising the FAP-binding agents and uses of the pharmaceutical compositions in the treatment of various diseases, including cancer, autoimmune diseases, and/or neurodegenerative diseases.

In some embodiments, the FAP-binding agents of the invention are used to target cancer-associated fibroblasts (CAF). For example, in various embodiments, FAP-binding agents of the invention target fibroblasts within the stroma of a tumor, e.g., in the treatment of cancers, e.g., cancers of epithelial origin, such as carcinoma. Since CAF plays a central role in regulating the dynamic and bidirectional interactions that occur between malignant epithelial cells, extracellular matrix (ECM), and numerous non-cancerous cells common in the tumor environment, including endothelial cells, adipocytes, inflammatory cells, and immune cells, the FAP-binding agents of the present invention provide a means to deliver critical anti-tumor therapies (e.g., modified cytokines and/or other targeting moieties as described elsewhere herein) to a target site. In various embodiments, FAP-binding agents of the invention target a stromal microenvironment consisting of activated fibroblasts, Endothelial Cells (ECs) involved in tubular adenogenesis, and extracellular matrix (ECM), which is constantly remodeling to accommodate tumor growth. Thus, for example in the case of chimeras (or chimeric proteins) with cytokines, optionally with additional targeting moieties, the FAP binding agents of the invention can deliver anti-tumor signals to the stromal microenvironment, which is crucial for tumor development. In various embodiments, the FAP-binding agents are used to target the membranes of cells critical for tumor niche formation in primary tumors or metastases (such as cancer-associated fibroblasts, MSCs, selected cancer cells, and endothelial cells).

FAP-binding agents

Fibroblast Activation Protein (FAP) is a melanoma membrane-bound gelatinase of 170kDa belonging to the serine protease family. FAP is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of osteosarcomas and soft tissue sarcomas. FAP is believed to be involved in controlling fibroblast growth or epithelial-mesenchymal interactions in the processes of development, tissue repair and epithelial carcinogenesis.

In some embodiments, the FAP-binding agents of the invention are protein-based agents capable of specifically binding to FAP. In some embodiments, the FAP-binding agents of the invention are protein-based agents capable of specifically binding to FAP without functionally modulating (e.g., partially or fully neutralizing) FAP.

In some embodiments, the FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that recognizes an epitope present on FAP. In some embodiments, the antigen recognition domain recognizes one or more linear epitopes present on FAP. As used herein, a linear epitope refers to any contiguous amino acid sequence present on FAP. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on FAP. As used herein, a conformational epitope refers to one or more segments of amino acids (which may be discontinuous) that form a three-dimensional surface having features and/or shape and/or tertiary structure that are capable of being recognized by an antigen recognition domain.

In some embodiments, FAP-binding agents of the present technology can bind to full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants, or mutants of human FAP. In some embodiments, FAP-binding agents of the present technology can bind to any form of human FAP, including monomeric, dimeric, heterodimeric, multimeric, and related forms. In one embodiment, the FAP-binding agent binds to a monomeric form of FAP. In another embodiment, the FAP-binding agent binds to a dimeric form of FAP. In another embodiment, the FAP-binding agent binds to a glycosylated form of FAP, which can be a monomeric form or a dimeric form.

In one embodiment, the FAP-binding agents of the invention comprise a targeting moiety with an antigen recognition domain that recognizes one or more epitopes present on human FAP. In some embodiments, the human FAP comprises the amino acid sequence:

MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLPTKYALWWSPNGKFLAYAEFNDKDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSRPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD(SEQ ID NO:1)。

in some embodiments, the FAP-binding agents of the invention comprise a targeting moiety capable of specific binding. In some embodiments, the FAP-binding agent comprises a targeting moiety, such as an antibody or derivative thereof, having an antigen recognition domain. In a fruit In embodiments, the FAP-binding agent comprises a targeting moiety, which is an antibody. In some embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain includes a variable region (e.g., V)_H) And at least three constant regions (e.g., CH)₁、CH₂And CH₃) And each light chain comprises a variable region (V)_L) And a constant region (C)_L). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions, also known as Complementarity Determining Regions (CDRs), flanked by four relatively conserved Framework Regions (FRs). The three CDRs (designated CDR1, CDR2, and CDR3) contribute to the antibody binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

In some embodiments, the FAP-binding agent comprises a targeting moiety that is an antibody derivative or antibody form. In some embodiments, the FAP-binding agents of the invention comprise a targeting moiety that is a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a miniprotein (cysteine knot protein, knottin), a DARPin; tetranectin (Tetranectin); affibody; trans body (Transbody); anti-transporter protein; AdNectin; affilin; affimer; a microtype (Microbody); an aptamer; austeres (alterase); a plastic antibody; ferulomer (phylomer); stradobody (stradobody); the macrocode (maxibody); the evibody (evibody); phenanthroibody (fynomer), armadillo repeat protein, Kunitz-type domain (Kunitz domain), avimer (avimer), atrazine (atrimer), prorobody (probody), immunomer (immunobody), tremelimumab (triomab), trojan (troybody); body of perps (pepbody); vaccine (vaccibody), monospecific (UniBody); bispecific (DuoBody), Fv, Fab ', F (ab')2, peptidomimetic molecules, or synthetic molecules, as described in the following U.S. patent nos. or patent publication nos.: US 7,417,130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446 and/or US 7,166,697, the contents of which patents are hereby incorporated by reference in their entirety. See also Storz mabs.2011, 5-6 months; 3(3):310-317.

In some embodiments, the FAP-binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH may be derived, for example, from an organism producing VHH antibodies, such as camel, shark, or the VHH may be a designed VHH. VHHs are therapeutic proteins of antibody origin that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camelids lacking the light chain. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH 3). VHH is available under the trademark NANOBODY or NANOBODIES.

In one embodiment, the FAP-binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

In some embodiments, the VHH comprises a fully human VH domain, such as HUMABODY (credence Biologics, Cambridge, UK). In some embodiments, the fully human VH domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain (e.g., HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Illustrative fully human VH domains (e.g., humabs) are described, for example, in WO 2016/113555 and WO 2016/113557, the entire disclosures of which are incorporated by reference.

By way of example, but not by way of limitation, in some embodiments, a human VHH FAP-binding agent comprises an amino acid sequence selected from the group consisting of:

2PE2：

QVQLQESGGGSVQVGGSLRLSCADSGSTFTINAMGWYRQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:2)

2PE5：

QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTSYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:3)

2PE7：

QVQLQESGGGLVHAGGSLRLSCADSGSTFSVNAVGWYRQAPGKRRDWVAGITSDGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:4)

2PE13：

QVQLQESGGGLVQVGGSLRLSCAASGSTFILNAMAWYRQAPGNRRELVAGISSGGDTNYPDSVKGRFTISRDNANNIVYLQMNSLKLEDTAVYYCNLWPPRASPSGRVYWGQGTQVTVSS(SEQ ID NO:5)

2PE14：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:6)

2PE17：

QVQLQESGGGLVQSGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:7)

2PE19：

QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMAWYRQAPGKRRELVAGISGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS(SEQ ID NO:8)

2PE20：

QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTVSRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:9)

2PE23：

QVQLQESGGGLVQPGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGTIYWGQGTQVTVSS(SEQ ID NO:10)

2PE25：

QVQLQESGGGLVQVGGSLRLSCAASGSSFIINAMGWYRQAPGKRRELVAGISSDGATHYPDSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNLWPPRASPSGRVYWGQGTQVTVSS(SEQ ID NO:11)

2PE27：

QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:12)

2PE28：

QVQLQESGGGLVQIGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGTIYWGQGTQVTVSS(SEQ ID NO:13)

2PE29：

QVQLQESGGGLVQPGGSLRLSCAASGSTSSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:14)

2PE30：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSINNAMGWYRQAPGKRRDWVAGITSSGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:15)

2PE32：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRRELVAGISSDDITYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRGYWGQGTQVTVSS(SEQ ID NO:16)

2PE33：

QVQLQESGGGLVQPGGSLRLSCADSGSTFSINSMGWYRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS(SEQ ID NO:17)

2PE36：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS(SEQ ID NO:18)

2PE38：

QVQLQESGGGLVQAGESLRLSCAASGSTFTINAMGWYRXAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:19)

2PE39：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSVNAVGWYRQAPGKRRDWVAGITSDGVTNYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:20)

2PE40：

QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:21)

2PE41：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSINSMGWYRQAPGKHRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS(SEQ ID NO:22)

2PE42：

QVQLQESGGGLVQAGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVRGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS(SEQ ID NO:23)

2PE43：

QVQLQESGGGLVQPGGSLTLACKGSGVELSRSAMAWYQQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDAAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:24)

2PE44：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLIPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:25)

2PE47：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:26)

2PE49：

QVQLQESGGGLVQAGGSLRLSCTASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:27)

2PE55：

QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNHPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:28)

2PE56：

QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMGWYRQAPGKRRDWVAGITSSGVTQYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:29)

2PE58：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRREWVAGISSSGPPHYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPMASPSGAIYWGQGTQVTVSS(SEQ ID NO:30)

2PE59：

QVQLQESGGGLVQPGGSLRLSCAVSGSIFSLNAMAWYRQAPGKRRELVAGISGGSVTNYPDSVKGRFTISRDSTKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:31)

2PE60：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:32)

2PE61：

QVQLQESGGGLVQPGGSLRLICAASGSTFSGNAMAWYRXAPGKRRELVAGISGGITTYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGLVYWGQGTQVTVSS(SEQ ID NO:33)

2PE62：

QVQLQESGGGLVQAGGSLRLSCADSSGSTFSINAMAWYRQAPGKRRDWVAGITSDSVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIDWGQGTQVTVSS(SEQ ID NO:34)

2PE63：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:35)

2PE67：

QVQLQESGGGLVQVGGSLRLSCAASGSTFILNAMGWYRQAPGNRRELVAGISSGGDTNYPDSVKGRFTISRDNANNIVYLQMNSLKLEDTAVYYCNLWPPRASPSGRPYWGQGTQVTVSS(SEQ ID NO:36)

2PE68：

QVQLQESGGGLVQPGGSLRLSCAASGSIFSTNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRAPPDGRVYWGQGTQVTVSS(SEQ ID NO:37)

2PE71：

QVQLQESGGGMVQSGRSLRLSCLASVNIVNLNSVGWYRQAPGQQRELVASITSAGSTNYAESVKGRFTISRDNSKNTVYLQMNSLKPSDTAVYYCNLWPPRVSPSGRGYWGQGTQVTVSS(SEQ ID NO:38)

2PE72：

QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMAWYRQAPGRRRELVAGIDGGGVTNYPDSVKGRFTISRDHAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:39)

2PE76：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFAISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:40)

2PE83：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSINSMGWYRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS(SEQ ID NO:41)

2PE84：

QVQLQESGGGLVQAGGSLRLSCAASGSISSLNAMGWYRQAPGKQREWVAGITSGGSTNYADSVKGRFTILRDNAKNTVYLQMSSLKFEDTAVYYCNLWPPRASPSGAVYWGQGTQVTVSS(SEQ ID NO:42)。

in various illustrative embodiments, the FAP-binding agent comprises an amino acid sequence selected from any of the sequences provided above, with or without a terminal histidine tag sequence (i.e., HHHHHHHH; SEQ ID NO: 43).

In various illustrative embodiments, the FAP-binding agent comprises an amino acid sequence selected from any of the sequences provided above, with or without an HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 44).

In various illustrative embodiments, the FAP-binding agent comprises an amino acid sequence selected from any of the sequences provided above, with or without an AAA linker (i.e., AAA).

In various illustrative embodiments, the FAP-binding agent comprises an amino acid sequence selected from any of the sequences provided above, with or without an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 45).

By way of example, but not by way of limitation, in some embodiments, a human VHH FAP-binding agent comprises an amino acid sequence selected from the group consisting of:

2PE86：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSINAMGWYRQAPGKRRDWVAGITSDGVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRVSPSGRIYWGQGTQVTVSS(SEQ ID NO:46)

2PE87：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAAYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:47)

2PE88：

QVQLQESGGGLVQPGGSLRLSCAASESTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNIVYLQMSSLKPEDTAVYYCNLWPPRAPPGGRVYWGQGTQVTVSS(SEQ ID NO:48)

2PE95：

QVQLQESGGGLVQVGGSLRLSCADSGSTFSINAMGWYRQAPGKRRDWVAGITSSGVTKYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRIYWGQGTQVTVSS(SEQ ID NO:49)

3PE1：

QVQLQESGGGLVQAGGSLKLSCAGSGSTFSINAMAWYRQAPGERRELVAGISGDNITNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:50)

3PE5：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGQRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:51)

3PE12：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSGNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRVSPGGGVYWGQGTQVTVSS(SEQ ID NO:52)

3PE21：

QVQLQESGGGLVQAGESLRLSCAASGRDFRDNSMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMDSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:53)

3PE28：

QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:54)

3PE42：

QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:55)

3PE43：

QVQLQESGGGLVQAGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS(SEQ ID NO:56)

3PE44：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAMAWYRQAPGKRRELVAGISGGDVTHYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:57)

3PE47：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLIPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:58)

3PE49：

QVQLQESGGGLVQPGGSLRLSCAGSGSTFSINAMAWYRQAPGERRELVAGISGDNITNYPNSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:59)

3PE57：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSVNAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWGQGTQVTVSS(SEQ ID NO:60)

3PE62：

QVQLQESGGGLVQPGGSLRLSCAASGSTFSSNAMAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGGVYWGQGTQVTVSS(SEQ ID NO:61)

3PE69：

QVQLQESGGGLVQPGGSLTLSCTTSEFTLAYFGVGWFRQAPGKRRDWVAGITTDGITKYPDSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGRLYWGQGTQVTVSS(SEQ ID NO:62)

3PE72：

QVQLQESGGGLVQAGGSLRLSCAASGSTFSGNAMAWYRRAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRVSPGGRVYWGQGTQVTVSS(SEQ ID NO:63)

3PE77：

QVQLQESGGGLVQPGGSLRLSCADSGSTFTINAMAWYRQAPGKRRELVAGISGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPDGRVYWSQGTQVTVSS(SEQ ID NO:64)

3PE82：

QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGKGTQVTVSS(SEQ ID NO:65)

3PE90：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINAMGWYRQAPGKRREWVAGITSGVTHYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:66)

3PE92：

QVQLQESGGGLVQVGGSLRLSCAASGSTFSINNAMGWYRQAPGKRREWVAGISSGGVTHYPDSVKGRFTISRDNAKNIVYLQMNSPKPEDTAVYYCNLWPPRASPSGSIYWGQGTQVTVSS(SEQ ID NO:67)

3PE93：

QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSMKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:68)

3PE94：

QVQLQESGGGLVQPEGSLRLSCAASGSISSINAMAWYRQAPGKRRELVAGIDGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:69)

2PE18：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRQTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:70)

2PE22：

QVQLQESGGGLVQPGGSLRLSCADSGSTFGLSAMGWYRQTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:71)

2PE26：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:72)

3PE4：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYSDSVKGRFTISRVNPKRIVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:73)

3PE16：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRLTPGKQRELVASITSDGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPDDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:74)

3PE22：

QVQLQESGGGLVQPGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:75)

3PE25：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLTAIGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVNPKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:76)

3PE26：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:77)

3PE33：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRENPKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:78)

3PE46：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVGAKRTVYLQMNSLRPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:79)

3PE55：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYADSVKGRFTISRVSAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:80)

3PE61：

QVQLQESGGGLVRPGGSLRLSCADSGSTFGLSAMGWYRQSPGKQRELVASIISDGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:81)

3PE63：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLSAMGWYRQSPGKQRELVASIISDGRTNYADSVKGRFTISRVNAKRTVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:82)

3PE70：

QVQLQESGGGLVQAGGSLRLSCADSGSTFGLGAMGWYRQSPGKQRELVASITSGGRTNYSDSVKGRFTISRVTPKRIVYLQMNSLKPEDTAVYVCNARFGIRDFWGQGTQVTVSS(SEQ ID NO:83)

2PE1：

QVQLQESGGGLVQAGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS(SEQ ID NO:84)

2PE35：

QVQLQESGGGLVQVGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS(SEQ ID NO:85)

3PE11：

QVQLQESGGGLVQPGGSLRLSCAASGSIFGINAVGWYRQAPGKQRELVATFTRGGDINYADSVKGRFTIFRDNAANTVYLQMNSLKAEDTAVYYCNTPPRIGRGYWGQGTQVTVSS (SEQ ID NO:86), by way of example and not by way of limitation, in some embodiments, the humanized VHH FAP binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

·P-1901：2PE14 QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1045)

P-1902: 2PE14_ opt1(Q1D _ Q5V _ A74S _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNKNTVYLQMSSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1046)

P-1903: 2PE14_ opt2(Q1D _ Q5V _ P60A _ A74S _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYDSVKGRFTISRDNKNTVYLQMSSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1047)

P-1904: 2PE14_ opt3(Q1D _ Q5V _ A74S _ S82N _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNKNTVYLQMNSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1048)

P-1905: 2PE14_ opt4(Q1D _ Q5V _ P60A _ A74S _ S82N _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYDSVKGRFTISRDNKNTVYLQMSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1049)

P-1906: 2PE14_ N32A (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1050)

P-1907: 2PE14_ N32D (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1051)

P-1908: 2PE14_ N32E (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1052)

P-1909: 2PE14_ N32F (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1053)

P-1910: 2PE14_ N32G (bold letters indicate mutations)

·QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1054)

P-1911: 2PE14_ N32H (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1055)

P-1912: 2PE14_ N32I (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1056)

P-1913: 2PE14_ N32K (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1057)

P-1914: 2PE14_ N32L (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1058)

P-1915: 2PE14_ N32P (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSI AVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1059)

P-1916: 2PE14_ N32Q (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1060)

P-1917: 2PE14_ N32R (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1061)

P-1918: 2PE14_ N32S (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1062)

P-1919: 2PE14_ N32T (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1063)

P-1920: 2PE14_ N32V (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1064)

P-1921: 2PE14_ N32W (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1065)

P-1922: 2PE14_ N32Y (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1066)

P-1923: 3PE42 (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS (SEQ ID NO:1067)

P-1924: 3PE42_ M31A (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1068)

P-1925: 3PE42_ M31D (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1069)

P-1926: 3PE42_ M31E (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1070)

P-1927: 3PE42_ M31F (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSS NAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1071)

P-1928: 3PE42_ M31G (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1072)

P-1929: 3PE42_ M31H (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1073)

P-1930: 3PE42_ M31I (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1074)

P-1931: 3PE42_ M31K (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1075)

P-1932: 3PE42_ M31L (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1076)

P-1933: 3PE42_ M31N (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1077)

P-1934: 3PE42_ M31P (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1078)

P-1935: 3PE42_ M31Q (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1079)

P-1936: 3PE42_ M31R (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1080)

P-1937: 3PE42_ M31S (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1081)

P-1938: 3PE42_ M31T (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1082)

P-1939: 3PE42_ M31V (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1083)

P-1940: 3PE42_ M31W (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSSNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1084)

P-1941: 3PE42_ M31Y (bold letters indicate mutations) QVQLQESGGGLVQPGESLRLSCAVSGSTSS NAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSS(SEQ ID NO:1085)

·P-2219：2PE14_OptA(Q1D_Q5V_N32W_A74S_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTFSIWAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNSKNTVYLQMSSLRPEDTAVYYCNLWPPRASPGGRVYWGQGTLVTVSS(SEQ ID NO:1086)

·P-2220：2PE14_OptB(Q1D_Q5V_N32W_A74S_S82N_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTFSIWAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNLWPPRASPGGRVYWGQGTLVTVSS(SEQ ID NO:1087)

·P-1923：3PE42 QVQLQESGGGLVQPGESLRLSCAVSGSTSSMNAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGGVYWGQGTQVTVSSHHHHHH(SEQ ID NO:1088)

·P-2221：3PE42_OptA(Q1D_Q5V_M31A_N32W_A74S_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTSSAWAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNSKNTVYLQMSSLRPEDTAVYYCNLWPPRASPGGGVYWGQGTLVTVSS(SEQ ID NO:1089)

·P-2222：3PE42_OptB(Q1D_Q5V_M31D_N32W_A74S_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTSSDWAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNSKNTVYLQMSSLRPEDTAVYYCNLWPPRASPGGGVYWGQGTLVTVSS(SEQ ID NO:1090)

·P-2223：3PE42_OptC(Q1D_Q5V_M31A_N32W_A74S_S82N_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTSSAWAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNLWPPRASPGGGVYWGQGTLVTVSS(SEQ ID NO:1091)

·P-2224：3PE42_OptD(Q1D_Q5V_M31D_N32W_A74S_S82N_K83R_Q108L)

DVQLVESGGGLVQPGGSLRLSCAASGSTSSDWAMAWYRQAPGKRRELVAGISGGGATNYPDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNLWPPRASPGGGVYWGQGTLVTVSS(SEQ ID NO:1092)

In some embodiments, the FAP binding agent comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementarity determining regions" or CDRs. As used herein, "framework region" or "FR" refers to the region of a variable domain that is located between CDRs. As used herein, "complementarity determining region" or "CDR" refers to the variable region of a VHH that contains an amino acid sequence capable of specifically binding to an antigenic target.

In some embodiments, the FAP-binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and/or CDR3 sequence. In some embodiments, the FAP-binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequences.

In some embodiments, the human FAP-binding agent comprises a CDR1 sequence selected from:

ATISSMNSMA(SEQ ID NO:87)；EFTLAYFGVG(SEQ ID NO:88)；ESTFSINAVA(SEQ ID NO:89)；GFIFRSTSMG(SEQ ID NO:90)；GGIFTIGPLG(SEQ ID NO:91)；GRDFRDNSMG(SEQ ID NO:92)；GSIFGINAVG(SEQ ID NO:93)；GSIFSLNAMA(SEQ ID NO:94)；GSIFSMG(SEQ ID NO:95)；GSIFSTNAMA(SEQ ID NO:96)；GSISSINAMA(SEQ ID NO:97)；GSISSLNAMG(SEQ ID NO:98)；GSISSRNAMG(SEQ ID NO:99)；GSSFIINAMG(SEQ ID NO:100)；GSTARLDAMG(SEQ ID NO:101)；GSTFGLGAMG(SEQ ID NO:102)；GSTFGLSAMG(SEQ ID NO:103)；GSTFGLTAIG(SEQ ID NO:104)；GSTFILNAMA(SEQ ID NO:105)；GSTFILNAMG(SEQ ID NO:106)；GSTFSGNAMA(SEQ ID NO:107)；GSTFSINAMA(SEQ ID NO:108)；GSTFSINAMG(SEQ ID NO:109)；GSTFSINAMM(SEQ ID NO:110)；GSTFSINAVA(SEQ ID NO:111)；GSTFSINNAMG(SEQ ID NO:112)；GSTFSINSMG(SEQ ID NO:113)；GSTFSSNAMA(SEQ ID NO:114)；GSTFSVNAVA(SEQ ID NO:115)。

in some embodiments, the human FAP-binding agent comprises a CDR2 sequence selected from:

AISSGGSTNYAASVKG(SEQ ID NO:116)；AVTSGGVTNYADSVKG(SEQ ID NO:117)；GIATDGRTNYAHSVKG(SEQ ID NO:118)；GIDGGGVTNYPDSVKG(SEQ ID NO:119)；GIDSADITDYARFVKG(SEQ ID NO:120)；GIIGSHSTNYADSVKG(SEQ ID NO:121)；GISGDNITNYPDSVKG(SEQ ID NO:122)；GISGDNITNYPNSVKG(SEQ ID NO:123)；GISGGDVTHYPDSVKG(SEQ ID NO:124)；GISGGGATNYPDSMKG(SEQ ID NO:125)；GISGGGATNYPDSVKG(SEQ ID NO:126)；GISGGGVTNHPDSVKG(SEQ ID NO:127)；GISGGGVTNYPDSVKG(SEQ ID NO:128)；GISGGGVTNYPDSVRG(SEQ ID NO:129)；GISGGGVTSYPDSVKG(SEQ ID NO:130)；GISGGITTYPDSVKG(SEQ ID NO:131)；GISGGSVTNYPDSVKG(SEQ ID NO:132)；GISGGVTNYPDSVKG(SEQ ID NO:133)；GISSDDITYYPDSVKG(SEQ ID NO:134)；GISSDGATHYPDSVKG(SEQ ID NO:135)；GISSGGDTNYPDSVKG(SEQ ID NO:136)；GISSGGVTHYPDSVKG(SEQ ID NO:137)；GISSSGPPHYPDSVKG(SEQ ID NO:138)；GITSDGITNYADSVKG(SEQ ID NO:139)；GITSDGLGNYVDFVKG(SEQ ID NO:140)；GITSDGLGNYVGFAKG(SEQ ID NO:141)；GITSDGVTKYPDSVKG(SEQ ID NO:142)；GITSDGVTNYPDSVKG(SEQ ID NO:143)；GITSDSVTKYPDSVKG(SEQ ID NO:144)。

in some embodiments, the human FAP-binding agent comprises a CDR3 sequence selected from:

AAVVTAKGMGAIQSRGY(SEQ ID NO:145)；ADSSRGKIYFSNYRSWNY(SEQ ID NO:146)；ARFGIRDF(SEQ ID NO:147)；FWPPLYGRP(SEQ ID NO:148)；FWPPPSDRPI(SEQ ID NO:149)；FWPPPSGRPI(SEQ ID NO:150)；KWPPSVPPN(SEQ ID NO:151)；LWPPMASPSGAIY(SEQ ID NO:152)；LWPPRAPPDGRVY(SEQ ID NO:153)；LWPPRAPPGGRVY(SEQ ID NO:154)；LWPPRASPDGGVY(SEQ ID NO:155)；LWPPRASPDGRVY(SEQ IDNO:156)；LWPPRASPGGGVY(SEQ ID NO:157)；LWPPRASPGGLVY(SEQ ID NO:158)；LWPPRASPGGRVY(SEQ ID NO:159)；LWPPRASPSGAVY(SEQ ID NO:160)；LWPPRASPSGRGY(SEQ ID NO:161)；LWPPRASPSGRID(SEQ ID NO:162)；LWPPRASPSGRIY(SEQ ID NO:163)；LWPPRASPSGRLY(SEQ ID NO:164)；LWPPRASPSGRPY(SEQ ID NO:165)；LWPPRASPSGRVY(SEQ ID NO:166)；LWPPRASPSGSIY(SEQ ID NO:167)；LWPPRASPSGTIY(SEQ ID NO:168)；LWPPRVSPGGGVY(SEQ ID NO:169)；LWPPRVSPGGRVY(SEQ ID NO:170)；LWPPRVSPSGRGY(SEQ ID NO:171)；LWPPRVSPSGRIY(SEQ ID NO:172)；LYPPASSGR(SEQ ID NO:173)；MYRPGTYDY(SEQ ID NO:174)；QWPPRALDA(SEQ ID NO:175)。

by way of example, but not by way of limitation, in some embodiments, a human VHH FAP-binding agent comprises an amino acid sequence selected from the group consisting of:

2PE48：

QVQLQESGGGLVQPGGSLRLSCAVSGTMLSRNAMGWYRQAPGKPRQWVAGITSDGLGNYVGFAKGRFTISRDNAKNTVYLQMNTLKPDDTAVYHCNFWPPPSGRPIWGQGTQVTVSS(SEQ ID NO:837)

3PE60：

QVQLQESGGGLVQPGGSLRLSCAVSGTMLSRNAMGWYRQAPGKQRQWVAGITSDGLGNYVDFVKGRFTISRDNARNTVYLQMNTLKPDDTAVYYCNFWPPPSDRPIWGQGTQVTVSS(SEQ ID NO:838)

2PE34：

QVQLQESGGGLVQAGGSLRLSCAVSGSTARLDAMGWYRQAPGKQREWVAGIDSADITDYARFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKWPPSVPPNWGHGTQVTVSS(SEQ ID NO:839)

3PE80：

QVQLQESGGGLVQAGGSLRLSCAVSGSTARLDAMGWYRQAPGKQREWVAGIDSADITDYARFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKWPPSVPPNWGQGTQVTVSS(SEQ ID NO:840)

2PE54：

QVQLQESGGGLVQAGGSLRLSCVHSGGIFTIGPLGWYRQAPGSQRELVATVTNGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAAVVTAKGMGAIQSRGYWGQGTQVTVSS(SEQ ID NO:841)

3PE81：

QVQLQESGGGLVQAGGSLRLSCAHSGGIFTIGPLGWYRQAPGSQRELVATVTNGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAAVVTAKGMGAIQSRGYWGQGTQVTVSS(SEQ ID NO:842)

2PE10：

QVQLQESGGGLVQAGGSLRLSCAASGSTFSINAMMWYRQAPGKQRELVASIGSGGNTYYADSVKGRFTISRDNGKSTLYLQMNSLKPEDTAVYYCKMYRPGTYDYWGQGTQVTVSS(SEQ ID NO:843)

3PE66：

QVQLQESGGGWVQPGGSLRLSCAASGSTFSINAMMWYRQAPGKQRELVASIGSGGNTYYADSVKGRFTISRDNGKSTLYLQMNSLKPEDTAVYYCKMYRPGTYDYWGQGTQVTVSS(SEQ ID NO:844)

2PE31：

QVQLQESGGGLVQAGGSLSVSCAASGSIFSMGWFRQAPGKQRELVAAVTSGGVTNYADSVKGRFTISRDNAKNTVYLQMKSLKPEDTAVYYCAADSSRGKIYFSNYRSWNYWGQGTQVTVSS(SEQ ID NO:845)

2PE79：

QVQLQESGGGLVQAGESLRLSCAVSATISSMNSMAWYRQAPGKQREWVAGLETGGRANYVDSVKGRFTISRDNARNTVLLQMNSLKPEDTAVYYCNRWPPLRSSWGQGTQVTVSS(SEQ ID NO:846)

2PE91：

QVQLQESGGGLVQPGESLRLSCAASGSISSRNAMGWYRQAPGKEREWVAGITSDGITNYADSVKGRFTISRDNAKNTVGLQMNSLKPDDTAVYYCNFWPPLYGRPWGQGTQVTVSS(SEQ ID NO:847)

3PE38：

QVQLQESGGGLVQPGGSLRLSCAASGFIFRSTSMGWYRQAPGKQREFVAGIIGSHSTNYADSVKGRFTISRDNAQNAVYLHMNTLKPEDTAVYYCNLYPPASSGRWGKGTQVTVSS(SEQ ID NO:848)

2PE57：

QVQLQESGGGLVQAGGSLRLSCAASLKISSINAMAWYRQAAGKQRELVAGIATDGRTNYAHSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNQWPPRALDAWGQGTQVTVSS(SEQ ID NO:849)

3PE15：

QVQLQESGGGLVQPGGSLRLSCAASGVTFGIGAMGWYRQTPENERELVAAISSGGSTNYAASVKGRFTISRDNAPNTVYLQMNSLKPEDTAIYYCNVRRGLAWYPGWGQGTQVTVSS (SEQ ID NO:850) in some embodiments, the FAP binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2 and/or CDR3 sequence. In some embodiments, the FAP-binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequences.

In some embodiments, the human FAP-binding agent comprises a CDR1 sequence selected from:

GSTFSVNAVG(SEQ ID NO:851)；GSTFTINAMA(SEQ ID NO:852)；GSTFTINAMG(SEQ ID NO:853)；GSTSSINAMA(SEQ ID NO:854)；GSTSSMNAMA(SEQ ID NO:855)；GTMLSRNAMG(SEQ ID NO:856)；GVELSRSAMA(SEQ ID NO:857)；GVTFGIGAMG(SEQ ID NO:858)；LKISSINAMA(SEQ ID NO:859)；SGSTFSINAMA(SEQ ID NO:860)；VNIVNLNSVG(SEQ ID NO:861)。

in some embodiments, the human FAP-binding agent comprises a CDR2 sequence selected from:

GITSGGSTNYADSVKG(SEQ ID NO:862)；GITSGVTHYPDSVKG(SEQ ID NO:863)；GITSSGVTKYPDSVKG(SEQ ID NO:864)；GITSSGVTNYPDSVKG(SEQ ID NO:865)；GITSSGVTQYPDSVKG(SEQ ID NO:866)；GITTDGITKYPDSLKG(SEQ ID NO:867)；GLETGGRANYVDSVKG(SEQ ID NO:868)；SIGSGGNTYYADSVKG(SEQ ID NO:869)；SIISDGRTNYADSVKG(SEQ ID NO:870)；SITSAGSTNYAESVKG(SEQ ID NO:871)；SITSDGRTNYADSVKG(SEQ ID NO:872)；SITSGGRTNYADSVKG(SEQ ID NO:873)；SITSGGRTNYSDSVKG(SEQ ID NO:874)；TFTRGGDINYADSVKG(SEQ ID NO:875)；TVTNGGGTYYADSVKG(SEQ ID NO:876)。

in some embodiments, the human FAP-binding agent comprises a CDR3 sequence selected from:

RWPPLRSS(SEQ ID NO:877)；TPPRIGRGY(SEQ ID NO:878)；VRRGLAWYPG(SEQ ID NO:879)。

in some embodiments, the human FAP-binding agent comprises a CDR1 sequence selected from:

GSTFSIAAVA(SEQ ID NO:1093)；GSTFSIDAVA(SEQ ID NO:1094)；GSTFSIEAVA(SEQ ID NO:1095)；GSTFSIFAVA(SEQ ID NO:1096)；GSTFSIGAVA(SEQ ID NO:1097)；GSTFSIHAVA(SEQ ID NO:1098)；GSTFSIIAVA(SEQ ID NO:1099)；GSTFSIKAVA(SEQ ID NO:1100)；GSTFSILAVA(SEQ ID NO:1101)；GSTFSIPAVA(SEQ ID NO:1102)；GSTFSIQAVA(SEQ ID NO:1103)；GSTFSIRAVA(SEQ ID NO:1104)；GSTFSISAVA(SEQ ID NO:1105)；GSTFSITAVA(SEQ ID NO:1106)；GSTFSIVAVA(SEQ ID NO:1107)；GSTFSIWAVA(SEQ ID NO:1108)；GSTFSIYAVA(SEQ ID NO:1109)；GSTSSANAMA(SEQ ID NO:1110)；GSTSSDNAMA(SEQ ID NO:1111)；GSTSSENAMA(SEQ ID NO:1112)；GSTSSFNAMA(SEQ ID NO:1113)；GSTSSGNAMA(SEQ ID NO:1114)；GSTSSHNAMA(SEQ ID NO:1115)；GSTSSINAMA(SEQ ID NO:1116)；GSTSSKNAMA(SEQ ID NO:1117)；GSTSSLNAMA(SEQ ID NO:1118)；GSTSSNNAMA(SEQ ID NO:1119)；GSTSSPNAMA(SEQ ID NO:1120)；GSTSSQNAMA(SEQ ID NO:1121)；GSTSSRNAMA(SEQ ID NO:1122)；GSTSSSNAMA(SEQ ID NO:1123)；GSTSSTNAMA(SEQ ID NO:1124)；GSTSSVNAMA(SEQ ID NO:1125)；GSTSSWNAMA(SEQ ID NO:1126)；GSTSSYNAMA(SEQ ID NO:1127)；GSTSSAWAMA(SEQ ID NO:1128)；GSTSSDWAMA(SEQ ID NO:1129)。

in some embodiments, the FAP-binding agents of the invention are human FAP antibodies (e.g., NI-206.82C2, NI-206.59B4, NI-206.22F7, NI-206.27E8, NI-206.12G4) or portions thereof (such as VH chain, VL chain, CDR1, CDR2, or CDR3) as described in international patent publication No. WO 2016/110598 (which is incorporated herein by reference in its entirety), wherein the amino acid sequences of the VH chain, VL chain and its CDRs are as follows:

FAP-binding agent NI-206.82C2

NI-206.82C2 VH：

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVTWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKGRITINPDTSKNQFYLQLKSVTPEDAAVYYCARDSSILYGDYWGQGTLVTVSS(SEQ ID NO:880)

NI-206.82C2 VL：

QAVLTQPSSLSASPGASASLTCTLPSGINVGTYRIFWFQQKPGSPPQYLLSYKSDSDNHQGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHSSAWVFGGGTKLTVL(SEQ ID NO:881)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.82C 2. The VH chain of NI-206.82C2 includes the following CDRs:

CDR1：GDSVSSNSVTWN(SEQ ID NO:882)

CDR2：RTYYRSKWYND(SEQ ID NO:883)

CDR3：DSSILYGDY(SEQ ID NO:884)

the VL chain of NI-206.82C2 includes the following CDRs:

CDR1：TLPSGINVGTYRIF(SEQ ID NO:915)

CDR2：KSDSDNH(SEQ ID NO:916)

CDR3：MIWHSSAWV(SEQ ID NO:917)

FAP-binding agent NI-206.59B4

NI-206.59B4 VH：

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTDYYIHWVRQAPGQGLEWMGWINPNRGGTNYAQKFQGRVTMTRDTSIATAYMELSRLRSDDTAVYYCATASLKIAAVGTFDCWGQGTLVTVSS(SEQ ID NO:918)

NI-206.59B4 VL：

SYELTQPPSVSVSPGQTARITCSGDALSKQYAFWFQQKPGQAPILVIYQDTKRPSGIPGRFSGSSSGTTVTLTISGAQADDEADYYCQSADSSGTYVFGTGTKVTVL(SEQ ID NO:919)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.59B 4. The VH chain of NI-206.59B4 includes the following CDRs:

CDR1：GYTFTDYYIH(SEQ ID NO:920)

CDR2：WINPNRGGTN(SEQ ID NO:921)

CDR3：ASLKIAAVGTFDC(SEQ ID NO:922)

the VL chain of NI-206.82C2 includes the following CDRs:

CDR1：SGDALSKQYAF(SEQ ID NO:923)

CDR2：QDTKRPS(SEQ ID NO:924)

CDR3：QSADSSGTYV(SEQ ID NO:925)

FAP-binding agent NI-206.22F7

NI-206.22F7 VH：

EVQLVETGGGVVQPGRSLRLSCAASGFSFSTHGMYWVRQPPGKGLEWVAVISYDGSDKKYADSVKGRFTISRDNSKNTVYLEMSSVRAEDTALYYCFCRRDAFDLWGQGTMVTVSS(SEQ ID NO:926)

NI-206.22F7 VL：

SYVLTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYEDTKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDSSGNYWVFGGGTEVTVL(SEQ ID NO:927)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.22F 7. The VH chain of NI-206.22F7 includes the following CDRs:

CDR1：GFSFSTHGMY(SEQ ID NO:928)

CDR2：VISYDGSDKK(SEQ ID NO:929)

CDR3：RRDAFDL(SEQ ID NO:930)

the VL chain of NI-206.22F7 includes the following CDRs:

CDR1：SGDALPKKYAY(SEQ ID NO:931)

CDR2：EDTKRPS(SEQ ID NO:932)

CDR3：YSTDSSGNYWV(SEQ ID NO:933)

FAP-binding agent NI-206.27E8

NI-206.27E8 VH：

EVQLVESGGGLVEPGGSLRLSCAASGFTFSDAWMNWVRQAPGKGLEWVGRIKTKSDGGTTDYAAPVRGRFSISRDDSKNTLFLEMNSLKTEDTAIYYCFITVIVVSSESPLDHWGQGTLVTVSS(SEQ ID NO:934)

NI-206.27E8 VL：

SYELTQPPSVSVSPGQTARITCSGDELPKQYAYWYQQKPGQAPVLVIYKDRQRPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSAYSINTYVIFGGGTKLTVL(SEQ ID NO:935)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.27E 8. The VH chain of NI-206.27E8 includes the following CDRs:

CDR1：GFTFSDAWMN(SEQ ID NO:936)

CDR2：RIKTKSDGGTTD(SEQ ID NO:937)

CDR3：TVIVVSSESPLDH(SEQ ID NO:938)

the VL chain of NI-206.27E8 includes the following CDRs:

CDR1：SGDELPKQYAY(SEQ ID NO:939)

CDR2：KDRQRPS(SEQ ID NO:940)

CDR3：QSAYSINTYVI(SEQ ID NO:941)

FAP-binding agents NI-206.12G4

NI-206.12G4 VH：

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWISYISSGSSYTNYADSVKGRFTISRDNAKKSVYLEVNGLTVEDTAVYYCARVRYGDREMATIGGFDFWGQGTLVTVSS(SEQ ID NO:942)

NI-206.12G4 VL：

SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQSPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSGGTSRIFGGGTKLTVL(SEQ ID NO:943)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.12G 4. The VH chain of NI-206.12G4 includes the following CDRs:

CDR1：GFTFSDYYMS(SEQ ID NO:944)

CDR2：YISSGSSYTN(SEQ ID NO:945)

CDR3：VRYGDREMATIGGFDF(SEQ ID NO:946)

the VL chain of NI-206.12G4 includes the following CDRs:

CDR1：SGDALPKQYAY(SEQ ID NO:947)

CDR2：KDSERPS(SEQ ID NO:948)

CDR3：QSADSGGTSRI(SEQ ID NO:949)

FAP-binding agents NI-206.17A6

NI-206.17A6 VH：

QVQLQESGPGLVRSTETLSLTCLVSGDSINSHYWSWLRQSPGRGLEWIGYIYYTGPTNYNPSLKSRVSISLGTSKDQFSLKLSSVTAADTARYYCARNKVFWRGSDFYYYMDVWGKGTTVTVSS(SEQ ID NO:950)

NI-206.17A6 VL：

EIVLTQSPGTLSLSLGERATLSCRASQSLANNYLAWYQQKPGQAPRLLMYDASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFVTSHHMYIFGQGTKVEIK(SEQ ID NO:951)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of NI-206.17a 6. The VH chain of NI-206.17A6 includes the following CDRs:

CDR1：GDSINSHYWS(SEQ ID NO:952)

CDR2：YIYYTGPTN(SEQ ID NO:953)

CDR3：NKVFWRGSDFYYYMDV(SEQ ID NO:954)

The VL chain of NI-206.17A6 includes the following CDRs:

CDR1：RASQSLANNYLA(SEQ ID NO:955)

CDR2：DASTRAT(SEQ ID NO:956)

CDR3：QQFVTSHHMYI(SEQ ID NO:957)

in some embodiments, the FAP binding agent of the invention is a FAP antibody (e.g., humanized F19) or portion thereof (such as a VH chain, VL chain, CDR1, CDR2, or CDR3) from international patent publication No. WO 1999/057151 (which is incorporated herein by reference in its entirety), wherein the amino acid sequences of the VH chain, VL chain, and CDRs thereof are as follows:

humanized F19 VH:

QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSS(SEQ ID NO:958)

humanized F19 VL:

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK(SEQ ID NO:959)

the FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH or VL chain of F19. The VH chain of F19 includes the following CDRs:

CDR1：RYTFTEYTIH(SEQ ID NO:960)

CDR2：GINPNNGIPN(SEQ ID NO:961)

CDR3：RRIAYGYDEGHAMDY(SEQ ID NO:962)

the VL chain of F19 includes the following CDRs:

CDR1：KSSQSLLYSRNQKNYLA(SEQ ID NO:963)

CDR2：WASTRES(SEQ ID NO:964)

CDR3：QQYFSYPLT(SEQ ID NO:965)

in some embodiments, the FAP binding agents of the invention are FAP antibodies (e.g., MFP5 mouse, MFP5 humanized variant 1, and MFP5 humanized variant 2) or portions thereof (such as VH chain, VL chain, CDR1, CDR2, or CDR3) from international patent publication No. WO 2007/077173 (which is incorporated herein by reference in its entirety), wherein the amino acid sequences of the VH chain, VL chain, and CDRs thereof are as follows:

FAP binder MP5 (mouse)

MFP5 VH (mouse):

QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSA(SEQ ID NO:966)

MFP5 VL (mouse):

QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKR(SEQ ID NO:967)

the FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH chain or VL chain of MFP5 (mouse). The VH chain of MFP5 includes the following CDRs:

CDR1：GYTFTNNGIN(SEQ ID NO:968)

CDR2：EIYPRSTNTL(SEQ ID NO:969)

CDR3：TLTAPFAF(SEQ ID NO:970)

the VL chain of MFP5 includes the following CDRs:

CDR1：SASSGVNFMH(SEQ ID NO:971)

CDR2：DTSKLAS(SEQ ID NO:972)

CDR3：QQWSFNPPT(SEQ ID NO:973)

FAP binder MP5 (humanised variant 1)

MFP5 (humanized variant 1) VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNNGINWLRQAPGQGLEWMGEIYPRSTNTLYAQKFQGRVTITADRSSNTAYMELSSLRSEDTAVYFCARTLTAPFAFWGQGTLVTVSS(SEQ ID NO:974)

MFP5 (humanized variant 1) VL:

QIVLTQSPATLSLSPGERATLSCSASSGVNFMHWYQQKPGQAPRRLIFDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSFNPPTFGQGTKVEIKR(SEQ ID NO:975)

the FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH chain or VL chain of MFP5 (humanized variant 1). The VH chain of MFP5 (humanized variant 1) includes the following CDRs:

CDR1：GYTFTNNGIN(SEQ ID NO:976)

CDR2：EIYPRSTNTL(SEQ ID NO:977)

CDR3：TLTAPFAF(SEQ ID NO:978)

the VL chain of MFP5 (humanized variant 1) includes the following CDRs:

CDR1：SASSGVNFMH(SEQ ID NO:979)

CDR2：DTSKLAS(SEQ ID NO:980)

CDR3：QQWSFNPPT(SEQ ID NO:981)

FAP binder MP5 (humanized variant 2)

MFP5 (humanized variant 2) VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNNGINWLRQAPGQGLEWMGEIYPRSTNTLYAQKFQGRVTITADRSSNTAYMELSSLRSEDTAVYFCARTLTAPFAFWGQGTLVTVSS(SEQ ID NO:982)

MFP5 (humanized variant 2) VL:

QIVLTQSPATLSLSPGERATLSCSASSGVNFMHWYQQKPGQAPKRLIFDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSFNPPTFGQGTKVEIKR(SEQ ID NO:983)

the FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH chain or VL chain of MFP5 (humanized variant 2). The VH chain of MFP5 (humanized variant 2) included the following CDRs:

CDR1：GYTFTNNGIN(SEQ ID NO:984)

CDR2：EIYPRSTNTL(SEQ ID NO:985)

CDR3：TLTAPFAF(SEQ ID NO:986)

the VL chain of MFP5 (humanized variant 2) included the following CDRs:

CDR1：SASSGVNFMH(SEQ ID NO:987)

CDR2：DTSKLAS(SEQ ID NO:988)

CDR3：QQWSFNPPT(SEQ ID NO:989)

in some embodiments, the FAP-binding agent of the invention is a FAP antibody (e.g., 4G8, 3F2, 28H1, 29B11, 14B3, and 4B9) or portion thereof (such as a VH chain, a VL chain, CDR1, CDR2, or CDR3) from international patent publication No. WO 2012/107417 (which is incorporated herein by reference in its entirety), wherein the amino acid sequences of the VH chain, the VL chain, and CDRs thereof are as follows:

FAP-binding agent 4G8

4G8 VH：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS(SEQ ID NO:990)

4G8 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKR(SEQ ID NO:991)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of 4G 8. The VH chain of 4G8 includes the following CDRs:

CDR1：GFTFSSYAMS(SEQ ID NO:992)

CDR2：AISGSGGSTY(SEQ ID NO:993)

CDR3：GWLGNFDY(SEQ ID NO:994)

the VL chain of 4G8 includes the following CDRs:

CDR1：RASQSVSRSYLA(SEQ ID NO:995)

CDR2：GASTRAT(SEQ ID NO:996)

CDR3：QQGQVIPPT(SEQ ID NO:997)

FAP-binding agent 3F2

3F2 VH：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS(SEQ ID NO:998)

3F2 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR(SEQ ID NO:999)

The FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH or VL chain of 3F 2. The VH chain of 3F2 includes the following CDRs:

CDR1：GFTFSSYAMS(SEQ ID NO:1000)

CDR2：AISGSGGSTY(SEQ ID NO:1001)

CDR3：GWFGGFNY(SEQ ID NO:1002)

the VL chain of 3F2 includes the following CDRs:

CDR1：RASQSVTSSYLA(SEQ ID NO:1003)

CDR2：VGSRRAT(SEQ ID NO:1004)

CDR3：QQGIMLPPT(SEQ ID NO:1005)

FAP-binding agent 28H1

28H1 VH：

QVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS(SEQ ID NO:1006)

28H1 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKR(SEQ ID NO:1007)

The FAP binding agents of the invention may comprise CDR1, CDR2 or CDR3 of the VH chain or VL chain of 28H 1. The VH chain of 28H1 includes the following CDRs:

CDR1：GFTFSSHAMS(SEQ ID NO:1008)

CDR2：AIWASGEQY(SEQ ID NO:1009)

CDR3：GWLGNFDY(SEQ ID NO:1010)

the VL chain of 28H1 includes the following CDRs:

CDR1：RASQSVSRSYLA(SEQ ID NO:1011)

CDR2：GASTRAT(SEQ ID NO:1012)

CDR3：QQGQVIPPT(SEQ ID NO:1013)

FAP-binding agent 29B11

29B11 VH：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS(SEQ ID NO:1014)

29B11 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR(SEQ ID NO:1015)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of 29B 11. The VH chain of 29B11 includes the following CDRs:

CDR1：GFTFSSYAMS(SEQ ID NO:1016)

CDR2：AIIGSGGITY(SEQ ID NO:1017)

CDR3：GWFGGFNY(SEQ ID NO:1018)

the VL chain of 29B11 includes the following CDRs:

CDR1：RASQSVTSSYLA(SEQ ID NO:1019)

CDR2：VGSRRAT(SEQ ID NO:1020)

CDR3：QQGIMLPPT(SEQ ID NO:1021)

FAP-binding agents 14B3

14B3 VH：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAILASGAITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS(SEQ ID NO:1022)

14B3 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR(SEQ ID NO:1023)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH or VL chain of 14B 3. The VH chain of 14B3 includes the following CDRs:

CDR1：GFTFSSYAMS(SEQ ID NO:1024)

CDR2：AILASGAITY(SEQ ID NO:1025)

CDR3：GWFGGFNY(SEQ ID NO:1026)

the VL chain of 14B3 includes the following CDRs:

CDR1：RASQSVTSSYLA(SEQ ID NO:1027)

CDR2：VGSRRAT(SEQ ID NO:1028)

CDR3：QQGIMLPPT(SEQ ID NO:1029)

FAP-binding agent 4B9

4B9 VH：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS(SEQ ID NO:1030)

4B9 VL：

EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKR(SEQ ID NO:1031)

The FAP binding agents of the invention may comprise the CDR1, CDR2 or CDR3 of the VH chain or VL chain of 4B 9. The VH chain of 4B9 includes the following CDRs:

CDR1：GFTFSSYAMS(SEQ ID NO:1032)

CDR2：AIIGSGASTY(SEQ ID NO:1033)

CDR3：GWFGGFNY(SEQ ID NO:1034)

the VL chain of 4B9 includes the following CDRs:

CDR1：RASQSVTSSYLA(SEQ ID NO:1035)

CDR2：VGSRRAT(SEQ ID NO:1036)

CDR3：QQGIMLPPT(SEQ ID NO:1037).

any of the FAP-binding agents disclosed herein can be an antibody. The FAP-binding agent as an antibody may be part of any of the chimeric proteins or chimeric protein complexes disclosed herein. As used herein, the term "antibody" refers to any immunoglobulin or antibody (e.g., human, hamster, feline, mouse, cartilaginous fish, or camelid antibody) and any derivative or conjugate thereof that specifically binds to an antigen. A variety of antibodies are known to those of skill in the art. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies (e.g., single domain antibodies, camelid antibodies, and cartilaginous fish antibodies), chimeric antibodies, feline antibodies, and feline humanized antibodies. Monoclonal antibodies are homogeneous populations of antibodies directed against a particular epitope of an antigen. Polyclonal antibodies are heterogeneous populations of antibody molecules contained in the serum of immunized animals. The term "antibody" also includes antibody derivatives and conjugates (e.g., antibodies conjugated to a stabilizing protein, detectable moiety, or therapeutic agent).

Any of the FAP-binding agents disclosed herein can be an antigen-binding fragment of an antibody. AsThe FAP-binding agent of the antigen-binding fragment of the antibody may be part of any of the chimeric proteins or chimeric protein complexes disclosed herein. An "antigen-binding fragment" is any portion of a full-length antibody that contains at least one variable domain capable of specifically binding to an antigen (e.g., a variable domain of a mammalian (e.g., feline, human, hamster, or mouse) heavy or light chain immunoglobulin, a camelid variable antigen-binding domain (VHH), or a cartilaginous fish immunoglobulin neo-antigen receptor (Ig-NAR) domain). Non-limiting examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. Additional antibody fragments containing at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies (mini-bodies), minibodies (micro-antibodies), sub-nano-antibodies (subnano-antibodies) and nanobodies, as well as any of the other forms of antibodies described, for example, in U.S. publication No. 2010/0092470.

Any of the FAP-binding agents disclosed herein can be an Fv fragment of an antibody. The FAP-binding agent that is an Fv fragment of an antibody can be part of any of the chimeric proteins or chimeric protein complexes disclosed herein. An "Fv fragment" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. In this configuration, the three Complementarity Determining Regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer.

Any of the FAP binding agents disclosed herein can be a Complementarity Determining Region (CDR) of an antibody. The FAP binding agent that is a CDR of an antibody may be part of any of the chimeric proteins or chimeric protein complexes disclosed herein. The term CDR refers to a region within an immunoglobulin (heavy or light chain immunoglobulin) that forms part of an antigen binding site in an antibody or antigen binding fragment thereof. As known in the art, each of the heavy and light chain immunoglobulins contains three CDRs, referred to as CDR1, CDR2, and CDR 3. In any antibody or antigen-binding fragment, the three CDRs from the heavy chain immunoglobulin and the three CDRs from the light chain immunoglobulin together form an antigen binding site in the antibody or antigen-binding fragment thereof. The Kabat database is a system used in the art for numbering CDR sequences present in a light chain immunoglobulin or a heavy chain immunoglobulin.

Together, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically with a lower affinity than the entire binding site. The "Fab fragment" also contains the constant domain of the light chain and the first constant domain of the heavy chain (C)_H1). "Fab fragment" differs from "Fab' fragment" in that it is in the heavy chain C_H1 domain has some residues added to the carboxy terminus, including one or more cysteines from the antibody hinge region. The "F (ab ')2 fragments" were originally generated as a pair of "Fab' fragments" with hinge cysteines between them. Methods of making such antibody fragments (such as papain or pepsin digestion) are known to those skilled in the art. For example, F (ab ')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of the F (ab')2 fragments. In some cases, Fab expression libraries can be constructed. See, e.g., Huse et al, Science,246:1275,1989. Once produced, the recognition of the TNFRSF25 polypeptide by the antibody or fragment thereof can be tested using standard immunoassay methods such as ELISA techniques, radioimmunoassays, and western blots. Referring to the description of the preferred embodiment, Short Protocols in Molecular BiologyChapter 11, Green Publishing Associates and John Wiley&Sons, Ausubel et al, eds., 1992.

The antibody may be of the IgA, IgD, IgE, IgG or IgM type, including IgG or IgM types, such as but not limited to IgG1, IgG2, IgG3, IgG4, IgM1 and IgM2 types. For example, in some cases, the antibody is of the IgG1 type, IgG2 type, or IgG4 type.

In some embodiments, an antibody as provided herein can be a fully human or humanized antibody. "human antibody" refers to an antibody encoded by a nucleic acid present in the human genome (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human antibodies can be produced in human cell cultures (e.g., feline hybridoma cells). In some embodiments, human antibodies can be produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies can be produced in bacterial or yeast cells.

Human antibodies can avoid certain problems associated with xenogeneic antibodies, such as antibodies having murine or rat variable and/or constant regions. For example, because the effector moiety is human, it may better interact with other parts of the human immune system, e.g., more efficiently destroy target cells through complement-dependent cytotoxicity or antibody-dependent cytotoxicity. Furthermore, the human immune system should not recognize the antibody as foreign. In addition, the half-life in the human circulation is similar to that of naturally occurring human antibodies, allowing smaller and less frequent doses to be administered. Methods for making human antibodies are known in the art.

As used herein, the term "humanized antibody" refers to a human antibody containing minimal sequences derived from a non-human (e.g., mouse, hamster, rat, rabbit, or goat) immunoglobulin. Humanized antibodies are typically chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, which carry human constant and/or variable region domains or changes in specificity. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient antibody are replaced by residues from an HVR of a non-human species (donor) antibody, such as a mouse, rat, rabbit, or goat antibody having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin can be replaced with corresponding non-human residues. In some embodiments, a humanized antibody may contain residues that are not present in the recipient antibody or the donor antibody. For example, such modifications may be made to improve antibody performance.

In some embodiments, a humanized antibody may contain substantially all, at least one, and typically two variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody may also contain at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin.

In some embodiments, a humanized antibody or antigen-binding fragment as provided herein can have reduced or minimal effector function (e.g., as compared to a corresponding non-humanized antibody) such that it does not stimulate effector cells to the same extent that a corresponding non-humanized antibody would produce.

Techniques for generating humanized antibodies are well known to those skilled in the art. In some embodiments, controlled rearrangement of antibody domains linked by protein disulfide bonds (to form new artificial protein molecules or "chimeric" antibodies) can be utilized (Konieczny et al, Haematologica (Budap.)14:95,1981). Recombinant DNA technology can be used to construct gene fusions between DNA sequences encoding the variable light and heavy domains of mouse antibodies and the light and heavy constant domains of human antibodies (Morrison et al, Proc Natl Acad Sci USA 81:6851,1984). For example, DNA sequences encoding the antigen-binding portions or CDRs of murine monoclonal antibodies can be molecularly grafted into DNA sequences encoding the heavy and light chain frameworks of human antibodies (Jones et al, Nature 321:522,1986; and Riechmann et al, Nature 332:323,1988). The expressed recombinant product is called a "reshaped" or humanized antibody and contains the framework of a human antibody light or heavy chain and the antigen recognition portion CDRs of a murine monoclonal antibody.

Other methods for designing heavy and light chains and for producing humanized antibodies are described in, for example, U.S. Pat. nos. 5,530,101; 5,565,332; 5,585,089; 5,639,641; 5,693,761; 5,693,762; and 5,733,743. Additional methods for humanizing antibodies are described, for example, in U.S. Pat. nos. 4,816,567; 4,935,496, respectively; 5,502,167, respectively; 5,558,864; 5,693,493, respectively; 5,698,417, respectively; 5,705,154, respectively; 5,750,078, respectively; and 5,770,403.

Can make it possible toThe antibodies disclosed herein are produced using standard methods. For example, the antibodies can be produced recombinantly, purified from a biological sample (e.g., a heterologous expression system), or chemically synthesized and used to immunize a host animal, including a rabbit, chicken, mouse, guinea pig, or rat. Various adjuvants that can be used to increase the immune response depend on the host species, including freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies can be prepared using, for example, FAP and standard hybridoma techniques. Specifically, monoclonal antibodies can be obtained by any technique, by the continuous cell line in culture (such as described by Kohler et al (Nature 256:495, 1975)), by the human B-cell hybridoma technique of Kosbor et al (Immunology Today,4:72,1983) or Cote et al (Proc. Natl. Acad. Sci. USA,80:2026,1983) and by Cole et al (Cole et al: (al.) (Nature 256:495, 1975)) Monoclonal Antibodies and Cancer TherapyThe EBV-hybridoma technology described by Alan r.liss, inc., pages 77-96, 1983) provides for the production of antibody molecules. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Hybridomas producing monoclonal antibodies can be cultured in vitro and in vivo.

In some embodiments, an antibody as provided herein has a heavy chain variable region comprising any of the VH amino acid sequences disclosed herein. In other embodiments, an antibody as provided herein has a heavy chain variable region comprising any of the VH amino acid sequences disclosed herein, but with 1 to 24 modifications (e.g., substitutions, additions or deletions) such that the amino acid sequence has, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three or twenty-four modifications. In some embodiments, an antibody as provided herein has a light chain variable region comprising any one of the VL amino acid sequences disclosed herein. In other embodiments, an antibody as provided herein has a light chain variable region that contains any one of the VL amino acid sequences disclosed herein, but has 1 to 24 modifications (e.g., substitutions, additions or deletions) such that the amino acid sequence has, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three or twenty-four modifications. This document also provides antibodies and antigen-binding fragments comprising the heavy chain variable region polypeptides and light chain variable region polypeptides as disclosed herein. For example, an antibody or antigen-binding fragment as disclosed herein can further comprise variable region heavy chain Framework (FW) sequences juxtaposed between the CDRs according to formulae (FW1) - (CDR1) - (FW2) - (CDR2) - (FW3) - (CDR3) - (FW 4). In some embodiments, the FW sequence may be a human sequence.

In some embodiments, for example, the FAP-binding agent has up to five modifications (substitutions, deletions, or insertions) in any of the amino acid sequences of the CDRs of the antibodies disclosed herein. For example, the FAP binding agent comprises up to five substitutions, deletions or insertions in any of the amino acid sequences of CDR1, CDR2, or CDR3 of the antibodies disclosed herein.

In some embodiments, amino acid substitutions to an antibody or FAP-binding agent disclosed herein can be made by selecting conservative substitutions that do not differ significantly in their effect on: maintaining (a) the structure of the peptide backbone in the substitution region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the side chain volume. For example, naturally occurring residues may be classified into the following groups based on side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine and arginine); (5) amino acids that affect chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups may be considered conservative substitutions. Non-limiting examples of conservative substitutions include, but are not limited to, valine to alanine substitutions, lysine to arginine substitutions, glutamine to aspartic acid substitutions, glutamic acid to aspartic acid substitutions, serine to cysteine substitutions, asparagine to glutamine substitutions, aspartic acid to glutamic acid substitutions, proline to glycine substitutions, arginine to histidine substitutions, leucine to isoleucine substitutions, isoleucine to leucine substitutions, arginine to lysine substitutions, leucine to methionine substitutions, leucine to phenylalanine substitutions, glycine to proline substitutions, threonine to serine substitutions, serine to threonine substitutions, tyrosine to tryptophan substitutions, phenylalanine to tyrosine substitutions, and/or leucine to valine substitutions. In some embodiments, the amino acid substitutions may be non-conservative, such that a member of one of the above classes of amino acids is exchanged for a member of another class. In some embodiments, the FAP-binding agent has at least 90% identity to any one of the amino acid sequences selected from SEQ ID NOs 2-42, 46-86, 837-850, 1045-1085 or 1086-1092 or the amino acid sequence of any of the FAP-binding agents disclosed herein. In some embodiments, the FAP-binding agent has about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity to any one of the amino acids selected from the group consisting of SEQ ID NOs 2-42, 46-86, 837-850, 1045-1085 or 1086-1092 or any of the FAP-binding agents disclosed herein.

In one embodiment, for example, the FAP-binding agent has up to five substitutions, deletions or insertions in any of the amino acid sequences selected from the group consisting of SEQ ID NOs 87-175 or 851-879 or any of the CDRs of the FAP-binding agents disclosed herein. For example, FAP-binding agents comprise up to five substitutions, deletions or insertions (e.g., one, two, three, four or five total amino acid substitutions, deletions or insertions) in any amino acid sequence selected from the group consisting of CDR1, e.g., SEQ ID NOS 87-115, 851-861, 1093-1129, 882, 915, 920, 923, 928, 931, 936, 939, 944, 947, 952, 955, 960, 963, 968, 971, 976, 979, 984, 987, 992, 995, 1000, 1003, 1008, 1011, 1016, 1019, 1024, 1027, 1032 or 1035. Similarly, in another embodiment, the FAP-binding agent comprises up to five substitutions, deletions or insertions (e.g., one, two, three, four or five total amino acid substitutions, deletions or insertions) in any amino acid sequence selected from the group consisting of CDR2, e.g., SEQ ID NO:116-144, 862-876, 883, 916, 921, 924, 929, 932, 937, 940, 945, 948, 953, 956, 961, 964, 969, 972, 977, 980, 985, 988, 993, 996, 1001, 1004, 1009, 1012, 1017, 1020, 1025, 1028, 1033 or 1036. Similarly, in another embodiment, the FAP binding agent comprises up to five substitutions, deletions or insertions (e.g., one, two, three, four or five total amino acid substitutions, deletions or insertions) in any amino acid sequence selected from the group consisting of CDR3, e.g., SEQ ID NOs 145-175, 877-879, 884, 917, 922, 925, 930, 933, 938, 941, 946, 949, 954, 957, 962, 965, 970, 973, 978, 981, 986, 989, 994, 997, 1002, 1005, 1010, 1013, 1018, 1021, 1026, 1029, 1034, 1037. Amino acid substitutions refer to the replacement of one or more amino acid residues in a peptide sequence with one or more other residues. Amino acid deletion refers to the removal of one or more amino acid residues from a peptide sequence. Amino acid insertion refers to the addition of one or more amino acid residues to a peptide sequence.

In various illustrative embodiments, the murine FAP binding agent has at least 90% identity to the amino acid sequence of sibrotuzumab (sibrotuzumab).

In some embodiments, the present technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the FAP-binding agents of the present technology as described herein. In some embodiments, the amino acid sequence of the FAP-binding agent further comprises an amino acid analog, amino acid derivative, or other non-canonical amino acid.

In some embodiments, the FAP-binding agent comprises a targeting moiety comprising a sequence at least 60% identical to any of the FAP sequences disclosed herein. For example, the FAP-binding agent can comprise a targeting moiety comprising at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 95% of any of the FAP sequences disclosed herein, At least about 96%, at least about 97%, at least about 98%, a sequence that is at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity to any of the sequences disclosed herein).

In some embodiments, the FAP-binding agent comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to any of the sequences disclosed herein. In some embodiments, the FAP-binding agent comprises a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations relative to any of the sequences disclosed herein. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions.

"conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobicity: met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln; (3) acidity: asp and Glu; (4) alkalinity: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; and (6) aromatic: trp, Tyr, Phe.

As used herein, "conservative substitutions" are defined as the exchange of one amino acid for another amino acid listed within the same one of the six standard amino acid groups shown above. For example, Asp is exchanged for Glu such that one negative charge is retained in the so-modified polypeptide. In addition, glycine and proline may be substituted for each other based on their ability to disrupt the a helix.

As used herein, a "non-conservative substitution" is defined as the exchange of one amino acid for another amino acid listed in a different one of the six standard amino acid groups (1) through (6) shown above.

In some embodiments, the substitution comprises a non-canonical amino acid. Illustrative non-classical amino acids generally include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, designer amino acids such as the beta methyl amino acid, C alpha-methyl amino acids, N alpha-methyl amino acids, and amino acid analogs.

In some embodiments, the one or more amino acid mutations are in a CDR (e.g., a CDR1 region, a CDR2 region, or a CDR3 region) of the targeting moiety. In another embodiment, the one or more amino acid mutations are in a Framework Region (FR) of the targeting moiety (e.g., FR1 region, FR2 region, FR3 region, or FR4 region).

Modification of the amino acid sequence can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in the following documents: sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,1989 and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

In some embodiments, the mutation does not substantially reduce the ability of a FAP-binding agent of the invention to specifically bind to FAP. In some embodiments, the mutation does not substantially reduce the ability of FAP-binding agents of the invention to specifically bind to FAP and not functionally modulate (e.g., partially or fully neutralize) FAP.

In some embodiments, an equilibrium dissociation constant (K) may be used_D) To describe the binding affinity of FAP binding agents of the present technology to full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or monomeric and/or dimeric forms of human FAP and/or any other naturally occurring or synthetic analogs, variants or mutants, including monomeric and/or dimeric forms. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and/or dimeric forms) of human FAP, wherein K is _DLess than about 1 μ M, about900nM, about 800nM, about 700nM, about 600nM, about 500nM, about 400nM, about 300nM, about 200nM, about 100nM, about 90nM, about 80nM, about 70nM, about 60nM, about 50nM, about 40nM, about 30nM, about 20nM, about 10nM, or about 5nM, or about 1 nM.

In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to an antigen of interest but does not functionally modulate (e.g., partially or fully neutralize) the antigen of interest, i.e., FAP. For example, in some embodiments, the targeting moiety of the FAP-binding agent targets only the antigen but does not substantially functionally modulate (e.g., partially or completely inhibit, reduce, or neutralize) a biological effect that the antigen has. In some embodiments, the targeting moiety of the FAP-binding agent binds to an epitope that is physically separated from an antigenic site that is important for the biological activity of the antigen (e.g., the active site of the antigen).

Such binding without significant functional modulation may be used in some embodiments of the present technology, including methods of using FAP-binding agents of the present invention to recruit active immune cells to a desired site, either directly or indirectly via an effector antigen. For example, in some embodiments, FAP-binding agents of the invention may be used in methods of reducing or eliminating a tumor so as to recruit dendritic cells to tumor cells directly or indirectly via FAP (e.g., the FAP-binding agent may comprise a targeting moiety having an anti-FAP antigen recognition domain and a targeting moiety having a recognition domain (e.g., an antigen recognition domain) for a tumor antigen or receptor). In such embodiments, it is desirable to directly or indirectly recruit dendritic cells but not functionally modulate or neutralize FAP activity. In these embodiments, FAP signaling is an important part of tumor reduction or elimination.

In some embodiments, the FAP-binding agent enhances antigen presentation by dendritic cells. For example, in some embodiments, the FAP-binding agents of the invention recruit dendritic cells to tumor cells, either directly or indirectly via FAP, wherein tumor antigens are subsequently endocytosed and presented on the dendritic cells in order to induce an effective humoral and cytotoxic T cell response.

In other embodiments (e.g., directed to treating cancer, autoimmune or neurodegenerative diseases), the FAP-binding agent comprises a targeting moiety that binds to and neutralizes an antigen of interest, FAP. For example, in some embodiments, the methods of the invention may inhibit or reduce FAP signaling or expression, e.g., to cause a reduction in an immune response.

Chimeras and fusions with signaling agents

In some embodiments, the FAP-binding agents of the present technology as disclosed herein are part of a chimera or fusion protein or Fc-based chimeric protein complex with one or more signaling agents. Thus, the present technology provides chimeric or fusion proteins or Fc-based chimeric protein complexes comprising, for example, a targeting moiety for FAP and one or more signaling agents. In some embodiments, the signaling agent is a wild-type signaling agent or a modified signaling agent.

In various embodiments, the chimera or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling agent relative to i) a signaling agent that is not fused to Fc; ii) or not in the context of a complex (such as but not limited to a heterodimeric complex) have improved target selectivity and safety. In various embodiments, the chimera or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling agent relative to i) a signaling agent that is not fused to Fc; ii) or a signaling agent not in the context of a complex (such as, but not limited to, a heterodimeric complex) has increased target-selective activity. In various embodiments, the Fc-based chimeric protein complex achieves conditional activity.

In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling agent having increased safety, such as reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to a signaling agent that is not fused to Fc or not within the context of the complex (e.g., without limitation, a heterodimeric complex). In various embodiments, increased safety means that the chimeras or fusion proteins or Fc-based chimeric proteins of the invention provide lower toxicity (e.g., systemic toxicity and/or tissue/organ related toxicity) as compared to signaling agents that are not fused to Fc or not within the context of a complex (e.g., without limitation, heterodimeric complexes); and/or reduce or substantially eliminate side effects; and/or increased tolerance, reduced or substantially eliminated adverse events; and/or a reduction or substantial elimination of off-target effects; and/or increased therapeutic window.

In some embodiments, the reduced affinity or activity at the receptor may be restored by attachment to one or more targeting moieties as described herein or after inclusion in an Fc-based chimeric protein complex as disclosed herein.

In various embodiments, the chimeras or fusion proteins or Fc-based chimeric protein complexes comprise a wild-type signaling agent with reduced, substantially reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., K)_D) And/or activation (e.g., when the modified signaling agent is an agonist of its receptor, measurable as, e.g., K_AAnd/or EC₅₀) And/or inhibition (e.g., as measurable by, e.g., K, when the modified signaling agent is an antagonist of its receptor_IAnd/or IC₅₀). In various embodiments, the reduced affinity at the signaling agent receptor allows for attenuation of activity. In such embodiments, the modified signaling agent has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% -20%, about 20%, compared to a signaling agent that is not fused to an Fc or a signaling agent that is not within the scope of a complex (e.g., without limitation, a heterodimeric complex) -40%, about 50%, about 40% -60%, about 60% -80%, about 80% -100% affinity. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-fold lower, 150-fold lower, about 150-fold lower, or more than 200-fold lower than a signaling agent that is not fused to Fc or not within the context of a complex (e.g., without limitation, heterodimer complexes).

In various embodiments, the chimeric or fusion protein or Fc-based chimeric protein complex comprises a wild-type signaling agent, such as a wild-type signaling agent that reduces the endogenous activity of the signaling agent to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% as compared to a signaling agent that is not fused to Fc or not within the scope of the complex (e.g., without limitation, a heterodimeric complex).

In some embodiments, the signaling agent is modified to have reduced affinity or activity for one or more of its receptors, thereby allowing for reduced activity (including agonism or antagonism) and/or preventing non-specific signaling or undesirable sequestration of the chimeric or fusion protein or Fc-based chimeric protein complex. In some embodiments, the signaling agent is antagonistic in its wild-type form and carries one or more mutations that attenuate its antagonistic activity. In some embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., an agonistic signaling agent is converted to an antagonistic signaling agent, and optionally, such converted signaling agent also carries one or more mutations that attenuate its antagonistic activity (e.g., as described in WO 2015/007520, the entire contents of which are hereby incorporated by reference).

Thus, in some embodiments, the signaling agent is a modified (e.g., mutated) form of the signaling agent having one or more mutations (e.g., a plurality of mutations). In some embodiments, the mutation allows the modified signaling agent to have one or more reduced activities, such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to the unmutated, i.e., wild-type, form of the signaling agent (e.g., comparing wild-type forms versus modified (e.g., mutated) forms of the same signaling agent). In some embodiments, the mutations that reduce or decrease binding or affinity include those that substantially reduce or eliminate binding or activity. In some embodiments, the mutations that reduce or decrease binding or affinity are different from those that substantially reduce or eliminate binding or activity. As a result, in some embodiments, the mutations allow for increased safety of signaling agents relative to unmutated, i.e., wild-type signaling agents, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects (e.g., comparing wild-type versus modified (e.g., mutated) forms of the same signaling agent).

As described herein, the agent may have improved safety due to one or more modifications (e.g., mutations). In some embodiments, increased safety means that the chimeric proteins or Fc-based chimeric protein complexes of the invention provide lower toxicity (e.g., systemic toxicity and/or tissue/organ related toxicity); and/or reduce or substantially eliminate side effects; and/or increased tolerance, reduced or substantially eliminated adverse events; and/or a reduction or substantial elimination of off-target effects; and/or increased therapeutic window.

In some embodiments, the signaling agent is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity provided by the wild-type signaling agent is agonism at the receptor (e.g., activation of a cellular effect at a treatment site). For example, the wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in a reduction or elimination of the activation activity of the modified signaling agent at the receptor. For example, the mutation may cause the modified signaling agent to deliver a reduced activation signal to the target cell, or may eliminate the activation signal. In some embodiments, the activity provided by the wild-type signaling agent is antagonism at the receptor (e.g., blocks or suppresses a cellular effect at the treatment site). For example, the wild-type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutation results in a reduction or elimination of the antagonistic activity of the modified signaling agent at the receptor. For example, the mutation may cause the modified signaling agent to deliver a reduced inhibitory signal to the target cell, or may eliminate the inhibitory signal. In some embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., converting an agonistic signaling agent into an antagonistic signaling agent (e.g., as described in WO 2015/007520, the entire contents of which are hereby incorporated by reference), and optionally, such a converted signaling agent also carries one or more mutations that reduce its binding affinity or activity for one or more of its receptors, or that substantially reduce or eliminate its binding affinity or activity for one or more of its receptors.

In some embodiments, the reduced affinity or activity at the receptor may be restored by linkage to one or more targeting moieties as described herein (e.g., targeting moieties for FAP) or after inclusion in an Fc-based chimeric protein complex as disclosed herein. In other embodiments, the reduced affinity or activity at the receptor is not substantially restored by the activity of the one or more targeting moieties or after inclusion in an Fc-based chimeric protein complex as disclosed herein.

In some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the present technology reduce off-target effects because their signaling agents have mutations that impair or eliminate binding affinity or activity at the receptor. In some embodiments, such a reduction in side effects is observed relative to, for example, wild-type signaling agents. In some embodiments, the signaling agent is active on the target cell because the one or more targeting moieties compensate for the lack of binding (e.g., without limitation and/or avidity) required for substantial activation. In some embodiments, the modified signaling agents are substantially inactive to the pathway of the therapeutically active site and their effects are substantially directed to the specifically targeted cell type, thereby substantially reducing undesirable side effects.

In some embodiments, the signaling agent may include one or more mutations that decrease or reduce binding or affinity to one receptor (i.e., the therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity at a second receptor. In such embodiments, the mutations may be at the same or different positions (i.e., the same mutation or mutations). In some embodiments, the one or more mutations that reduce binding and/or activity at one receptor are different from one or more mutations that substantially reduce or eliminate binding and/or activity at another receptor. In some embodiments, the one or more mutations that reduce binding and/or activity at one receptor are the same as the one or more mutations that substantially reduce or eliminate binding and/or activity at another receptor. In some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the invention have a modified signaling agent that combines mutations that attenuate binding and/or activity at a therapeutic receptor and thus allow for a more controlled on-target therapeutic effect (e.g., relative to a wild-type signaling agent) with mutations that substantially reduce or eliminate binding and/or activity at another receptor and thus reduce side effects (e.g., relative to a wild-type signaling agent).

In some embodiments, the substantial reduction or elimination of binding or activity is substantially not recoverable by a targeting moiety (e.g., a targeting moiety for FAP or any other targeting moiety described herein) or after inclusion in an Fc-based chimeric protein complex as disclosed herein. In some embodiments, a substantial reduction or elimination of binding or activity can be restored by the targeting moiety or after inclusion in an Fc-based chimeric protein complex as disclosed herein. In some embodiments, substantially reducing or eliminating binding or activity at the second receptor may also prevent adverse effects mediated by another receptor. Alternatively or additionally, substantially reducing or eliminating binding or activity at another receptor improves therapeutic efficacy, as the reduced or eliminated sequestration of the therapeutic chimeric protein is away from the site of therapeutic action. For example, in some embodiments, this circumvents the need for high doses of the chimeric proteins or Fc-based chimeric protein complexes of the invention that can compensate for loss at another receptor. This ability to reduce the dose also provides a lower potential for side effects.

In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced, substantially reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., KD) and/or activation (e.g., measurable as, e.g., KA and/or EC50 when the modified signaling agent is an agonist of its receptor) and/or inhibition (e.g., measurable as, e.g., Ki and/or 1050 when the modified signaling agent is an antagonist of its receptor). In some embodiments, reduced affinity at the immunomodulatory agent receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling agent has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% -20%, about 20% -40%, about 50%, about 40% -60%, about 60% -80%, about 80% -100% relative to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 150-fold lower, or more than 200-fold lower relative to the wild-type signaling agent.

In embodiments where the modified signaling agent has a mutation that reduces binding at one receptor and substantially reduces or eliminates binding at a second receptor, the modified signaling agent has a reduced or reduced degree of binding affinity for one receptor that is less than a substantial reduction or elimination of affinity for another receptor. In some embodiments, the reduction or decrease in binding affinity of the modified signaling agent for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantial decrease or elimination of the affinity for another receptor. In some embodiments, a substantial reduction or elimination refers to a greater reduction in binding affinity and/or activity than a reduction or elimination.

In some embodiments, the modified signaling agent comprises one or more mutations that reduce the endogenous activity of the signaling agent, e.g., to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1% relative to the wild-type signaling agent.

In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced affinity for its receptor than the binding affinity of the one or more targeting moieties for its receptor or receptors is lower. In some embodiments, this difference in binding affinity exists between the signaling agent/receptor and the targeting moiety/receptor on the same cell. In some embodiments, this difference in binding affinity allows the signaling agent (e.g., a mutated signaling agent) to have a localized on-target effect and minimize off-target effects that underlie the side effects observed with wild-type signaling agents. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 150-fold lower.

Receptor binding activity can be measured using methods known in the art. For example, affinity and/or binding activity can be assessed by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard,1949) or by reflection interference spectroscopy under flow-through conditions as described by Brecht et al (1993), the entire contents of all of which are hereby incorporated by reference.

In various embodiments, the additional signaling agent is selected from wild-type or modified versions of cytokines, growth factors, and hormones. Illustrative examples of such cytokines, growth factors and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones (such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone); parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and tumor necrosis factor-beta; a mullerian inhibitor; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and insulin-like growth factor-II; an osteoinductive factor; interferons such as, for example, interferon- α, interferon- β, and interferon- γ (as well as type I, type II, and type III interferons), Colony Stimulating Factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as, for example, IL-1 β, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; tumor necrosis factors such as, for example, TNF- α or TNF- β; and other polypeptide factors including, for example, LIF and Kit Ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced by recombinant bacterial, eukaryotic, or mammalian cell culture systems, as well as biologically active equivalents of the native sequence cytokines.

In some embodiments, the additional signaling agent is a wild-type or modified version of a growth factor selected from, but not limited to, Transforming Growth Factors (TGF) such as (TGF- α and TGF- β), Epidermal Growth Factor (EGF), insulin-like growth factors (such as insulin-like growth factor-I and insulin-like growth factor-II), Fibroblast Growth Factor (FGF), genetic regulatory protein (heregulin), platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor (VEGF).

In one embodiment, the growth factor is a wild-type or modified version of a Fibroblast Growth Factor (FGF). Illustrative FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF 23.

In one embodiment, the growth factor is a wild-type or modified version of a Transforming Growth Factor (TGF). Illustrative TGF include, but are not limited to, TGF-alpha and TGF-beta and subtypes thereof, including various subtypes of TGF-beta, including TGF-beta 1, TGF-beta 2, and TGF-beta 3.

In some embodiments, the additional signaling agent is a wild-type or modified version of a hormone selected from, but not limited to: human chorionic gonadotropin, gonadotropin releasing hormone, androgen, estrogen, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyroid stimulating hormone releasing hormone, growth hormone releasing hormone, adrenocorticotropic hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids, mineralocorticoids, epinephrine, norepinephrine, progesterone, insulin, glycitin, pullulan, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, Erythropoietin (EPO), and angiotensinogen.

In some embodiments, the signaling agent is an immunomodulator, such as one or more of an interleukin, an interferon, and a tumor necrosis factor.

In some embodiments, the signaling agent is a wild-type interleukin or a modified interleukin, including, for example, IL-1; IL-1 β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or their fragments, variants, analogs or family members. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of proliferation of immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: matthews et al, Lymphokines and interferences, A Practical Approach, edited by Clemens et al, IRL Press, Washington, D.C.1987, page 221-; and Orencole and Dinarello (1989) Cytokine 1, 14-20.

In some embodiments, the signaling agent is a wild-type interferon or a modified version of an interferon, such as a type I interferon, a type II interferon, and a type III interferon. Illustrative interferons include, for example, interferon-a-1, interferon-a-2, interferon-a-4, interferon-a-5, interferon-a-6, interferon-a-7, interferon-a-8, interferon-a-10, interferon-a-13, interferon-a-14, interferon-a-16, interferon-a-17 and interferon-a-21, interferon- β and interferon- γ, interferon κ, interferon ε, interferon τ, interferon δ, IFN-v, and interferon ω.

In some embodiments, the signaling agent is wild-type Tumor Necrosis Factor (TNF) or a protein in the Tumor Necrosis Factor (TNF) or TNF family, including but not limited to TNF- α, TNF- β, LT- β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and modified versions of TRAIL.

The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Thus, in some embodiments, the modified signaling agent comprises a polypeptide having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, to the known wild-type amino acid sequence of the signaling agents described herein, Or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96% >, or, Or about 97%, or about 98%, or about 99% sequence identity).

In some embodiments, the modified signaling agent comprises a peptide having at least about 60%, or at least about 61%, 65%, or at least about 66%, 70%, or at least about 71%, 75%, or at least about 76%, 80%, or at least about 81%, 85%, or at least about 86%, 90%, or at least about 91%, or at least about 62%, or at least about 67%, or at least about 72%, or at least about 77%, or at least about 82%, or at least about 87%, or at least about 92%, or at least about 63%, or at least about 68%, or at least about 73%, or at least about 78%, or at least about 83%, or at least about 88%, or at least about 93%, or at least about 64%, or at least about 69%, or at least about 74%, or at least about 79%, or at least about 84%, or at least about 89%, or a portion of the amino acid sequence of the signaling agents described herein, Or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96% >, or, Or about 97%, or about 98%, or about 99% sequence identity).

In some embodiments, the modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions as described elsewhere herein. In some embodiments, the substitution may also include a non-classical amino acid as described elsewhere herein.

As described herein, the modified signaling agents carry mutations that affect affinity and/or activity at one or more receptors. In some embodiments, there is reduced affinity and/or activity for a therapeutic receptor, e.g., a receptor through which a desired therapeutic effect (agonism or antagonism) is mediated. In some embodiments, the modified signaling agent carries a mutation that substantially reduces or eliminates affinity and/or activity at a receptor, e.g., by which the desired therapeutic effect is not mediated (e.g., as a result of hybridization of binding). Receptors for any of the modified signaling agents as described herein, e.g., one of cytokines, growth factors, and hormones, are known in the art.

Illustrative mutations that provide reduced affinity and/or activity (e.g., agonism) at the receptor are found in WO 2013/107791 and PCT/EP2017/061544 (e.g., for interferons), WO 2015/007542 (e.g., for interleukins), and WO 2015/007903 (e.g., for TNF), the entire contents of each of which are hereby incorporated by reference. Illustrative mutations that provide reduced affinity and/or activity (e.g., antagonism) at receptors are found in WO2015/007520, the entire contents of which are hereby incorporated by reference.

In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and/or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor in the Tumor Necrosis Factor Receptor (TNFR) superfamily, a TGF- β receptor, a receptor in the immunoglobulin (Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.

In some embodiments, the receptor for the signaling agent is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2(β subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and Thrombopoietin (TPO), prolactin, and growth hormone. Illustrative type I cytokine receptors include, but are not limited to, the GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

In some embodiments, the receptor of the signaling agent is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are receptors primarily used for interferons. This family of receptors includes, but is not limited to, receptors for interferon-a, interferon-13 and interferon-y, IL10, IL22, and tissue factor. Illustrative type II cytokine receptors include but are not limited to IFN-a receptors (e.g., IFNAR1 and IFNAR2), IFN-3 receptors, IFN-y receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

In some embodiments, the receptor of the signaling agent is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors that have seven transmembrane structures and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, the CC chemokine receptor, the CXC chemokine receptor, the CX3C chemokine receptor, and the XC chemokine receptor (XCR 1). Illustrative chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCRs, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCRs, CSCR6, CXCR7, XCR1, and CX3CR 1.

In some embodiments, the receptor of the signaling agent is a TNFR family member. Tumor Necrosis Factor Receptor (TNFR) family members share a cysteine-rich domain (CRD) formed by three disulfide bonds surrounding the CXXCXXC core motif, forming an elongated molecule. Illustrative tumor necrosis factor receptor family members include: CDI 20a (tnfrsfia), CD120b (tnfrsbb), lymphotoxin beta receptor (LTBR, TNFRSF b), CD 134(TNFRSF b), CD b (CD b, TNFRSFs), FAS (FAS, TNFRSF b), TNFRSF6b (TNFRSF6b), CD b (CD b, TNFRSF b), CD b (TNFRSF b), CD137(TNFRSF b), tnfrfioa (tnfrsfioa), TNFRSFIOB, (TNFRSFIOB), tnfrsioc (tnfrsioc), tnfrsfiod (tnfrsfiod), tnfrsfiod (tnfrsfia), bone protector (TNFRSFIOB), tnfrsff 12 b (tnfrsfrs3672), tnfrsfrs3672 (tnfrsfrsf b), tnfrsfrsf b (tnfrsfrsf b), tnfrsfrsf b, tnfrs b, tnfrsf3672, tnfrsfrsf b, tnfrs b. In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120b (TNFRSBB) or TNF-R2.

In some embodiments, the receptor for the signaling agent is a TGF- β receptor. TGF-beta receptors are single-pass serine/threonine kinase receptors. TGF- β receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR 3.

In some embodiments, the receptor of the signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

In some embodiments, the receptor of the signaling agent is a receptor of the tyrosine kinase superfamily. Receptors in the tyrosine kinase superfamily are well known in the art. There are approximately 58 known Receptor Tyrosine Kinases (RTKs), divided into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR 5.

In some embodiments, the wild-type or modified signaling agent is interferon alpha. In some embodiments, the modified IFN- α agents have reduced affinity and/or activity for IFN- α/β receptors (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified IFN- α agents have substantially reduced or eliminated affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., the IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified IFN- α agent is a modified human IFN- α agent.

Mutant forms of interferon are known to those skilled in the art. By way of example, and not by way of limitation, in some embodiments the modified signaling agent is the allelic form of IFN- α 2a having the amino acid sequence SEQ ID NO: 176.

By way of example, and not by way of limitation, in some embodiments the modified signaling agent is the allelic form of IFN- α 2b having the amino acid sequence SEQ ID NO:177 that differs from IFN- α 2a at amino acid position 23.

In some embodiments, the modified IFN- α 2 signaling agent is a human IFN- α 2 mutant (IFN- α 2a or IFN- α 2 b). In some embodiments, the human IFN-. alpha.2mutant (IFN-. alpha.2a or IFN-. alpha.2b) is mutated at one or more of amino acids 144-154, such as, for example, amino acid positions 148, 149 and/or 153. In some embodiments, the human IFN- α 2 mutant comprises one or more mutations selected from L153A, R149A, and M148A.

In some embodiments, the IFN- α 2 mutant has reduced affinity and/or activity for IFNAR 1. In some embodiments, the IFN- α 2 mutant is a human IFN- α 2 mutant comprising one or more mutations selected from the group consisting of F64A, N65A, T69A, L80A, Y85A, and Y89A as described in W02010/030671, the entire contents of which are hereby incorporated by reference.

In some embodiments, the IFN- α 2 mutant is a human IFN- α 2 mutant comprising one or more mutations selected from K133A, R144A, R149A, and L153A as described in W02008/124086, the entire contents of which are hereby incorporated by reference.

In some embodiments, the IFN- α 2 mutant is a human IFN- α 2 mutant comprising one or more mutations selected from R120E and R120E/K121E as described in W02015/007520 and W02010/030671, the entire contents of which are hereby incorporated by reference.

In some embodiments, the IFN- α 2 mutant antagonizes wild-type IFN- α 2 activity. In some embodiments, the mutant IFN- α 2 has reduced affinity and/or activity for IFNAR1 while retaining affinity and/or activity for IFNR 2.

In some embodiments, the human IFN- α 2 mutant comprises (1) one or more mutations selected from R120E and R120E/K121E that produce an antagonistic effect, without wishing to be bound by theory; and (2) one or more mutations selected from K133A, R144A, R149A, and L153A, which, without wishing to be bound by theory, allow for attenuation of effects at, for example, IFNAR 2. In some embodiments, the human IFN- α 2 mutant comprises R120E and L153A.

In some embodiments, the human IFN- α 2 mutant comprises one or more mutations selected from L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, a145G, a145M, M148A, R149A, S152A, L153A and N156A as disclosed in WO 2013/059885, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or L30A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutants comprise mutations H57Y, E58N, Q61S, and/or R33A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or M148A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or L153A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutants comprise the mutations N65A, L80A, Y85A and/or Y89A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutants comprise the mutations N65A, L80A, Y85A, Y89A and/or D114A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises the mutation R144X ₁、A145X₂And R33A, wherein X₁Selected from A, S, T, Y, L and I, and wherein X₂Selected from G, H, Y, K and D. In some embodiments, relative to the amino acid sequence of SEQ ID NO:176 or 177, the mutant human IFN alpha 2 has one or more selected from the group consisting of R33A, T106X₃、R120E、R144X₁A145X₂M148A, R149A and L153A, wherein X₁Selected from A, S, T, Y, L and I, wherein X₂Selected from G, H, Y, K and D, and wherein X₃Selected from A and E.

In some embodiments, the signaling agent is wild-type interferon alpha 1 or modified interferon alpha 1. In some embodiments, the invention provides a chimeric protein or Fc-based chimeric protein complex comprising a wild-type IFN α 1. In various embodiments, the wild-type IFN α 1 comprises the amino acid sequence:

CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHEL

IQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAV

KKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ ID NO:1042)。

in various embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the invention comprise a modified version of IFN α 1, i.e., IFN α 1 variants (including IFN α 1 mutants), as a signaling agent. In various embodiments, the IFN α 1 variants encompass mutants, functional derivatives, analogs, precursors, isoforms, splice variants or fragments of the interferon.

In some embodiments, the IFN α 1 interferon is modified to have mutations at one or more amino acids at positions L15, a19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157, relative to SEQ ID NO: 1042. The mutation may optionally be a hydrophobic mutation and may, for example, be selected from alanine, valine, leucine and isoleucine. In some embodiments, the IFN alpha 1 interferon is modified to have one or more mutations selected from the group consisting of: l15, a19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157. In some embodiments, relative to SEQ ID NO:1042, the IFN alpha 1 mutants contain one or more mutations selected from the group consisting of: L30A/H58Y/E59N _ Q62S, R33A/H58Y/E59N/Q62S, M149A/H58Y/E59N/Q62S, L154A/H58Y/E59N/Q62S, R145A/H58Y/E59N/Q62S, D115A/R121A, L118A/R121A, L118A/R121A/K122A, R121A/K122A and R121E/K122E.

In some embodiments, IFN- α 1 is a polypeptide comprising one or more mutations that reduce undesired disulfide bond pairing, wherein the one or more mutations are, for example, at amino acid position C1, C29, C86, C99, or C139 of SEQ ID NO: 1042. In some embodiments, the mutation at position C86 may be, for example, C86S or C86A or C86Y. These C86 mutants of IFN-. alpha.1 are referred to as reduced cysteine-based aggregation mutants. In some embodiments, the IFN α 1 variant comprises mutations at positions C1, C86, and C99, relative to SEQ ID NO: 1042.

In one embodiment, the wild-type or modified signaling agent is interferon-beta. In such embodiments, the modified interferon beta agent has reduced affinity and/or activity for an IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and/or activity for an IFN-alpha/beta receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain.

In an illustrative embodiment, the modified signaling agent is IFN- β. In some embodiments, the IFN- β encompasses a functional derivative, analog, precursor, isoform, splice variant, or fragment of IFN- β. In some embodiments, the IFN- β encompasses IFN- β derived from any species. In one embodiment, the chimeric protein or Fc-based chimeric protein complex comprises a modified version of mouse IFN- β. In another embodiment, the chimeric protein or Fc-based chimeric protein complex comprises a modified version of human IFN- β. Human IFN- β is a polypeptide comprising 166 amino acid residues and having a molecular weight of about 22 kDa. The amino acid sequence of human IFN-beta is shown in SEQ ID NO: 178.

In some embodiments, the human IFN- β is an IFN- β la in a glycosylated form of human IFN- β. In some embodiments, the human IFN- β is IFN- β -lb in the non-glycosylated form of human IFN- β having a Met-1 deletion and a Cys-17 to Ser mutation.

In some embodiments, the modified IFN- β has one or more mutations that reduce the binding or affinity of the modified IFN- β to the IFNAR1 subunit of IFNAR. In some embodiments, the modified IFN- β has reduced affinity and/or activity at IFNAR 1.

In some embodiments, the modified IFN- β having reduced affinity and/or activity at IFNAR1 is a human IFN- β and has one or more mutations at positions F67, R71, L88, Y92, 195, N96, K123, and R124. In some embodiments, the one or more mutations is a substitution selected from the group consisting of F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, 195A, N96G, K123G, and R124G. In some embodiments, the modified human IFN- β comprises the F67G mutation. In some embodiments, the modified human IFN- β comprises a K123G mutation. In some embodiments, the modified human IFN- β comprises F67G and R71A mutations. In some embodiments, the modified human IFN- β comprises L88G and Y92G mutations. In some embodiments, the modified human IFN- β comprises Y92G, 195A, and N96G mutations. In some embodiments, the modified human IFN- β comprises K123G and R124G mutations. In some embodiments, the modified human IFN- β comprises F67G, L88G, and Y92G mutations. In some embodiments, the modified human IFN- β comprises F67S, L88S, and Y92S mutations.

In some embodiments, the modified IFN- β has one or more mutations that reduce the binding or affinity of the modified IFN- β to the IFNAR2 subunit of IFNAR. In some embodiments, the modified IFN- β has reduced affinity and/or activity at IFNAR 2.

In some embodiments, the modified IFN- β having reduced affinity and/or activity at IFNAR2 is a human IFN- β and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations is a substitution selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In some embodiments, the modified human IFN- β comprises the W22G mutation. In some embodiments, the modified human IFN- β comprises the L32A mutation. In some embodiments, the modified human IFN- β comprises the L32G mutation. In some embodiments, the modified human IFN- β comprises the R35A mutation. In some embodiments, the modified human IFN- β comprises the R35G mutation. In some embodiments, the modified human IFN- β comprises the V148G mutation. In some embodiments, the modified human IFN- β comprises the R152A mutation. In some embodiments, the modified human IFN- β comprises the R152G mutation. In some embodiments, the modified human IFN- β comprises the Y155G mutation. In some embodiments, the modified human IFN- β comprises the W22G and R27G mutations. In some embodiments, the modified human IFN- β comprises L32A and R35A mutations. In some embodiments, the modified human IFN- β comprises L151G and R152A mutations. In some embodiments, the modified human IFN- β comprises the V148G and R152A mutations.

In some embodiments, the modified IFN- β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, a141Y, a142T, E149K and R152H. In some embodiments, the modified IFN- β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, a141Y, a142T, E149K, and R152H, in combination with C175 or MA.

In some embodiments, the modified IFN- β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, a141Y, a142T, E149K, and R152H, in combination with any other IFN- β mutation described herein.

The crystal structure of human IFN- β is known and described in Karpusas et al, (1998) PNAS,94(22):1181311818. Specifically, the structure of human IFN- β has been shown to include five alpha helices (i.e., A, B, C, D and E) and four loop regions connecting these helices (i.e., AB, BC, CD and DE loops). In some embodiments, the modified IFN- β has one or more mutations in the A, B, C, D, E helix and/or in the AB, BC, CD, and DE loops that reduce the binding affinity or activity of the modified IFN- β at a therapeutic receptor, such as an IFNAR. Illustrative mutations are described in WO 2000/023114 and US 20150011732, the entire contents of which are hereby incorporated by reference.

In one illustrative embodiment, the modified IFN- β is a human IFN- β comprising an alanine substitution at amino acid positions 15, 16, 18, 19, 22, and/or 23. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising an alanine substitution at amino acid positions 71-73. In illustrative embodiments, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 92, 96, 99, and 100. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 128, 130, 131 and 134. In an illustrative embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at W22 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at R27, wherein the mutation is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at W22, wherein the mutation is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R27, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at R35 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at L32 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R35, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at R71 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at F67 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R71, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at F67 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at L88 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at I95 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (1), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at N96 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at Y92 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at 195 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), methionine (M), and valine (V); and a mutation at N96, which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at R124 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at K123 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R124 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at R152 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at L151 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at R152 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at V148 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R152 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN- β comprises SEQ ID NO 178 and a mutation at Y155 that is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

In some embodiments, the present technology relates to a chimeric protein or Fc-based chimeric protein complex comprising: (a) 178 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue; and (b) one or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest (e.g., FAP), said modified IFN- β and said one or more targeting moieties optionally being linked to one or more linkers. In some embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M and V. In some embodiments, the mutation at position W22 is G.

Other illustrative IFN- β mutants are provided in PCT/EP2017/061544, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the signaling agent is wild-type or modified interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and/or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and/or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains.

For example, the mutant IFN- γ may comprise a mutation, as a non-limiting example a truncation. In various embodiments, the mutant IFN- γ has a truncation at the C-terminus of, for example, about 5 to about 20 amino acid residues, or about 16 amino acid residues, or about 15 amino acid residues, or about 14 amino acid residues, or about 7 amino acid residues, or about 5 amino acid residues. In various embodiments, the mutant IFN- γ has one or more mutations at positions Q1, V5, E9, K12, H19, S20, V22, a23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, a118, E119, and K125. In various embodiments, the mutant IFN- γ has one or more mutations selected from the group consisting of V5E, S20E, V22A, a23G, a23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and a118G substitutions. In various embodiments, the mutant IFN- γ comprises the V22A mutation. In various embodiments, the mutant IFN- γ comprises the a23G mutation. In various embodiments, the mutant IFN- γ comprises the D24G mutation. In various embodiments, the mutant IFN- γ comprises a H111A mutation or a H111D mutation. In various embodiments, the mutant IFN- γ comprises the I114A mutation. In various embodiments, the mutant IFN- γ comprises the Q115A mutation. In various embodiments, the mutant IFN- γ comprises the a118G mutation. In various embodiments, the mutant IFN- γ comprises an a23G mutation and a D24G mutation. In various embodiments, the mutant IFN- γ comprises the I114A mutation and the a118G mutation. IFN- γ is shown in SEQ ID NO 1043 below and all mutations are relative to SEQ ID NO 1043:

MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ(SEQ ID NO:1043)。

In one embodiment, the wild-type or modified signaling agent is consensus interferon. In some embodiments, the consensus interferon comprises the amino acid sequence SEQ ID NO 179.

In some embodiments, the consensus interferon is modified to have one or more amino acids at positions 33 and/or 145-155 (such as amino acid positions 145, 146, 149, 179, relative to SEQ ID NO:179,150 and/or 154) has a mutation. In some embodiments, the consensus interferon is modified to have a mutation at one or more amino acids at positions 33 and/or 145-155 (such as amino acid positions 145, 146, 149, 150 and/or 154) relative to SEQ ID NO 179, the substitutions optionally being hydrophobic and selected from alanine, valine, leucine and isoleucine. In some embodiments, the consensus interferon mutant comprises a sequence selected from R33A, R145X, relative to SEQ ID NO 179₁、A146X₂One or more mutations of M149A, R150A and L154A, wherein X₁Selected from A, S, T, Y, L and I, and wherein X₂Selected from G, H, Y, K and D.

In one embodiment, the consensus interferon is modified to have a mutation at amino acid position 121 (i.e., K121) relative to SEQ ID NO: 179. In one embodiment, the consensus interferon comprises the K121E mutation relative to SEQ ID NO 179.

In one embodiment, the modified signaling agent comprises a consensus interferon variant as disclosed in U.S. patent nos. 4,695,623, 4,897,471, 5,541,293, and 8,496,921, all of which are hereby incorporated by reference in their entirety. For example, the consensus interferon variant may comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. patent nos. 4,695,623, 4,897,471, and 5,541,293.

In some embodiments, the wild-type or modified signaling agent is Vascular Endothelial Growth Factor (VEGF). VEGF is a potent growth factor that plays a major role in physiological and pathological angiogenesis, regulates vascular permeability, and can act as a growth factor on cells expressing VEGF receptors. Additional functions include, among others, stimulating cell migration in macrophage lineages and endothelial cells. There are several members of the VEGF growth factor family, and at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family may bind to and activate more than one VEGFR type. For example, VEGF-A binds to VEGFR-1 and VEGFR-2, while VEGF-C binds to VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 activation regulates angiogenesis, while VEGFR-3 activation is associated with lymphangiogenesis. The main angiogenic signals are produced by VEGFR-2 activation. VEGFR-1 activation has been reported to be associated with a negative effect in angiogenesis. VEGFR-1 signaling has also been reported to be important for in vivo tumor progression via bone marrow derived VEGFR-1 positive cells (contributing to the formation of the pre-metastatic niche in bone). Several VEGF-a directed/neutralizing therapeutic antibody-based therapies have been developed, primarily for the treatment of various human tumors that are dependent on angiogenesis. These therapies are not without side effects. This may not be surprising given that these therapies act as general non-cell/tissue specific VEGFNEGFR interaction inhibitors. Therefore, it is desirable to limit VEGF (e.g., VEGF-A)/NEGFR-2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).

In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D or VEGF-E and their isoforms, including various isoforms of VEGF-A, such as VEGF121, VEGF121b, VEGF145, VEGF165b, VEGF189, and VEGF 206. In some embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-1(Flt-1) and/or VEGFR-2 (KDR/Flk-1). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1(Flt-1) and/or VEGFR-2 (KDR/Flk-1). In one embodiment, the modified signaling agent has reduced affinity and/or activity for VEGFR-2(KDR/Flk-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1). Such embodiments may be useful, for example, in methods of wound healing or in the treatment of ischemia-related diseases (which, without wishing to be bound by theory, are mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In some embodiments, binding to VEGFR-1(Flt-1) associated with cancer and pro-inflammatory activity is avoided. In some embodiments, VEGFR-1(Flt-1) acts as a decoy receptor, thus substantially reducing or eliminating affinity at the receptor, thereby avoiding sequestration of the therapeutic agent. In one embodiment, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1(Flt-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-2 (KDR/Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-3. Alternatively, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-3.

Pro-angiogenic therapies are also important in various diseases (e.g., ischemic heart disease, hemorrhage, etc.) and include VEGF-based therapies. VEGFR-2 activation has a pro-angiogenic effect (on endothelial cells). Activation of VEFGR-1 stimulates the migration of inflammatory cells (including, for example, macrophages) and results in inflammation-associated high vascular permeability. Activation of VEFGR-1 can also promote bone marrow-associated tumor niche formation. Therefore, in such a situation, it would be desirable to select a VEGF-based therapeutic for VEGFR-2 activation. In addition, it is desirable for the cells to specifically target, for example, endothelial cells.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., antagonism) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. For example, when targeting tumor vascular endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA, etc.), such constructs specifically inhibit VEGFR-2 activation on such marker positive cells, but do not activate VEGFR-1 (if activity is abolished) en route and on target cells, thereby eliminating the induction of an inflammatory response. This would provide a more selective and safer anti-angiogenic therapy for many tumor types compared to VEGF-a neutralization therapy.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonism) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. In some embodiments, such constructs promote angiogenesis without inducing VEGFR-1-associated inflammatory responses by targeting vascular endothelial cells. Thus, such a construct would have a targeted pro-angiogenic effect and substantially reduce the risk of side effects caused by systemic activation of VEGFR-2 as well as VEGFR-1.

In an illustrative embodiment, the wild-type or modified signaling agent is VEGF165 (wild-type), the VEGF165 having the amino acid sequence SEQ ID NO: 180.

In another illustrative embodiment, the wild-type or modified signaling agent is VEGF165b (wild-type), the VEGF165b having the amino acid sequence SEQ ID NO: 181.

In these embodiments, the modified signaling agent has a mutation at amino acid 183 (e.g., a substitution mutation at 183, such as 183K, 183R, or 183H). Without wishing to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, for example, U.S. patent No. 9,078,860, which is hereby incorporated by reference in its entirety.

In one embodiment, the wild-type or modified signaling agent is TNF- α. TNF is a pleiotropic cytokine with many diverse functions including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and migration, inflammation, and septic shock. It binds to two distinct membrane receptors on target cells: TNFR1(p55) and TNFR2(p 75). TNFR1 shows a very broad expression pattern, whereas TNFR2 is preferentially expressed on certain populations of lymphocytes, tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Very distinct biological pathways are activated in response to receptor activation, but there is also some overlap. Generally, without wishing to be bound by theory, TNFR1 signaling is associated with induction of apoptosis (cell death), while TNFR2 signaling is associated with activation of cell survival signals (e.g., activation of the nfkb pathway).

Administration of TNF has systemic toxicity and this is primarily due to the adaptor of TNFR 1. It should be noted, however, that activation of TNFR2 is also associated with a variety of activities, and as with TNFR1, control of TNF targeting and activity is important in the context of the development of TNF-based therapeutics.

In some embodiments, the modified signaling agent has reduced affinity and/or activity for TNFR1 and/or TNFR 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR 2. TNFR1 is expressed in most tissues and is involved in cell death signaling, whereas TNFR2 is involved in cell survival signaling, by contrast. Thus, in embodiments of the methods for treating cancer, the modified signaling agent has reduced affinity and/or activity for TNFR1 and/or substantially reduced or eliminated affinity and/or activity for TNFR 2. In these embodiments, the chimeric protein or Fc-based chimeric protein complex can be targeted to cells in need of apoptosis, such as tumor cells or tumor vascular endothelial cells. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for treating a neurodegenerative disorder, the modified signaling agent has a reduced affinity and/or activity for TNFR2 and/or a substantially reduced or eliminated affinity and/or activity for TNFR 1. In other words, in some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the invention comprise a modified TNF-a agent that allows for a favorable death or survival signal.

In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a modified TNF with reduced affinity and/or activity for TNFR1 and/or with substantially reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, such chimeras or Fc-based chimeric protein complexes are more potent inducers of apoptosis than wild-type TNF and/or chimeras or Fc-based chimeric protein complexes that carry only one or more mutations that contribute to reduced affinity and/or activity for TNFR 1. In some embodiments, such chimeras or Fc-based chimeric protein complexes can be used to induce tumor cell death or tumor vascular endothelial cell death (e.g., for the treatment of cancer). Furthermore, in some embodiments, for example, these chimeric or Fc-based chimeric protein complexes avoid or reduce Treg cell activation via TNFR2, thus further supporting TNFR 1-mediated anti-tumor activity in vivo.

In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a modified TNF with reduced affinity and/or activity for TNFR2 and/or with substantially reduced or eliminated affinity and/or activity for TNFR 1. In some embodiments, such chimeras or Fc-based chimeric protein complexes are more potent activators of cell survival in some cell types that may be a specific therapeutic target under various disease settings, including but not limited to stimulation of neurogenesis. In addition, such chimeras preferring TNFR2 or Fc-based chimeric protein complexes may also be used to treat autoimmune diseases (e.g., crohn's disease, diabetes, MS, colitis, and the like, and numerous other autoimmune diseases described herein). In some embodiments, the chimera or Fc-based chimeric protein complex targets autoreactive T cells. In some embodiments, the chimera or Fc-based chimeric protein complex promotes Treg cell activation and indirect suppression of cytotoxic T cells.

In some embodiments, the chimeras or Fc-based chimeric protein complexes cause death of autoreactive T cells, for example, by activating TNFR2 and/or avoiding TNFR1 (e.g., modified TNF having reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR 1). Without wishing to be bound by theory, these autoreactive T cells have their apoptosis/survival signals altered, for example, by changes in NF κ B pathway activity/signaling. In some embodiments, the chimeras or Fc-based chimeric protein complexes cause death of autoreactive T cells with nfkb pathway impairment or modifications that underlie an imbalance in cell death (apoptosis)/survival signaling properties of the autoreactive T cells and optionally alter susceptibility to certain death-inducing signals (e.g., TNFR2 activation).

In some embodiments, TNFR 2-based chimeras or Fc-based chimeric protein complexes have additional therapeutic applications in a variety of diseases including various autoimmune diseases, cardiac diseases, demyelinating and neurodegenerative disorders, as well as infectious diseases and the like.

In one embodiment, the wild-type TNF- α has the amino acid sequence of SEQ ID NO 182.

In such embodiments, the modified TNF- α agent has a mutation at one or more of amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, thereby producing a modified TNF- α with reduced receptor binding affinity. See, for example, U.S. patent No. 7,993,636, which is hereby incorporated by reference in its entirety.

In some embodiments, the modified human TNF-a portion has a mutation at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, a109, P113, Y115, E127, N137, D143, a145 and E146 as described, for example, in WO/2015/007903, the entire contents of which are hereby incorporated by reference (numbering according to the human TNF sequence, Genbank accession number BAG70306, version BAG70306.1, GI: 197692685). In some embodiments, the modified human TNF-a moiety has a substitution mutation selected from the group consisting of L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, a109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, a145G, a145R, a145T, E146D, E146K, and S147D. In one embodiment, the human TNF- α moiety has a mutation selected from the group consisting of Y87Q, Y87L, Y87A, Y87F and Y87H. In another embodiment, the human TNF- α moiety has a mutation selected from 197A, 197Q and 197S. In another embodiment, the human TNF- α moiety has a mutation selected from the group consisting of Y115A and Y115G. In one embodiment, the human TNF- α portion has the E146K mutation. In one embodiment, the human TNF- α portion has the Y87H and E146K mutations. In one embodiment, the human TNF- α portion has the Y87H and a145R mutations. In one embodiment, the human TNF- α portion has the R32W and S86T mutations. In one embodiment, the human TNF- α moiety has the R32W and E146K mutations. In one embodiment, the human TNF- α portion has the L29S and R32W mutations. In one embodiment, the human TNF- α portion has the D143N and a145R mutations. In one embodiment, the human TNF- α portion has the D143N and a145R mutations. In one embodiment, the human TNF- α moiety has the a145T, E146D, and S147D mutations. In one embodiment, the human TNF- α portion has the a145T and S147D mutations.

In some embodiments, the modified TNF-alpha agent has one or more mutations selected from N39Y, S147Y, and Y87H as described in WO 2008/124086, the entire contents of which are hereby incorporated by reference.

In some embodiments, the modified human TNF- α moiety has a mutation that provides receptor selectivity as described in PCT/IB2016/001668, the entire contents of which are hereby incorporated by reference. In some embodiments, the TNF mutation is TNF-R1 selective. In some embodiments, the TNF-R1-selective TNF mutation is at one or more of positions R32, S86, and E146. In some embodiments, the TNF-R1-selective TNF mutation is one or more of R32W, S86T, and E146K. In some embodiments, the TNF-R1-selective TNF mutation is one or more of R32W, R32W/S86T, R32W/E146K, and E146K. In some embodiments, the TNF mutation is TNF-R2 selective. In some embodiments, the TNF-R2-selective TNF mutation is at one or more of positions A145, E146, and S147. In some embodiments, the TNF-R2-selective TNF mutation is one or more of a145T, a145R, E146D, and S147D. In some embodiments, the TNF-R2 selective TNF mutation is one or more of A145R, A145T/S147D, and A145T/E146D/S147D.

In some embodiments, the wild-type or modified signaling agent is TNF- β. TNF-beta forms homotrimers or heterotrimers with LT-beta (LT-. alpha.1ss.2). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR2 and/or a herpes virus entry mediator (HEVM) and/or LT- β R.

In one embodiment, the wild-type TNF- β has the amino acid sequence of SEQ ID NO 183.

In such embodiments, the modified soluble agent may comprise a mutation at one or more of amino acids 106-113, thereby generating a modified TNF- β with reduced receptor binding affinity for TNFR 2. In one embodiment, the modified soluble agent has one or more substitution mutations at amino acid positions 106-113. In some embodiments, the substitution mutation is selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E/S106E, Q107E/S106D, Q107D/S106E, and Q107D/S106D. In another embodiment, the modified soluble agent has an insertion of about 1 to about 3 amino acids at position 106 and 113.

In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which can be a single chain trimer form as described in WO 2015/007903, the entire contents of which are incorporated by reference.

In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) having reduced affinity and/or activity at TNFR1, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has substantially reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) having reduced affinity and/or activity at TNFR2, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has substantially reduced or eliminated affinity and/or activity for TNFR 1. The constructs of such embodiments may be used, for example, in methods of suppressing a TNF response in a cell-specific manner. In some embodiments, the antagonistic TNF family member (e.g., TNF- α, TNF- β) is a single chain trimeric form as described in WO 2015/007903.

In some embodiments, the wild-type or modified signaling agent is TRAIL. In some embodiments, the modified TRAIL agent has reduced affinity and/or activity for DR4(TRAIL-RI) and/or DR5(TRAIL-RII) and/or DcR1 and/or DcR 2. In some embodiments, the modified TRAIL agent has substantially reduced or eliminated affinity and/or activity for DR4(TRAIL-RI) and/or DR5(TRAIL-RII) and/or DcR1 and/or DcR 2.

In one embodiment, the wild type TRAIL has the amino acid sequence SEQ ID NO 184.

In such embodiments, the modified TRAIL agent may comprise mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264 and H270-E271 (numbering according to the human sequence, Genbank accession number NP-003801, version 10 NP-003801.1, GI: 4507593; see above).

In some embodiments, the modified TRAIL agent may comprise one or more mutations that substantially reduce the affinity and/or activity of the modified TRAIL agent for TRAIL-R1. In such embodiments, the modified TRAIL agent may specifically bind to TRIL-R2. Illustrative mutations include mutations at one or more of amino acid positions Y189, R191, Q193, H264, 1266, and D267. For example, the mutation may be one or more of Y189Q, R191K, Q193R, H264R, 1266L, and D267Q. In one embodiment, the modified TRAIL agent comprises the mutations Y189Q, R191K, Q193R, H264R, 1266L and D267Q.

In some embodiments, the modified TRAIL agent may comprise one or more mutations that substantially reduce the affinity and/or activity of the modified TRAIL agent for TRAIL-R2. In such embodiments, the modified TRAIL agent may specifically bind to TRIL-R1. Illustrative mutations include mutations at one or more of amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutation may be one or more of G131R, R1491, S159R, N199R, K201H, and S215D. In one embodiment, the modified TRAIL agent comprises the mutations G131R, R1491, S159R, N199R, K201H and S215D. Additional TRAIL mutations are described, for example, in trebin et al, (2014) Cell Death and Disease,5: e1035, the entire disclosure of which is hereby incorporated by reference.

In some embodiments, the wild-type or modified signaling agent is TGF α. In such embodiments, the modified TGF α agents have reduced affinity and/or activity for Epidermal Growth Factor Receptor (EGFR). In some embodiments, the modified TGF α agents have substantially reduced or eliminated affinity and/or activity for Epidermal Growth Factor Receptor (EGFR).

In some embodiments, the wild-type or modified signaling agent is TGF β. In such embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TGFBR1 and/or TGFBR 2. In some embodiments, the modified signaling agent optionally has reduced or substantially reduced or eliminated affinity and/or activity for TGFBR3, which, without wishing to be bound by theory, may act as a reservoir for a ligand for the TGF- β receptor. In some embodiments, TGF is more prevalent with TGFBR1 relative to TGFBR2 or TGFBR2 relative to TGFBR 1. Similarly, without wishing to be bound by theory, LAP may act as a reservoir for ligands of TGF- β receptors. In some embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2 and/or substantially reduced or eliminated affinity and/or activity for a latent form associated peptide (LAP). In some embodiments, such chimeras or Fc-based chimeric protein complexes may be used for kamurati-Engelmann disease (Camurati-Engelmann disease) or other diseases associated with inappropriate TGF β signaling.

In some embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β) that has reduced affinity and/or activity, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520) at one or more of TGFBR1, TGFBR2, TGFBR3, the entire contents of which are hereby incorporated by reference herein). In these embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β), which also optionally has substantially reduced or eliminated affinity and/or activity at one or more of TGFBR1, TGFBR2, TGFBR 3.

In some embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β) that has reduced affinity and/or activity, i.e., antagonistic activity, at TGFBR1 and/or TGFBR2 (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β), which also optionally has substantially reduced or eliminated affinity and/or activity at TGFBR 3.

In some embodiments, the wild-type or modified signaling agent is an interleukin. In one embodiment, the wild-type or modified signaling agent is IL-1. In some embodiments, the wild-type or modified signaling agent is IL-1 α or IL-1 β. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R1 and/or IL-1 RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R1 and/or IL-1 RAcP.

In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R 2. For example, in some embodiments, the modified IL-1 β agents of the present invention avoid the interaction at IL-1R2 and thus substantially reduce their function as attractants and/or acceptors for therapeutic agents.

In one embodiment, the wild-type IL-1 β has the amino acid sequence SEQ ID NO 185.

IL-1 β is a proinflammatory cytokine and an important immune system modulator. It is a potent activator of CD 4T cell responses, increasing the proportion of Th17 cells and the expansion of IFN γ and IL-4 producing cells. IL-1 β is also a potent modulator of CD8+ T cells, enhancing antigen-specific CD8+ T cell expansion, differentiation, migration to the periphery, and memory. IL-1 beta receptors include IL-1R1 and IL-1R 2. Binding to IL-1R1 and signaling via IL-1R1 constitute the mechanism by which IL-1 mediates a wide variety of its biological (and pathological) activities. IL1-R2 may function as decoy receptors, thereby reducing the availability of IL-1 for interaction and signaling via IL-1R 1.

In some embodiments, the modified IL-1 β has reduced affinity and/or activity (e.g., agonistic activity) for IL-1R 1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is inducible or recoverable IL-1 β/IL-1R1 signaling and loss of therapeutic chimera at IL-R2 is prevented, and thus the dose of IL-1 β required is reduced (e.g., relative to wild-type or chimeras carrying only attenuating mutations for IL-R1). Such constructs may be used, for example, in methods of treating cancer, including, for example, stimulating the immune system to mount an anti-cancer response.

In some embodiments, the modified IL-1 β has reduced affinity and/or activity (e.g., antagonistic activity, such as natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference) for IL-1R 1. In some embodiments, the modified IL-1 β has substantially reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is unrecoverable IL-1 β/IL-1R1 signaling and loss of therapeutic chimera at IL-R2 is prevented, and thus the dose of IL-1 β required is reduced (e.g., relative to wild-type or chimeras carrying only attenuating mutations for IL-R1). Such constructs may be used, for example, in methods of treating autoimmune diseases, including, for example, suppression of the immune system.

In such embodiments, the modified signaling agent has a deletion of amino acids 52-54, which results in a modified human IL-1 β having reduced binding affinity for type I IL-1R and reduced biological activity. See, for example, WO 1994/000491, the entire contents of which are hereby incorporated by reference. In some embodiments, the modified human IL-1 β has a substitution selected from a group consisting of a117G/P118G, R120X, L122A, T125G/L126G, R127G, Q130G, Q131G, K132G, S137G/Q138G, L145G, H146G, L145G/L147G, Q148G/Q150G, Q150G/D151G, M152G, F162G/Q36164, F166G, Q164G/E167G, N169G/D170G, I172G, V174G, K208G, K0003672, K209G/K210, K219, K G, E36221, E G/N G, N G/N224, N245K G/K G, N G, K0003672, K G/K G, K209/K G, NP G, NP-G, and NP-G, wherein the conservative substitutions are based on a number of the amino acid change in a number of a non-conservative change as described in a conservative change in a non-NP-G, e.g. a conservative change, e.g. a non-G, NP-G, wherein said modified human IL-G, NP-G, and a change is incorporated by a conservative change (e.g. a change is based on a conservative change, NP-G, e.g. a change is cited, GI: 10835145). In some embodiments, the modified human IL-1 β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In one embodiment, the modified human IL-1 β comprises mutations Q131G and Q148G. In one embodiment, the modified human IL-1 β comprises the mutations Q148G and K208E. In one embodiment, the modified human IL-1 β comprises the mutations R120G and Q131G. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146A. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146N. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146R. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146E. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146G. In one embodiment, the modified human IL-1 β comprises the mutations R120G and K208E. In one embodiment, the modified human IL-1 β comprises the mutations R120G, F162A, and Q164E.

In some embodiments, the wild-type or modified signaling agent is IL-2. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-2R α and/or IL-2R β and/or IL-2R γ. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-2R β and/or IL-2R γ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-2 ra. Such embodiments may relate to the treatment of cancer, for example, when the modified IL-2 is agonistic at IL-2R β and/or IL-2R γ. For example, the constructs of the invention may be beneficial in attenuating the activation of CD8+ T cells with IL2 receptors β and γ (which may provide an anti-tumor effect), while being detrimental to tregs with IL2 receptors α, β and γ (which may provide an immunosuppressive, pro-tumor effect). Furthermore, in some embodiments, preference for IL-2R β and/or IL-2R γ over IL-2R α avoids a number of IL-2 side effects, such as pulmonary edema. Furthermore, IL-2-based chimeras or Fc-based chimeric protein complexes may be used for the treatment of autoimmune diseases, for example when the modified IL-2 has antagonistic properties at IL-2R β and/or IL-2R γ (e.g.natural antagonistic activity or antagonistic activity due to one or more mutations, see for example WO 2015/007520, the entire contents of which are hereby incorporated by reference). For example, the constructs of the invention may be beneficial in attenuating inhibition (and thus suppressing immune responses) of CD8+ T cells with IL2 receptors β and γ, while being detrimental to tregs with IL2 receptors α, β and γ. Alternatively, in some embodiments, a chimera or Fc-based chimeric protein complex carrying IL-2 favors activation of tregs, and thus immunosuppression, while disfavoring activation of CD8+ T cells. For example, these constructs may be used to treat diseases or diseases that would benefit from immune suppression, such as autoimmune diseases.

In some embodiments, the chimeric protein or Fc-based chimeric protein complex has a targeting moiety for FAP + dendritic cells as described herein and a modified IL-2 agent with reduced affinity and/or activity for IL-2R β and/or IL-2R γ and/or substantially reduced or eliminated affinity and/or activity for IL-2R α. In some embodiments, these constructs provide targeted FAP + dendritic cell activity and are generally inactive (or have substantially reduced activity) against Treg cells. In some embodiments, such constructs have an enhanced immunostimulatory effect (e.g., without wishing to be bound by theory, by not stimulating tregs) compared to wild-type IL-2, while eliminating or reducing systemic toxicity associated with IL-2.

In some embodiments, the wild-type IL-2 has the amino acid sequence SEQ ID NO: 186.

In such embodiments, the modified IL-2 agent has one or more mutations at amino acids L72(L72G, L72A, L725, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), F42(F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and Y45(Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). Without wishing to be bound by theory, it is believed that these modified IL-2 agents have reduced affinity for the high affinity IL-2 receptor and retain affinity for the high affinity IL-2 receptor compared to the wild-type IL-2. See, for example, U.S. patent publication No. 2012/0244112, the entire contents of which are hereby incorporated by reference.

In some embodiments, the modified IL-2 agent has one or more mutations at amino acids R38, F42, Y45, and E62. For example, the modified IL-2 agent may comprise one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL-2 agent may comprise a mutation at C125. For example, the mutation may be C125S. In such embodiments, The modified IL-2 agent may have a substantially reduced affinity and/or activity for IL-2R α, as described, for example, in Carmenate et al (2013) The Journal of Immunology,190: 6230-. In some embodiments, the modified IL-2 agent having a mutation at R38, F42, Y45, and/or E62 is capable of inducing expansion of effector cells, including CD8+ T cells and NK cells, but not Treg cells. In some embodiments, the modified IL-2 agent having a mutation at R38, F42, Y45, and/or E62 is less toxic than a wild-type IL-2 agent. Chimeric proteins or Fc-based chimeric protein complexes comprising said modified IL-2 agents having substantially reduced affinity and/or activity for IL-2 ra may be used, for example, in oncology.

In other embodiments, the modified IL-2 agent may have substantially reduced affinity and/or activity for IL-2R β, as described, for example, in WO 2016/025385, the entire disclosure of which is hereby incorporated by reference. In such embodiments, the modified IL-2 agent may induce expansion of Treg cells but not effector cells (such as CD8+ T cells and NK cells). Chimeric proteins or Fc-based chimeric protein complexes comprising said modified IL-2 agent having substantially reduced affinity and/or activity for IL-2R β may for example be used in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 agent may comprise one or more mutations at amino acids N88, D20, and/or a 126. For example, the modified IL-2 agent may comprise one or more of N88R, N881, N88G, D20H, Q126L, and Q126F.

In some embodiments, the modified IL-2 agent may comprise a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, modified IL-2 having a mutation at D109 or C125 can be used to attach to a PEG moiety.

In some embodiments, the wild-type or modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and/or activity for an IL-3 receptor that is a heterodimer with a unique alpha chain paired with a common beta (β c or CD131) subunit. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for an IL-3 receptor that is a heterodimer with a unique alpha chain paired with a common beta (β c or CD131) subunit.

In some embodiments, the wild-type or modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and/or activity for a type 1 and/or type 2 IL-4 receptor. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for a type 1 and/or type 2 IL-4 receptor. The type 1 IL-4 receptor is composed of an IL-4 Ra subunit with a common gamma chain and specifically binds IL-4. The type 2 IL-4 receptor includes an IL-4 Ra subunit that binds to a different subunit known as IL-13 Ra 1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for a type 2 IL-4 receptor.

In some embodiments, the wild-type IL-4 has the amino acid sequence SEQ ID NO: 187.

In such embodiments, the modified IL-4 agent has one or more mutations at amino acids R121(R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122(E122F), Y124(Y124A, Y124Q, Y124R, Y124S, Y124T), and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents maintain type I receptor-mediated activity, but significantly reduce other receptor-mediated biological activity. See, for example, U.S. patent No. 6,433,157, which is hereby incorporated by reference in its entirety.

In some embodiments, the wild-type or modified signaling agent is IL-6. IL-6 signals through a cell surface type I cytokine receptor complex that includes a ligand-binding IL-6R chain (CD126) and a signal transduction component, gp 130. IL-6 can also bind to IL-6R soluble form (sIL-6R), the latter is IL-6R extracellular portion. The sIL-6R/IL-6 complex may be involved in neurite outgrowth and neuronal survival, and may therefore be important in nerve regeneration by remyelination. Thus, in some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-6R/gp130 and/or sIL-6R. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-6R/gp130 and/or sIL-6R.

In some embodiments, the wild-type IL-6 has the amino acid sequence of SEQ ID NO: 188.

In such embodiments, the modified signaling agent has one or more mutations at amino acids 58, 160, 163, 171, or 177. Without wishing to be bound by theory, it is believed that these modified IL-6 agents exhibit reduced binding affinity to IL-6R- α and reduced biological activity. See, for example, WO 97/10338, the entire contents of which are hereby incorporated by reference.

In some embodiments, the wild-type or modified signaling agent is IL-10. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-10 receptor 1 and IL-10 receptor 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-10 receptor 1 and IL-10 receptor 2.

In some embodiments, the wild-type or modified signaling agent is IL-11. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-11R α and/or IL-11R β and/or gp 130. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-11R α and/or IL-11R β and/or gp 130.

In some embodiments, the wild-type or modified signaling agent is IL-12. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-12R β 1 and/or IL-12R β 2. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-12R β 1 and/or IL-12R β 2.

In some embodiments, the wild-type or modified signaling agent is IL-13. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-4 receptor (IL-4R α) and IL-13R α 1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-4 receptor (IL-4R α) or IL-13R α 1.

In some embodiments, the wild-type IL-13 has the amino acid sequence SEQ ID NO: 189.

In such embodiments, the modified IL-13 agent has one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. Without wishing to be bound by theory, it is believed that these modified IL-13 agents exhibit reduced biological activity. See, for example, WO 2002/018422, the entire contents of which are hereby incorporated by reference.

In one embodiment, the signaling agent is wild-type or modified IL-15. In various embodiments, the modified IL-15 has reduced affinity and/or activity for interleukin 15 receptor.

In one embodiment, the wild-type IL-15 has the following amino acid sequence:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:1044)。

in such embodiments, the modified IL-15 agent has one or more mutations at amino acids S7, D8, K10, K11, E46, L47, V49, I50, D61, N65, L66, I67, I68, L69, N72, Q108 relative to SEQ ID NO: 1044.

In some embodiments, the wild-type or modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-18R α and/or IL-18R β. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-18R α and/or IL-18R β. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for type II IL-18 ra, i.e., an isoform of IL-18 ra that lacks the TIR domain required for signaling.

In some embodiments, the wild-type IL-18 has the amino acid sequence SEQ ID NO 190.

In such embodiments, the modified IL-18 agent may comprise one or more mutations at an amino acid or region of amino acids selected from the group consisting of Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-a97, N27-K129, Q139-M149, K165-K171, R183, and Q190-N191 as described in WO/2015/007542, the entire contents of which are hereby incorporated by reference (numbering based on human IL-18 sequences, Genbank accession No. AAV38697, version AAV38697.1, GI: 54696650).

In some embodiments, the wild-type or modified signaling agent is IL-33. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the ST-2 receptor and IL-1 RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for the ST-2 receptor and IL-1 RAcP.

In some embodiments, the wild-type IL-33 has the amino acid sequence SEQ ID NO 191.

In such embodiments, the modified IL-33 agent may comprise one or more mutations at an amino acid or region of amino acids selected from the group consisting of 1113-Y122, 5127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269 as described in WO/2015/007542, the entire contents of which are hereby incorporated by reference (numbering based on human sequence, Genbank accession No. NP-254274, version NP-254274.1, GI: 15559209).

In some embodiments, the wild-type or modified signaling agent is Epidermal Growth Factor (EGF). EGF is a member of the efficient growth factor family. Members include EGF, HB-EGF, and other members such as TGF alpha, amphiregulin, neuregulin, epithelial regulatory protein, beta cell protein. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB 4. These receptors may act as homodimeric and/or heterodimeric receptor subtypes. Different EGF family members exhibit different selectivity for various receptor subtypes. For example, EGF is associated with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2, and some other heterodimeric isoforms. HB-EGF has a similar pattern, but it is also associated with ErbB 4/4. The positive or negative modulation of EGF (EGF-like) growth factor signalling is of considerable therapeutic interest. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers where EGFR signaling constitutes the primary growth promoting signal. Alternatively, stimulation of EGFR signaling is of therapeutic interest, for example, in promoting wound healing (acute and chronic), oral mucositis (a major side effect of various cancer therapies, including but not limited to radiation therapy).

In some embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB 4. Such embodiments may be used, for example, in methods of treating wounds. In some embodiments, the modified signaling agent binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of these receptors. In such embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4, which allows antagonism of the activity of these receptors in an attenuated manner. Such embodiments may be used, for example, in the treatment of cancer. In one embodiment, the modified signaling agent has reduced affinity and/or activity for ErbB 1. ErbB1 is a therapeutic target for kinase inhibitors-most of these kinase inhibitors have side effects because of their poor selectivity (e.g., gefitinib, erlotinib, afatinib, bugatinib, and icotinib). In some embodiments, the reduced antagonistic ErbB1 signaling is more targeted and has fewer side effects than other agents that target the EGF receptor.

In some embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1 (e.g., antagonistic, e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference) and/or substantially reduced or eliminated affinity and/or activity for ErbB4 or other subtypes that may interact therewith. By specific targeting via a targeting moiety, cell-selective suppression of ErbB1/ErbB1 receptor activation (antagonism, e.g., natural antagonism or antagonism due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference) will be achieved without involvement of other receptor subtypes that may be associated with inhibition of the associated side effects. Thus, such constructs will provide cell-selective (e.g., tumor cells with activated EGFR signaling due to receptor amplification, overexpression, etc.) anti-EGFR (ErbB1) drug effects with reduced side effects, as compared to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in vivo.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonism) for ErbB4 and/or other subtypes with which it may interact. By targeting specific target cells via a targeting moiety, selective activation of ErbB1 signaling (e.g., epithelial cells) is achieved. In some embodiments, such constructs are useful for treating wounds (promoting healing) with reduced side effects, particularly for treating chronic conditions and for applications other than topical application of therapeutics (e.g., systemic wound healing).

In one embodiment, the wild-type or modified signaling agent is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has reduced affinity and/or activity for the insulin receptor and/or IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has substantially reduced or eliminated affinity and/or activity for the insulin receptor and/or IGF1 or IGF2 receptor. The attenuated response at the insulin receptor allows control of diabetes, obesity, metabolic disorders, etc., while the direct distance from the IGF1 or IGF2 receptor avoids the effects of pre-cancer.

In one embodiment, the wild-type or modified signaling agent is insulin-like growth factor I or insulin-like growth factor II (IGF-1 or IGF-2). In one embodiment, the modified signaling agent is IGF-1. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the insulin receptor and/or IGF1 receptor. In one embodiment, the modified signaling agent may bind to IGF1 receptor and antagonize the activity of the receptor. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the IGF1 receptor, which allows antagonism of the activity of the receptor in a reduced manner. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for insulin receptor and/or IGF1 receptor. In some embodiments, the modified signaling agent has reduced affinity and/or activity for the IGF2 receptor, which allows antagonism of the activity of the receptor in a reduced manner. In one embodiment, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for the insulin receptor and, thus, does not interfere with insulin signaling. In some embodiments, this is suitable for cancer treatment. In some embodiments, the agents of the invention can prevent IR isoform a from causing resistance to cancer therapy.

In one embodiment, the wild-type or modified signaling agent is EPO. In various embodiments, the modified EPO agents have reduced affinity and/or activity for an EPO receptor (EPOR) receptor and/or an ephrin receptor (EphR) relative to wild-type EPO-or other EPO-based agents described herein. In some embodiments, the modified EPO agent has substantially reduced or eliminated affinity and/or activity for an EPO receptor (EPOR) receptor and/or an Eph receptor (EphR). Illustrative EPO receptors include, but are not limited to, EPOR homodimers or EPOR/CD131 heterodimers. Also included as EPO receptors are beta-shared receptors (β cR). Illustrative Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB 6. In some embodiments, the modified EPO protein comprises one or more mutations that result in the EPO protein having a reduced affinity for a receptor comprising one or more different EPO receptors or Eph receptors (e.g., heterodimers, heterotrimers, and the like, including but not limited to EPOR-EPHB4, EPOR- β cR-EPOR). Receptors of european patent publication No. 2492355, including but not limited to NEPOR, are also provided, the entire contents of which are hereby incorporated by reference.

The structure of the human EPO protein is expected to comprise four helix bundles including helix a, helix B, helix C and helix D. In various embodiments, the modified EPO protein comprises one or more mutations located in the four regions of the EPO protein important for biological activity, namely amino acid residues 10-20, 44-51, 96-108 and 142-156. In some embodiments, the one or more mutations are at residues 11-15, 44-51, 100-151 and 147-151. These residues are restricted to helix A (Val11, Arg14 and Tyr15), helix C (Ser100, Arg103, Ser104 and Leu108), helix D (Asn147, Arg150, Gly151 and Leu155) and the A/B connecting loop (residues 42-51). In some embodiments, the modified EPO protein comprises mutations between amino acids 41-52 and at residues 147, 150, 151, and 155. Without wishing to be bound by theory, it is believed that mutations of these residues have a substantial effect on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein comprises mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, it is believed that mutations of these residues have a moderate effect on receptor binding activity and a much greater effect on in vitro biological activity. Illustrative substitutions include, but are not limited to, one or more of the following: val11Ser, Arg14Ala, Arg14Gln, Tyr15lle, Pro42Asn, Thr44lle, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104lle, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala and Leu155 Ala.

In some embodiments, the modified EPO protein comprises a mutation that affects biological activity without affecting binding, e.g., Eliot et al Mapping of the Active Site of Recombinant Human erythropoetin 1997 1, 15; those mutations listed in Blood:89(2), the entire contents of which are hereby incorporated by reference.

In some embodiments, the modified EPO protein comprises one or more mutations involving surface residues in the EPO protein involved in receptor contact. Without wishing to be bound by theory, it is believed that mutations of these surface residues are less likely to affect protein folding, thereby retaining some biological activity. Illustrative surface residues that may be mutated include, but are not limited to, residues 147 and 150. In illustrative embodiments, the mutation is a substitution, including one or more of N147A, N147K, R150A, and R150E.

In some embodiments, the modified EPO protein comprises one or more mutations at residues N59, E62, L67, and L70 and one or more mutations that affect disulfide bond formation. Without wishing to be bound by theory, it is believed that these mutations affect folding and/or are predicted to be in the embedded position, and thus indirectly affect biological activity.

In one embodiment, the modified EPO protein comprises a K20E substitution that significantly reduces receptor binding. See, e.g., Elliott et al, (1997) Blood,89:493-502, the entire contents of which are hereby incorporated by reference.

Other EPO mutations that can be incorporated into the chimeric EPO proteins of the invention are disclosed, for example, in the following documents: elliott et al, (1997) Blood,89:493-502, the entire contents of which are hereby incorporated by reference; and Taylor et al, (2010) PEDS,23(4): 251-.

In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the invention has (i) a targeting moiety for FAP and (ii) a targeting moiety for a tumor cell, and any of the wild-type or modified or mutated signaling agents described herein. In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the invention has a targeting moiety for FAP on dendritic cells and a second targeting moiety for PD-L1 or PD-L2 on tumor cells.

In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the invention has (i) a targeting moiety for FAP and (ii) a targeting moiety for a checkpoint inhibitor marker, and any of the wild-type or modified or mutated interferons described herein. In one embodiment, the chimeric protein or Fc-based chimeric protein complex of the invention has a targeting moiety for FAP on dendritic cells and a second targeting moiety for PD-1.

In some embodiments, the signaling agent is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Illustrative toxins or toxic enzymes include, but are not limited to, diphtheria toxin, pseudomonas toxin, anthrax toxin, Ribosome Inactivating Proteins (RIP) such as ricin and saporin, cucurbitacin toxin, abrin, gelonin, and pokeweed antiviral protein. Additional toxins include those disclosed in Mathew et al, (2009) Cancer Sci 100(8):1359-65, the entire disclosure of which is hereby incorporated by reference. In such embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the present technology can be used to induce cell death in a cell-type specific manner. In such embodiments, the toxin may be modified, e.g., mutated, to reduce the affinity and/or activity of the toxin in order to attenuate the effect, as described for other signaling agents herein.

Fc domains

Fragment crystallizable domains (Fc domains) are tail regions of antibodies that interact with Fc receptors located on the cell surface of cells involved in the immune system (e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells). In IgG, IgA and IgD antibody isotypes, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody. In IgM and IgE antibody isotypes, the Fc domain comprises three heavy chain constant domains (C) per polypeptide chain _HDomains 2-4).

In some embodiments, the Fc-based chimeric protein complexes of the present technology comprise an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG 4.

In some embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from human IgG1, IgG2, IgG3, or IgG 4.

In some embodiments, the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from IgG1, IgG2, IgG3, or IgG 4.

In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the one or more mutations of the Fc domain reduce or eliminate effector function of the Fc domain. In some embodiments, the mutant Fc domain has reduced affinity or binding to a target receptor. As an example, in some embodiments, the mutation of the Fc domain reduces or eliminates binding of the Fc domain to Fc γ R. In some embodiments, the fcyr is selected from fcyri; fc gamma RIIa, 131R/R; fc gamma RIIa, 131H/H, Fc gamma RIIb; and Fc γ RIII. In some embodiments, the mutation of the Fc domain reduces or eliminates binding to a complement protein (such as, for example, C1 q). In some embodiments, the mutation of the Fc domain reduces or eliminates binding to both Fc γ R and complement proteins (such as, for example, C1 q).

In some embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate effector function of the Fc domain. By way of example, in some embodiments, the LALA mutations include L234A and L235A substitutions in human IgG (e.g., IgG1) (where numbering is based on the common numbering of CH2 residues of human IgG1 according to the EU convention (PNAS, Edelman et al, 1969; 63(1) 78-85).

In some embodiments, the Fc domain of human IgG comprises a mutation at one or more of L234, L235, K322, D265, P329, and P331 to reduce or eliminate the effector function of the Fc domain. As an example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

In some embodiments, the Fc domain comprises a FALA mutation to reduce or eliminate effector function of the Fc domain. As an example, in some embodiments, the FALA mutations comprise F234A and L235A substitutions in human IgG 4.

In some embodiments, the Fc domain of human IgG4 comprises a mutation at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate the effector function of the Fc domain. As an example, in some embodiments, the mutation is selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

In some embodiments, the one or more mutations of the Fc domain stabilize a hinge region in the Fc domain. As an example, in some embodiments, the Fc domain comprises a mutation at S228 of a human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.

In some embodiments, the one or more mutations of the Fc domain promote chain pairing of the Fc domain. In some embodiments, chain pairing is facilitated by ion pairing (also known as charged pairs, ionic bonds, or pairs of charged residues).

In some embodiments, the Fc domain comprises mutations at one or more of the following amino acid residues of IgG to promote ion pairing: d356, E357, L368, K370, K392, D399 and K409.

As an example, in some embodiments, a human IgG Fc domain comprises one of the combinations of mutations in table 1 to facilitate ion pairing.

In some embodiments, chain pairing is facilitated via knob-into-hole mutations. In some embodiments, the Fc domain comprises one or more mutations to achieve knob-in-hole interactions in the Fc domain. In some embodiments, the first Fc chain is engineered to express a "knob" and the second Fc chain is engineered to express a complementary "hole". As an example, in some embodiments, a human IgG Fc domain comprises the mutations of table 2 to achieve knob-into-hole interactions.

In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology comprises any combination of the mutations disclosed above. As an example, in some embodiments, the Fc domain comprises a mutation that promotes ion pairing and/or knob-into-hole interactions. As an example, in some embodiments, the Fc domain comprises a mutation having one or more of the following properties: promote ion pairing, induce knob-into-hole interactions, reduce or eliminate effector function of the Fc domain, and cause Fc stabilization (e.g., at the hinge).

As an example, in some embodiments, a human IgG Fc domain comprises a mutation disclosed in table 3 that promotes ion pairing in the Fc domain and/or promotes knob-into-hole interactions.

As an example, in some embodiments, a human IgG Fc domain comprises a mutation disclosed in table 4 that promotes ion pairing of the Fc domain, promotes knob-into-hole interactions, or a combination thereof. In various embodiments, "chain 1" and "chain 2" of table 4 can be interchanged (e.g., chain 1 can have Y407T, and chain 2 can have T366Y).

As an example, in some embodiments, a human IgG Fc domain comprises a mutation disclosed in table 5 that reduces or eliminates Fc γ R and/or complement binding in the Fc domain. In various embodiments, there are mutations of table 5 in both strands.

In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a homodimer, i.e., the Fc domain in the chimeric protein complex comprises two identical protein fragments.

In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a heterodimer, i.e., the Fc domain comprises two distinct protein fragments.

In some embodiments, the heterodimeric Fc domain is engineered using ion pairing and/or knob-into-hole mutations described herein. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans-orientation/configuration. In trans-orientation/configuration, in various embodiments, the targeting moiety and signaling agent are not found on the same polypeptide chain of the Fc-based chimeric protein complex of the invention.

In trans orientation, in various embodiments, the targeting moiety and signaling agent are not found on the same polypeptide chain of the Fc-based chimeric protein complex of the invention. In the cis orientation, in various embodiments, the targeting moiety and signaling agent are found on separate polypeptide chains of the Fc-based chimeric protein complex. In the cis orientation, in various embodiments, the targeting moiety and the signaling agent are found on the same polypeptide chain of the Fc-based chimeric protein complex.

In some embodiments, where more than one targeting moiety is present in a heterodimeric protein complex described herein, one targeting moiety may be in a trans orientation (relative to the signaling agent) while another targeting moiety may be in a cis orientation (relative to the signaling agent). In some embodiments, the signaling agent and the target moiety are on the same end/side (N-terminus or C-terminus) of the Fc domain. In some embodiments, the signaling agent and targeting moiety are on different sides/ends (N-and C-termini) of the Fc domain.

In some embodiments, where more than one targeting moiety is present in a heterodimeric protein complex described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties are trans relative to each other as they are on different Fc chains). In some embodiments, where more than one targeting moiety is present on the same Fc chain, the targeting moieties may be on the same or different sides/ends (N-terminus or/and C-terminus) of the Fc chain.

In some embodiments, where more than one signaling agent is present in a heterodimeric protein complex described herein, the signaling agents can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the signaling agents are trans relative to each other as they are on different Fc chains). In some embodiments, where more than one signaling agent is present on the same Fc chain, the signaling agents may be on the same or different sides/ends (N-terminus or/and C-terminus) of the Fc chain.

In some embodiments, where more than one signaling agent is present in a heterodimeric protein complex described herein, one signaling agent may be in a trans orientation (e.g., with respect to the targeting moiety) while the other signaling agent may be in a cis orientation (e.g., with respect to the targeting moiety).

In some embodiments, the Fc domain comprises or begins with a core hinge region of wild-type human IgG1, the core hinge region comprising the sequence Cys-Pro-Cys. In some embodiments, the Fc domain further comprises an upper hinge or portion thereof (e.g., DKTHTCPPC; see WO 2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC; see Lo et al, Protein Engineering, Vol.11, No. 6, pp.495-500, 1998)).

Fc-based chimeric protein complexes

The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one Signaling Agent (SA) disclosed herein, and at least one Targeting Moiety (TM) disclosed herein.

The Fc-based chimeric protein complexes of the invention may encompass a complex of two fusion proteins, each comprising an Fc domain. In some embodiments, the Fc-based chimeric protein complex is homodimeric.

In some embodiments, the Fc-based chimeric protein complex is heterodimeric. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans-orientation/configuration. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation/configuration. In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise a signaling agent and a targeting moiety on a single polypeptide.

In some embodiments, the Fc-based chimeric protein has an improved half-life in vivo relative to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex. In some embodiments, the Fc-based chimeric protein has improved solubility, stability, and other pharmacological properties relative to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex.

The heterodimeric Fc-based chimeric protein complex is composed of two different polypeptides. In the embodiments described herein, the targeting domain is located on a different polypeptide than the signaling agent, and thus, the protein contains only one copy of the targeting domain, and only one copy of the signaling agent can be prepared (this provides a configuration in which potential interference with the desired property can be controlled). Furthermore, in various embodiments, only one targeting domain (e.g., VHH) may avoid cross-linking of antigens on the cell surface (which may, in some cases, trigger adverse effects). Furthermore, in various embodiments, a signaling agent may mitigate molecular "crowding" and potential interference with the restoration of effector function that is mediated depending on the avidity of the targeting domain. Furthermore, in various embodiments, the heterodimeric Fc-based chimeric protein complex can have two targeting moieties, and these targeting moieties can be placed on two different polypeptides. For example, in various embodiments, the C-termini of two targeting moieties (e.g., VHHs) may be masked to avoid potential autoantibodies or pre-existing antibodies (e.g., VHH autoantibodies or pre-existing antibodies). Furthermore, in various embodiments, for example, an Fc-based chimeric protein complex having a targeting domain on a different polypeptide than a heterodimer of a signaling agent (e.g., a wild-type signaling agent), may facilitate "cross-linking" of two cell types (e.g., tumor cells and immune cells). Furthermore, in various embodiments, the heterodimeric Fc-based chimeric protein complex may have two signaling agents, each on a different polypeptide to allow for more complex effector responses.

Furthermore, in various embodiments, for example, Fc-based chimeric protein complexes having targeting domains on different polypeptides rather than heterodimers of signaling agents, in a practical manner provides combinatorial diversity of targeting moieties and signaling agents. For example, in various embodiments, a polypeptide having any of the targeting moieties described herein can be combined "off-the-shelf" with a polypeptide having any of the signaling agents described herein to allow for rapid generation of various combinations of targeting moieties and signaling agents in a single Fc-based chimeric protein complex.

In some embodiments, the Fc-based chimeric protein complex comprises one or more linkers. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting an Fc domain, one or more signaling agents, and one or more targeting moieties. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each signaling agent and a targeting moiety (or connecting a signaling agent to one of the targeting moieties if more than one targeting moiety is present). In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each signaling agent to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting each targeting moiety to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting the targeting moiety to another targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects the signaling agent to another signaling agent.

In some embodiments, the Fc-based chimeric protein complex comprises two or more targeting moieties. In such embodiments, the targeting moieties may be the same targeting moiety or they may be different targeting moieties.

In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling agents. In such embodiments, the signaling agents may be the same targeting moiety or they may be different targeting moieties.

As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two Signaling Agents (SA), and at least two Targeting Moieties (TM), wherein the Fc domain, signaling agents, and targeting moieties are selected from any of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is homodimeric.

In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 5A-5F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 6A-6H. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 7A-7H. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 8A-8D. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 9A-9F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 10A-10J. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 11A-11D. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 12A-12F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 13A-13J. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 14A-14F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 15A-15L. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 16A-16L. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 17A-17F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 18A-18L. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of figures 19A-19L. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 20A-20J. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 21A-21J. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 22A-22F. In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 23A-23F.

In some embodiments, the signaling agent is attached to the targeting moiety and the targeting moiety is attached to the same end of the Fc domain (see fig. 5A-5F). In some embodiments, the Fc domain is homodimeric.

In some embodiments, the signaling agent and the targeting moiety are linked to an Fc domain, wherein the targeting moiety and the signaling agent are linked on the same terminus (see fig. 5A-5F). In some embodiments, the Fc domain is homodimeric.

In some embodiments, the targeting moiety is attached to a signaling agent and the signaling agent is attached to the same end of the Fc domain (see fig. 5A-5F). In some embodiments, the Fc domain is homodimeric.

In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties are linked to the Fc domain, and the signaling agents are linked to the same end of the targeting moieties (see fig. 6A-6H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, two targeting moieties are attached to the Fc domain and two targeting moieties are attached to the signaling agent that is attached to the same end of the Fc domain (see fig. 6A-6H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, and one targeting moiety in each pair is linked to the same end of the Fc domain, and the signaling agents are linked to the same end of the Fc domain (see fig. 6A-6H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, wherein one targeting moiety of each pair is linked to the signaling agent and the other targeting moiety of the pair is linked to the Fc domain, wherein the targeting moieties linked to the Fc domain are linked on the same terminus (see fig. 6A-6H). In some embodiments, the Fc domain is homodimeric.

In some embodiments, the homodimeric Fc-based chimeric protein complex has two or more signaling agents. In some embodiments, where there are four signaling agents and two targeting moieties, the two signaling agents are linked to each other, and one signaling agent of the pair is linked to the same end of the Fc domain, and the targeting moieties are linked to the same end of the Fc domain (see fig. 7A-7H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four signaling agents and two targeting moieties, two signaling agents are attached to the same end of the Fc domain, and two of the signaling agents are each attached to a targeting moiety, wherein the targeting moieties are attached to the same end of the Fc domain (see fig. 7A-7H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four signaling agents and two targeting moieties, the two signaling agents are linked to each other and one signaling agent of the pair is linked to the targeting moiety and the targeting moieties are linked to the same end of the Fc domain (see fig. 7A-7H). In some embodiments, the Fc domain is homodimeric.

As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, wherein the Fc domain comprises one or more ion-pairing mutations and/or one or more knob-into-hole mutations; at least one signaling agent; and at least one targeting moiety, wherein the ion-pairing motif and/or knob hole-in motif, signaling agent, and targeting moiety are selected from any of the ion-pairing group and/or knob hole-in motif, signaling agent, and targeting moiety disclosed herein. In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, the signaling agent is linked to the targeting moiety, which is linked to the Fc domain (see fig. 14A-14F and 17A-17F). In some embodiments, the targeting moiety is linked to the signaling agent, which is linked to the Fc domain (see fig. 14A-14F and 17A-17F).

In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, the signaling agent and the targeting moiety are linked to the Fc domain (see fig. 8A-8D, fig. 11A-11D, fig. 14A-14F, and fig. 17A-17F). In some embodiments, the targeting moiety and the signaling agent are attached to the same end of different Fc chains (see fig. 8A-8D and fig. 11A-11D). In some embodiments, the targeting moiety and the signaling agent are attached to different ends of different Fc chains (see fig. 8A-8D and fig. 11A-11D). In some embodiments, the targeting moiety and the signaling agent are linked to the same Fc chain (see fig. 14A-14F and fig. 17A-17F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the signaling agent is linked to the Fc domain, and the two targeting moieties may: 1) linked to each other, wherein one targeting moiety is linked to an Fc domain; or 2) each linked to an Fc domain (see fig. 9A-9F, fig. 12A-12F, fig. 15A-15L, fig. 18A-18L, fig. 20A-20J, and fig. 21A-21J). In some embodiments, the targeting moiety is linked to one Fc chain and the signaling agent is on the other Fc chain (see fig. 9A-9F and fig. 12A-12F). In some embodiments, the targeting moiety and the signaling agent in a pair are linked to the same Fc chain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, one targeting moiety is attached to the Fc domain, the other targeting moiety is attached to the signaling agent, and the pair of targeting moieties are attached to the Fc domain (see fig. 15A-15L, fig. 18A-18L, fig. 20A-20J, and fig. 21A-21J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to the same Fc chain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to different Fc chains (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are attached to the same terminus (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the targeting moieties are linked to the signaling agent, the signaling agent is linked to the Fc domain, and the unpaired targeting moieties are linked to the Fc domain (see fig. 15A-15L, fig. 18A-18L, fig. 20A-20J, and fig. 21A-21J). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked to the same Fc chain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked to different Fc chains (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the paired signaling agent and the unpaired targeting moiety are linked on the same end (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the targeting moieties are linked together and the signaling agent is linked to one of the targeting moieties in the pair, wherein the targeting moiety not linked to the signaling agent is linked to the Fc domain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the targeting moieties are linked together and the signaling agent is linked to one of the targeting moieties in the pair, wherein the signaling agent is linked to the Fc domain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the targeting moieties are both linked to the signaling agent, wherein one of the targeting moieties is linked to the Fc domain (see fig. 15A-15L and fig. 18A-18L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there is one signaling agent and two targeting moieties, the targeting moiety and the signaling agent are linked to the Fc domain (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the targeting moiety is attached to the terminus (see fig. 20A-20J and fig. 21A-21J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are attached to the same end of the Fc domain and the targeting moiety is attached to the Fc domain (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the signaling agent is linked to an Fc domain on the same Fc chain, and the targeting moiety is linked to another Fc chain (see fig. 22A-22F and fig. 23A-23F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agent is linked to the targeting moiety, which is linked to the Fc domain, and the other signaling agent is linked to the Fc domain (see fig. 10A-10J, fig. 13A-13J, fig. 16A-16L, and fig. 19A-19L). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the targeting moiety and the unpaired signaling agent are attached to the same end of different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the targeting moiety and the unpaired signaling agent are attached to different ends of different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the targeting moiety and the unpaired signaling agent are linked to the same Fc chain (see fig. 16A-16L and fig. 19A-19L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the targeting moiety is linked to the signaling agent, which is linked to the Fc domain, and the other signaling agent is linked to the Fc domain (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the paired signaling agent and the unpaired signaling agent are linked to different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the paired signaling agent and the unpaired signaling agent are attached to the same end of different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the paired signaling agent and the unpaired signaling agent are attached to different ends of different Fc chains (see fig. 10A-10J and fig. 13A-13J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and the targeting moiety is linked to one of the paired signaling agents, wherein the targeting moiety is linked to the Fc domain (see fig. 16A-16L and fig. 19A-19L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and one of the signaling agents is linked to the Fc domain and the targeting moiety is linked to the Fc domain (see fig. 16A-16L, fig. 19A-19L, fig. 22A-22F, and fig. 23A-23F). In some embodiments, the pair of signaling agent and targeting moiety are linked to the same Fc chain (see fig. 16A-16L and fig. 19A-19L). In some embodiments, the pair of signaling agent and targeting moiety are linked to different Fc chains (see fig. 22A-22F and fig. 23A-23F). In some embodiments, the pair of signaling agent and targeting moiety are attached to the same end of different Fc chains (see fig. 22A-22F and fig. 23A-23F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are both linked to the targeting moiety, wherein one of the signaling agents is linked to the Fc domain (see fig. 16A-16L and fig. 19A-19L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.

In some embodiments, where there are two signaling agents and one targeting moiety, the signaling agents are linked together and one of the signaling agents is linked to the targeting moiety and the other signaling agent is linked to the Fc domain (see fig. 16A-16L and fig. 19A-19L).

In some embodiments, where there are two signaling agents and one targeting moiety, each signaling agent is linked to an Fc domain, and the targeting moiety is linked to one of the signaling agents (see fig. 16A-16L and fig. 19A-19L). In some embodiments, the signaling agents are linked to the same Fc chain (see fig. 16A-16L and fig. 19A-19L).

In some embodiments, the targeting moiety or signaling agent is linked to a peptide comprising C_H2 and C_H3 and optionally an Fc domain of a hinge region. For example, vectors encoding targeting moieties, signaling agents, or combinations thereof linked as a single nucleotide sequence to an Fc domain can be used to prepare such polypeptides.

Multispecific chimeras and fusions with signaling agents

In some embodiments, the FAP-binding agents of the present technology are part of a chimera or fusion or Fc-based chimeric protein complex with one or more signaling agents as described herein and/or one or more additional targeting moieties. Accordingly, the present technology provides a chimeric or fusion protein or Fc-based chimeric protein complex comprising one or more signaling agents and a targeting moiety for FAP and/or one or more additional targeting moieties.

In some embodiments, the FAP-binding agents of the present technology are multispecific, i.e., the FAP-binding agents comprise two or more targeting moieties with recognition domains that recognize and bind to two or more targets (e.g., antigens or receptors or epitopes). In such embodiments, the FAP-binding agents of the present technology may comprise two or more targeting moieties having recognition domains that recognize and bind to two or more epitopes on the same antigen or on different antigens. In some embodiments, such multispecific FAP-binding agents exhibit a number of advantageous properties, such as increased affinity and/or increased selectivity. In one embodiment, the FAP binding agents of the present technology comprise two targeting moieties and are bispecific, i.e., bind to and recognize two epitopes on the same antigen or on different antigens.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise two or more targeting moieties, each targeting moiety being an antibody or antibody derivative as described herein. In one embodiment, the multispecific FAP-binding agents of the present technology comprise at least one VHH comprising an antigen recognition domain for FAP and one antibody or antibody derivative comprising an antigen recognition domain for a tumor antigen.

In some embodiments, the multispecific FAP-binding agents of the invention have two or more targeting moieties that target different antigens or receptors, and one targeting moiety may attenuate its antigen or receptor, e.g., the targeting moiety binds its antigen or receptor with low affinity or avidity (including, e.g., with lower affinity or avidity than the other targeting moiety has for its antigen or receptor, e.g., the difference between binding affinities may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold, or 5000-fold; e.g., a lower affinity or avidity targeting moiety may bind its antigen or receptor with a KD in the range of medium to high nM or low to medium pM, while a higher affinity or avidity targeting moiety may bind its antigen or receptor with a KD in the range of medium to high pM or low to medium nM). For example, in some embodiments, multispecific FAP-binding agents of the invention comprise an attenuated targeting moiety directed against a confounding antigen or receptor, such that targeting of target cells (e.g., via other targeting moieties) can be improved and the effects of multiple types of cells, including those not targeted by therapy (e.g., by binding to the confounding antigen or receptor with higher affinity than provided in these embodiments) can be prevented.

Multispecific FAP-binding agents of the present technology can be constructed using methods known in the art, see, e.g., U.S. patent No. 9,067,991, U.S. patent publication No. 20110262348, and WO 2004/041862, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, multispecific FAP-binding agents comprising two or more targeting moieties of the present technology can be constructed by chemical crosslinking, e.g., by reacting amino acid residues with an organic derivatizing agent as described by Blattler et al, Biochemistry 24,1517-1524 and EP294703, the entire contents of which are hereby incorporated by reference. In another illustrative embodiment, a multispecific FAP-binding agent comprising two or more targeting moieties is constructed by genetic fusion, i.e., construction of a single polypeptide comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct may be formed that encodes a first VHH having an antigen recognition domain for FAP and a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen. One method for generating bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO96/34103, the entire content of which is hereby incorporated by reference. In another illustrative embodiment, multispecific FAP-binding agents of the present technology can be constructed by using linkers. For example, the carboxy terminus of a first VHH having an antigen recognition domain for FAP may be linked to the amino terminus of a second antibody or antibody derivative having an antigen recognition domain for a tumor antigen (or vice versa). Illustrative linkers that may be used are described herein. In some embodiments, the components of the multispecific FAP-binding agents of the present technology are directly linked to each other without the use of a linker.

In some embodiments, the multispecific FAP-binding agents of the present technology recognize and bind to FAP and one or more antigens found on one or more immune cells, which may include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, T helper cells, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or a subset thereof. In some embodiments, the FAP-binding agent specifically binds to an antigen of interest and is effective to recruit, directly or indirectly, one or more immune cells.

In some embodiments, the multispecific FAP-binding agents of the present technology recognize and bind to FAP and one or more antigens found on tumor cells. In these embodiments, FAP-binding agents of the invention can recruit immune cells directly or indirectly to tumor cells or the tumor microenvironment. In some embodiments, FAP-binding agents of the invention may recruit, directly or indirectly, immune cells, e.g., immune cells (e.g., CTLs) that can kill and/or suppress tumor cells, to a site of action (such as a tumor microenvironment, as a non-limiting example).

In some embodiments, the FAP-binding agents of the invention are capable of or useful in methods involving altering immune cell balance in favor of immune attack by a tumor. For example, FAP-binding agents of the invention can alter immune cell ratios at clinically significant sites in favor of cells that can kill and/or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, T helper cells, Natural Killer (NK) cells, natural killer T (nkt) cells, anti-tumor macrophages (e.g., M1 macrophages), neutrophils, B cells, dendritic cells, or subsets thereof, and against cells that protect tumors (e.g., bone marrow-derived suppressor cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof).

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety having an antigen recognition domain that specifically binds to an antigen associated with a tumor cell. In some embodiments, the targeting moiety recruits tumor cells directly or indirectly. For example, in some embodiments, tumor cells are recruited to one or more effector cells (e.g., immune cells described herein) that can kill and/or suppress the tumor cells. In some embodiments, the targeting moiety recruits T cells to tumor cells, e.g., directly or indirectly, through two targeting moieties that interact with their respective antigens on tumor and FAP positive immune cells (e.g., dendritic cells).

Tumor cells or cancer cells refer to uncontrolled growth of cells or tissues and/or abnormal increase in cell survival time and/or inhibition of apoptosis that interferes with the normal function of body organs and systems. Tumor cells include, for example, benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. Illustrative tumor cells include, but are not limited to, the following: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; liver cancer; hepatoma; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphomas (NHLs), Small Lymphocytic (SL) NHLs, intermediate grade/follicular NHLs, intermediate grade diffuse NHLs, high grade immunoblastic NHLs, high grade lymphoblastic NHLs, high grade small non-nuclear blastoid NHLs, large lumpy NHLs (bulk disease NHLs), mantle cell lymphomas, AIDS-related lymphomas, and Waldenstrom's Macroglobulinemia, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), and abnormal vascular dysplasias associated with nevus and macular tumors (e.g. edema, edema associated with brain tumors) and Meigs' syndrome.

Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes, including, for example, adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas, sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myelogenous, acute lymphoblastic, chronic myelogenous, chronic lymphocytic, and hairy cells), lymphomas and myelomas (including, for example, hodgkin's and non-hodgkin's lymphomas, light chain, non-secretory, MGUS, and plasmacytomas), and central nervous system cancers (including, for example, brain (e.g., gliomas (e.g., astrocytomas, oligogliomas, and ependymomas), meningiomas, pituitary adenomas, and neuromas, and spinal cord tumors (e.g., meningiomas and fibroneuromas).

Illustrative tumor antigens include, but are not limited to, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD 3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A2, Al-Al 2, MAGE-A5, MAGE-A7, MAGE-A5, MAGE-A3, MAGE-2, MAGE-A-B, MAGE-A-B-E3, and C-B-E, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), GAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, a-fetoglobulin, E-cadherin, a-linked protein, 3-linked-protein, p120ctn, PR111100, polyp-7, gp7, APC-linked protein, Pmac 27, and sarcoidosis, The cell lining proteins, connexin 37, Ig idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family tumor antigens, Imp-1, NA, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA and TNFRSF 17. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of these tumor antigens.

In some embodiments, the multispecific FAP-binding agents of the present invention recognize and bind to FAP and antigens on tumor cells. In some embodiments, the multispecific FAP-binding agent recruits CTLs, directly or indirectly, to the tumor cell or tumor microenvironment.

In some embodiments, the multispecific FAP-binding agents of the present invention have targeting moieties that target two different cells (e.g., to form a synapse) or the same cell (e.g., to obtain a more concentrated signaling agent effect).

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a T cell. In some embodiments, the targeting moiety recruits T cells directly or indirectly. In one embodiment, the antigen recognition domain specifically binds to effector T cells. In some embodiments, the antigen recognition domain recruits effector T cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location of a cell having one or more disease cells or cells modulated for therapeutic effect). Illustrative effector T cells include cytotoxic T cells (e.g., α β TCR, CD3+, CD8+, CD45R0 +); CD4+ effector T cells (e.g., α β TCR, CD3+, CD4+, CCR7+, CD62L high, IL-7R/CD127 +); CD8+ effector T cells (e.g., α β TCR, CD3+, CD8+, CCR7+, CD62L high, IL-7R/CD127 +); effector memory T cells (e.g., CD62L low, CD44+, TCR, CD3+, IL-7R/CD127+, IL-15R +, CCR7 low); central memory T cells (e.g., CCR7+, CD62L +, CD27 +; or CCR7 high, CD44+, CD62L high, TCR, CD3+, IL-7R/CD127+, IL-15R +); CD62L + effector T cells; CD8+ effector memory T cells (TEM), including early effector memory T cells (CD27+ CD62L-) and late effector memory T cells (CD27-CD62L-) (TemE and TemL, respectively); CD127(+) CD25 (low /) effector T cells; CD127(-) CD25(-) effector T cells; CD8+ stem cell memory effector cells (TSCMs) (e.g., CD44 (low) CD62L (high) CD122 (high) sca (+); TH1 effector T cells (e.g., CXCR3+, CXCR6+ and CCR5 +; or α β TCR, CD3+, CD4+, IL-12R +, IFN γ R +, CXCR3+), TH2 effector T cells (e.g., CCR3 ', CCR 4' and CCR8 +; or α β TCR, CD3+, CD4+, IL-4R +, IL-33R +, CCR4+, IL-17RB +, CRTH2 +); TH9 effector T cells (e.g., α β TCR, CD3+, CD4 +); TH17 effector T cells (e.g.. alpha. beta.TCR, CD3+, CD4+, IL-23R +, CCR6+, IL-1R +); CD4+ CD45RO + CCR7+ effector T cells, ICOS + effector T cells; CD4+ CD45RO + CCR7(-) effector T cells; and IL-2, IL-4 and/or IFN-gamma secreting effector T cells.

Illustrative T cell antigens of interest include, for example (and where appropriate extracellular domains): CD8, CD3, SLAMF4, IL-2R alpha, 4-1BB/TNFRSF9, IL-2R beta, ALCAM, B7-1, IL-4R, B7-H3, BLAME/SLAMF, CEACAM1, IL-6R, CCR3, IL-7R alpha, CCR4, CXCRI/IL-S RA, CCR5, CCR6, IL-10R alpha, CCR 7, IL-10R beta, CCRS, IL-12R beta 1, CCR9, IL-12R beta 2, CD9, IL-13R alpha 1, IL-13, CD9, INTEGRILT 9/9, I CDS 9/CDS 5 9, ILT 9/CDS 5 9, Tellin (Tellin) a 4/CD lugrn) a, IL-2R alpha, CDS 72/CDS 72, CD 9/CDS 9, CD 9/LT 11R beta, CDS 11/11, CDS 11/S11, CDS3, CDS 9, CDS3, CDS 9, CDS3, CDS 9, CDS3, CDS 9, CDS3, CDS 9, CDS3, and CDS3, and CDS3, CDS3, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSF, KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R4, CDS4/SLAMF 4, NCAM-L4, CD4, NKG2 4, CD229/SLAMF 4, NKG2 4, CD2 4-10/SLAMF 4, NT-4, CD4, NTB-A/SLAMF 4, common gamma chain/IL-2R gamma, Fasthrostatin, FastCC/4, CRACAM 1-CX-1, CTCR 3, CTCR 1/TCAM 3, CTCR 4, CTLA-TCAM-4, CTLA-11, CTLA-TCAM 3, CTCR 4, CTLA-TCAM-3, CTCR 1-4, CTLA-11, CTLA-TCCR 3, CTLA-4, CTLA-TCCR 3, CTMA-4, CTLA-4, CTMA-TCCR 3-4, CTMA-TCMA-4, CTMA-11-4, CTMA-X-11-4, CTMA-11-36X-11, CTMA-4, CTMA-TCCR 3-11-4, CTMA-3-11, CTMA-TCCR 3, CTMA-X-4, CTMA-3, CTMA-4, CTMA-X-4, CTMA-3, CTMA-4, CTMA-X-4, CTMA-X-4, CTMA-X-36X-X-3-X-4, CTMA-X, Fas ligand/TNFSF 6, TIM-4, Fcy RIII/CD16, TIM-6, TNFR1/TNFRSF1A, granulysin, TNF RIII/TNFRSF1B, TRAI L RI/TNFRFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAILR3/TNFRSF10C, IFN-. gamma.R 1, TRAILR4/TNFRSF10D, IFN-. gamma.R 2, TSLP, IL-1R1 and TSLP. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of these illustrative T cell antigens.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety to CD8, which is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementarity determining regions" or CDRs. As used herein, "framework region" or "FR" refers to the region of a variable domain that is located between CDRs. As used herein, "complementarity determining region" or "CDR" refers to the variable region of a VHH that contains an amino acid sequence capable of specifically binding to an antigenic target.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a VHH directed to CD8 that has a variable domain comprising at least one CD8 CDR1, CD8 CDR2, and/or CD8 CDR3 sequence.

In some embodiments, the CD8 CDR1 sequence is selected from SEQ ID NO:192 or SEQ ID NO: 193.

In some embodiments, the CD8 CDR2 sequence is selected from SEQ ID NO:194 or SEQ ID NO: 195.

In some embodiments, the CD8 CDR3 sequence is selected from SEQ ID NO:196 or SEQ ID NO:197 or SEQ ID NO: 198.

In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the group consisting of seq id nos: r3HCD27(SEQ ID NO: 199); or R3HCD129(SEQ ID NO: 200); or R2HCD26(SEQ ID NO: 201).

In some embodiments, the CD8 targeting moiety comprises a VHH having a variable domain comprising at least one CD8 CDR1, CD8 CDR2 and/or CD8 CDR3 sequence as described below.

In some embodiments, the CD8 CDR1 sequence is selected from SEQ ID NO:202 to SEQ ID NO: 270.

In some embodiments, the CD8 CDR2 sequence is selected from SEQ ID NOs 271 to 339.

In some embodiments, the CD8 CDR3 sequence is selected from SEQ ID NO:340 to SEQ ID NO: 408.

In some embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from the group consisting of seq id nos: 1CDA 7(SEQ ID NO: 409); or 1CDA 12(SEQ ID NO: 410); or 1CDA 14(SEQ ID NO: 411); or 1CDA 15(SEQ ID NO: 412); or 1CDA 17(SEQ ID NO: 413); or 1CDA 18(SEQ ID NO: 414); or 1CDA 19(SEQ ID NO: 415); or 1CDA 24(SEQ ID NO: 416); or 1CDA 26(SEQ ID NO: 417); or 1CDA 28(SEQ ID NO: 418); or 1CDA 37(SEQ ID NO: 419); or 1CDA 43(SEQ ID NO: 420); or 1CDA 45(SEQ ID NO: 421); or 1CDA 47(SEQ ID NO: 422); or 1CDA 48(SEQ ID NO: 423); or 1CDA 58(SEQ ID NO: 424); or 1CDA 65(SEQ ID NO: 425); or 1CDA 68(SEQ ID NO: 426); or 1CDA 73(SEQ ID NO: 427); or 1CDA 75(SEQ ID NO: 428); or 1CDA 86(SEQ ID NO: 429); or 1CDA 87(SEQ ID NO: 430); or 1CDA 88(SEQ ID NO: 431); or 1CDA 89(SEQ ID NO: 432); or 1CDA 92(SEQ ID NO: 433); or 1CDA 93(SEQ ID NO: 434); or 2CDA 1(SEQ ID NO: 435); or 2CDA 5(SEQ ID NO: 436); or 2CDA 22(SEQ ID NO: 437); or 2CDA 28(SEQ ID NO: 438); or 2CDA 62(SEQ ID NO: 439); or 2CDA 68(SEQ ID NO: 440); or 2CDA 73(SEQ ID NO: 441); or 2CDA 74(SEQ ID NO: 442); or 2CDA 75(SEQ ID NO: 443); or 2CDA 77(SEQ ID NO: 444); or 2CDA 81(SEQ ID NO: 445); or 2CDA 87(SEQ ID NO: 446); or 2CDA 88(SEQ ID NO: 447); or 2CDA 89(SEQ ID NO: 448); or 2CDA 91(SEQ ID NO: 449); or 2CDA 92(SEQ ID NO: 450); or 2CDA 93(SEQ ID NO: 451); or 2CDA 94(SEQ ID NO: 452); or 2CDA 95(SEQ ID NO: 453); or 3CDA 3(SEQ ID NO: 454); or 3CDA 8(SEQ ID NO: 455); or 3CDA 11(SEQ ID NO: 456); or 3CDA 18(SEQ ID NO: 457); or 3CDA 19(SEQ ID NO: 458); or 3CDA 21(SEQ ID NO: 459); or 3CDA 24(SEQ ID NO: 460); or 3CDA 28(SEQ ID NO: 461); or 3CDA 29(SEQ ID NO: 462); or 3CDA 31(SEQ ID NO: 463); or 3CDA 32(SEQ ID NO: 464); or 3CDA 33(SEQ ID NO: 465); or 3CDA 37(SEQ ID NO: 466); or 3CDA 40(SEQ ID NO: 467); or 3CDA 41(SEQ ID NO: 468); or 3CDA 48(SEQ ID NO: 469); or 3CDA 57(SEQ ID NO: 470); or 3CDA 65(SEQ ID NO: 471); or 3CDA 70(SEQ ID NO: 472); or 3CDA 73(SEQ ID NO: 473); or 3CDA 83(SEQ ID NO: 474); or 3CDA 86(SEQ ID NO: 475); or 3CDA 88(SEQ ID NO: 476); or 3CDA 90(SEQ ID NO: 477).

In various illustrative embodiments, the CD8 targeting moiety comprises an amino acid sequence selected from any of the above sequences without a terminal histidine tag sequence (i.e., HHHHHHHH; SEQ ID NO: 43).

In some embodiments, the CD8 targeting moiety comprises an amino acid sequence described in U.S. patent publication No. 2014/0271462, which is incorporated by reference in its entirety. In some embodiments, the CD8 targeting moiety comprises an amino acid sequence described in table 0.1, table 0.2, table 0.3, and/or fig. 1A-121 of U.S. patent publication No. 2014/0271462, which is incorporated by reference in its entirety. In some embodiments, the CD8 targeting moiety comprises HCDR1 of SEQ ID NO:478 or 479 and/or HCDR2 of SEQ ID NO:478 or 479 and/or HCDR3 of SEQ ID NO:478 or 479 and/or LCDR1 of SEQ ID NO:480 and/or LCDR2 of SEQ ID NO:480 and/or LCDR3 of SEQ ID NO: 480.

In some embodiments, the present technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the targeting moiety to CD8 as described herein. In some embodiments, the amino acid sequence of the targeting moiety directed to CD8 further comprises an amino acid analog, amino acid derivative, or other non-canonical amino acid.

In some embodiments, the targeting moiety recognizes and/or binds FMS-like tyrosine kinase 3(Flt3) or is a natural ligand for Flt3, such as FMS-like tyrosine kinase 3 ligand (Flt3L) or a truncated region thereof (e.g., which is capable of binding Flt 3). In some embodiments, the targeting moiety is the extracellular domain of Flt 3L. In some embodiments, the targeting moiety comprises a Flt3L domain, wherein the Flt3L domain is a single chain dimer, optionally wherein one Flt3L domain is linked to another Flt3L domain via one or more linkers, wherein the linkers are flexible linkers.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a macrophage/monocyte. In some embodiments, the targeting moiety recruits macrophages/monocytes directly or indirectly, e.g., to a treatment site (e.g., a site with one or more disease cells or cells modulated for therapeutic effect). By way of example and not limitation, in some embodiments, the macrophage/monocyte antigen includes, for example, SIRP1a, B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common 3-chain, integrin a 4/CD49d, BLAME/SLAMF8, integrin a X/CDllc, CCL a X/C a X, integrin 32/CD a X, CD155/PVR, integrin 33/CD a X, CD a X/PECAM-1, latex, CD a X/SR-B a X, leukotriene B a X R a X, CD a X/TNFRSF a X, a X-B a X, CD a X, LMIR a X/CD a X, CD a X/CD a X, CRA 3/CD a X, CRA AMF a X/CD a X, CRA 3/CD a X, CRA a X/CD a X, CD a X/CD a X, CRA-CD a X, CD a X/CD a X, LMIR a X, CRA-CD a X, CD a X/CD a X, CRA-CD a X, CD a X/CD a X, CRA-CD a X, CD a X/CD a X, CRA-L a X, CRA-CD a X, CRA-L a X, CRA-CD a X, CRA-C a X, CRA-L a X, CRA-L a X, and CD a X, CRA-C a X, CRA-3/CD a X, CRA-C a X, CRA-a X, and CD a X, CRA-C a X, CRA-3/CD a X, CRA-L a X, and CD a X, MD-2, EMMPRIN/CD147, MGL2, endothelin/CD 105, osteoactivin/GPNMB, Fc-y RI/CD64, osteomodulin, Fc-y RIIB/CD32B, PD-L2, Fc-y RIIC/CD32c, Siglec-3/CD33, Fc-y RIIA/CD32a, SIGNR1/CD209, Fc-y RIII/CD16, SLAM, GM-CSF Ra, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-yRI, TLR4, IFN-gamma R2, TREM-I, IL-I RII, TREM-2, ILT2/CD 4685, TREM-3, ILT 48 46/CD 85, TREM 1/TLT-1, 2B4/SLAMF 4, IL 10-ALC 24, PEPT R p, NAL R p/CD R p, ANAM 598, NAL/CD 573 9, NAIL-5985, CAMM-CD 102, TREM-3, LAM-Y-D-3, LAM-D-8, LID-D-III, Common 3 chain, ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85 5, CCR5, CD206, integrin a 4/CD49 5, CCR5, integrin 5/CDII B, CCR5, integrin 5/CDllc, CD155/PVR, integrin 32/CD 5, integrin 133/CD 5, CD 5/SR-B5, LAIR 5, CD5, leukotriene B5-R5, CD 5-B5, CD 5/SLAMF, LMIR5/CD300, LMIR5/CD 5, LMIR 36163, LMIR5/CD300, blood coagulation factor III/CSF 300, tissue factor III/CSF 300, CLCX 1/CX 72, EMR 5/CD 5, LMIR5/CD 5, LMIR 5/CSF 1/CSR 5, LMIR5/CD 5, LMIR5, CD 5/CSF 1/CSR 5, LMIR5, CD5, LMIR5/CD 5, LMIR5, CD5, LMIR 5/CSF 1-CR 147, MCR 5, CD5, MCCR 1-CR 1-CXCR 1, CD5, LMIR5, CD5, MCCR 1/CXCR 1-CR 1-CX 5, CD5, LMIR5, CD5, MCR 24, LMIR5, CD5, LMIR5, CD5, LMIR5, MCR 24, LMIR5, MCR 24, LMIR5, LMIR 36, Fc-y RI/CD64, PSGL-1, Fc-y RIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Ra, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-I, IL-6R, TREM-2, CXCRI/IL-8RA, TREM-3, and TREMLITTLT-1. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of the macrophage/monocyte antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a dendritic cell. In some embodiments, the targeting moiety recruits dendritic cells directly or indirectly, e.g., to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). By way of example and not limitation, in some embodiments, the Dendritic Cell (DC) antigen includes, for example, FAP, XCR1, RANK, CD36/SRB3, LOX-1/SR-EI, CD68, MARCO, CD163, SR-A68/MSR, CD5 68, SREC-1, CL-PI/C0LEC 68, SREC-II, LIMPIIISRB 68, RP105, TLR 68, 4-IBB ligand/TNFSF 68, IL-12/IL-23p 68, 4-amino-1, 8-naphthamide, ILT 68/CD 85 68, CCL 68/6 Ckine, ILT 68/CD 85, 8-oxo-dG, CD 72/CD 3685, CD 6D, LARG 68/CD 72, LARG 68/CAAGRON 68, LARG 68/GLC 68, LARG 68/CARG 68, LARG 68/GEN 68, LARG 68/CARG 68, LARG 68, and LARG 68, Leukotriene B4 RI, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/SIGlec-4-a, CD43, MCAM, CD45, MD-1, CD68, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAMLI, CD2F-10/SLAMF9, bone activating elements GPNMB, Chern 23, PD-L9, CLEC-1, CLEC 105, CLEC-2, CLEC-H3, CLEC 2-DEC-72, SLAMC-GCC-36205, CRA-11, SLAMC-3695, SLECC-3/SIGC-36205, CRA-3, SLECC-36205, SLECC-3, SLECC-3695, CRA-TCC-3, SLECC-36205, CD 3653, CD 3695, CD 3660, CD 3695, CD-TCEC-3, CD 36205, CD 3653, CD 3660, CD8, CD 3660, CDC 205, CD-TCEC, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc-y R1/CD64, TLR3, Fc-y RIIB/CD32b, TREM-1, Fc-y RIIC/CD32c, TREM-2, Fc-y RIIA/CD32a, TREM-3, Fc-y RIII/CD16, TREML1/TLT-1, ICAM-2/CD102, DEC205, and capsaicin R1. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of the DC antigens disclosed above.

In some embodiments, a chimeric protein or Fc-based chimeric protein complex of the invention comprises a targeting moiety comprising an amino acid sequence having at least 60% identity to any one of the sequences disclosed herein. For example, in some embodiments, the chimeric protein or Fc-based chimeric protein complex comprises a targeting moiety comprising a peptide having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, or a combination thereof, with any one of the sequences disclosed herein, At least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity (e.g., with about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98% >, to any of the sequences disclosed herein, About 99% or about 100% sequence identity).

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, a megakaryocyte, platelet, erythrocyte, mast cell, basophil, neutrophil, and eosinophil. In some embodiments, the antigen recognition domain recruits, directly or indirectly, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, and eosinophils, e.g., to a treatment site (e.g., a site of a cell having one or more disease cells or being modulated for therapeutic effect).

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and/or platelets. By way of example and not by way of limitation, in some embodiments, the megakaryocyte and/or platelet cell antigens include, for example, GP11b/111a, GP1b, vWF, PF4, and TSP. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of the megakaryocyte and/or platelet antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with red blood cells. By way of example, and not by way of limitation, in some embodiments, the red blood cell antigens include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein 11b/111a), CD41b (GPllb), CD71 (transferrin receptor), CD105, glycophorin a, glycophorin C, c-kit, HLA-DR, H2(MHC-I1), and rhesus antigens. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the red blood cell antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a mast cell. By way of example and not by way of limitation, in some embodiments, the mast cell antigen includes, for example, SCFR/CD117, Fca, CD2, CD25, CD35, CD88, CD203C, C5R1, CMAI, FCERIA, FCER2, TPSABI. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the mast cell antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with a basophil. By way of example and not by way of limitation, in some embodiments, the basophil antigen includes, for example, Fca, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the basophil antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety having an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with neutrophils. By way of example, and not by way of limitation, in some embodiments, the neutrophil antigen includes, for example, 7D5, CD 10/cala, CD13, CD16(FcRlll), CD18 protein (LFA-1, CR3 and p150, 95), CD45, CD67, and CD 177. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the above-disclosed neutrophil antigens.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a target (e.g., antigen, receptor) associated with an eosinophil. By way of example, and not by way of limitation, in some embodiments, the eosinophil antigen comprises, for example, CD35, CD44, and CD 69. In some embodiments, the FAP-binding agent comprises a targeting moiety that binds one or more of the eosinophil antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to any suitable antigen or receptor or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. By way of example and not by way of limitation, in some embodiments, the tissue-specific markers include, but are not limited to, endothelial cell surface markers (such as, for example, ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELE, SELP, TEK, THBD, VCAMI, VWF); smooth muscle cell surface markers (such as, for example, ACTA2, MYHIO, MYHI 1, MYH9, MYOCD); fibroblast (matrix) cell surface markers (such as, for example, ALCAM, CD34, COLIAI, COL1a2, COL3a1, FAP, PH-4); epithelial cell surface markers (such as, for example, CDID, K61RS2, KRTIO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCI, tactdi); neovascular markers (such as, for example, CD13, TFNA, α -v β -3(av33), E-selectin); and adipocyte surface markers (such as, for example, ADIPOQ, FABP4, and RETN). In some embodiments, the FAP-binding agent comprises a targeting moiety that binds to one or more of the antigens disclosed above.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to a checkpoint marker expressed on a T cell. In some embodiments, the checkpoint marker is one or more checkpoint markers selected from PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD4OL, TIM3, and A2 aR.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety having an antigen recognition domain that specifically binds to a checkpoint marker. In some embodiments, the checkpoint marker is one or more checkpoint markers selected from PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86, ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX40/OX4OL, CD27, CD4OL, TIM3/Ga19, and A2 aR.

By way of example, and not by way of limitation, in some embodiments, the multispecific FAP-binding agents of the present invention comprise (i) a targeting moiety for CD 8; (ii) targeting moieties directed to checkpoint markers expressed on T cells, such as one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, 0X40, CD27, CD4OL, TIM3, and A2 aR; and/or (iii) a targeting moiety directed against a tumor cell, and any of the modified (e.g., mutated) signaling agents described herein.

In some embodiments, the multispecific FAP-binding agents of the invention have one or more targeting moieties for PD-1. In some embodiments, the FAP-binding agent has one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the FAP-binding agent comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a VHH for PD-1 that has a variable domain comprising at least one PD-1CDR1, PD-1CDR2, and/or PD-1CDR3 sequence.

In some embodiments, the PD-1CDR1 sequence is selected from SEQ ID NO:481 through SEQ ID NO: 494.

In some embodiments, the PD-1CDR2 sequence is selected from SEQ ID NO:495 to SEQ ID NO: 508.

In some embodiments, the PD-1CDR3 sequence is selected from SEQ ID NO 509 to SEQ ID NO 521.

In various illustrative embodiments, the PD-1 targeting moiety comprises an amino acid sequence selected from the group consisting of seq id nos: 2PD 23: (SEQ ID NO: 522); or 2PD 26: (SEQ ID NO: 523); or 2PD 90: (SEQ ID NO: 524); or 2 PD-106: (SEQ ID NO: 525); or 2 PD-16: (SEQ ID NO: 526); or 2PD 71: (SEQ ID NO: 527); or 2 PD-152: (SEQ ID NO: 528); or 2 PD-12: (SEQ ID NO: 529); or 3PD 55: (SEQ ID NO: 530); or 3PD 82: (SEQ ID NO: 531); or 2PD 8: (SEQ ID NO: 532); or 2PD 27: (SEQ ID NO: 533); or 2PD 82: (SEQ ID NO: 534); or

3PD36：(SEQ ID NO:535)。

In various illustrative embodiments, the PD-1 targeting moiety comprises an amino acid sequence selected from any of the above without a terminal histidine tag sequence (i.e., without HHHHHHHH; SEQ ID NO: 43).

In some embodiments, the targeting moiety comprises an anti-PD-1 antibody, pelrolizumab (aka MK-3475, Myxobolus, and/or Myxobolus, and optionally a pharmaceutically acceptable carrier,) Or a fragment thereof. In some embodiments, the targeting moiety is one or more of pentolizumab and other humanized anti-PD-1 antibodies, which are disclosed in Hamid et al (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and WO 2009/114335, the entire disclosures of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, the pentalizumab or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence (SEQ ID NO:536) and/or a light chain comprising an amino acid sequence (SEQ ID NO: 537).

In some embodiments, the targeting moiety comprises the anti-PD-1 antibody nivolumab (nivolumab) (in turn)BMS-936558, MDX-1106, ONO-4538, and,) Or a fragment thereof. In some embodiments, the targeting moiety is one or more of nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1, which are disclosed in US 8,008,449 and WO 2006/121168, the entire disclosures of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, nivolumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence (SEQ ID NO:538) and/or a light chain comprising an amino acid sequence (SEQ ID NO: 539).

In some embodiments, the targeting moiety comprises an anti-PD-1 antibody pidilizumab (pidilizumab) (also known as CT-011, hBAT, or hBAT-1), or a fragment thereof. In some embodiments, the dermadilizumab and the other humanized anti-PD-I monoclonal antibody are selected from the group consisting of dermadilizumab and other humanized anti-PD-I monoclonal antibodies disclosed in: US 2008/0025980 and WO 2009/101611, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises one or more light chain variable regions comprising an amino acid sequence selected from the group consisting of the following sequences disclosed in US 2008/0025980: (SEQ ID NO: 540); (SEQ ID NO: 541); (SEQ ID NO: 542); and (SEQ ID NO: 543); and/or a heavy chain comprising an amino acid sequence selected from the group consisting of the following sequences disclosed in US 2008/0025980: (SEQ ID NO: 544); (SEQ ID NO: 545); (SEQ ID NO: 546); (SEQ ID NO: 547); and (SEQ ID NO: 548).

In some embodiments, the targeting moiety comprises a light chain comprising (SEQ ID NO:549) and a heavy chain comprising (SEQ ID NO: 550).

In some embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

In some embodiments, the targeting moiety comprises PD-L2-Fc fusion protein AMP-224 or a fragment thereof, which is disclosed in WO 2010/027827 and WO 2011/066342, the entire disclosures of which are hereby incorporated by reference. In some embodiments, the targeting moiety comprises (SEQ ID NO:551) and/or B7-DC fusion protein containing (SEQ ID NO: 552).

In some embodiments, the targeting moiety comprises the peptide AUNP 12 or any other peptide disclosed in US 2011/0318373 or US 8,907,053. By way of example and not by way of limitation, in some embodiments, the targeting moiety comprises the following AUNP 12 sequence:

SNTSESFK(SNTSESF)FRVTQLAPKAQIKE—NH₂(SEQ ID NO:553)

(i.e. compound 8 of US 2011/0318373).

In some embodiments, the targeting moiety comprises the anti-PD-1 antibody 1E3 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:554) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 555).

In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:556) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 557).

In some embodiments, the targeting moiety comprises the anti-PD-1 antibody 1H3 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO: 558);

and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 559).

In some embodiments, the targeting moiety comprises a VHH for PD-1 disclosed in US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, the VHH for PD-1 comprises one or more of the following sequences disclosed in US 8,907,065: (SEQ ID NO: 560); (SEQ ID NO: 561); (SEQ ID NO: 562); (SEQ ID NO: 563); or (SEQ ID NO: 564).

In some embodiments, the targeting moiety comprises any of the anti-PD-1 antibodies or fragments thereof disclosed in US 2011/0271358 and WO 2010/036959, the entire contents of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 565); (SEQ ID NO: 566); (SEQ ID NO: 567); (SEQ ID NO: 568); or (SEQ ID NO: 569); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 570); (SEQ ID NO: 571); (SEQ ID NO: 572); or (SEQ ID NO: 573).

In some embodiments, the multispecific FAP-binding agents of the invention comprise one or more antibodies to PD-1 or antibody fragments thereof selected from TSR-042(Tesaro, Inc.), regen 2810(Regeneron Pharmaceuticals, Inc.), PDR001(Novartis Pharmaceuticals) and BGB-a317(BeiG ene Ltd.).

In some embodiments, the multispecific FAP-binding agents of the invention have one or more targeting moieties to PD-L1. In some embodiments, the FAP-binding agent has one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the FAP-binding agent comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to PD-L1 polypeptide.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a VHH directed to PD-L1 that has a variable domain comprising at least one PD-L1CDR 1, PD-L1CDR2, and/or PD-L1CDR 3 sequence.

In some embodiments, the PD-L1CDR 1 sequence is selected from SEQ ID NO:574 to SEQ ID NO: 604.

In some embodiments, the PD-L1CDR2 sequence is selected from SEQ ID NO 605 to SEQ ID NO 635.

In some embodiments, the PD-L1CDR 3 sequence is selected from SEQ ID NO 636 to SEQ ID NO 666.

In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from the group consisting of seq id nos: 2LIG 2: (SEQ ID NO: 667); or 2LIG 3: (SEQ ID NO: 668); or 2LIG 16: (SEQ ID NO: 669); or 2LIG 22: (SEQ ID NO: 670); or 2LIG 27: (SEQ ID NO: 671); or 2LIG 29: (SEQ ID NO: 672); or 2LIG 30: (SEQ ID NO: 673); or 2LIG 34: (SEQ ID NO: 674); or 2LIG 35: (SEQ ID NO: 675); or 2LIG 48: (SEQ ID NO: 676); or 2LIG 65: (SEQ ID NO: 677); or 2LIG 85: (SEQ ID NO: 678); or 2LIG 86: (SEQ ID NO: 679); or 2LIG 89: (SEQ ID NO: 680); or 2LIG 97: (SEQ ID NO: 681); or 2LIG 99: (SEQ ID NO: 682); or 2LIG 109: (SEQ ID NO: 683); or 2LIG 127: (SEQ ID NO: 684); or 2LIG 139: (SEQ ID NO: 685); or 2LIG 176: (SEQ ID NO: 686); or 2LIG 189: (SEQ ID NO: 687); or 3LIG 3: (SEQ ID NO: 688); or 3LIG 7: (SEQ ID NO: 689); or 3LIG 8: (SEQ ID NO: 690); or 3LIG 9: (SEQ ID NO: 691); or 3LIG 18: (SEQ ID NO: 692); or 3LIG 20: (SEQ ID NO: 693); or 3LIG 28: (SEQ ID NO: 694); or 3LIG 29: (SEQ ID NO: 695); or 3LIG 30: (SEQ ID NO: 696); or 3LIG 33: (SEQ ID NO: 697).

In some embodiments, the PD-L1 targeting moiety comprises an amino acid sequence selected from any of the above sequences without a terminal histidine tag sequence (i.e., HHHHHHHH; SEQ ID NO: 43).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody MED14736 (also known as durvalumab) or a fragment thereof. MED14736 is selective for PD-L1 and blocks the binding of PD-L1 to PD-1 and CD80 receptors. In some embodiments, MED14736 and antigen-binding fragments thereof used in the methods provided herein comprise heavy and light chains or heavy and light chain variable regions. The sequence of MED14736 is disclosed in WO/2016/06272, the entire contents of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments MED14736, or an antigen-binding fragment thereof, used in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO:698) and/or a light chain comprising the amino acid sequence (SEQ ID NO: 699).

In some embodiments, MED14736, or antigen-binding fragment thereof, used in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:885) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 886).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (atezolizumab) (also known as MPDL3280A, RG7446) or a fragment thereof. By way of example, but not by way of limitation, in some embodiments, the atuzumab or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain comprising an amino acid sequence (SEQ ID NO:887) and/or a light chain comprising an amino acid sequence (SEQ ID NO: 888).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody aviluzumab (avelumab) (also known as MSB0010718C) or a fragment thereof. By way of example, but not by way of limitation, in some embodiments, avizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence (SEQ ID NO:889) and/or a light chain comprising an amino acid sequence (SEQ ID NO: 890).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12a4, MDX-1105) disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments BMS-936559 or an antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO:891) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 892).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3G10 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 3G10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:893) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 894).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 10a5 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 10a5 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:895) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 896).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 5F8 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:897) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 898).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 10H10 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:899) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 900).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1B12 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:901) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 902).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 7H1 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:903) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 904).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 11E6 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:905) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 906).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 12B7 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:907) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 908).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 13G4 disclosed in US 2013/0309250 and WO 2007/005874, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:909) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 910).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1E12 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, 1E12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO: 911);

and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 912).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 1F4 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 1F4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO:913) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 914).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2G11 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 2G11 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:700) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 701).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3B6 disclosed in US 2014/0044738, or a fragment thereof, the entire disclosure of which is hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO: 702);

and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 703).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3D10 disclosed in US 2014/0044738 and WO 2012/145493, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:704) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 705).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in US 2011/0271358 and WO 2010/036959, the entire contents of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 706); (SEQ ID NO: 707); (SEQ ID NO: 708); (SEQ ID NO: 709); or (SEQ ID NO: 710); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 711); (SEQ ID NO: 712); (SEQ ID NO: 713); or (SEQ ID NO: 714).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.7a4 or fragment thereof disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 2.7a4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:715) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 716).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10 disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 2.9D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:717) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 718).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9 disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 2.14H9 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:719) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 720).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.20a8 or fragment thereof disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 2.20A8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:721) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 722).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8 disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 3.15G8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:723) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 724).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1 disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, or fragments thereof, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments 3.18G1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:725) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 726).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or fragments thereof, disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, 2.7A4OPT, or antigen-binding fragments thereof, for use in the methods provided herein comprise a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:727) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 728).

In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody 2.14H90PT or a fragment thereof as disclosed in WO 2011/066389, US 8,779,108, and US 2014/0356353, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, 2.14H90PT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO:729) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 730).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO 2016/061142, the entire contents of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in WO 2016/061142: (SEQ ID NO: 731); (SEQ ID NO: 732); (SEQ ID NO: 733); (SEQ ID NO: 734); (SEQ ID NO: 735); (SEQ ID NO: 736); (SEQ ID NO: 737); (SEQ ID NO: 738); or (SEQ ID NO: 739); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in WO 2016/061142: (SEQ ID NO: 740); (SEQ ID NO: 741); (SEQ ID NO: 742); (SEQ ID NO: 743); (SEQ ID NO: 744); (SEQ ID NO: 745); (SEQ ID NO: 746); (SEQ ID NO: 747); or (SEQ ID NO: 748).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2016/022630, the entire contents of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in WO 2016/022630: (SEQ ID NO: 749); (SEQ ID NO: 750); (SEQ ID NO: 751); (SEQ ID NO: 752); (SEQ ID NO: 753); (SEQ ID NO: 754); (SEQ ID NO: 755); (SEQ ID NO: 756); (SEQ ID NO: 757); (SEQ ID NO: 758); (SEQ ID NO: 759); (SEQ ID NO: 760); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in WO 2016/022630: (SEQ ID NO: 761); (SEQ ID NO: 762); (SEQ ID NO: 763); (SEQ ID NO: 764); (SEQ ID NO: 765); (SEQ ID NO: 766); (SEQ ID NO: 767); (SEQ ID NO: 768); (SEQ ID NO: 769); (SEQ ID NO: 770); (SEQ ID NO: 771); and (SEQ ID NO: 772).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO 2015/112900, the entire contents of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in WO 2015/112900: (SEQ ID NO: 773); (SEQ ID NO: 774); (SEQ ID NO: 775); or (SEQ ID NO: 776); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in WO 2015/112900: (SEQ ID NO: 777); (SEQ ID NO: 778); (SEQ ID NO: 779); (SEQ ID NO: 780); (SEQ ID NO: 781); (SEQ ID NO: 782); (SEQ ID NO: 783); (SEQ ID NO: 784); or (SEQ ID NO: 785).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO 2010/077634 and US 8,217,149, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising an amino acid sequence (SEQ ID NO:786) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 787).

In some embodiments, the targeting moiety comprises any of the anti-PD-L1 antibodies obtainable from a hybridoma obtained under CNCM accession numbers CNCM 1-4122, CNCM 1-4080, and CNCM 1-4081 as disclosed in US 20120039906, the entire disclosure of which is hereby incorporated by reference.

In some embodiments, the targeting moiety comprises a VHH against PD-L1 disclosed in US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, the VHH for PD-L1 includes one or more of the following sequences in US 8,907,065: (SEQ ID NO: 788); (SEQ ID NO: 789); (SEQ ID NO: 790); (SEQ ID NO: 791); (SEQ ID NO: 792); and (SEQ ID NO: 793).

In some embodiments, the multispecific FAP-binding agents of the invention have one or more targeting moieties to PD-L2. In some embodiments, the FAP-binding agent has one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the FAP-binding agent comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to PD-L2 polypeptide.

In some embodiments, the targeting moiety comprises a VHH against PD-L2 disclosed in US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. By way of example, and not by way of limitation, in some embodiments, the VHH for PD-1 comprises one or more of the following sequences in US 8,907,065: (SEQ ID NO: 794); (SEQ ID NO: 795); (SEQ ID NO: 796); (SEQ ID NO: 797); (SEQ ID NO: 798); (SEQ ID NO: 799); and (SEQ ID NO: 800).

In some embodiments, the targeting moiety comprises any of the anti-PD-L2 antibodies as disclosed in US2011/0271358 and W02010/036959, the entire contents of which are hereby incorporated by reference. By way of example, but not by way of limitation, in some embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 801); (SEQ ID NO: 802); (SEQ ID NO: 803); (SEQ ID NO: 804); or (SEQ ID NO: 805); and/or a light chain comprising one or more amino acid sequences selected from the following sequences in US 2011/0271358: (SEQ ID NO: 806); (SEQ ID NO: 807); (SEQ ID NO: 808); or (SEQ ID NO: 809).

In some embodiments, targeting moieties of the present technology comprise a peptide having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, or any one of the sequences disclosed herein, At least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity to any one of the sequences disclosed herein) targeted to PD-1, PD-L1 and/or PD-L2.

In some embodiments, the targeting moiety of the present technology may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, Complementarity Determining Regions (CDRs), and framework region sequences that target PD-1, PD-L1, and/or PD-L2 as disclosed herein.

In some embodiments, the targeting moiety is one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and/or PD-L2, which are disclosed in: WO 2011/066389, US 2008/0025980, US 2013/0034559, US 8,779,108, US 2014/0356353, US 8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO 2011,/066342, US 8,907,065, WO 2016/062722, WO 2009/101611, W02010/027827, WO 2011/066342, WO 2007/005874, WO 2001/014556, US2011/0271358, WO 2010/036959, WO 2010/077634, US 8,217,149, US 2012/0039906, WO 2012/145493, US 2011/0318373, US patent No. 8,779,108, US 20140044738, WO 2009/089149, WO 2007/00587, WO 2016061142, WO 2016,02263, WO 2010/077634 and WO 2015/112900, the entire disclosures of which are hereby incorporated by reference.

In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety with an antigen recognition domain that specifically binds to XCR1, e.g., on a dendritic cell. In some embodiments, the multispecific FAP-binding agents of the present technology comprise a targeting moiety having an antigen recognition domain comprising all or part of XCL 1.

In some embodiments, the multispecific FAP-binding agent presents a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen, receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not a component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-protein, non-cellular marker, including but not limited to nucleic acids, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.

In some embodiments, the target of interest (e.g., antigen, receptor) is a part of or a marker associated with a non-cellular component of the matrix or extracellular matrix (ECM). As used herein, stroma refers to the connecting and supporting framework of a tissue or organ. The matrix may include a collection of cells such as fibroblasts/myofibroblasts, glia, epithelium, fat, immune, vascular, smooth muscle and immune cells, as well as extracellular matrix (ECM) and extracellular molecules. In some embodiments, the target of interest (e.g., antigen, receptor) is part of a non-cellular component of a matrix such as an extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. The ECM is composed of a large collection of biochemically distinct components, including but not limited to proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are typically produced by neighboring cells and secreted into the ECM via exocytosis. Once secreted, ECM components tend to aggregate to form complex macromolecular networks. In some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the present technology comprise a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-proteinaceous molecule) located on any component of the ECM. Illustrative components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, such as polysaccharides and/or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, such as polysaccharides, nucleic acids, and/or lipids).

In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on the ECM proteoglycan. Proteoglycans are glycosylated proteins. The basic proteoglycan unit includes a core protein having one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge, thereby attracting positively charged sodium ions (Na +), which attract water molecules via osmosis, thereby keeping the ECM and resident cells hydrated. Proteoglycans may also help to capture and store growth factors within the ECM. Illustrative proteoglycans that the chimeric proteins or Fc-based chimeric protein complexes of the present technology can target include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In one embodiment, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide, such as hyaluronic acid.

In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on the ECM fiber. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists in the ECM in the form of fibrillar proteins and provides structural support to retain cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present within the ECM, including, but not limited to, fibrillar collagen (type I, type II, type il, type V, type XI), fibril associated collagen (facit collagen) (type IX, type XII, type XIV), short chain collagen (type VIII, type X), basement membrane collagen (type IV), and/or type VI, type VII, or type XIII collagen. Elastin fibers provide elasticity to the tissue, allowing them to stretch when needed and then return to their original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

In some embodiments, the targeting moiety recognizes one or more ECM proteins, including but not limited to tenascin, fibronectin, fibrin, laminin (laminin), or nidogen/entactin (enteractin).

In some embodiments, the targeting moiety recognizes and binds to tenascin. The glycoproteins of the Tenascin (TN) family include at least four members, tenascin C, tenascin R, tenascin X and tenascin W. The primary structure of tenascin comprises several common motifs in sequence in the same contiguous sequence: an amino-terminal heptad repeat, an Epidermal Growth Factor (EGF) -like repeat, a fibronectin type III domain repeat, and a carboxy-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of EGF-like repeats and fibronectin type III repeats. Isoform variants also exist in particular with respect to tenascin C. More than 27 splice variants and/or isoforms of tenascin-C are known. In a particular embodiment, the targeting moiety recognizes and binds to tenascin CA 1. Similarly, tenascin R also has different splice variants and isoforms. Tenascin R is usually present in dimeric or trimeric form. tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin W exists in the form of a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on tenascin. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric and/or trimeric and/or hexameric forms of tenascin.

In one embodiment, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that links cells to collagen fibers in the ECM, allowing the cells to move through the ECM. Upon binding to the integrin, fibronectin unfolds to form a functional dimer. In some embodiments, the targeting moiety recognizes a monomeric and/or dimeric form of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. By way of example and not by way of limitation, in some embodiments, the targeting moiety recognizes fibronectin extracellular domain a (eda) or fibronectin extracellular domain b (edb). Elevated EDA levels are associated with a variety of diseases and conditions, including psoriasis, rheumatoid arthritis, diabetes and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing EDA isoforms and can be used to target the chimeric protein or Fc-based chimeric protein complex to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing EDB isoforms. In some embodiments, such targeting moieties may be used to target the chimeric protein or Fc-based chimeric protein complex to tumor cells, including tumor neovasculature.

In some embodiments, the targeting moiety recognizes and binds to fibrin. Fibrin is another proteinaceous substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which causes fibrin polymerization. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric as well as polymeric forms of fibrin.

In some embodiments, the targeting moiety recognizes and binds to laminin. Laminin is the major component of the basal layer that underlies the protein network of cells and organs. Laminins are heterotrimeric proteins containing an a chain, a 3 chain, and a y chain. In some embodiments, the targeting moiety recognizes one or more epitopes on a laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

In some embodiments, the targeting moiety recognizes and binds to nestin or an catenin. Nestin/catenin is a family of highly conserved sulfated glycoproteins. They constitute the major structural component of the basement membrane and function to link laminin and collagen IV network in the basement membrane. Members of this family include nestin-1 and nestin-2. In some embodiments, the targeting moiety recognizes an epitope on nestin-1 and/or nestin-2.

In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein. In some embodiments, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any contiguous amino acid sequence present on the protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to one or more segments of amino acids (which may be discontinuous) that form a three-dimensional surface having features and/or shape and/or tertiary structure that are capable of being recognized by an antigen recognition domain.

In some embodiments, the targeting moiety binds to a full-length and/or mature form and/or isoform and/or splice variant and/or fragment and/or any other naturally occurring or synthetic analog, variant or mutant of any one described herein. In some embodiments, the targeting moiety can bind to any form of a protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In some embodiments, the targeting moiety may bind to any post-translational modified form of the proteins described herein, such as glycosylated and/or phosphorylated forms.

In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule, such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In some embodiments, the DNA is shed from necrotic or apoptotic tumor cells or other diseased cells into the extracellular space.

In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibrolipid (fibrofatty) plaques are characterized by accumulation of lipid-laden cells under the intima of the artery. Beneath the endothelium is a fibrous cap covering the atheromatous core of the plaque. The core comprises lipid-laden cells (macrophages and smooth muscle cells), fibrin, proteoglycans, collagen, elastin, and cellular debris with elevated tissue cholesterol and cholesterol ester content. In late-stage plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells) that form regions of cholesterol crystals with hollow needle-like fissures. At the periphery of the plaque are younger foam cells and capillaries. Fibrous plaque is also localized under the intima, within the arterial wall, causing thickening and distension of the wall, and sometimes multiple plaques of the lumen with some degree of muscular layer atrophy. Fibrous plaques contain collagen fibers (eosinophilic) that precipitate calcium (hematoxylin-philic) and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, cellular debris, and calcium or other inorganic deposits or precipitates. In some embodiments, the cellular debris is nucleic acid, such as DNA or RNA, released from dead cells.

In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative disease. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located in amyloid plaques found in the brain of alzheimer's disease patients. For example, the targeting moiety can recognize and bind to the peptide amyloid β, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more acellular structures located in brain plaques found in huntington's disease patients. In some embodiments, the targeting moiety recognizes and binds to one or more acellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases such as dementia with lewy bodies and inclusion body myositis.

Linkers and functional groups

In some embodiments, the FAP-binding agent may comprise one or more functional groups, residues, or moieties. In some embodiments, the one or more functional groups, residues, or moieties are linked or genetically fused to any of the signaling agents or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desired properties or functionalities to FAP-binding agents of the present technology. Examples of such functional groups and techniques for introducing them into FAP binders are known in the art, see, for example, Remington's Pharmaceutical Sciences, 16 th edition, Mack Publishing co., Easton, Pa (1980).

In some embodiments, the FAP-binding agent may extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties by conjugation and/or fusion with another agent. In some embodiments, the FAP binding agent may be fused or conjugated to one or more of PEG, XTEN (e.g., in the form of rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In some embodiments, each of the individual chimeric proteins or Fc-based chimeric protein complexes is fused to one or more of the agents described in BioDrugs (2015)29:215-239, the entire contents of which are hereby incorporated by reference.

In some embodiments, the functional group, residue or moiety comprises a suitable pharmacologically acceptable polymer, such as poly (ethylene glycol) (PEG) or a derivative thereof (such as methoxy poly (ethylene glycol) or mPEG). In some embodiments, attachment of a PEG moiety increases half-life and/or reduces immunogenicity of the FAP-binding protein. In general, any suitable form of pegylation may be used, such as pegylation used in the art for antibodies and antibody fragments (including but not limited to single domain antibodies, such as VHH); see, e.g., Chapman, nat. biotechnol.,54,531-545 (2002); veronese and Harris, adv. drug Deliv. Rev.54,453-456 (2003); harris and Chess, nat. rev. drug. discov.,2, (2003) and W004060965, the entire contents of which are hereby incorporated by reference. Various reagents for the pegylation of proteins are also available from the market, for example, Nektar Therapeutics in the united states. In some embodiments, site-directed pegylation is used, in particular, via a cysteine residue (see, e.g., Yang et al, Protein Engineering,16,10,761-770(2003), the entire contents of which are hereby incorporated by reference). For example, PEG may be attached to cysteine residues naturally occurring in FAP-binding agents of the present technology for this purpose. In some embodiments, FAP-binding agents of the present technology are modified to appropriately introduce one or more cysteine residues for attachment of PEG, or amino acid sequences comprising one or more cysteine residues for attachment of PEG may be fused to the amino and/or carboxy terminus of the FAP-binding agent using techniques known in the art.

In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation. In some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and/or post-translational modification.

In some embodiments, the functional group, residue or moiety comprises one or more detectable labels or other signal generating groups or moieties. Suitable labels and techniques suitable for linking, using and detecting them are known in the art and include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamines and fluorescent metals such as Eu or other lanthanide metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, thermoacridinium ester, imidazole, acridinium salt, oxalate, dioxetane or GFP and their analogs), radioisotopes, metals, metal chelates or metal cations or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnostics and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, Eu or other lanthanide metals, and other metals or metal cations) delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalytic enzyme, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. Such labelled VHH and polypeptides of the present technology may for example be used for in vitro, in vivo or in situ assays (including per se known immunoassays such as ELISA, RIA, EIA and other "sandwich assays" etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

In some embodiments, the functional group, residue, or moiety comprises a tag linked or genetically fused to the FAP-binding agent. In some embodiments, the FAP-binding agent may comprise a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or prevent binding of the FAP-binding agent to FAP or any other antigen of interest, such as a tumor antigen. In some embodiments, the tag is at least about: three to five amino acids long, five to eight amino acids long, eight to twelve amino acids long, twelve to fifteen amino acids long, or fifteen to twenty amino acids long. Illustrative labels are described, for example, in U.S. patent publication No. US 2013/0058962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) and histidine (His) tag. In one embodiment, the FAP-binding agent comprises a His-tag.

In some embodiments, the functional group, residue, or moiety comprises a chelating group, e.g., to chelate a metal or one of the metal cations. By way of example, and not by way of limitation, in some embodiments, the chelating group is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In some embodiments, the functional group, residue, or moiety comprises a functional group that is part of a specific binding pair, such as a biotin- (strept) avidin binding pair. Such functional groups may be used to link the FAP-binding agent of the present technology to another protein, polypeptide, or chemical compound that is bound to (i.e., by forming a binding pair with) the other half of the binding pair. For example, in some embodiments, the FAP-binding agents of the present technology may be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such conjugated FAP-binding agents may be used as reporter genes, e.g. in diagnostic systems, wherein a detectable signal generating agent is conjugated to avidin or streptavidin. Such binding pairs may also be used, for example, to bind FAP-binding agents to carriers, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described by Cao and Suresh, Journal of Drug Targeting,8,4,257 (2000). Such binding pairs may also be used to link a therapeutically active agent to FAP-binding agents of the present technology.

In some embodiments, FAP-binding agents of the invention optionally comprise one or more linkers. In some embodiments, the FAP-binding agents comprise a linker connecting the binding regions and/or targeting moieties. In some embodiments, the FAP-binding agent comprises a linker connecting each signaling agent and a targeting moiety (or connecting a signaling agent to one of the targeting moieties if more than one targeting moiety is present). In some embodiments, the linker may be used to link various functional groups, residues, or moieties as described herein to FAP-binding agents. In some embodiments, the linker is a single amino acid or a plurality of amino acids that do not affect or reduce the stability, orientation, binding, neutralization, and/or clearance characteristics of the binding region and binding protein. In some embodiments, the linker is selected from a peptide, a protein, a sugar, or a nucleic acid.

In some embodiments, the FAP-binding agents of the invention comprise a linker connecting the targeting moiety and the signaling agent. In other embodiments, the Fc-based chimeric protein complex comprises a linker connecting the targeting moiety to the Fc domain or a linker connecting the signaling moiety to the Fc domain. In some embodiments, the chimeric protein or Fc-based chimeric protein complex of the invention comprises a linker within the signaling agent (e.g., in the case of single-chain TNF, the chimeric protein may comprise two linkers to produce a trimer).

The present technology contemplates the use of a variety of linker sequences. In some embodiments, the linker may be derived from a naturally occurring multidomain protein or be an empirical linker as described, for example, in: chichil et al, (2013), Protein Sci.22(2): 153-; chen et al, (2013), Adv Drug Deliv Rev.65(10): 1357-. In some embodiments, the linker may be designed using a linker design database and a computer program, such as those described in the following documents: chen et al, (2013), Adv Drug Deliv Rev.65(10): 1357-. In some embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and/or stability, improve expression, improve pharmacokinetics, and/or improve biological activity of the FAP-binding agents of the invention.

In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids long. For example, in some embodiments, the linker is less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids long. For example, in some embodiments, the linker is more than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In another embodiment, the joint is rigid.

In some embodiments, the linker length allows for efficient binding of the targeting moiety and signaling agent to its receptor. For example, in some embodiments, the linker length allows for effective binding of one of the targeting moieties and the signaling agent to a receptor on the same cell and effective binding of the other targeting moiety to another cell. Illustrative cell pairs are provided elsewhere herein.

In some embodiments, the linker is at least as long as the minimum distance between one of the targeting moieties and the binding site of the signaling agent to a receptor on the same cell. In some embodiments, the linker length is at least two, or three, or four, or five, or ten, or twenty, or 25, or 50, or one hundred, or more times the minimum distance between one of the targeting moieties and the binding site of the signaling agent to a receptor on the same cell.

In some embodiments, one linker connects two targeting moieties to each other and has a shorter length, while one linker connects a targeting moiety and a signaling agent and is longer than the linker connecting the two targeting moieties. For example, in some embodiments, the difference in amino acid length between the linker connecting the two targeting moieties and the linker connecting the targeting moiety and the signaling agent can be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids. In some embodiments, the linker is flexible. In another embodiment, the joint is rigid.

In some embodiments, the linker consists essentially of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser)_nWherein n is from about 1 to about 8, such as 1, 2, 3, 4, 5, 6, 7 or 8(SEQ ID NO:810 to SEQ ID NO: 817). In some embodiments, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 818). In some embodiments, the linker includes, but is not limited to, a linker having the sequence LE, GGGGS (SEQ I)D NO:810)、(GGGGS)_n(n＝1-4)(SEQ ID NO:810-813)、(Gly)₈(SEQ ID NO:819)、(Gly)₆(SEQ ID NO:820)、(EAAAK)_n(n＝1-3)(SEQ ID NO:821-823):、A(EAAAK)_nA(n＝2-5)(SEQ ID NO:824-827)、AEAAAKEAAAKA(SEQ ID NO:824)、A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO:828), PAPAP (SEQ ID NO:829), KESGSVSSEQLAQFRSLD (SEQ ID NO:830), EGKSSGSGSESKST (SEQ ID NO:831), GSAGSAAGSGEF (SEQ ID NO:832) and (XP)_nWherein X represents any amino acid, e.g., Ala, Lys, or Glu. In some embodiments, the linker is a GGS.

In some embodiments, the linker is one or more of GGGSE (SEQ ID NO:833), GSESG (SEQ ID NO:834), GSEGS (SEQ ID NO:835), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO:836), and a linker with G, S and E randomly placed every four amino acid intervals.

In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). Without wishing to be bound by theory, in some embodiments, the hinge region found in IgG, IgA, IgD, and IgE class antibodies acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant region, the hinge domains are structurally diverse, differing in both sequence and length from immunoglobulin class and subclass to immunoglobulin class and subclass. For example, the length and flexibility of the hinge region vary from IgG subclass to subclass. The hinge region of IgG1 encompasses amino acids 216 and 231 and, because it is free to flex, the Fab fragment can rotate about its axis of symmetry and move within a sphere centered on the first of the two inter-heavy chain disulfide bridges.

IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional inter-heavy chain disulfide bridges. These properties constrain the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (up to about four times the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible polyproline double helix. In IgG3, the Fab fragment is relatively distant from the Fc fragment, thereby imparting greater flexibility to the molecule. The elongate hinge in IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG 2. The flexibility of the hinge region was reported in descending order as IgG3> IgG1> IgG4> IgG 2.

According to crystallographic studies, immunoglobulin hinge regions can be further functionally subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin et al, 1992Immunological Reviews 130: 87. The upper hinge region includes a hinge from C_H1Is the first residue in the hinge that constrains motion, typically the amino acid that forms the first cysteine residue of the interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the segment of the antibody. The core hinge region contains heavy interchain disulfide bridges, while the lower hinge region joins C_H2Amino terminus of Domain and including C_H2The residue of (1). The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Cys, which when dimerized by disulfide bond formation yields a cyclic octapeptide that is thought to act as a pivot, thus imparting flexibility. In some embodiments, the linkers of the invention comprise one or two or three of the upper, core and lower hinge regions of any antibody (e.g., IgG, IgA, IgD and IgE, including subclasses such as IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA 2). The hinge region may also contain one or more glycosylation sites, including numerous types of structurally distinct sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance to enteroproteases to hinge region polypeptides, which is considered an advantageous property of secretory immunoglobulins. In some embodiments, the linker of the present technology comprises one or more glycosylation sites. In some cases In embodiments, the linker is the hinge-CH 2-CH3 domain of human IgG4 antibody.

In some embodiments, the FAP-binding agents of the invention are linked to a peptide comprising C_H2 and C_H3 and optionally an antibody Fc domain of a hinge region. For example, vectors encoding FAP-binding agents of the present technology linked as a single nucleotide sequence to an Fc domain can be used to prepare such polypeptides.

In some embodiments, the linker is a synthetic linker, such as PEG.

In some embodiments, the linker may be functional. For example, in some embodiments, the linker functions to improve folding and/or stability, improve expression, improve pharmacokinetics, and/or improve biological activity of FAP-binding agents of the invention. In another example, the linker may function to target the FAP-binding agent to a particular cell type or location.

Modification and production of FAP-binding agents

In some embodiments, the FAP-binding agent comprises a targeting moiety that is a VHH. In some embodiments, the VHH is not limited to a particular biological source or a particular method of preparation. For example, the VHH may be obtained generally by: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of naturally occurring VHH domains or by expression of nucleic acids encoding such humanized VHH domains; (4) by "camelisation" of naturally occurring VH domains from any animal species, such as from a mammalian species, such as from a human, or by expression of nucleic acids encoding such camelised VH domains; (5) by "camelization" of "domain antibodies" or "Dab" as described in the art, or by expression of nucleic acids encoding such camelized VH domains; (6) preparing proteins, polypeptides or other amino acid sequences known in the art by using synthetic or semi-synthetic techniques; (7) preparing a nucleic acid encoding a VHH by using nucleic acid synthesis techniques known in the art, followed by expression of the nucleic acid so obtained; and/or (8) by any combination of one or more of the foregoing.

In one embodiment, the FAP-binding agent comprises a VHH corresponding to the VHH domain of a naturally occurring heavy chain antibody directed against human FAP. In some embodiments, such VHH sequences may be generated or obtained generally by: by suitably immunizing a camelid with a FAP molecule (i.e. so as to generate an immune response and/or heavy chain antibodies against FAP); by obtaining a suitable biological sample (such as a blood sample or any B cell sample) from the camel; and generating a VHH sequence for FAP by using any suitable known technique starting from the sample. In some embodiments, a naturally occurring VHH domain directed to FAP may be obtained from a natural library of camelid VHH sequences, for example, by screening such library using FAP or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 9937681, WO 0190190, WO 03025020 and WO 03035694, the entire contents of which are hereby incorporated by reference. In some embodiments, improved synthetic or semi-synthetic libraries derived from natural VHH libraries may be used, such as VHH libraries obtained from natural VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as described for example in WO 0043507, the entire contents of which are hereby incorporated by reference. In some embodiments, another technique for obtaining a VHH sequence for FAP comprises suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., so as to generate an immune response and/or heavy chain antibodies to FAP), obtaining a suitable biological sample (such as a blood sample, or any B cell sample) from the transgenic mammal, and then using any suitable known technique, starting with the sample, to generate a VHH sequence for FAP. For example, for this purpose, mice expressing heavy chain antibodies as described in WO 02085945 and in WO 04049794, as well as other methods and techniques, can be used (the entire contents of which are hereby incorporated by reference).

In one embodiment, the FAP-binding agent comprises a VHH that has been "humanized", i.e. by substitution of one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence (and in particular in the framework sequence) to one or more amino acid residues present at corresponding positions in the VH domain of a conventional 4 chain antibody from human. This can be done using humanization techniques known in the art. In some embodiments, possible humanized substitutions or combinations of humanized substitutions may be determined by methods known in the art, e.g., by comparing the sequence of the VHH to the sequence of a naturally occurring human VH domain. In some embodiments, the humanized substitutions are selected such that the resulting humanized VHH still retains favorable functional properties. In general, as a result of humanization, VHHs of the present technology may become more "human-like" compared to the corresponding naturally occurring VHH domains, while still retaining advantageous properties, such as reduced immunogenicity. In some embodiments, the humanized VHH of the present technology may be obtained in any suitable manner known in the art and is therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising a naturally occurring VHH domain as a starting material.

In some embodiments, the FAP-binding agent comprises a VHH that has been "camelised", i.e. by substituting one or more amino acid residues in the amino acid sequence from a naturally occurring VH domain of a conventional 4 chain antibody for one or more amino acid residues present at corresponding positions in the VHH domain of a camelid heavy chain antibody. In some embodiments, such "camelised" substitutions are inserted at amino acid positions formed and/or present at the VH-VL interface and/or at so-called camelid marker residues (see, for example, WO 9404678, the entire contents of which are hereby incorporated by reference). In some embodiments, the VH sequences used as starting materials or points for generating or designing camelized VHHs are VH sequences from mammals, for example, human VH sequences such as VH3 sequences. In some embodiments, the camelized VHH may be obtained in any suitable manner known in the art (i.e. as indicated at points (1) - (8) above) and is therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising a naturally occurring VH domain as a starting material.

In some embodiments, "humanization" and "camelization" may be performed by: nucleotide sequences encoding a naturally occurring VHH domain or VH domain, respectively, are provided and then one or more codons in the nucleotide sequence are altered in a manner known in the art in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. Such nucleic acids can then be expressed in a manner known in the art in order to provide the desired VHH of the present technology. Alternatively, the amino acid sequence of a desired humanized or camelized VHH of the present technology may be designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using peptide synthesis techniques known in the art. Furthermore, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding a desired humanized or camelized VHH may be designed and then synthesized de novo using nucleic acid synthesis techniques known in the art, after which the nucleic acid so obtained may be expressed in a manner known in the art in order to provide the desired VHH of the present technology. Other suitable methods and techniques for obtaining a VHH of the present technology and/or nucleic acid encoding said VHH starting from a naturally occurring VH sequence or VHH sequence are known in the art and may for example comprise combining one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences) and/or one or more synthetic or semi-synthetic sequences in a suitable manner so as to provide a VHH of the present technology or a nucleotide sequence or nucleic acid encoding said VHH.

Methods for generating FAP binders of the present technology are described herein. For example, DNA sequences encoding FAP-binding agents of the present technology can be chemically synthesized using methods known in the art. The synthetic DNA sequence may be linked to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct encoding the desired FAP-binding agent. Thus, in some embodiments, the present technology provides an isolated nucleic acid comprising a nucleotide sequence encoding a FAP-binding agent of the present technology.

Nucleic acids encoding FAP-binding agents of the present technology can be incorporated (linked) into expression vectors that can be introduced into host cells by transfection, transformation, or transduction techniques. For example, a nucleic acid encoding a FAP-binding agent of the present technology can be introduced into a host cell by retroviral transduction. Illustrative host cells are E.coli cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney 293(HEK 293) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cell may be grown under conditions that allow the host cell to express the gene encoding the FAP-binding agent of the present technology. Thus, in some embodiments, the present technology provides expression vectors comprising a nucleic acid encoding a FAP-binding agent of the present technology. In some embodiments, the present technology additionally provides host cells comprising such expression vectors.

The specific expression and purification conditions will vary depending on the expression system employed. For example, if a gene is expressed in E.coli, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, secretion signals, enhancers, and various introns. The genetic construct may be introduced into the host cell using transfection, transformation or transduction techniques.

The FAP-binding agents of the present technology can be produced by growing a host cell transfected with an expression vector encoding the FAP-binding agent under conditions that allow expression of the protein. After expression, the protein may be collected and purified using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags or by chromatography. In one embodiment, the FAP-binding agent comprises a His-tag. In some embodiments, the FAP-binding agent comprises a His-tag and a proteolytic cleavage site that allows cleavage of the His-tag.

Thus, in some embodiments, the present technology provides a nucleic acid encoding a FAP-binding agent of the present technology. In some embodiments, the present technology provides a host cell comprising a nucleic acid encoding a FAP-binding agent of the present technology.

In some embodiments, the FAP-binding agents of the invention or chimeric proteins or Fc-based chimeric protein complexes comprising the same may be expressed in vivo, e.g., in a patient. For example, in some embodiments, the FAP-binding agents of the invention or chimeric proteins or Fc-based chimeric protein complexes comprising the same may be administered in the form of a nucleic acid encoding the FAP-binding agents of the invention or chimeric proteins or Fc-based chimeric protein complexes comprising the same. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the FAP-binding agents of the invention or chimeric proteins or Fc-based chimeric protein complexes comprising the same are encoded by modified mrnas, i.e., mrnas comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in U.S. patent No. 8,278,036, the entire contents of which are hereby incorporated by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, s2U, Ψ, and 2' -O-methyl-U. In some embodiments, the present technology relates to administering modified mRNA encoding one or more chimeric proteins or Fc-based chimeric protein complexes of the present invention. In some embodiments, the present technology relates to gene therapy vectors comprising the modified mRNA. In some embodiments, the present technology relates to gene therapy methods comprising the gene therapy vectors. In some embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, newcastle disease virus, or vaccinia.

Pharmaceutically acceptable salts and excipients

The FAP-binding agents described herein (and/or any other therapeutic agent) may have a sufficiently basic functional group that can react with an inorganic or organic acid, or a carboxyl group that can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, the pharmaceutically acceptable salts listed in the following references: journal of Pharmaceutical Science,66,2-19(1977) and The Handbook of Pharmaceutical Salts; properties, Selection, and use, p.h.stahl and c.g.wermuth (ed.), Verlag, zurich (switzerland)2002, which are hereby incorporated by reference in their entirety.

As non-limiting examples, pharmaceutically acceptable salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, salt, salts of benzoic acid, salts of benzoic acid, salts of acid, salts of acid, salts of acid, salts of acids, salts of acid, salts of acids, salts of acid, salts of acids, salts of acid, salts of acids, salts of acid, Naphthalene-2-benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne-1, 4-dicarboxylate, hexyne-1, 4-dicarboxylate, decanoate, octanoate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, phthalate, terephthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-isethionate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1, 5-sulfonate, xylenesulfonate, and tartrate.

The term "pharmaceutically acceptable salt" also refers to a salt of a composition of the present technology having an acidic functional group, such as a carboxylic acid functional group, with a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxy-substituted monoalkylamines, dialkylamines or trialkylamines, dicyclohexylamines; tributylamine; pyridine; n-methyl, N-ethylamine; a diethylamine; triethylamine; mono (2-OH-lower alkylamine), bis (2-OH-lower alkylamine) or tris (2-OH-lower alkylamine) (such as mono (2-hydroxyethyl) amine, bis (2-hydroxyethyl) amine or tris (2-hydroxyethyl) amine), 2-hydroxy-tert-butylamine or tris (hydroxymethyl) methylamine, N-di-lower alkyl-N- (hydroxy-lower alkyl) -amine (such as N, N-dimethyl-N- (2-hydroxyethyl) amine) or tris (2-hydroxyethyl) amine; N-methyl-D-glucosamine; and amino acids such as arginine, lysine, and the like.

In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

Pharmaceutical compositions and formulations

In some embodiments, the present technology pertains to pharmaceutical compositions comprising FAP-binding agents (and/or any other therapeutic agent) described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present technology pertains to pharmaceutical compositions comprising FAP-binding agents of the present invention. In another embodiment, the present technology pertains to a pharmaceutical composition comprising any of the other therapeutic agents described herein. In another embodiment, the present technology pertains to pharmaceutical compositions comprising FAP-binding agents of the present invention in combination with any other therapeutic agent described herein. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise suitable amounts of pharmaceutically acceptable excipients in order to provide a form for suitable administration.

In some embodiments, the pharmaceutical excipient may be a liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipient may be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliaries, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Physiological saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, as desired. Further examples of suitable Pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-.

The present technology encompasses various formulation forms of the described pharmaceutical compositions (and/or other therapeutic agents). Any of the inventive pharmaceutical compositions (and/or other therapeutic agents) described herein can be in the form of a solution, suspension, emulsion, drop, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dried powder, or any other suitable form. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated in a gelatin capsule. In another embodiment, the pharmaceutical composition is formulated as a liquid.

In some embodiments, the pharmaceutical compositions (and/or other agents) of the present invention may also include a solubilizing agent. The agent may also be delivered with a suitable vehicle or delivery device as known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device.

Formulations of the present technology comprising the pharmaceutical compositions (and/or other agents) of the present invention may suitably be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing into association the therapeutic agent with the carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation dosage form (e.g., wet or dry granulation, powder blend, etc., followed by tableting using conventional methods known in the art).

In some embodiments, any of the pharmaceutical compositions (and/or other agents) described herein are formulated according to conventional procedures as compositions suitable for the mode of administration described herein.

In some embodiments, routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or topical. Administration may be local or systemic. In some embodiments, the administering is effected orally. In another embodiment, the administration is by parenteral injection. The mode of administration may be left to the discretion of the physician and will depend in part on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

In some embodiments, the FAP-binding agents described herein are formulated according to conventional procedures as compositions suitable for oral administration. By way of example, and not by way of limitation, in some embodiments, the composition for oral delivery is in the form of a tablet, lozenge, aqueous or oily suspension, granule, powder, emulsion, capsule, syrup, or elixir. In some embodiments, the orally administered composition comprises one or more agents, for example, a sweetening agent such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, oil of wintergreen, or cherry; a colorant; and preservatives to provide pharmaceutically palatable preparations. In some embodiments, when in tablet or pill form, the composition is coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. Selectively permeable membranes surrounding any FAP-binding agent driven by osmotic activity described herein are also suitable for use in compositions for oral administration. In these latter platforms, fluid from the environment surrounding the capsule is drawn in by the driving compound which expands to displace the agent or agent composition through the orifice. These delivery platforms can provide an essentially zero order delivery profile, as opposed to the sharp peak profile of immediate release formulations. In some embodiments, the oral composition comprises a time delay material, such as, for example, glyceryl monostearate or glyceryl stearate in some embodiments, the oral composition comprises standard excipients, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipient is of pharmaceutical grade. Suspensions, as well as the active compounds, can contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) which may be dissolved or suspended in a sterile injectable medium immediately prior to use. They may contain, for example, suspending or dispersing agents as known in the art. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and tonicity adjusting agents such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or Phosphate Buffered Saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved against the effects of microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

The compositions provided herein can be formulated, alone or in combination with other suitable components, into aerosol formulations (i.e., "sprays") for administration via inhalation. The aerosol formulation may be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

Any of the inventive pharmaceutical compositions (and/or other agents) described herein may be administered by controlled or sustained release means or by delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. patent nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated by reference herein in its entirety. Such dosage forms may be used to provide controlled or sustained release of one or more active ingredients using, for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide a desired release profile at varying ratios. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The present technology thus provides single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, caplets and caplets suitable for controlled or sustained release.

The controlled or sustained release of the active ingredient may be stimulated by various conditions including, but not limited to, a change in pH, a change in temperature, by the wavelength of appropriate light, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

In another embodiment, the Controlled Release system may be placed in the vicinity of the target area to be treated, thus requiring only a fraction of the total body dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol.2, pp.115-138 (1984)). Other controlled release systems discussed in the review by Langer,1990, Science 249: 1527-.

The pharmaceutical formulation is preferably sterile. Sterilization may be achieved, for example, by filtration through sterile filtration membranes. In the case of compositions that are lyophilized, filter sterilization may be performed before or after lyophilization and reconstitution.

Administration and dosage

It will be appreciated that the actual dosage of the FAP-binding agent administered in accordance with the present techniques and/or any of the therapeutic agents described herein will vary depending on the particular dosage form and mode of administration. One skilled in the art can consider many factors (e.g., body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic predisposition, and response sensitivity) that can modulate the effects of FAP-binding agents. Administration can be continuous or in one or more discrete doses within the maximum tolerated dose. One skilled in the art can use conventional dose administration testing to determine the optimal rate of administration for a given set of conditions.

In some embodiments, a suitable dose of the FAP-binding agent and/or any of the therapeutic agents described herein is in the range of about 0.01mg/kg to about 10g/kg of subject body weight, about 0.01mg/kg to about 1g/kg of subject body weight, about 0.01mg/kg to about 100mg/kg of subject body weight, about 0.01mg/kg to about 10mg/kg of subject body weight, e.g., about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.08mg/kg, about 0.09mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, About 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, about 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, 1.9mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg body weight, about 100mg/kg body weight, about 1g/kg body weight, about 10g/kg body weight, including all values and ranges therebetween.

In some embodiments, individual doses of the FAP-binding agent and/or any therapeutic agent described herein are in a dosage form containing, for example, about 0.01mg to about 100g, about 0.01mg to about 75g, about 0.01mg to about 50g, about 0.01mg to about 25g, about 0.01mg to about 10g, about 0.01mg to about 7.5g, about 0.01mg to about 5g, about 0.01mg to about 2.5g, about 0.01mg to about 1g, about 0.01mg to about 100mg, about 0.1mg to about 90mg, from about 0.1mg to about 80mg, from about 0.1mg to about 70mg, from about 0.1mg to about 60mg, from about 0.1mg to about 50mg, from about 0.1mg to about 40mg of active ingredient, from about 0.1mg to about 30mg, from about 0.1mg to about 20mg, from about 0.1mg to about 10mg, from about 0.1mg to about 5mg, from about 0.1mg to about 3mg, from about 0.1mg to about 1mg or from about 5mg to about 80mg per unit dosage form. For example, a unit dosage form may be about 0.01mg, about 0.02mg, about 0.03mg, about 0.04mg, about 0.05mg, about 0.06mg, about 0.07mg, about 0.08mg, about 0.09mg, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 5mg, about 50mg, about 5g, about 100g, about 5g, and all values between these.

In one embodiment, the FAP-binding agent and/or any therapeutic agent described herein is about 0.01mg to about 100g per day, about 0.01mg to about 75g per day, about 0.01mg to about 50g per day, about 0.01mg to about 25g per day, about 0.01mg to about 10g per day, about 0.01mg to about 7.5g per day, about 0.01mg to about 5g per day, about 0.01mg to about 2.5g per day, about 0.01mg to about 1g per day, about 0.01mg to about 100mg per day, about 0.1mg to about 95mg per day, about 0.1mg to about 90mg per day, about 0.1mg to about 85mg per day, about 0.1mg to about 80mg per day, about 0.1mg to about 75mg per day, about 0.1mg to about 70mg per day, about 0.65 mg to about 0.1mg per day, about 0.1mg to about 0.55 mg per day, about 0.1mg to about 0.5mg per day, about 0.1mg to about 1mg per day, about 0.5mg per day, about 10mg per day, about 1mg per day, or about 0.5mg per day, From about 0.1mg to about 35mg per day, from about 0.1mg to about 30mg per day, from about 0.1mg to about 25mg per day, from about 0.1mg to about 20mg per day, from about 0.1mg to about 15mg per day, from about 0.1mg to about 10mg per day, from about 0.1mg to about 5mg per day, from about 0.1mg to about 3mg per day, from about 0.1mg to about 1mg per day, or from about 5mg to about 80mg per day. In some embodiments, the FAP-binding agent is administered at a dosage of about 0.01mg, about 0.02mg, about 0.03mg, about 0.04mg, about 0.05mg, about 0.06mg, about 0.07mg, about 0.08mg, about 0.09mg, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 200mg, about 500mg, about 1g, about 2.5g, about 5g, about 7.5g, about 10g, about 25g, about 50g, about 75g, about 100g of a daily dose administration, including all values and ranges between these values.

In accordance with certain embodiments of the present technology, a pharmaceutical composition comprising the FAP-binding agent and/or any therapeutic agent described herein can be administered, e.g., more than once per day (e.g., about two, about three, about four, about five, about six, about seven, about eight, about nine, or about ten times per day), about once per day, about once every other day, about once every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once per year.

Combination therapy and other therapeutic agents

In some embodiments, the pharmaceutical compositions of the present technology are co-administered in combination with one or more additional therapeutic agents. In some embodiments, the co-administration may be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and the FAP-binding agent of the present technology are administered to the subject simultaneously. The term "simultaneously" as used herein means that the other therapeutic agent and the FAP-binding agent are administered no more than about 60 minutes apart, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the additional therapeutic agent and the FAP-binding agent can be performed by simultaneous administration of a single formulation (e.g., a formulation comprising the additional therapeutic agent and the FAP-binding agent) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the FAP-binding agent).

Co-administration does not require simultaneous administration of each therapeutic agent, as long as the time course of administration is such that the pharmacological activities of the other therapeutic agent and the FAP-binding agent overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the FAP-binding agent may be administered sequentially. The term "sequentially" as used herein means that the administration time interval of the additional therapeutic agent and the FAP-binding agent is more than about 60 minutes. For example, the time between sequential administration of the additional therapeutic agent and the FAP-binding agent may be separated by more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, or more than about 2 weeks, or more than about one month. The optimal time of administration will depend on the metabolic rate, the rate of excretion, and/or the pharmacokinetic activity of the other therapeutic agent and FAP-binding agent administered. The additional therapeutic agent or FAP-binding agent may be administered first.

Co-administration also does not require that the therapeutic agent be administered to the subject by the same route of administration. In fact, each therapeutic agent may be administered by any suitable route, e.g., parenterally or non-parenterally.

In some embodiments, the FAP-binding agents described herein act synergistically when co-administered with another therapeutic agent. As used herein, "synergistically" refers to a greater than additive therapeutic effect resulting from the combination of at least two agents and over what would otherwise result from administration of each agent alone. For example, a disease or disorder can be treated with a lower dose of one or more agents such that the efficacy of the treatment is increased and side effects are reduced. In such embodiments, the FAP-binding agent and other therapeutic agent may be administered at doses lower than those employed when each agent is used in a monotherapy setting.

In some embodiments, the present technology pertains to chemotherapeutic agents as other therapeutic agents. For example, but not by way of limitation, such combinations of FAP-binding agents of the invention and chemotherapeutic agents may be used to treat cancer, as described elsewhere herein. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa (thiotepa) and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meteredopa), and uretepa (uredpa); ethyleneimine and methylmelamine including altretamine, tritylamine, triethylenephosphoramide, triethylenethiophosphoryl Amines and trimethylolmelamine (trimethlomelamine); polyacetylene (acetogenin) (e.g., bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin; sponge statin (cally statin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (e.g., cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB 1-TM 1); eislobin (eleutherobin); coprinus atrata base (pancratistatin); sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine oxide hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neoentizine (novembichin), benzene mustard cholesterol (phenyleneterester), prednimustine (prednimustine), trofosfamide (trofosfamide), uramustine (uracil); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranirnustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ l and calicheamicin ω l (see, e.g., Agnew, chem. Intl. Ed. Engl.,33:183-186 (1994))); daptomycin (dynemicin), including daptomycin a; bisphosphonates, such as clodronate (clodronate); epothilones (esperamicins); and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin (aclacinomycin), actinomycin (actinomycin), amphenicol (aurramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), karabicin (carabicin), carminomycin (caminomycin), carcinotrophin (carzinophilin), chromomycin (chromomycins), actinomycin D (dactinomycin), daunorubicin (daunorubicin), and daunorubicin Mycin (daunorubicin), ditorexin (detortucin), 6-diazo-5-oxo-L-norleucine, ADRIAMICIN doxorubicin (including morpholino-doxorubicin), cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), maricomycin (marcellomycin), mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), nogamycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), doxycycline (potfiromycin), puromycin (puromycin), quinomycin (quelemycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), stanin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin (denopterin), methotrexate, pteropterin (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamiazine (thiamiprine), thioguanine; pyrimidine analogs such as cyclocytidine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as caridotestosterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitioandrostanol (epitiostanol), mepiquitane (mepiquitazone), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (minoglutethimide), mitotane (mitotane), trostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bessburyl (beslabucil); bisantrene; edatrexate (edatraxate); colchicine (demecolcine); diazaquinone (diaziqutone); iloxanel (elformithine); ammonium etiolate (ellitinium acet) ate); epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; mushroom polysaccharides (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); azofurans (sizofurans); germanium spiroamines (spirogyranium); tenuizonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes) such as T-2 toxin, myxomycin a (veracurin a), bacillus a (roridin a), and serpentin (anguidine)); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (manomostine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa; taxoids (toxoids) (e.g., TAXOL, paclitaxel (paclitaxel) (Bristol-Myers squirb Oncology, Princeton, n.j.)), Albumin engineered nanoparticle formulations of paclitaxel without polyoxyethylated castor oil (Cremophor) (American Pharmaceutical Partners, Schaumberg,111.) and TAXOTERE docetaxel (doxetaxel) (Rhone-Poulenc ror, Antony, France)); chlorambucil; GEMZAR (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (such as, for example, cisplatin (cissplatin), oxaliplatin (oxaliplatin), and carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine); NAVELBINE (vinorelbine); norfloxacin (novantrone);teniposide (teniposide); edatrexate (edatrexate); daunorubicin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (including irinotecan with 5-FU regimens) and leucovorin (leucovorin)); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid (retinic acid); capecitabine (capecitabine); combretastatin (combretastatin); folinic acid (LV); oxaliplatin (oxaliplatin), including oxaliplatin treatment regimen (FOLFOX); lapatinib (typerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib) (T) that reduce cell proliferation ) ) and VEGF-A; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additionally, in some embodiments, the method of treatment further comprises the use of radiation. Additionally, in some embodiments, the method of treatment further comprises the use of photodynamic therapy.

Thus, in some embodiments, the present technology relates to combination therapy using the FAP-binding agents and chemotherapeutic agents. In some embodiments, the present technology relates to administering the FAP-binding agent to a patient being treated with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a DNA intercalating agent, such as, but not limited to, doxorubicin, cisplatin, daunorubicin, and epirubicin. In some embodiments, the DNA intercalating agent is doxorubicin.

In some embodiments, the FAP-binding agent acts synergistically when co-administered with doxorubicin. In some embodiments, the FAP-binding agent acts synergistically when co-administered with doxorubicin for the treatment of a tumor or cancer. For example, co-administration of the FAP-binding agent and doxorubicin may act synergistically to reduce or eliminate the tumor or cancer, or slow the growth and/or progression and/or metastasis of the tumor or cancer. In some embodiments, the combination of the FAP-binding agent and doxorubicin may exhibit improved safety profiles when compared to the agent used alone in the context of monotherapy. In some embodiments, the FAP-binding agent and doxorubicin are administered at a lower dose than the dose employed when the agent is used in the context of monotherapy. In some embodiments, the FAP-binding agent comprises a mutated interferon, such as a mutated IFN α. In some embodiments, the mutant IFN α comprises one or more mutations relative to SEQ ID NO:688 or SEQ ID NO:689 at positions 148, 149 and 153, such as the substitutions M148A, R149A and L153A.

In some embodiments, the present technology relates to combination therapies utilizing one or more immune modulators, such as, but not limited to, agents that modulate immune checkpoints. In some embodiments, the immunomodulator targets one or more of PD-1, PD-L1, and PD-L2. In some embodiments, the immunomodulator is a PD-1 inhibitor. In some embodiments, the immunomodulator is an antibody specific for one or more of PD-1, PD-L1, and PD-L2. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piezolidumab (CT-011, CURE TECH), MK-3475(MERCK), BMS 936559(BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE). In some embodiments, the immunomodulator targets one or more of CD137 or CD 137L. In some embodiments, the immunomodulatory agent is an antibody specific for one or more of CD137 or CD 137L. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, ureluzumab (also known as BMS-663513 and anti-4-1 BB antibodies). In some embodiments, the chimeric proteins or Fc-based chimeric protein complexes of the invention are combined with umeitumumab (optionally with one or more of nivolumab, liriluzab, and umeitumumab) to treat solid tumors and/or B-cell non-hodgkin's lymphoma and/or head and neck cancer and/or multiple myeloma. In some embodiments, the immunomodulatory agent is an agent that targets one or more of: CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R 5A. In some embodiments, the immunomodulator is an antibody specific for one or more of: CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R 5A. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). In some embodiments, the chimeric protein or Fc-based chimeric protein complex of the invention is combined with ipilimumab, optionally with bavituximab (bavituximab), to treat one or more of melanoma, prostate cancer, and lung cancer. In some embodiments, the immunomodulator is targeted to CD 20. In some embodiments, the immunomodulator is an antibody specific for CD 20. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, ofatumumab (genemab), obinutuzumab (obinutuzumab) (GAZYVA), AME-133v (applied MOLECULAR evoution), orelizumab (GENENTECH), TRU-015 (trubiton/EMERGENT), veltuzumab (imum-106).

In some embodiments, the present technology relates to combination therapies using the FAP-binding agents and checkpoint inhibitors. In some embodiments, the present technology relates to administering the FAP-binding agent to a patient being treated with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an agent that targets one or more of PD-1, PD-L1, PD-L2, and CTLA-4 (including any of the anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4 agents described herein). In some embodiments, the checkpoint inhibitor is nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidoclizumab (CT-011, CURE TECH), MK-3475(MERCK), BMS 936559(BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE), ipilimumab (MDX-010, MDX-101, Yvoery, BMS), and tremelimumab (Pfizer). In some embodiments, the checkpoint inhibitor is an antibody directed to PD-L1.

In some embodiments, the FAP-binding agent acts synergistically when co-administered with the anti-PD-L1 antibody. In some embodiments, the FAP-binding agent acts synergistically when co-administered with the anti-PD-L1 antibody for treating a tumor or cancer. For example, co-administration of the FAP-binding agent and the anti-PD-L1 antibody may act synergistically to reduce or eliminate the tumor or cancer, or slow the growth and/or progression and/or metastasis of the tumor or cancer. In some embodiments, the combination of the FAP-binding agent and the anti-PD-L1 antibody may exhibit improved safety profiles when compared to the agent used alone in the context of monotherapy. In some embodiments, the FAP-binding agent and the anti-PD-L1 antibody may be administered at a lower dose than that employed when the agent is used in the context of monotherapy. In some embodiments, the FAP-binding agent comprises a mutated interferon, such as a mutated IFN α. In an illustrative embodiment, the mutated IFN α comprises one or more mutations relative to SEQ ID NO:688 or SEQ ID NO:689 at positions 148, 149 and 153, such as the substitutions M148A, R149A and L153A.

In some embodiments, the present technology relates to combination therapies using the FAP-binding agents and immunosuppressive agents. In some embodiments, the present technology relates to administering the FAP-binding agent to a patient being treated with an immunosuppressive agent. In some embodiments, the immunosuppressive agent is TNF.

In illustrative embodiments, the FAP-binding agent acts synergistically when co-administered with TNF. In an illustrative embodiment, the FAP-binding agent acts synergistically when co-administered with TNF for the treatment of a tumor or cancer. For example, co-administration of the FAP-binding agent and TNF may act synergistically to reduce or eliminate the tumor or cancer, or slow the growth and/or progression and/or metastasis of the tumor or cancer. In some embodiments, the combination of the FAP-binding agent and TNF may exhibit improved safety profiles when compared to the agent used alone in the context of monotherapy. In some embodiments, the FAP-binding agent and TNF may be administered at a lower dose than that employed when the agent is used in the context of monotherapy. In some embodiments, the FAP-binding agent comprises a mutated interferon, such as a mutated IFN α. In illustrative embodiments, the mutated IFNa comprises one or more mutations relative to SEQ ID NO:688 or SEQ ID NO:689 at positions 148, 149 and 153, such as the substitutions M148A, R149A and L153A.

In some embodiments, the FAP-binding agent acts synergistically when used in combination with Chimeric Antigen Receptor (CAR) T cell therapy. In an illustrative embodiment, the FAP-binding agent acts synergistically when used in combination with CAR T cell therapy to treat a tumor or cancer. In one embodiment, the FAP-binding agent acts synergistically when used in combination with CAR T cell therapy to treat a blood-based tumor. In one embodiment, the FAP-binding agent acts synergistically when used in combination with CAR T cell therapy to treat a solid tumor. For example, use of the FAP-binding agent and CAR T cells may act synergistically to reduce or eliminate the tumor or cancer, or slow the growth and/or progression and/or metastasis of the tumor or cancer. In some embodiments, the FAP-binding agents of the present technology induce CAR T cell division. In some embodiments, the FAP-binding agents of the present technology induce CAR T cell proliferation. In some embodiments, the FAP-binding agents of the present technology prevent CAR T cells from failing to respond.

In some embodiments, the CAR T cell therapy comprises T cell therapies that target antigens (e.g., tumor antigens) such as, but not limited to, carbonic anhydrase ix (caix), 5T4, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CS1, CD138, Lewis-Y, L1-CAM, MUC16, ROR-1, IL13Ra2, gp100, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), B Cell Maturation Antigen (BCMA), human papilloma virus type 16E 6(HPV-16E6), CD171, folate receptor alpha (FR-a), GD2, human epidermal growth factor receptor 2(HER2), mesothelin, egfrvlll, Fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), and vascular endothelial growth factor receptor 2(VEGF-R2), as well known in the art for other tumors. Other illustrative tumor antigens include, but are not limited to, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin B, colorectal-associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, T cell receptor/CD 3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-Al2, MAGE-A2 (MAGE-A8678B-2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE family tumor antigens (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21, RCAS1, a-fetoprotein, E-cadherin, a-catenin, 3-and y-catenin, p120ctn, 100 gp Pme1117, AME, NY-catenin, CDSO-27, CDPC-catenin, and BYPD-catenin, Ig idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family tumor antigens, Imp-1, NA, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1 and PD-L2.

Illustrative CAR T cell therapies include, but are not limited to, JCAR014(Juno Therapeutics), JCAR015(Juno Therapeutics), JCAR017(Juno Therapeutics), JCAR018(Juno Therapeutics), JCAR020(Juno Therapeutics), JCAR023(Juno Therapeutics), JCAR024(Juno Therapeutics), CTL019(Novartis), KTE-C19(Kite Pharma), BPX-401 (Bellim Pharmaceuticals), BPX-601 (Bellium Pharmaceuticals), BPX 2121 (Blubbird Bio), CD-19 sleep human cells (Zipamarty), CAR 19 (Belllium Therapeutics), cells 86123 (Green cells), UCC 38 (UC 32), cells developed by Ocotus 1, and cells developed by Ocotus officinalis (Ocotus officinalis).

In some embodiments, the present technology relates to combination therapies utilizing one or more of the chimeric agents described in WO 2013/10779, WO 2015/007536, WO 2015/007520, WO 2015/007542, and WO 2015/007903, the entire contents of which are hereby incorporated by reference in their entirety.

In some embodiments, including but not limited to infectious disease applications, the present technology relates to anti-infective agents as other therapeutic agents. In some embodiments, the anti-infective agent is an antiviral agent, including, but not limited to, Abacavir (Abacavir), Acyclovir (Acyclovir), Adefovir (Adefovir), Amprenavir (Amprenavir), Atazanavir (Atazanavir), Cidofovir (Cidofovir), Darunavir (daunarvir), Delavirdine (Delavirdine), Didanosine (Didanosine), Docosanol (Docosanol), Efavirenz (Efavirenz), elvitevir (Elvitegravir), Emtricitabine (Emtricitabine), entufvirtide (envirtide), etravirdine (etravirtide), Etravirine (Etravirine), Famciclovir (Famciclovir) and Foscarnet (Foscarnet). In some embodiments, the anti-infective agent is an antibacterial agent, including, but not limited to, cephalosporins (cefotaxin, cefuroxime, cefazamide, cefazolin, cefalothin, cefaclor, cefamandole, cefoxitin, cefprozil, cefditoren, cefuroxime, cefaclor, cefuroxime axetil, cefuroxime axetil, cefuroxime; fluoroquinolone antibiotics (ciprofloxacin), levofloxacin (Levaquin), ofloxacin (floxin), gatifloxacin (tequin), moxifloxacin (avelox), and norfloxacin (norflox)); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and deoxycycline); penicillin antibiotics (amoxicillin), ampicillin (ampicillin), penicillin V, dicloxacillin (dicloxacillin), carbenicillin (carbenicillin), vancomycin (vancomycin), and methicillin (methicillin)); a monoamide ring antibiotic (aztreonam); and carbapenem antibiotics (ertapenem), doripenem (doripenem), imipenem (imipenem)/cilastatin (cilastatin), and meropenem (meropenem)). In some embodiments, the anti-infective agent includes antimalarials (e.g., chloroquine (chloroquine), quinine (quinine), mefloquine (mefloquine), primaquine (primaquine), doxycycline (doxycycline), artemether/lumefantrine (lumefantrine), atovaquone (atovaquone)/proguanil (proguanil), and sulfadoxine (sulfadoxine)/pyrimethamine (pyrimethanmine)), metronidazole (metronidazole), tinidazole (tinidazole), ivermectin (ivermectin), pyrantel pamoate (pyrantel pamoate), and albendazole (albendazole).

In some embodiments, including but not limited to autoimmune applications, the other therapeutic agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenal corticosteroids and their synthetic analogs are well known in the art. Examples of corticosteroids that may be used in the present technology include, but are not limited to, hydroxytryptaminolone (hydroxytryptaminolone), alpha-methyl dexamethasone (alpha-methydexamethasone), beta-methyl dexamethasone, beclomethasone dipropionate (beclomethasone dipropionate), betamethasone benzoate (betamethasone benzoate), betamethasone dipropionate, betamethasone valerate, clobetasol valerate (clobetasol valerate), desonide (desonide), desoximetasone (desoxymethythasone), dexamethasone (desoxymethasone), diflorasone diacetate (diflorasone diacetate), diflucortolone (diflucortolone valerate), fludrolone hydrofluoride (diflucortolone), fluocinonide (fludroxylone acetonide), fluocinonide (fluocinonide/fluocinonide), fluocinonide (fluocinonide), fluocinonide (fluocinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cotolone, fluocinolone acetonide, fludrocortisone, diflorasone diacetate, fludrolone acetonide, medrysone, amcinafelon, amcinamide, betamethasone and its corresponding esters, prednisolone chloride, clocortolone (clocotrione), cinolone (clesinolone), dichloropine (dichlorrisone), difluprednate (difluprednate), fluocinolone (fluclone ide), flunisolide (flutolide), fluorometholone (fluorometholone), fluoropolylone (fluperolone), flupredone (fluprednilone), hydrocortisone (hydrocortisone), methylprednisolone (meprednisone), paramethasone (paramethasone), prednisolone (prednisone), prednisone (prednisone), betamethasone dipropionate. (NSAIDs) that may be used in the present technology include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen (ibuprofen), sulindac (fulindac), naproxen (naproxen), ketoprofen (ketoprofen), etofenamate (etofenamate), phenylbutazone (phenylbutazone), and indomethacin (indomethacin). In some embodiments, the immunosuppressive agent can be a cytostatic agent, such as an alkylating agent, an antimetabolite (e.g., thioxathixate, methotrexate), a cytotoxic antibiotic, an antibody (e.g., basiliximab, daclizumab, and muromab), an anti-immunophilin (anti-immunophilin) (e.g., cyclosporine, tacrolimus, sirolimus), interferon, an opioid, a TNF binding protein, a mycophenolate mofetil, and an small biological agent (e.g., fingolimod, myriocin). Other anti-inflammatory agents are described, for example, in U.S. patent No. 4,537,776, the entire contents of which are hereby incorporated by reference.

In some embodiments, FAP-binding agents described herein include modified derivatives, i.e., by covalently linking any type of molecule to the composition such that the covalent linkage does not interfere with the activity of the composition. By way of example, but not limitation, derivatives include compositions modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be made using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

In other embodiments, the FAP-binding agents described herein further comprise a cytotoxic agent, which in illustrative embodiments comprises a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to the compositions described herein.

Thus, the FAP-binding agents described herein may undergo post-translational modifications to add effector moieties, such as chemical linkers; a detectable moiety such as a fluorescent dye, an enzyme, a substrate, a bioluminescent material, a radioactive material, and a chemiluminescent moiety; or a functional moiety such as streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and a radioactive material.

Illustrative cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopterin, 6-thioguanterin, cytarabine, 5-fluorouracil, dacarbazine; alkylating agents such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), cisplatin, and carboplatin (burdine); anthracyclines, including daunorubicin (formerly daunorubicin), doxorubicin (adriamycin), mitorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene; antibiotics, including actinomycin D (dactinomycin/actinomycin D), bleomycin, calicheamicin, mithramycin and Amphenicol (AMC); and antimitotic agents such as vinblastines (vinca alkaloid), vincristine (vincristine), and vinblastine (vinblastine). Other cytotoxic agents include paclitaxel (paclitaxel), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emidine (emetine), etoposide, teniposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), interferons, and mixtures of these cytotoxic agents.

Other cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platinum, paclitaxel, irinotecan, 5-fluorouracil, gemcitabine, folinic acid, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine), molestanes, tyrosine kinase inhibitors, radiation therapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g., IL-12 or IL-2), IL-12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigen-specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD 20, Rituxan, aurilizumab, ofatumumab, DXL625, Rituxan, erbitumumab, and erbitumumab,Or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin and pseudomonas toxin, may be conjugated to these therapeutic agents (e.g., antibodies) to produce cell type specific killing agents (Youle et al, proc. nat ' l acad. sci. usa 77:5483 (1980); gililand et al, proc. nat ' l acad. sci. usa 77:4539 (1980); Krolick et al, proc. nat ' l acad. sci. usa 77:5419 (1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described by golden in U.S. patent No. 6,653,104. Embodiments of the present technology also relate to radioimmunoconjugates in which an alpha or beta particle emitting radionuclide is stably coupled to a FAP binding agent, with or without the use of a complex forming agent. Such radionuclides include beta-emitters such as phosphorus-32, scandium-47, copper-67, gallium-67, yttrium-88, yttrium-90, iodine-125, iodine-131, samarium-153, lutetium-177, rhenium-186, or rhenium-188; and alpha emitters such as astatine-211, lead-212, bismuth-213, or actinium-225.

Illustrative detectable moieties also include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Other illustrative fluorescent materials include, but are not limited to, rhodamine, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Other illustrative chemiluminescent moieties include, but are not limited to, luminol. Other illustrative bioluminescent materials include, but are not limited to, fluorescein and aequorin. Other illustrative radioactive materials include, but are not limited to, iodine-125, carbon-14, sulfur-35, tritium, and phosphorus-32.

Method of treatment

The methods and compositions described herein can be applied to the treatment of various diseases and disorders, including but not limited to cancer, infections, immune disorders, fibrotic diseases, inflammatory diseases or disorders, anemia, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders.

In addition, any of the agents of the invention can be used for the treatment of, or the manufacture of a medicament for the treatment of, a variety of diseases and disorders, including, but not limited to, cancer, infection, immune disorders, inflammatory diseases or disorders, fibrotic diseases, and autoimmune diseases.

In some embodiments, the invention relates to the treatment of patients suffering from or suffering from one or more of the following diseases: chronic granulomatous Disease, osteopetrosis, idiopathic pulmonary fibrosis, Friedreich's ataxia, atopic dermatitis, Chagas Disease, cancer, heart failure, autoimmune diseases, sickle cell Disease, thalassemia, blood loss, transfusion reactions, diabetes, vitamin B12 deficiency, collagen vascular Disease, schwakman syndrome, thrombocytopenic purpura, celiac Disease, endocrine deficient states such as hypothyroidism or edison's Disease, autoimmune diseases such as Crohn's Disease, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerative colitis immune disorders such as eosinophilic fasciitis, low immunoglobulin leukemia or tumor/cancer, graft-versus-host Disease, leukemia, and autoimmune diseases, Non-hematologic syndromes (e.g., Down's syndrome, Dubowwitz syndrome, seeker syndrome, felter syndrome, hemolytic uremic syndrome, myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria, myelofibromas, pancytopenia, pure red blood cell aplasia, Schoenlein-Henoch purpura, malaria, protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis, hypometabolic state, and congestive heart failure).

In some embodiments, the invention relates to the treatment of patients suffering from or suffering from one or more of the following diseases: chronic granulomatous disease, osteopetrosis, idiopathic pulmonary fibrosis, friedrich's ataxia, atopic dermatitis, chagas disease, mycobacterial infection, cancer, scleroderma, hepatitis c, septic shock and rheumatoid arthritis.

In some embodiments, the present technology relates to the treatment of cancer or patients with cancer. As used herein, cancer refers to any uncontrolled cell growth that may interfere with the normal function of body organs and systems, and includes both primary and metastatic tumors. A primary tumor or cancer migrates from its original location and the seed in a vital organ may eventually lead to death of the subject by a decline in function of the affected organ. Metastasis is a cancer cell or group of cancer cells that appear at a location remote from the primary tumor due to dissemination of the cancer cells from the primary tumor to other parts of the body. The metastasis may eventually lead to death of the subject. For example, cancer may include benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

Illustrative cancers that may be treated include, but are not limited to, leukemias (including, e.g., acute myelogenous, acute lymphoblastic, chronic myelogenous, chronic lymphocytic and hairy cells), lymphomas and myelomas (including, e.g., hodgkin's and non-hodgkin's lymphomas, light chain, non-secretory, MGUS and plasmacytomas), and central nervous system cancers (including, e.g., brain (e.g., gliomas (e.g., astrocytomas, oligogliomas and ependymomas), meningiomas, pituitary adenomas and neuromas, and spinal cord tumors (e.g., meningiomas and fibroneuromas).

Illustrative cancers that may be treated include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; liver cancer; hepatoma; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphomas (NHLs), Small Lymphocytic (SL) NHLs, intermediate grade/follicular NHLs, intermediate grade diffuse NHLs, high grade immunoblastic NHLs, high grade lymphoblastic NHLs, high grade nonnucleated NHLs, massive NHLs, mantle cell lymphomas, AIDS-related lymphomas, and Waldenstrom's macroglobulinemia, Chronic Lymphocytic Leukemia (CLLs), Acute Lymphoblastic Leukemia (ALL), Acute Myelogenous Leukemia (AML), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular hyperplasia, edema associated with mole hamartoma (e.g., edema associated with brain tumors) and meigs' syndrome.

In some embodiments, the present technology provides FAP-binding agents for use in the treatment of cancer that are part of a chimera or Fc-based chimeric protein complex that further comprises a modified signaling agent. In some embodiments, FAP-binding agents of the present technology significantly reduce and/or eliminate tumors. In some embodiments, the FAP-binding agents of the invention significantly reduce and/or eliminate tumors when administered to a subject in combination with other anti-cancer agents, such as chemotherapeutic agents, checkpoint inhibitors, and immunosuppressive agents. In some embodiments, the combination of the FAP-binding agent and the other anti-cancer agent synergistically reduces tumor size and/or eliminates tumor cells.

In some embodiments, the present technology relates to combination cancer therapy with FAP-binding agents that are part of a chimera or Fc-based chimeric protein complex comprising one or more targeting moieties and one or more modified signaling agents. Thus, the present technology provides chimeric or fusion proteins or Fc-based chimeric protein complexes comprising, for example, a targeting moiety for FAP and one or more signaling agents, and their use in combination with anti-cancer agents.

For example, in some embodiments, the present technology pertains to cancer combination therapies involving chimeras or Fc-based chimeric protein complexes of FAP binding agents described herein with modified signaling agents (including but not limited to mutated human interferons, such as IFN α, including human interferon α 2).

In other embodiments, the FAP-binding agents of the invention are part of a chimera or Fc-based chimeric protein complex that comprises multiple targeting moieties and thus exists in a bispecific or trispecific form. For example, in some embodiments, the present technology pertains to cancer combination therapies involving FAP-binding agents chimeric or Fc-based chimeric protein complexes with checkpoint inhibitor binding agents described herein (e.g., anti-PD-L1, anti-PD-1, anti-PD-L2, or anti-CTLA) and modified signaling agents (including but not limited to mutated human interferons, such as IFN α, including human interferon α 2).

In some embodiments, the present technology relates to the treatment of patients suffering from microbial and/or chronic infections, or patients suffering from microbial and/or chronic infections. Illustrative infections include, but are not limited to, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus (Epstein-Barr virus) or parvovirus, T-cell leukemia virus, bacterial overgrowth syndrome, fungal or parasitic infections.

In some embodiments, the compositions of the invention are used to treat or prevent one or more inflammatory diseases or conditions, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, glycerol kinase deficiency, Familial Eosinophilia (FE), autosomal recessive spasmodic ataxia, inflammatory disorders of the throat; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.

In some embodiments, the present technology is applicable to the treatment of autoimmune diseases and/or neurodegenerative diseases.

In some embodiments, compositions of the invention are used to treat or prevent one or more conditions characterized by undesirable CTL activity and/or conditions characterized by high levels of cell death. For example, in some embodiments, compositions of the invention are used to treat or prevent one or more disorders associated with uncontrolled or overactive immune responses.

In some embodiments, the compositions of the invention are used to treat or prevent one or more autoimmune and/or neurodegenerative diseases or disorders, such as MS, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, Guillain-Barre syndrome (Guillain-Barre syndrome), scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, rasmassen's encephalitis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, nieer's syndrome; transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In some embodiments, the present technology is used to treat or prevent various autoimmune and/or neurodegenerative diseases. In some embodiments, the autoimmune Disease and/or neurodegenerative Disease is selected from MS, alzheimer's Disease (including but not limited to early onset alzheimer's Disease, late onset alzheimer's Disease, and Familial Alzheimer's Disease (FAD), parkinson's Disease, and parkinson's Disease (including but not limited to idiopathic parkinson's Disease, vascular parkinson's Disease, drug-induced parkinson's Disease, lewy body dementia, hereditary parkinson's Disease, juvenile parkinson's Disease), huntington's Disease, amyotrophic lateral sclerosis (ALS, including but not limited to sporadic ALS, familial ALS, western pacific ALS, juvenile ALS, west-type malaya Disease).

In one embodiment, the invention provides a method for treating or preventing one or more liver disorders selected from the group consisting of viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, alcoholic liver disease, fatty liver disease, steatosis, steatohepatitis, non-alcoholic fatty liver disease, drug-induced liver disease, cirrhosis, fibrosis, liver failure, drug-induced liver failure, metabolic syndrome, hepatocellular carcinoma, cholangiocarcinoma, primary biliary cirrhosis (primary biliary cholangitis), micro-cholangiocarcinoma, Gilbert's syndrome, jaundice, and any other hepatotoxicity-related signs. In some embodiments, the present invention provides methods for treating or preventing liver fibrosis. In some embodiments, the present invention provides methods for treating or preventing Primary Sclerosing Cholangitis (PSC), chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis c infection, alcoholic liver disease, liver injury (optionally caused by progressive fibrosis and liver fibrosis). In some embodiments, the present invention provides methods for treating or preventing nonalcoholic steatohepatitis (NASH). In some embodiments, the present invention provides methods of reducing or preventing fibrosis. In some embodiments, the present invention provides methods of reducing or preventing cirrhosis of the liver. In some embodiments, the present invention provides methods of reducing or preventing hepatocellular carcinoma.

In some embodiments, the invention provides a method of treating or preventing a fibrotic disease, optionally selected from liver fibrosis, lung fibrosis, Primary Sclerosing Cholangitis (PSC), chronic liver disease, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis c infection, alcoholic liver disease, liver injury, cirrhosis, and myelodysplastic syndrome.

In various embodiments, the present invention provides methods for treating or preventing cardiovascular disease, such as diseases or conditions affecting the heart and blood vessels, including, but not limited to, Coronary Heart Disease (CHD), cerebrovascular disease (CVD), aortic valve stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), Transient Ischemic Attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, vascular disease, and/or congestive heart failure. In various embodiments, the present invention provides methods for treating or preventing cardiovascular diseases involving inflammation.

In various embodiments, the present invention provides methods for treating or preventing one or more respiratory diseases, such as asthma, Chronic Obstructive Pulmonary Disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergy, respiratory obstruction, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, Hantavirus lung syndrome (HPS), lueffler's syndrome, Goodpasture's syndrome, pleuritis, pneumonia, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory syncytial virus infection, and other respiratory diseases.

In some embodiments, the methods of the present technology can be used to treat a human subject. In some embodiments, the human is a child. In other embodiments, the human is an adult. In other embodiments, the human is an elderly human. In other embodiments, the human may be referred to as a patient. In some embodiments, the human is a female. In some embodiments, the human is a male.

In certain embodiments, the age of the human is in the range of about 1 to about 18 months of age, about 18 to about 36 months of age, about 1 to about 5 years of age, about 5 to about 10 years of age, about 10 to about 15 years of age, about 15 to about 20 years of age, about 20 to about 25 years of age, about 25 to about 30 years of age, about 30 to about 35 years of age, about 35 to about 40 years of age, about 40 to about 45 years of age, about 45 to about 50 years of age, about 50 to about 55 years of age, about 55 to about 60 years of age, about 60 to about 65 years of age, about 65 to about 70 years of age, about 70 to about 75 years of age, about 75 to about 80 years of age, about 80 to about 85 years of age, about 85 to about 90 years of age, about 90 to about 95 years of age, or about 95 to about 100 years of age. In some embodiments, the human is over 30 years of age.

Immunomodulation

In some embodiments, the compositions of the invention are capable of or useful in methods of immunomodulation. For example, in some embodiments, the treatment methods of the invention may comprise immunomodulation as described herein. In some embodiments, the immunomodulation comprises IFN signaling in the context of Dendritic Cells (DCs), including modified IFN signaling.

In some embodiments, multispecific FAP-binding agents are provided. In some embodiments, such multispecific FAP-binding agents of the present technology recognize and bind to FAP and one or more antigens found on one or more immune cells, which may include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, T helper cells, natural killer cells), B lymphocytes, plasma cells, dendritic cells, or a subset thereof. In some embodiments, the FAP-binding agent specifically binds to an antigen of interest and is effective to recruit, directly or indirectly, one or more immune cells.

In some embodiments, the FAP-binding agent specifically binds to an antigen of interest and is effective to directly or indirectly recruit one or more immune cells to cause immune suppression, e.g., the FAP-binding agent directly or indirectly recruits an immune suppressive immune cell. In some embodiments, the immunosuppressive immune cell is a regulatory T cell (or "Treg," as used herein, refers to a subpopulation of T cells that modulate the immune system, eliminate autoimmune diseases, maintain tolerance to self-antigens, and block anti-tumor immune responses). Other immunosuppressive immune cells include bone marrow suppressor cells (or "MSCs," as used herein, refers to a heterogeneous population of cells defined by their bone marrow origin, immature state, and ability to effectively suppress T cell responses); tumor-associated neutrophils (or "TAN", as used herein, refers to a subset of neutrophils that are capable of suppressing an immune response); tumor-associated macrophages (or "TAMs," as used herein, refers to a subset of macrophages that can reduce the immune response), M2 macrophages, and/or tumor-inducing mast cells (as used herein, refers to a subset of bone marrow-derived long-lived heterogeneous cell populations). In addition, immunosuppressive immune cells include Th2 cells and Th17 cells. In addition, immunosuppressive immune cells include immune cells, such as CD4+ and/or CD8+ T cells, that express one or more checkpoint inhibitory receptors (e.g., receptors expressed on immune cells that prevent or inhibit an uncontrolled immune response, including CTLA-4, B7-H3, B7-H4, TIM-3). See, Stagg, J.et al, immunological adaptive approach in triple-negative breakthrough cancer. Ther Adv Med Oncol. (2013)5(3): 169-.

In some embodiments, the FAP-binding agent stimulates the regulation of T cell (Treg) proliferation. Treg cells are characterized by the expression of Foxp3 (forkhead box p3) transcription factor. The majority of Treg cells are CD4+ and CD25+ and can be considered a subset of helper T cells, but a small population may be CD8 +. Thus, the immune response to be modulated by the methods of the present technology may comprise inducing the proliferation of Treg cells, optionally in response to an antigen. Thus, the methods can comprise administering to a subject a composition comprising the antigen, wherein the antigen is associated with a binding agent having affinity for FAP. The antigen may be administered with an adjuvant that promotes the proliferation of Treg cells.

As long as this method involves stimulation of the proliferation and differentiation of Treg cells in response to a particular antigen, it can be considered as a method of stimulating an immune response. However, whereas Treg cells are capable of otherwise modulating the response of other cells of the immune system to an antigen, e.g., suppressing or suppressing their activity, the effect on the immune system as a whole may modulate (e.g., suppress or suppress) the response to that antigen. Thus, the methods of this aspect of the present technology may also be considered to be methods of modulating (e.g., inhibiting or suppressing) an immune response to an antigen.

In some embodiments, the methods therapeutically or prophylactically inhibit or suppress an undesirable immune response to a particular antigen, even in a subject that has pre-existing immunity or sustained immune response to that antigen. This may be particularly useful, for example, in the treatment of autoimmune diseases.

Under certain conditions, it is also possible to tolerize a subject to a particular antigen by targeting the antigen to FAP-expressing antigen presenting cells. The present technology thus provides a method for inducing tolerance to an antigen in a subject, the method comprising administering to the subject a composition comprising the antigen, wherein the antigen is associated with a binding agent having affinity for FAP, and wherein the antigen is administered in the absence of an adjuvant. In this context, tolerance typically involves the depletion of immune cells that would otherwise be able to respond to the antigen or the induction of a sustained reduction in the response of such immune cells to the antigen.

It may be particularly desirable to generate a Treg response against an antigen that exhibits or is at risk of developing an undesirable immune response in a subject. For example, it may be an autoantigen against which an immune response occurs in an autoimmune disease. Examples of autoimmune diseases for which specific antigens have been identified as potentially important for pathogenesis include multiple sclerosis (myelin basic protein), insulin dependent diabetes (glutamate decarboxylase), insulin resistant diabetes (insulin receptor), celiac disease (prolamin), bullous pemphigoid (type ii collagen), autoimmune hemolytic anemia (Rh protein), autoimmune thrombocytopenia (GpIlb/ila), myasthenia gravis (acetylcholine receptor), graves 'disease (thyroid stimulating hormone receptor), glomerulonephritis such as goodpasture's disease (α 3(IV) NC1 collagen), and pernicious anemia (intrinsic factor). Alternatively, the target antigen may be an exogenous antigen that stimulates a response that also elicits destruction of host tissue. For example, acute rheumatic fever is caused by an antibody response to streptococcal antigens that cross-react with cardiomyocyte antigens. These antigens, or specific fragments or epitopes thereof, may therefore be suitable antigens for use in the present technology.

In some embodiments, the agents of the invention or methods of using these agents disrupt FAP signaling (e.g., via neutralization of FAP), e.g., by reducing or inhibiting binding of FAP to its ligand. Some autoimmune diseases are characterized by abnormally high levels of cell death, and it is thought that the immune response to self-antigens associated with these cells may contribute to the pathogenesis of these conditions. Thus FAP antagonists can be used to prevent FAP from binding to exposed ligands in dead and dying cells (e.g., those undergoing immunogenic cell death), and can thus inhibit or prevent stimulation of immune responses to these antigens.

In some embodiments, the agents of the present technology or methods of using these agents reduce or suppress autoreactive T cells. In some embodiments, the multispecific FAP-binding agent causes such immune suppression, optionally by interferon signaling, in the case of a chimera or Fc-based chimeric protein complex. In some embodiments, the multispecific FAP-binding agent stimulation may suppress PD-L1 or PD-L2 signaling and/or expression of autoreactive T cells. In some embodiments, the FAP-binding agent causes this immune suppression, optionally by interferon signaling, in the case of a chimera or Fc-based chimeric protein complex. In some embodiments, the FAP-binding agent stimulation may suppress PD-L1 or PD-L2 signaling and/or expression of autoreactive T cells.

In some embodiments, the methods of the invention comprise modulating the ratio of regulatory T cells to effector T cells to facilitate immune suppression, e.g., to treat an autoimmune disease. For example, in some embodiments, the methods of the invention reduce and/or inhibit one or more of: cytotoxic T cells; effector memory T cells; central memory T cells; CD8+ stem cell memory effector cells; TH1 effector T cells; TH2 effector T cells; TH9 effector T cells; TH17 effector T cells. For example, in some embodiments, the methods of the invention increase and/or stimulate one or more of: CD4+ CD25+ FOXP3+ regulatory T cells, CD4+ CD25+ regulatory T cells, CD4+ CD 25-regulatory T cells, CD4+ CD25 high regulatory T cells, TIM-3+ PD-1+ regulatory T cells, lymphocyte activator gene-3 (LAG-3) 'regulatory T cells, CTLA-4/CD152+ regulatory T cells, neuropilin-1 (Nrp-1) + -regulatory T cells, CCR4+ CCR8+ regulatory T cells, CD62L (L-selectin)' regulatory T cells, CD45 'RB low regulatory T cells, CD127 low regulatory T cells, RC32/GARP + regulatory T cells, CD39+ regulatory T cells, GITR + regulatory T cells, LAP' regulatory T cells, 1B11+ regulatory T cells, BTLA + regulatory T cells, T cells of type 1 (Tr 1) T cells, T cells of type 2 (Th 2) helper T cells, and NKT cells, CD8+ regulatory T cells, CD8+ CD 28-regulatory T cells, and/or regulatory T cells that secrete IL-10, IL-35, TGF-3, TNF-a, galectin-1, IFN-y, and/or MCP 1.

In some embodiments, the methods of the invention facilitate an immunosuppressive signal over an immunostimulatory signal. In some embodiments, the methods of the invention allow for the reversal or suppression of immune activation or co-stimulatory signals. In some embodiments, the methods of the invention allow for the provision of an immunosuppressive signal. For example, in some embodiments, the agents and methods of the invention to reduce the effect on immunostimulatory signals are not limited to one or more of the following: 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS ligand, CD137 ligand, and TL 1A. Furthermore, in some embodiments, the agents of the invention and methods of increasing the effect on immunosuppressive signals are not limited to one or more of the following: CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7.

Medicine box

The present technology also provides kits for administering any of the FAP-binding agents described herein (e.g., with or without other therapeutic agents). The kit is a combination of materials or components that includes at least one pharmaceutical composition of the invention described herein. Thus, in some embodiments, the kit contains at least one pharmaceutical composition described herein.

The exact nature of the components to be disposed in the kit will depend on their intended purpose. In one embodiment, the kit is configured for the purpose of treating a human subject.

Instructions for use may be included in the kit. Instructions for use typically include tangible representations that describe the techniques to be employed in using the components of the kit to achieve a desired therapeutic result, such as in the treatment of cancer. Optionally, the kit also contains other useful components readily apparent to those skilled in the art, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings or other useful accessories.

The materials and components assembled in the kit may be provided to the practitioner for storage in any convenient and suitable manner that maintains their operability and utility. For example, these components may be provided at room temperature, refrigeration temperature, or freezing temperature. These components are typically contained in a suitable packaging material. In some embodiments, the packaging material is constructed by well-known methods, preferably to provide a sterile, non-contaminating environment. The packaging material may have an external label indicating the contents and/or purpose of the kit and/or its components.

Definition of

As used herein, "a/an" or "the" may mean one or more than one.

Furthermore, the term "about" when used in conjunction with a numerical indication of a reference means that the referenced numerical indication adds or subtracts up to 10% of the referenced numerical indication. For example, the language "about 50" covers the range of 45 to 55.

As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount such that a disease or disorder or one or more signs or symptoms associated with a disease or disorder is prevented or reduced. In the case of therapeutic or prophylactic use, the amount of the composition administered to a subject will depend on the extent, type and severity of the disease, as well as the characteristics of the individual (such as general health, age, sex, weight and drug resistance). The skilled person will be able to determine the appropriate dosage in view of these and other factors. The compositions may also be administered in combination with one or more other therapeutic compounds. In the methods described herein, the therapeutic compound may be administered to a subject suffering from one or more signs or symptoms of a disease or disorder.

As used herein, a property is "reduced" if the readout of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be appreciated by one of ordinary skill in the art, in some embodiments, the activity is reduced and some downstream readouts will be reduced but other downstream readouts may be increased.

Conversely, an activity is "increased" if the readout of activity and/or effect is increased by a significant amount, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold in the presence of an agent or stimulus relative to the absence of such agent or stimulus.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "comprise," and variations thereof, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the term "may" and variations thereof are intended to be non-limiting, such that a listing that an embodiment may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

Although the open-ended term "comprising" is used herein as a term such as comprising, containing, or having synonyms for describing and claiming the present technology, the present technology or embodiments thereof may alternatively be described using alternative terms such as "consisting of … …" or "consisting essentially of … …".

As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

As used herein, a "therapeutically effective amount" or a "pharmacologically effective dose" of a compound refers to the level of the compound at which at least the physiological effects of a disease or disorder are ameliorated. Therapeutic benefit also includes interrupting or slowing the progression of the underlying disease or condition, regardless of whether an improvement is achieved. A therapeutically effective amount may be administered in one or more administrations. The amount of compound that constitutes a therapeutically effective amount will vary depending on the compound, the condition and its severity and the general health, age, sex, weight and tolerance of the subject to be treated, but can be routinely determined by one of ordinary skill in the art.

Effective amount, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining LD50 (the dose lethal to about 50% of the population) and ED50 (the dose therapeutically effective in about 50% of the population). The dosage may vary depending on the dosage form used and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED 50. In some embodiments, compositions and methods that exhibit a greater therapeutic index are preferred. The therapeutically effective dose can be initially estimated by in vitro assays, including, for example, cell culture assays. In addition, the dose may be formulated in animal models to achieve a circulating plasma concentration range that includes IC50 as determined in cell culture or in an appropriate animal model. The content of the described composition in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect.

In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will cause a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes interrupting or slowing the progression of the underlying disease or condition, regardless of whether an improvement is achieved.

As used herein, "method of treatment" is equally applicable to the use of a composition for the treatment of a disease or condition described herein and/or to one or more uses of a composition for the manufacture of a medicament for the treatment of a disease or condition described herein.

Examples

The term actaferon (afn) is occasionally used herein to refer to the chimeric proteins described herein (detailed information on the form of the chimeric protein is provided in the examples).

Example 1. humanFAP Generation of VHH libraries

Llamas were injected subcutaneously at day 0, day 7, day 14, day 21, day 28, and day 35, with about 200 μ g of recombinant human FAP α extracellular domain per injection. On day 40, approximately 100ml of anticoagulated blood was collected from llamas for lymphocyte preparation. VHH libraries were constructed from these lymphocytes to screen for the presence of antigen-specific VHH. The library was subjected to 3 rounds of panning on solid phase coated antigen (100. mu.g/ml, 100mM NaHCO3 pH 8.2). Based on the sequence data of positive colonies, 96 different full-length VHHs were distinguished, which belong to 13 different sets of CDR3 (see Table 6, SEQ ID Nos: 2-42, 46-86 and 837-850).

Example 2: human FAP VHH characterization

An expression vector encoding 96 human FAP-binding VHH from example 1 (pMECS) was transformed into WK6 cells. After overnight IPTG stimulation, VHH (with C-terminal His-tag) was expressed in periplasmic extracts. These extracts were used in FACS binding assays: HEK293T cells were transiently transfected with a plasmid encoding human FAP (pMET7 FLAG-hfp), a plasmid encoding mouse FAP (pMET7 FLAG-mfp) or an empty vector (MOCK) as a control. Two days after transfection, cells were resuspended and incubated in FACS buffer (PBS +0.5mM EDTA + 3% FBS) with 1/5 diluted periplasmic extract. VHH binding was detected using FITC-conjugated anti-His Ab (GENSCRIPT). Samples were collected using a macSQuant X instrument (MILTENYI BIOTEC) and analyzed using FlowLogic software (MILTENYI BIOTEC). The data are summarized in fig. 1.

The binding of the 11 selected VHHs to human, mouse and cynomolgus FAP as described above was studied in more detail. Briefly, HEK293T cells transfected in a different manner were incubated with serially diluted purified VHH based on metal affinity for two hours. Binding was detected in FACS using FITC-conjugated anti-His Ab (Genscript). Surprisingly, VHH is cross-reactive to humans, mice and cynomolgus monkeys. Data showing cross-reactivity of VHH with human, mouse and cynomolgus FAP are summarized in figure 2. Data for 2PE14 is shown in fig. 2A, data for 2PE17 is shown in fig. 2B, data for 2PE36 is shown in fig. 2C, data for 2PE40 is shown in fig. 2D, data for 2PE42 is shown in fig. 2E, data for 2PE44 is shown in fig. 2F, data for 3PE12 is shown in fig. 2G, data for 3PE42 is shown in fig. 2H, data for 3PE57 is shown in fig. 2I, data for 3PE93 is shown in fig. 2J, and data for 3PE94 is shown in fig. 2K.

Example 3: human FAP ActaFeron (AFN)

Based on affinity for human, mouse and cynomolgus FAP, three FAP VHHs were selected for evaluation in the context of "knob-in-hole Fc' AFN of heterodimers: 2PE14, 3PE12 and 3PE 42. To this end, the FAP VHH sequence was fused via a flexible 20 × GGS linker and in pcdna3.4 expression vector to a human IgG1 Fc sequence containing L234A _ L235A _ K322Q effector mutations and "pore" modifications Y349C _ T366S _ L368A _ Y407V (see sequence below). The second AFN partner (also cloned into pcdna3.4 vector), consisting of a fusion between the human IgG1 Fc sequence containing L234A _ L235A _ K322Q effector mutation and the "knob" modification S354C _ T366W and the hIFNa2 sequence with AFN mutation R149A and O-glycosylation mutation T106E.

To generate the heterodimeric "knob into hole" AFN, a combination of the two plasmids was transfected into ExpiCHO cells (thermofibres) according to the manufacturer's instructions. Seven days after transfection, recombinant proteins were purified using a protein a rotating plate (thermafipher), quantified, and purity tested using SDS-PAGE.

The biological activity of the resulting AFN (2PE14-AFN, 3PE12-AFN and 3PE42-AFN) was measured on parental HL116 cells (IFN-responsive cell lines stably transfected with a p6-16 luciferase reporter) and derived stably transfected HL116-hFAP cells. Cells were seeded overnight and stimulated with serial dilutions of FAP VHH AFN for 6 hours. Luciferase activity was measured on an EnSight multimode microplate reader (Perkin Elmer). The data in figure 3 clearly show that FAP targets lead to IFN-like signaling in HL 116-hfp cells, while hardly any reporter activation was observed in the parental HL116 cells. Notably, HL116 and HL 116-hfp cells were equally sensitive to wild-type IFNa 2.

Structure of 2PE14 AFN:

·hFAP VHH 2PE14-20*GGS-hIgG1 Fc_L234A_L235A_K322Q_Y349C_T366S_L368A_ Y407V

amino acid sequence of 2PE14 AFN (amino acid sequence of hfp VHH 2PE14 is shown in bold letters, amino acid sequence of 20 × GGS linker is shown in italic letters, and amino acid sequence of hIgG1Fc _ L234A _ L235A _ K322Q _ Y349C _ T366S _ L368A _ Y407V is shown in underlined letters):

structure of 3PE12 AFN:

·hFAP VHH 3PE12-20*GGS-hIgG1 Fc_L234A_L235A_K322Q_Y349C_T366S_L368A_ Y407V

amino acid sequence of 3PE12 AFN (amino acid sequence of hfp VHH 3PE12 is shown in bold letters, amino acid sequence of 20 × GGS linker is shown in italic letters, and amino acid sequence of hI ggg 1Fc _ L234A _ L235A _ K322Q _ Y349C _ T366S _ L368A _ Y407V is shown in underlined letters):

structure of 3PE42 AFN:

·hFAP VHH 3PE42-20*GGS-hIgG1 Fc_L234A_L235A_K322Q_Y349C_T366S_L368A_ Y407V

amino acid sequence of 3PE12 AFN (amino acid sequence of hfp VHH 3PE42 is shown in bold letters, amino acid sequence of 20 × GGS linker is shown in italic letters, and amino acid sequence of hIgG1Fc _ L234A _ L235A _ K322Q _ Y349C _ T366S _ L368A _ Y407V is shown in underlined letters):

·hIgG1 Fc_L234A_L235A_K322Q_S354C_T366W-20*GGS-hIFNa2_T106E_R149A

example 4: humanization of FAP VHH sequences

In this example, the sequences of the two FAP VHH 2PE14 and 3PE42 are humanized. Humanization was evaluated using four "opt" variants (sequences P-1902 to P-1905) containing the following combinations of mutations: Q1D, Q5V, P60A, a74S, S82N, K83R, and Q108L. In addition, both VHHs contained deamidation sites in CDR1 (N32), while VHH 3PE42 also contained an additional oxidation motif in CDR1 (M31). The instability of both sequences was eliminated by a single mutation to any other amino acid (except C or P; sequences P-1906 to P-1922 for deamidation, and P-1924 to P-1941 for oxidation motifs). Sequences ordered by Twist Biosciences were cloned in pET28 vector with C-terminal His-tag and transformed into BL21 cells. VHH was expressed after overnight IPTG stimulation and purified from periplasmic extracts using hispu Cobalt rotor Plates (ThermoFisher) according to the manufacturer's guidelines. The affinity of the resulting VHH for FAP was measured on an Octet RED96 instrument (ForteBio) using the biolayer interferometry (BLI) technique.

Briefly, recombinant FAP protein (BioLegend) was biotinylated using IP with an antibody biotinylation kit (Pierce) and used to load streptavidin sensors (ForteBio). The association and dissociation of FAP VHH was monitored at three concentrations (10, 5 and 2.5nM) and used to calculate association and dissociation constants and hence affinity. The results for FAP VHH variants are summarized in table 7. Preferred mutations of N32 include F, Q, R and W. Preferred mutations for M31 include A, D, K, L, N, Q, R, S and W.

Table 7:

the sequence is as follows:

·P-1901：2PE14 QVQLQESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1045)

p-1902: 2PE14_ opt1(Q1D _ Q5V _ A74S _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNKNTVYLQMSSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1046)

P-1904: 2PE14_ opt3(Q1D _ Q5V _ A74S _ S82N _ K83R _ Q108L; bold letters indicate mutations)VQLESGGGLVQPGGSLRLSCAASGSTFSINAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNKNTVYLQMSLPEDTAVYYCNLWPPRASPGGRVYWGQGTVTVSS(SEQ ID NO:1048)

P-1906: 2PE14_ N32A (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1050)

P-1907: 2PE14_ N32D (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1051)

P-1908: 2PE14_ N32E (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1052)

P-1909: 2PE14_ N32F (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSI AVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1053)

P-1910: 2PE14_ N32G (bold letters indicate mutations)

·QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1054)

P-1911: 2PE14_ N32H (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1055)

P-1912: 2PE14_ N32I (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1056)

P-1913: 2PE14_ N32K (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1057)

P-1914: 2PE14_ N32L (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1058)

P-1915: 2PE14_ N32P (bold letters indicate mutations) QVQLQESGGGLVQPGGSLRLSCAASGSTFSIAVAWYRQAPGKRRELVAGISGGGVTNYPDSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNLWPPRASPGGRVYWGQGTQVTVSS(SEQ ID NO:1059)