阅读说明：本技术 用于治疗癌症的、lif抑制剂和pd-1轴抑制剂的组合 (Combination of LIF inhibitors and PD-1 axis inhibitors for the treatment of cancer ) 是由 J·塞奥内苏亚雷斯 J·阿尼多富尔盖拉 J·弗兰森 R·M·哈勒特 M·帕斯夸尔加西亚 E 于 2019-04-11 设计创作，主要内容包括：本文描述了使用白血病抑制因子(LIF)结合多肽和PD-1轴抑制剂的组合治疗癌症的方法。(Described herein are methods of treating cancer using a combination of Leukemia Inhibitory Factor (LIF) binding polypeptides and PD-1 axis inhibitors.)

1. Use of a Leukemia Inhibitory Factor (LIF) -binding antibody in combination with a PD-1, PDL-1 or PDL-2 signaling inhibitor to treat cancer in an individual, wherein the LIF-binding antibody comprises:

a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and

f) Immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

2. The use of claim 1, wherein the LIF-binding antibody comprises:

a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4;

c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6;

d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

3. The use of claim 1, wherein the LIF-binding antibody comprises:

a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5;

c) Immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7;

d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

4. The use of any one of claims 1-3, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations.

5. The use of any one of claims 1-3, wherein the LIF-binding antibody and the PD-1, PDL-1, or PDL-2 signaling inhibitor are administered to the individual in the same formulation.

6. The use of any one of claims 1-4, wherein the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual.

7. The use of any one of claims 1-4, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to administration of the LIF-binding antibody to the individual.

8. The use of any one of claims 1-4, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual.

9. The use of any one of claims 1-8, wherein the LIF-binding antibody is humanized.

10. The use of any one of claims 1-9, wherein the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

11. The use of claim 10, wherein the VH sequence is identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

12. The use of any one of claims 1 to 11, wherein the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic ductal adenocarcinoma, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, non-small cell lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

13. The use of claim 12, wherein the cancer comprises non-small cell lung cancer.

14. The use of claim 12, wherein the cancer comprises pancreatic ductal adenocarcinoma.

15. The use of any one of claims 1 to 14, wherein prior treatment of the cancer with a checkpoint inhibitor was unsuccessful.

16. The use of any one of claims 1-15, wherein prior treatment of the cancer with a LIF-binding antibody was unsuccessful.

17. The use of claim 15 or 16, wherein the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signalling inhibitor.

18. The use of any one of claims 1 to 17, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is an antibody or fragment thereof that binds to PD-1.

19. Use of a Leukemia Inhibitory Factor (LIF) -binding antibody in combination with a PD-1, PDL-1, or PDL-2 signaling inhibitor for treating cancer in an individual, wherein the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

20. The use of claim 19, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations.

21. The use of claim 19, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation.

22. The use of claim 19 or 20, wherein the LIF-binding antibody is administered to the individual prior to administering the inhibitor of PD-1, PDL-1 or PDL-2 signaling to the individual.

23. The use of claim 19 or 20, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to the administration of the LIF binding antibody to the individual.

24. The use of any one of claims 19-21, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual at the same time as the LIF-binding antibody is administered to the individual.

25. The use of any one of claims 19-24, wherein the LIF binding antibody is humanized.

26. The use of any one of claims 19 to 25, wherein the VH sequence is identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

27. The use of any one of claims 19-26, wherein the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic ductal adenocarcinoma, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, non-small cell lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

28. The use of claim 27, wherein the cancer comprises non-small cell lung cancer.

29. The use of claim 27, wherein the cancer comprises pancreatic ductal adenocarcinoma.

30. The use of any one of claims 19-29, wherein prior treatment of the cancer with a checkpoint inhibitor was unsuccessful.

31. The use of any one of claims 19-30, wherein prior treatment of the cancer with a LIF-binding antibody was unsuccessful.

32. The use of claim 30 or 31, wherein the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signalling inhibitor.

33. The use of any one of claims 19 to 32, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is an antibody or fragment thereof that binds to PD-1.

34. A method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

an immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 9 or 10;

immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 11 or 12; and

an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and

b) PD-1, PDL-1 or PDL-2 signaling inhibitors.

35. The method of claim 34, wherein the LIF-binding antibody comprises:

a) Immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4;

c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6;

d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

36. The method of claim 34, wherein the LIF-binding antibody comprises:

a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5;

c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7;

d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10;

e) Immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

37. The method of any one of claims 34-36, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations.

38. The method of any one of claims 34-36, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation.

39. The method of any one of claims 34-37, wherein the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual.

40. The method of any one of claims 34-37, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to administering the LIF binding antibody to the individual.

41. The method of any one of claims 34-37, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual at the same time as the LIF-binding antibody is administered to the individual.

42. The method of any one of claims 34-41, wherein the LIF-binding antibody is humanized.

43. The method of any one of claims 34-42, wherein the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

44. The method of claim 43, wherein the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

45. The method of any one of claims 34-44, wherein the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic ductal adenocarcinoma, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, non-small cell lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

46. The method of claim 45, wherein the cancer comprises non-small cell lung cancer.

47. The method of claim 45, wherein the cancer comprises pancreatic ductal adenocarcinoma.

48. The method of any one of claims 34 to 45, wherein prior treatment of the cancer with a checkpoint inhibitor was unsuccessful.

49. The method of any one of claims 34-45, wherein prior treatment of the cancer with a LIF-binding antibody was unsuccessful.

50. The method of claim 48 or 49, wherein the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor.

51. The method of any one of claims 34 to 50, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

52. The method of claim 51, wherein the antibody comprises Pabolizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

53. The method of claim 51, wherein the antibody specifically binds PDL-1 or PDL-2.

54. The method of claim 53, wherein the antibody comprises Devolumab, Attributab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

55. The method of any one of claims 34 to 50, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

56. The method of claim 55, wherein the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

57. The method of any one of claims 34 to 50, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

58. The method of claim 57, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises one or more of: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

59. A method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and

an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46; and

b) PD-1, PDL-1 or PDL-2 signaling inhibitors.

60. The method of claim 59, wherein the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

61. The method of claim 59 or 60, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations.

62. The method of claim 59 or 60, wherein the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation.

63. The method of any one of claims 59 to 61, wherein the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual.

64. The method of any one of claims 59 to 61, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to administration of the LIF-binding antibody to the individual.

65. The method of any one of claims 59 to 61, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual.

66. The method of any one of claims 59-65, wherein the LIF-binding antibody is humanized.

67. The method of any one of claims 59 to 66, wherein the cancer comprises advanced solid tumor, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic ductal adenocarcinoma, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, non-small cell lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

68. The method of claim 67, wherein the cancer comprises non-small cell lung cancer.

69. The method of claim 67, wherein the cancer comprises pancreatic ductal adenocarcinoma.

70. The method of any one of claims 59 to 69, wherein prior treatment of the cancer with a checkpoint inhibitor was unsuccessful.

71. The method of any one of claims 59 to 69, wherein prior treatment of the cancer with a LIF-binding antibody was unsuccessful.

72. The method of claim 70 or 71, wherein the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor.

73. The method of any one of claims 59 to 72, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

74. The method of claim 73, wherein the antibody comprises Pabolizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

75. The method of claim 73, wherein the antibody specifically binds PDL-1 or PDL-2.

76. The method of claim 75, wherein the antibody comprises Devolumab, Attributab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

77. The method of any one of claims 59 to 72, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

78. The method of claim 77, wherein the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

79. The method of any one of claims 59 to 72, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

80. The method of claim 79, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises one or more of: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

81. The use of claim 18, wherein the antibody comprises pabulizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

82. The use of claim 18, wherein the antibody specifically binds PDL-1 or PDL-2.

83. The use of claim 82, wherein the antibody comprises Devolumab, Attributumab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

84. The use of any one of claims 1 to 17, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

85. The use of claim 84, wherein the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

86. The use of any one of claims 1 to 17, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

87. The use of claim 86, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises one or more of: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

88. The use of claim 33, wherein the antibody comprises palivizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

89. The use of claim 33, wherein the antibody specifically binds PDL-1 or PDL-2.

90. The use of claim 89, wherein the antibody comprises Devolumab, Attributumab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

91. The use of any one of claims 19 to 32, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

92. The use of claim 91, wherein the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

93. The use of any one of claims 19 to 32, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

94. The use of claim 93, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises one or more of: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

Background

Leukemia Inhibitory Factor (LIF), an interleukin 6(IL-6) -type cytokine, is involved in a variety of biological activities, including inhibiting cell differentiation. Human LIF is a 202 amino acid polypeptide that exerts biological effects by binding to gp130 heterodimeric cell surface LIF receptors (LIFR or CD 118). This results in activation of growth-promoting signaling pathways such as the mitogen-activated protein kinase (MAPK) and Janus-activated kinase (JAK/STAT) pathways. High expression levels and high serum levels of LIF have been shown to be associated with poor prognosis in many types of cancer.

Programmed cell death protein 1, also known as PD-1 and CD279, is a cell surface receptor expressed by activated T cells and B cells and plays an important role in down-regulating the immune system and promoting self-tolerance by inhibiting the inflammatory activity of T cells. PD-1 has been shown to bind to two different ligands PDL-1(CD274) and PDL-2(CD 273). Signaling through this PD-1 axis is an important mechanism for tumor growth and metastasis by allowing escape from immune surveillance. Recently, many different types of tumors have been shown to express PDL-1 and PDL-2, facilitating escape from immune surveillance, resulting in increased tumor growth and metastasis.

Disclosure of Invention

Described herein are methods of treating or preventing cancer, tumors, or other neoplasms in an individual. Methods and compositions of matter include combinations of LIF binding polypeptides and PD-1 axis inhibitors. These methods can utilize anti-LIF antibodies that antagonize or block LIF activity and polypeptides that inhibit the binding activity of PD-1, PDL-1, and PDL-2 or PD-1, PDL-1, and PDL-2 signaling. In certain embodiments, the PD-1 axis inhibitor binds and inhibits the interaction between PD-1 and PDL-1 or PDL-2. In particular, these combinations exhibit surprising synergy when compared to anti-LIF antibodies or PD-1 axis inhibitors alone.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling inhibitor to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF-binding antibody comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the LIF-binding antibody is deimmunized. In certain embodiments, the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling inhibitor to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; (c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6; (d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; and (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF-binding antibody comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the LIF-binding antibody is deimmunized. In certain embodiments, the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling inhibitor to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5; (c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; (d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF-binding antibody comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the LIF-binding antibody is deimmunized. In certain embodiments, the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling inhibitor to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF-binding antibody comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the LIF-binding antibody is deimmunized. In certain embodiments, the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding polypeptide in combination with an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling to treat cancer in an individual. In certain embodiments, the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding polypeptide is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to administering the LIF-binding polypeptide to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding polypeptide is administered to the individual. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binds to more than one Peptides include antibodies that specifically bind to LIF. In certain embodiments, the antibody that specifically binds LIF comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab)₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs 41, 42, 44, or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and are And the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar_DAnd (4) combining. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabulizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDl-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzene)And [ b ]][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer. In certain embodiments, the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a PD-1, PDL-1 or PDL-2 signaling inhibitor administered as a monotherapy.

In another aspect, described herein is the use of an antibody that specifically binds Leukemia Inhibitory Factor (LIF) in combination with a PD-1 binding antibody to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

In another aspect, described herein is a method of treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) leukemia Inhibitory Factor (LIF) binding polypeptides; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling. In certain embodiments, the method comprises administering an effective amount of the LIF-binding polypeptide to the individual having cancer. In certain embodiments, the method comprises administering an effective amount of the PD-1 inhibitor to the individual having cancer. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds LIF comprises at least one framework region derived from a framework region of a human antibody. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab) ₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determiningRegion 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar _DAnd (4) combining. In certain embodiments, the LIF-specific binding isThe bound antibody has a K of less than about 100 pmol_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabulizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-dasparagine-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer. In certain embodiments, the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of an LIF-binding polypeptide inhibitor. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of an inhibitor of PD-1, PDL-1 or PDL-2 signaling. In certain embodiments, the Leukemia Inhibitory Factor (LIF) -binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered separately. In certain embodiments, the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered simultaneously. In certain embodiments, the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered in a single composition.

In another aspect, described herein is a method of treating an individual having cancer, the method comprising: administering to the individual having cancer an effective amount of a Leukemia Inhibitory Factor (LIF) -binding polypeptide, wherein a therapeutic amount of an inhibitor of PD-1(CD279), PDL-1(CD274), or PDL-2(CD-273) signaling has been administered to the individual. In certain embodiments, the method inhibits growth or metastasis of the cancer. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunizedIn (1). In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab) ₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In some embodiments, the and Antibodies to which LIF specifically binds include: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar_DAnd (4) combining. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabollizumab, nivolumab, AMP-514, or sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazine-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer. In certain embodiments, the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of an inhibitor of PD-1, PDL-1 or PDL-2 signaling.

In another aspect, described herein is a method of treating an individual having cancer, the method comprising: administering to the individual having cancer; an effective amount of an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling, wherein a therapeutic amount of a Leukemia Inhibitory Factor (LIF) binding polypeptide has been administered to the individual. In certain embodiments, the method inhibits growth or metastasis of the cancer. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the LIF-specific binding isThe antibody is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab) ₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is identical to the amino acid set forth in SEQ ID NO:42 The amino acid sequences are identical; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar_DAnd (4) combining. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabollizumab, nivolumab, AMP-514, sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling comprise: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer. In certain embodiments, the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of an LIF-binding polypeptide inhibitor.

In another aspect, described herein is a method of treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and (b) a PD-1 binding antibody.

In another aspect, described herein is a kit comprising: (a) leukemia Inhibitory Factor (LIF) binding polypeptides; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab) ₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) immunoglobulin light chain complementarity determiningRegion 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar _DAnd (4) combining. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or antigen-binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabollizumab, nivolumab, AMP-514, or sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibodyIncluding Devolumab, Attempuzumab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the kit further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, described herein is a composition comprising: (a) leukemia inhibitory factorA child (LIF) -binding polypeptide; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab)₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having a sequence as set forth in any one of SEQ ID NOS: 45-48 An amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical in amino acid sequence. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID No. 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the antibody that specifically binds LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 200 picomolar _DAnd (4) combining. In certain embodiments, the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PD-1. In certain embodiments, the antibody comprises pabollizumab, nivolumab, AMP-514, or sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In some embodimentsSmall molecule inhibitors of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ]) group ]-3-yl) methoxy]Pyridin-3-yl } methyl) amino]Ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b)][1,4]Dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenylyl ] carbonyl]-3-yl) methoxy]Phenyl radical]Methyl radical]-2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof. In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient, carrier or diluent.

In another aspect, described herein is a method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and PD-1 or PDL-1 binding antibodies. In certain embodiments, the method comprises administering to the individual having cancer an effective amount of the antibody that specifically binds LIF. In certain embodiments, the method comprises administering an effective amount of the PD-1 or PDL-1 binding antibody to the individual having cancer. In certain embodiments, the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately. In certain embodiments, the cancer is glioblastoma multiforme (GBM), NSCLC (non-small cell lung cancer), ovarian cancer, colorectal cancer, thyroid cancer, or pancreatic cancer.

In another aspect, described herein is a method of reducing pre-Tumor Associated Macrophages (TAMs) in an individual having cancer, comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and PD-1 or PDL-1 binding antibodies. In certain embodiments, the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately. In certain embodiments, the TAM exhibits cell surface expression of any 1, 2, or 3 molecules selected from the list consisting of CD11b, CD206, and CD 163.

In another aspect, described herein is a method of generating immune memory in an individual having cancer, comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and PD-1 or PDL-1 binding antibodies. In certain embodiments, the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately. In certain embodiments, the immunological memory is mediated by CD8+ T cells. In certain embodiments, the immunological memory is mediated by CD4+ T cells.

In another aspect, described herein is a method of increasing the amount of T lymphocytes in a tumor comprising administering to an individual having the tumor an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and PD-1 or PDL-1 binding antibodies. In certain embodiments, the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately. In certain embodiments, the T lymphocytes comprise CD8+ T cells. In certain embodiments, the T lymphocytes comprise CD4+ T cells.

In another aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1, PDL-1, or PDL-2 signaling inhibitor to treat cancer in an individual, wherein the LIF binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, wherein the LIF binding antibody is humanized. In certain embodiments, wherein the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, wherein the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, wherein the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the cancer has been previously treated with an LIF antibody. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1.

In another aspect, described herein is a method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and (b) a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the LIF-binding antibody comprises: (a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; (c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6; (d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; and (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the LIF-binding antibody comprises: (a) immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5; (c) immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; (d) immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the LIF-binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, wherein the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the cancer has been previously treated with an LIF antibody. In certain embodiments, wherein the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody comprises pabulizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

In another aspect, described herein is a method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and (ii) an immunoglobulin light chain variable region (VL) that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46; and (b) a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations. In certain embodiments, the LIF-binding antibody and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation. In certain embodiments, the LIF-binding antibody is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual. In certain embodiments, the PD-1, PDL-1, or PDL-2 signaling inhibitor is administered to the individual prior to administering the LIF-binding antibody to the individual. In certain embodiments, the inhibitor of PD-1, PDL-1, or PDL-2 signaling is administered to the individual at the same time that the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF binding antibody is humanized. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof. In certain embodiments, the cancer comprises non-small cell lung cancer. In certain embodiments, the cancer comprises pancreatic ductal adenocarcinoma. In certain embodiments, the cancer has been previously treated with a checkpoint inhibitor. In certain embodiments, the cancer has been previously treated with an LIF antibody. In certain embodiments, the checkpoint inhibitor is a PD-1, PDL-1 or PDL-2 signaling inhibitor. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody comprises pabulizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof. In certain embodiments, the antibody specifically binds to PDL-1 or PDL-2. In certain embodiments, the antibody comprises Devolumab, Attempuzumab, Avermezumab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor. In certain embodiments, small molecule inhibitors of PD-1, PDL-1 or PDL-2 signaling include: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

Drawings

Fig. 1 depicts a western blot showing inhibition of LIF-induced STAT3 phosphorylation by different anti-LIF humanized antibodies.

Fig. 2A and 2B depict western blots showing inhibition of LIF-induced STAT3 phosphorylation by humanized and parent 5D8 antibodies.

FIG. 3A shows IC for LIF inhibition in U-251 cells using the h5D8 antibody₅₀。

FIG. 3B shows representative IC of r5D8 and h5D8 inhibition of pSTAT3 under endogenous LIF stimulation conditions₅₀Dose response curves. Representative curves are shown (n ═ 1h5D8, n ═ 2r5D 8).

Fig. 4 depicts a western blot showing the inhibition of LIF-induced STAT3 phosphorylation by different monoclonal antibodies described in the present disclosure.

Figure 5 depicts immunohistochemical staining and quantification of LIF expression in glioblastoma multiforme (GBM), NSCLC (non-small cell lung cancer), ovarian cancer, colorectal cancer, and pancreatic tumors from human patients. Bars represent mean +/-SEM.

Fig. 6A is a diagram showing an experiment performed in a non-small cell lung cancer mouse model using a humanized 5D8 antibody.

Fig. 6B is a diagram showing an experiment performed in a non-small cell lung cancer mouse model using the r5D8 antibody.

Figure 7A shows the inhibitory effect of r5D8 on U251 cells in GBM orthotopic mouse model. Quantification on day 26 is shown.

Figure 7B shows data from mice inoculated with luciferase-expressing human U251 GBM cells and then treated twice weekly with 100, 200, or 300 μ g of h5D8 or vehicle. Tumor size was determined by bioluminescence (Xenogen IVIS spectra) on day 7. The figure shows individual tumor measurements, the horizontal bars represent mean ± SEM. Statistical significance was calculated using the unpaired nonparametric Mann-Whitney U test.

FIG. 8A shows the inhibitory effect of r5D8 on ovarian cancer cell growth in a syngeneic mouse model.

Figure 8B shows individual tumor measurements at day 25.

Figure 8C demonstrates that h5D8 shows a significant reduction in tumor growth (p <0.05) when administered at 200 μ g/mouse twice weekly. Symbols are mean + SEM, statistical significance compared to vehicle (unpaired nonparametric Mann-Whitney U test).

Figure 9A shows the inhibition of colorectal cancer cell growth by r5D8 in a syngeneic mouse model.

Figure 9B shows individual tumor measurements at day 17.

Fig. 10A shows the reduction of macrophage infiltration to tumor sites, representative images and quantification of CCL22+ cells in GBM orthotopic mouse model.

Figure 10B shows the reduction of macrophage infiltration in a human organotypic tissue slice culture model. Shown are representative images (left) and quantitative (right).

Figure 10C shows the reduction of macrophage infiltration into tumor sites, representative images and quantification of CCL22+ cells in an ovarian cancer syngeneic mouse model.

Figure 10D shows the reduction of macrophage infiltration to tumor sites in colorectal cancer syngeneic mouse models, representative images and quantification of CCL22+ cells.

Figure 10E shows the inflammatory phenotype of tumor-associated macrophages (TAMs) harvested from tumors treated with h5D8(15mg/kg, 2QW) on day 25 (end point). In the treated tumors, TAMs were polarized towards the M1 pro-inflammatory phenotype. Statistical significance was determined by unpaired t-test.

Fig. 10F shows gene expression data for monocytes cultured with LIF knockdown conditioned medium of cells.

Figure 11A shows the increase of non-myeloid effector cells in an ovarian cancer syngeneic mouse model after treatment with r5D 8.

Figure 11B shows the increase of non-myeloid effector cells in a colorectal cancer syngeneic mouse model after treatment with r5D 8.

FIG. 11C shows CD4+ T in a mouse model of NSCLC cancer after treatment with r5D8_REGThe percentage of cells decreased.

Figure 12 shows data for CT26 tumor-bearing mice intraperitoneally administered either PBS (control) or r5D8 twice weekly treated in the presence or absence of anti-CD 4 and anti-CD 8 depleting antibodies. The figure shows individual tumor measurements at d13, expressed as mean tumor volume + SEM. Statistical differences between groups were determined by unpaired nonparametric Mann-Whitney U test. R5D8 inhibited the growth of CT26 tumor (p < 0.05). In the presence of anti-CD 4 and anti-CD 8 depleting antibodies, the growth inhibitory effect of r5D8 on tumors was significantly reduced (. p.. 0.0001).

Fig. 13A shows an overview of the co-crystal structure of h5D8 Fab complexed with LIF. gp130 interaction site maps on the surface of LIF (dark shading).

FIG. 13B illustrates the detailed interaction between LIF and h5D8, showing salt bridge forming residues and a buried surface area greater thanResidue h5D 8.

Fig. 14A shows a superposition of five h5D8 Fab crystal structures and indicates that despite crystallization under different chemical conditions, they are highly similar.

Fig. 14B shows a broad network of van der waals interactions mediated by unpaired Cys 100. This residue is ordered, participates in shaping the conformation of HCDR1 and HCDR3, and is not involved in unwanted disulfide mismatches. The distance between residues is shown and marked with a dashed line.

Figure 15A demonstrates the binding of the h5D 8C 100 mutant to human LIF by ELISA.

Figure 15B demonstrates the binding of the h5D 8C 100 mutant to mouse LIF by ELISA.

Fig. 16A shows by Octet that h5D8 does not block the binding between LIF and LIFR. Sequential binding of h5D8 to LIF followed by LIFR.

FIGS. 16B and 16C show ELISA assays for LIF/mAb complexes bound to immobilized LIFR or gp 130. Species-specific peroxidase-conjugated anti-IgG antibody signal ((-) and h5D8 anti-human antibodies, r5D8 and B09 anti-rat antibodies) the antibody portion of mAb/LIF complex bound to immobilized LIFR (fig. 16B) or gp130 (fig. 16C) coated plates was detected.

FIGS. 17A and 17B show mRNA expression of LIF (FIG. 16A) or LIFR (FIG. 16B) in 72 different human tissues.

Figures 18A-D show data that h5D8 and anti-PD-1 treated CT26 tumor mice showed significantly slower growth compared to anti-PD-1 treated tumors. Similar results were obtained in three independent CT26 efficacy experiments. 18A shows the time course of tumor growth, while 18B shows individual data points at day 24. Statistical significance was determined by the Mann-Whitney test. 18C shows a Kaplan-Meier survival plot for mice bearing CT26 or MC38 (starting on day 10 with anti-PD 1) tumors treated with anti-PD 1(RMP1-14) and h5D 8/anti-PD 1. The Kaplan-Meier survival plots for mice bearing CT26 or MC38 tumors treated with control (IgG) and h5D8 monotherapy at the bottom. Tumor volume in long-term tumor-free CT26 survivors after 18D tumor reimplantation.

FIGS. 19A-D show flow cytometry analyses to detect the abundance of functional CD8T cells in h5D 8/anti-PD-1 treated tumors. 19A shows the production of IFN γ in response to a tumor associated peptide (GP70) based thereonAbility to define CD8T cell function. A representative FAC data diagram is shown at 19B. Significance was determined by unpaired t-test. The CD8+ TIL frequency of total CD45+ immune infiltrates in CT26 tumors after treatment is shown on the left of 19C (n-7/group); on the right is H2-L from MuLV expressed with CT26 tumor ^dThe frequency of IFN-. gamma. + CD8+ TIL after in vitro stimulation (n 7/group) of the peptide corresponding to the limiting gp70 (a.a.423-431; AH1) epitope. 19D the CD8+ TIL frequency of total CD45+ immune infiltrates in MC38 tumors after treatment (n-6-7/group) is shown on the left. On the right is H2-K from MuLV expressed with MC38 tumor^bThe frequency of IFN- γ + CD8+ TIL after in vitro stimulation with the peptide corresponding to the restrictive p15e (a.a.604-611) epitope (n-6-7/group).

Figures 20A-P show that LIF blockade reduces tumor growth and modulates immune cell infiltration in GBM and ovarian cancer models expressing high levels of LIF. 20A shows the distribution of LIF mRNA expression (log2 RSEM) in 28 different solid tumors. The black line represents the estimated cut-off between low expression/background noise. The bottom panel shows the correlation between ssGSEA, LIF expression and the relative abundance of TAM and Treg based on immune cell type gene signature (Pearson R)²Value). Only if the correlation is significant (adjusted P-value)<0.1), the correlation value is shown. 20B shows linear regression plots of LIF expression and TAM and Treg relative abundance (rescaling ssGSEA from 0 to 1 for visualization purposes) in GBM, prostate adenocarcinoma (PRAD), thyroid cancer (THCA) and ovarian cancer (OV) cohorts. The shading represents the confidence interval of the regression estimation. 20C, 20H and 20K show the total flux (p/s) or abdominal volume (mm), respectively ³) To measure tumor growth in the GL261N (20C), RCAS (20H) and ID8(20K) models. A schedule representative of the experimental procedure is shown. anti-LIF (r5D8) or isotype control (IgG) treatment was initiated on the day of surgery (GL261N and RCAS) or 14 days after vaccination (dpi) (ID 8). 20D and 20L show representative p-STAT3, Ki67, CC3 and CD8 IHC percentages for GL261N (20D) and ID8(20L) tumor staining. 20E-20F, 20I-20J and 20M-20N show CD11b analyzed by flow cytometry⁺F4/80⁺CD163⁺CD206⁺MHCII^{Is low in}TAM (20E, 20M), or CD11b⁺Ly6G^-Ly6C^-CD163⁺CD206⁺MHCII^{Is low in}(20I) And CD8 of GL261N (20F), RCAS (20J) and ID8(20N) tumors⁺T cell (CD 3)⁺CD8⁺) Percentage (D). 20G and 20P show the overall survival of the GL261N (20G) and ID8(20P) models treated with anti-LIF (r5D8) or IgG. Time course of abdominal volume in ID8 mice treated with anti-LIF (r5D8) or IgG (20O). Data are mean ± SEM. Statistical analysis was performed by the Mann-Whitney T test and the log rank test. P<0.05；**P<0.01；***P<0.001；****P<0.0001。

Fig. 21A-G show LIF modulating CXCL9, CCL2, CD206, and CD163 in TAMs. 21A shows isolated CD11b from anti-LIF (r5D8) treated ID8 mice and controls⁺Differential expression analysis of cells. Representing significant overexpression of the gene (Q value)<0.1) and a significantly low expression volcanic pattern. The heatmap represents the expression values of the indicated genes, each column representing one sample and each row representing one gene. The last column represents the log2 fold change in gene expression (log2 FC). 21B shows isolated CD11B from anti-LIF (r5D8) treated or untreated ID8 and GL261N tumors ⁺mRNA expression of the indicator gene in the cell. 21C shows TAM (CD11 b) from anti-LIF (r5D8) treated or untreated GL261N tumors⁺Ly6G^-Ly6C^-) Medium CCL2⁺And CXCL9⁺Percent of (a) and average fluorescence intensity (MFI). 21D shows the percentage of double positive cells relative to TAM marker positive cells. CXCL9 quantification is relative to total cell number. 21E shows IHC quantification of the indicative markers from 20 GBM tumors. Correlations between LIF staining (y-axis) and CCL2, CD206, CD163, and CXCL9 staining (x-axis) were calculated, showing the R-squared coefficient (R |)²). 21F shows GL261N at CXCL9^-/-And CCL2^-/-Tumor growth in mice or mice treated with the indicated antibodies is shown as total flux (p/s). 21G shows tumor-infiltrating CD8 in the indicated treatment⁺Fold increase of T cells (FI). Data are mean ± SEM. Statistical analysis was performed by the Mann-Whitney T test. P<0.05；**P<0.01；***P<0.001；****P<0.0001。

Figures 22A-H show that LIF represses CXCL9 through epigenetic silencing and induces CCL2 through activation of STAT 3.22A and 22B show qRT-PCR analysis of indicator genes in BMDM. BMDM was preincubated with 20ng/ml LIF for 72h, then stimulated with 5ng/ml IFN γ or 10 μ g/ml IL4 over a 6h period (22A) or with 0.1, 0.5, 1 and 5ng/ml IFN' y for 24h (22B). 22C shows a CXCL9 ELISA of BMDM pre-incubated with 20ng/ml LIF and then stimulated with 0.1ng/ml IFN γ for 24 h. 22D shows human CD11b from human GBM cultured with 20ng/ml LIF for 72h and then with 0.1ng/ml IFN γ for 24h ⁺Sorting cells (77% CD11 b)⁺CD14⁺See fig. 29A) for CXCL9 ELISA. 22E shows ChIP for trimethyl-histone H3(H3K27me3), EZH2 and acetyl-histone 4(H4ac) in BMDM treated with 20ng/ml LIF for 72H. The schedule shows the CXCL9 promoter region analyzed. 22F shows CCL2 ELISA and CCL2 mRNA levels in BMDM treated with 20ng/ml LIF for 6 and 24 h. 22G shows p-STAT3 ChIP in BMDM stimulated with 20ng/ml LIF for 15 minutes. A schematic representation of the STAT Binding Site (SBS) on the CCL2 promoter is depicted. Data are mean ± SD and statistically analyzed by student t-test. 22H shows the relationship to Iba1⁺Percentage of cells that were double positive, and CXCL9 in GBM organic sections (patients 1, 2, 3) incubated with anti-LIF (r5D8) for 3 days at 10. mu.g/ml relative to the total number of cells⁺Percentage of cells. Data are mean ± SEM of all patients. Statistical analysis was performed by the Mann-Whitney T test. P<0.05，**P<0.01；***P<0.001；****P<0.0001。

FIGS. 23A-I show LIF blockade inducing CD8 in human GBM⁺T cell tumor infiltration and its combination with anti-PD 1 promoted tumor regression. 23A shows a schematic representation of the experimental procedure performed with GBM patient-derived xenografts and human organotypic models. 23B shows CXCL9 and CCL2 mRNA expression levels in organotypic samples (patients 4, 5, 6) treated with anti-LIF (r5D8) for 72h and then cultured with PBMC for 24 h. 23C shows CD8 in organotypic tissue (patients 4, 5, 6) treated with anti-LIF (r5D8) for 72h and then cultured with PBMCs for 48h as detected by flow cytometry ⁺FI of T-infiltrating cells. 23D shows GBM organs detected by flow cytometry under treatment with anti-LIF (r5D8) and/or anti-CXCL 9 for 72h and then culture with PBMC for 48hCD8 in type tissue⁺FI of T-infiltrating cells. 23E shows CD8⁺T-infiltrating cells were entered into GBM samples implanted subcutaneously in NSG mice. Bar graph representation of CD8 detected in tissue by flow cytometry⁺T cells and CD8 detected in blood of the same animal⁺Ratio of T cells. There were four patients (7, 8, 9, 10) and their corresponding PBMCs. 23F shows survival of GL261N mice treated with anti-LIF (r5D8), anti-PD 1, or the combination. The overall survival as determined by the Kaplan-Meier curve is shown. 23G shows tumor growth of the treated GL261N model, expressed as fold change in tumor size between 13dpi and 6 dpi. 23H shows a schedule representing 3X10 inoculation in 6 mice⁵Experimental procedure for complete regression of GL261N cells and by anti-LIF (r5D8), anti-PD 1 combination treatment. Parallel inoculation of 10 naive mice with 3x10⁵GL261N cells. 23I shows LIF CD8⁺Schematic representation of the effect of T cell tumor infiltration. Statistical analysis was performed by the Mann-Whitney T test or the log rank test. P <0.05；**P<0.01；***P<0.001。

FIGS. 24A-D show LIF expression in tumors. In 24A, LIF IHC was performed in tissue microarrays of human GBM and the degree of staining was quantified using the H-score method. 24B shows LIF ELISA of supernatants from neurosphere cultures of 15 patients. 24C and 24D show CD45 isolated from human GBM tumor (24C) and GL261N tumor (24D)⁺And CD45^-qRT-PCR analysis of indicator genes in cells.

FIGS. 25A-J show that LIF blockade inhibits tumor growth in a mouse model. 25A shows the results of qRT-PCR and ELISA of LIF on GL261, GL261N, and GL261N CRISPR/LIF cells. 25B shows tumor growth as total flux (p/s) at 12dpi in mice vaccinated with GL261N and GL261N CRISPR/LIF. 25C and 25D show that ID8 cells were infected with pLKO.1 or two independent pLKO.1-shLIF lentiviruses. LIF expression was determined by qRT-PCR and ELISA (25C). 25D shows the results of inoculation of ID8 cells in the peritoneum of mice. A treatment schedule is shown. Abdominal volume (mm) was measured at 40dpi³). 25E shows treatment at 12dpi with anti-LIF (r5D8) or control IgGGrowth of GL261 tumor in mice. 25F shows the NOD SCID and RAG1 in C57BL/6 mice and two immunodeficiency models ^-/-The result of inoculation of GL261N cells. A treatment schedule is shown. Tumor growth was measured at 12 dpi. 25G-25J show CD4 in GL261N (25G-25H) and ID8(25I-25J) tumors as determined by flow cytometry⁺T cell population gated NK cells (CD 335)⁺) And Treg (CD 3)⁺CD4⁺FoxP3⁺) Percentage of (c). Data are presented as mean ± SEM. Statistical analysis was performed by the Mann-Whitney T test. P<0.05；**P<0.01；***P<0.001；****P<0.0001。

FIGS. 26A-H show the characterization of immune cell infiltrates after treatment with anti-LIF (r5D 8). 26A shows CD8 of GL261N tumor⁺Percentage of GZMA and MFI in T cell populations. 26B and 26C show PD1 in GL261N (26B) and ID8(26C) model tumors⁺CD8⁺Percentage of T cells. 26D shows invasive TAM (CD11 b) in GL261N and RCAS tumors in response to anti-LIF (r5D8) treatment⁺Ly6G^-Ly6C^-CD49d⁺) Percentage of (c). Flow cytometry gating strategies are shown. 26E shows dendritic cell population (DC) in GL261N tumor (CD11 b)⁺CD11c⁺MHCII⁺) And antigen presentation, as determined by MHCII expression. 26F shows ELISA of IL-12 and IL-10 in GL261N tumors. 26G shows the results of anti-LIF (r5D8) treated GL261N tumor-bearing mice at 8 dpi. Tumor volumes were measured as total flux (p/s) at 13 dpi. 26H shows CD8 in tumors determined by flow cytometry ⁺T cell (CD 3)⁺CD8⁺) Percentage of (c). Data are presented as mean ± SEM. Statistical analysis was performed by the Mann-Whitney T test. P<0.05；**P<0.01。

FIG. 27A shows TAM (CD11 b) as determined by flow cytometry⁺Ly6G^-Ly6C^-) And CD8⁺T cell (CD 3)⁺CD8⁺) Percentage of CCR2, CXCR3, and LIFR expression on the population. Data are presented as mean ± SEM. Statistical analysis was performed by the Mann-Whitney T test. P<0.05. 27B shows CD11B selected from GL261N tumors⁺Ly6G^-Ly6C^-And CD11b^-Ly6G^-Ly6C^-qRT-PCR analysis of the indicated genes in the cells.

Figure 28 shows the correlation between LIF and CD163, CD206 and CCL2 expression in GBM and ovarian cancer. Regression plots between LIF and CD163, CD206, CCL2 expression (in log2 RSEM) in GBM and ovarian cancer (OV) TCGA tumor cohorts.

Fig. 29A and 29B show the modulation of CXCL9 by LIF in murine and human macrophages. FIG. 29A shows determination of CD11b in culture by flow cytometry⁺CD14⁺A cell. Data are presented as mean ± SD. Statistical analysis was performed by student t-test. P<0.01；***P<0.001. FIG. 29B shows that BMDM was preincubated with LIF (20ng/ml) for 18h, followed by stimulation with 1 μ g/ml LPS for 6 h.

Figure 30 shows the anti-tumor response to combined treatment with anti-LIF (r5D8) and anti-PD 1 in GL261N, RCAS and ID8 models. Figure 30 shows GL261N, RCAS and ID8 tumor-bearing mice treated with anti-LIF (r5D8) and/or anti-PD 1 (treatment schedules shown). Tumor growth in total flux (p/s) (GL261N and RCAS) or abdominal volume (mm) ³) (ID8) measurement is performed. Statistical analysis was performed by the Mann-Whitney T test. P<0.05；**P<0.01。

Detailed Description

Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and/or" unless otherwise indicated.

In one aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding polypeptide in combination with an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD-273) signaling to treat cancer in an individual.

In another aspect, described herein is the use of a Leukemia Inhibitory Factor (LIF) binding antibody in combination with a PD-1 binding antibody to treat cancer in an individual, wherein the LIF binding antibody comprises: (a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

In another aspect, described herein is a method of treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) leukemia Inhibitory Factor (LIF) binding polypeptides; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling.

In another aspect, described herein is a method of treating an individual having cancer, the method comprising: administering to the individual having cancer an effective amount of a Leukemia Inhibitory Factor (LIF) -binding polypeptide, wherein a therapeutic amount of an inhibitor of PD-1(CD279), PDL-1(CD274), or PDL-2(CD-273) signaling has been administered to the individual.

In another aspect, described herein is a method of treating an individual having cancer, the method comprising: administering to the individual having cancer; an effective amount of an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling, wherein a therapeutic amount of a Leukemia Inhibitory Factor (LIF) binding polypeptide has been administered to the individual.

In another aspect, described herein is a method of reducing pre-Tumor Associated Macrophages (TAMs) in a tumor of an individual having cancer, comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF); and (b) a PD-1, PDL-1 or PDL-2 signaling inhibitor.

In another aspect, described herein is a method of generating immune memory in an individual having cancer, comprising administering to an individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF); and (b) a PD-1, PDL-1 or PDL-2 signaling inhibitor.

In another aspect, described herein is a method of increasing the amount of T lymphocytes in a tumor in an individual comprising administering to the individual having the tumor an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF); and (b) a PD-1, PDL-1 or PDL-2 signaling inhibitor.

In another aspect, described herein is a method of treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) a Leukemia Inhibitory Factor (LIF) binding antibody, comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and (b) a PD-1 binding antibody.

In another aspect, described herein is a kit comprising: (a) leukemia Inhibitory Factor (LIF) binding polypeptides; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling.

In another aspect, described herein is a composition comprising: (a) leukemia Inhibitory Factor (LIF) binding polypeptides; and (b) an inhibitor of PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling.

In another aspect, described herein is a method of reducing pre-Tumor Associated Macrophages (TAMs) in an individual having cancer, comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and a PD-1 axis inhibitor.

In another aspect, described herein is a method of generating immune memory in an individual having cancer, comprising administering to the individual having cancer an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and a PD-1 axis inhibitor.

In another aspect, described herein is a method of increasing the amount of T lymphocytes in a tumor comprising administering to an individual having the tumor an effective amount of a combination of: (a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), comprising: (i) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5; (iii) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8; (iv) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10; (v) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and (vi) an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and a PD-1 axis inhibitor.

As used herein, the terms "individual," "subject," and "patient" are used interchangeably and include a human diagnosed with or suspected of having a tumor, cancer, or other neoplasm. Individuals may also include mammals such as mice, rats, dogs, cats, pigs, sheep, cows, horses, goats, llamas, alpacas, or yaks.

As used herein, the term "about" refers to an amount that varies by 10% about the stated amount.

As used herein, the term "treatment" refers to an intervention in a physiological or disease state in an individual that is designed or intended to alleviate at least one sign or symptom associated with the physiological or disease state. Reference herein to treatment of cancer refers to an intervention aimed at inducing a complete response, a partial response, delayed progression of the cancer or tumor being treated, reduction in tumor size or tumor burden, or delay in tumor growth or tumor burden. Treatment also refers to intervention aimed at reducing metastasis or malignancy of the cancer or tumor. One skilled in the art will recognize that not all individuals in a heterogeneous population of individuals with a disease will respond equally or completely to a given treatment. Nevertheless, these individuals are still considered to have received treatment. Unsuccessful treatment often leads to disease progression, and additional treatment with other therapeutic approaches is therefore necessary. In certain aspects, the antibodies and methods described herein can be used to maintain remission of cancer or to prevent recurrence of the same cancer or a different cancer associated with the cancer being treated.

As used herein, the term "combination" or "combination therapy" may refer to the simultaneous administration of the items to be combined or the sequential administration of the items to be combined. As described herein, when a combination refers to sequential administration of articles, the articles may be administered in any temporal order.

The terms "cancer" and "tumor" relate to a physiological condition in mammals characterized by dysregulated cell growth. Cancer is a class of diseases in which a group of cells exhibit uncontrolled or unwanted growth. Cancer cells can also spread to other locations, which can lead to the formation of metastases. The spread of cancer cells in the body can occur, for example, through lymph or blood. Uncontrolled growth, invasion and metastasis formation are also known as malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which typically do not invade or metastasize.

As used herein, the term "effective amount" refers to the amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, inhibiting or blocking receptor-ligand interactions (e.g., LIF-LIFR, PD-1-PDL1/PDL-2), inhibiting signaling pathways (e.g., STAT3 phosphorylation), reducing tumor growth, reducing metastasis of a tumor, or prolonging survival of a tumor-bearing animal. "therapeutic amount" is the concentration of drug calculated to exert a therapeutic effect. Therapeutic amounts include dosage ranges capable of inducing a therapeutic response in a population of individuals. The mammal may be a human subject. The human subject may have or be suspected of having a tumor.

As used herein, a "checkpoint inhibitor" refers to a drug that inhibits a biomolecule produced by an organism (a "checkpoint molecule"), which negatively modulates the anti-tumor/cancer activity of T cells in the organism. Checkpoint molecules include, but are not limited to, PD-1, PDL-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, and NOX 2.

As used herein, unless otherwise indicated, the term "antibody" includes antigen-binding fragments of an antibody, i.e., antibody fragments that retain the antigen-specific binding ability to bind to a full-length antibody, e.g., fragments that retain one or more CDR regions. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; heavy chain antibodies, single chain antibody molecules, e.g., single chain variable fragments (scFv), nanobodies, and multispecific antibodies, such as bispecific antibodies, formed from antibody fragments with different specificities. In certain embodiments, the antibodies are humanized in a manner that reduces the immune response of the individual to the antibody. For example, the antibody can be chimeric, e.g., a non-human variable region with human constant regions, or CDR grafted, e.g., a non-human CDR region with human constant region and variable region framework sequences. In certain embodiments, the humanized antibody is deimmunized. Deimmunization involves removing or mutating one or more T cell epitopes in the constant region of the antibody. In certain embodiments, the antibodies described herein are monoclonal. As used herein, a "recombinant antibody" is an antibody that includes amino acid sequences derived from two different species or from two different sources, and includes synthetic molecules, e.g., an antibody that includes non-human CDRs and a human framework or constant region. In certain embodiments, the recombinant antibodies of the invention are produced or synthesized from recombinant DNA molecules.

Percent (%) sequence identity with respect to a reference polypeptide or antibody sequence is the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide or antibody sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are known, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. Suitable parameters for aligning the sequences can be determined, including the algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the use of the sequence comparison computer program ALIGN-2 results in% amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genettech, Inc., and the source code has been filed with the user document in the U.S. copyright office of Washington, D.C. 20559, registered under U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from GeneTak corporation, san Francisco, Calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including the digital UNIX V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and were unchanged.

In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a to/and/or relative to a given amino acid sequence B (alternatively this may be expressed by the phrase as to/and/or relative to a given amino acid sequence a having or comprising a particular% amino acid sequence identity) is calculated as follows: 100 times the score X/Y, where X is the number of amino acid residues in the program alignment of A and B that are scored as identical matches by the sequence alignment program ALIGN-2, where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless otherwise specifically stated, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "epitope" includes any determinant capable of being bound by an antigen binding protein, such as an antibody. An epitope is a region of an antigen that is bound by an antigen binding protein that targets the antigen, and when the antigen is a protein, an epitope includes specific amino acids that directly contact the antigen binding protein. Epitopes are most often located on proteins, but in some cases may be located on other kinds of molecules (such as sugars or lipids). Epitope determinants may include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. Generally, an antibody having specificity for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

As used herein, the term "TAM" or "tumor-associated macrophages" includes macrophage or monocyte-derived immune cells that are abundantly present in the microenvironment of a solid tumor. TAM includes but is not limited to expression of CD11b⁺、Ly6G^-、Ly6C^-、CD206⁺、CD163⁺、MHCII^{Is low in}、CD49d⁺Or any combination thereof.

Structural Properties of the antibodies described herein

Complementarity determining regions ("CDRs") are part of the immunoglobulin (antibody) variable regions, which are primarily responsible for the antigen binding specificity of antibodies. CDR regions are highly variable from one antibody to another even though the antibodies specifically bind the same target or epitope. The heavy chain variable region comprises three CDR regions, abbreviated VH-CDR1, VH-CDR2 and VH-CDR 3; the light chain variable region comprises three CDR regions, abbreviated as VL-CDR1, VL-CDR2 and VL-CDR 3. These CDR regions are arranged contiguously in the variable region, with CDR1 being the N-most terminal and CDR3 being the C-most terminal. Interspersed between the CDRs are framework regions that contribute to the structure and show much less variability than the CDR regions. The heavy chain variable region comprises four framework regions, abbreviated VH-FR1, VH-FR2, VH-FR3 and VH-FR 4; the light chain variable region comprises four framework regions, abbreviated as VL-FR1, VL-FR2, VL-FR3 and VL-FR 4. A complete full-size bivalent antibody comprising two heavy and light chains would include: 12 CDRs having three unique heavy chain CDRs and three unique light chain CDRs; 16 FR regions having four distinct heavy chain FR regions and four distinct light chain FR regions. In certain embodiments, an antibody described herein comprises a minimum of three heavy chain CDRs. In certain embodiments, an antibody described herein comprises a minimum of three light chain CDRs. In certain embodiments, an antibody described herein comprises a minimum of three heavy chain CDRs and three light chain CDRs. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known protocols, including Kabat et al (1991), "Sequences of proteins of immunological Interest [ protein Sequences of immunological Interest ]," public health service of national institute of health ("Kabat" numbering scheme), Besserda, U.S. 5 th edition; Al-Lazikani et Al, (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al, J.mol.biol. [ J.M. J.Biol. [ J.M. Biol. ]262:732-745(1996), "Antibody-antigen interactions: Contact analysis and binding site morphology ]," ("Contact" numbering scheme); lefranc MP et al, "IMGT unique number for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains [ coded for by IMGT unique immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains ]," Dev Comp Immunol [ developmental and comparative immunology ], month 1 2003; 27(1) 55-77 ("IMGT" numbering scheme); and Honegger A and Plouckthun A, "Yeast amino number scheme for immunoglobulin variable domains" an automatic modeling and analysis tool ". A. an automatic modeling and analysis tool". JMolBiol [ journal of molecular biology ], 6.8 days 2001; 309(3) 657-70 ("Aho" numbering scheme). CDRs are identified herein from variable sequences provided using different numbering systems, either using kabat, IMGT, the georgia numbering system, or any combination of the three. The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the kabat approach is based on structural alignment, while the georgia approach is based on structural information. The numbering of both the kabat and geodesia schemes is based on the most common length of the antibody region sequences, with insertions by insertion letters (e.g., "30 a"), and deletions revealed in some antibodies. These two schemes place certain insertions and deletions ("indels") at different locations, resulting in different numbers. The contact protocol is based on an analysis of complex crystal structures and is similar in many respects to the joxiya numbering scheme. In some embodiments, the CDRs of the present disclosure are determined by the kabat method, the IMGT method, the geodesia method, or any combination thereof.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) typically have similar structures, each domain comprising four conserved Framework Regions (FRs) and three CDRs (see, e.g., Kindt et al KubyImmunology, sixth edition, w.h.freeman and Co. [ w.h. virhmann, p 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to isolate antibodies that bind a particular antigen from antibodies that bind the antigen, to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al, J.Immunol. [ J.Immunol ]150:880-887 (1993); Clarkson et al, Nature [ Nature ]352:624-628 (1991)). In certain embodiments, the antibodies described herein are humanized. In certain embodiments, the antibodies described herein are chimeric. In certain embodiments, the antibodies described herein comprise rat-derived variable regions. In certain embodiments, the antibodies described herein comprise rat-derived CDRs. In certain embodiments, the antibodies described herein comprise mouse-derived variable regions. In certain embodiments, the antibodies described herein comprise mouse-derived CDRs.

Alterations (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in CDR-encoding codons at high mutation rates during somatic cell maturation (see, e.g., Chowdhury, methods mol. biol. [ molecular biology methods ]207: 179. 196(2008)), and the resulting variants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, e.g., Hoogenboom et al Methods in molecular biology [ Methods of molecular biology ]178:1-37 (2001)). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science [ Science ],244:1081-1085 (1989)). CDR-H3 and CDR-L3 are specifically targeted in general. Alternatively or additionally, the crystal structure of the antigen-antibody complex is analyzed to identify the contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired property.

In certain embodiments, the antibodies described herein comprise a constant region in addition to a variable region. The heavy chain constant region (C) _H) Comprising four domains, abbreviated C_H1、C_H2、C_H3 and C_H4, at the C-terminus of the complete heavy chain polypeptide, i.e., at the C-terminus of the variable region. The light chain constant region (C)_L) C is greater than C_HMuch smaller and located at the C-terminus of the intact light chain polypeptide, i.e., the C-terminus of the variable region. The constant region is highly conserved and includes different isoforms associated with slightly different functions and properties. In certain embodiments, the constant region is dispensable for an antibody that binds a target antigen. In certain embodiments, the constant region, heavy chain, and light chain of the antibody are optional. In certain embodiments, the antibodies described herein lack one or more of a light chain constant region, a heavy chain constant region, or both. Most monoclonal antibodies are of the IgG isotype; further divided into four subclasses of IgG₁、IgG₂、IgG₃And IgG₄. In certain embodiments, the antibodies described herein comprise any IgG subclass. In certain embodiments, the IgG subclass comprises IgG₁. In certain embodiments, the IgG subclass comprises IgG₂. In certain embodiments, the IgG subclass comprises IgG₃. In certain embodiments, the IgG subclass comprises IgG₄。

Antibodies include a fragment crystallizable region (Fc region) that is responsible for binding to complement and Fc receptors. The Fc region comprises C of the antibody molecule _H2、C_H3 and C_HZone 4. The Fc region of an antibody is responsible for activating complement and antibody-dependent cellular cytotoxicity (ADCC). The Fc region also contributes to the serum half-life of the antibody. In certain embodiments, the Fc region of an antibody described herein comprises one or more amino acid substitutions that promote complement-mediated cell lysis. In certain embodiments, the Fc region of an antibody described herein comprises one or more amino acid substitutions that promote ADCC. In some instancesIn embodiments, the Fc region of an antibody described herein comprises one or more amino acid substitutions that reduce complement-mediated cell lysis. In certain embodiments, the Fc region of an antibody described herein comprises one or more amino acid substitutions that increase binding of the antibody to an Fc receptor. In certain embodiments, the Fc receptor comprises Fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIIA (CD16a), Fc γ RIIIB (CD16b), or any combination thereof. In certain embodiments, the Fc region of an antibody described herein comprises one or more amino acid substitutions that increase the serum half-life of the antibody. In certain embodiments, the one or more amino acid substitutions that increase the serum half-life of the antibody increases the affinity of the antibody for neonatal Fc receptor (FcRn).

In some embodiments, antibodies of the disclosure are variants with some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to demonstrate the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods may be employed (e.g., ACTI)^TMAnd CytotoxNon-radioactive cytotoxicity assay). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMCs), monocytes, macrophages, and Natural Killer (NK) cells.

Antibodies may have increased half-life and improved binding to neonatal Fc receptor (FcRn) (see e.g. US 2005/0014934). Such antibodies may include Fc regions having one or more substitutions therein that improve binding of the Fc region to FcRn, and include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (according to the EU numbering system) (see, e.g., U.S. patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature [ Nature ]322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94/29351).

Clinically useful antibodies are often "humanized" to reduce immunogenicity in human subjects. Humanized antibodies improve the safety and efficacy of monoclonal antibody therapy. One common method of humanization is to generate monoclonal antibodies in any suitable animal (e.g., mouse, rat, hamster) and replace the constant regions with human constant regions, and antibodies engineered in this manner are referred to as "chimeric". Another common method is "CDR-grafting", which replaces non-human V-FR with human V-FR. In the CDR-grafting method, all residues except the CDR region are of human origin. In certain embodiments, the antibodies described herein are humanized. In certain embodiments, the antibodies described herein are chimeric. In certain embodiments, the antibodies described herein are CDR grafted.

Humanization generally reduces or has little effect on the overall affinity of the antibody. Described herein are antibodies that unexpectedly have greater affinity for their target after humanization. In certain embodiments, humanization increases the affinity for the antibody by 10%. In certain embodiments, humanization increases the affinity for the antibody by 25%. In certain embodiments, humanization increases the affinity for the antibody by 35%. In certain embodiments, humanization increases the affinity for the antibody by 50%. In certain embodiments, humanization increases the affinity for the antibody by 60%. In certain embodiments, humanization increases the affinity for the antibody by 75%. In certain embodiments, humanization increases the affinity for the antibody by 100%. Affinity is suitably measured using Surface Plasmon Resonance (SPR). In certain embodiments, the affinity is measured using glycosylated human LIF. In certain embodiments, the glycosylated human LIF is immobilized to the surface of the SPR chip. In some cases In embodiments, the antibody has a K of less than about 300 nanomolar, 200 nanomolar, 150 nanomolar, 125 nanomolar, 100 nanomolar, 90 nanomolar, 80 nanomolar, 70 nanomolar, 60 nanomolar, 50 nanomolar, 40 nanomolar or less_DAnd (4) combining.

The compositions and methods described herein comprise a combination of a LIF-binding polypeptide and a PD-1 axis inhibitor. In certain embodiments, the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide is an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding antibody comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is Fab, F (ab) ₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies. In certain embodiments, the LIF-binding antibody is the h5D8 antibody described herein (SEQ ID NO:42 and SEQ ID NO:46), or a heavy chain cysteine mutant (SEQ ID NO:66) or an antibody having the CDRs of h5D8 or a cysteine mutant form thereof.

In certain embodiments described herein, the antibody utilized or administered in combination with a PD-1 inhibitor is the h5D8 antibody. The h5d8 antibody specifically binds LIF and includes VH-CDR1 as set forth in any one of SEQ ID NOs 1-3, VH-CDR2 as set forth in any one of SEQ ID NOs 4 or 5, and VH-CDR3 as set forth in any one of SEQ ID NOs 6-8. In certain embodiments described herein, h5D8 specifically binds LIF and includes VL-CDR1 as set forth in any one of SEQ ID NO. 9 or 10, VL-CDR2 as set forth in SEQ ID NO. 11 or 12, and VL-CDR3 as set forth in SEQ ID NO. 13. In certain embodiments described herein, h5D8 specifically binds LIF and includes VH-CDR1 set forth in any one of SEQ ID NOs 1-3, VH-CDR2 set forth in any one of SEQ ID NOs 4 or 5, and VH-CDR3 set forth in any one of SEQ ID NOs 6-8, VL-CDR1 set forth in any one of SEQ ID NOs 9 or 10, VL-CDR2 set forth in any one of SEQ ID NOs 11 or 12, and VL-CDR3 set forth in SEQ ID NOs 13.

In certain embodiments, the antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 14-17, a VH-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 18 or 19, a VH-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 20-22, or a VH-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 26-29, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 30-33, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 34-37, or a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence at least about 80%, about 90%, or about 95% identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 20, a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 24, at least about 80% to the amino acid sequence set forth in SEQ ID NO. 27, and/or a VH-FR3 amino acid sequence at least about 80%, or a VH-FR4 amino acid sequence at least about 80%, or a, A VL-FR1 amino acid sequence that is 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:31, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:35, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:38, and a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 26 to 29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 30 to 33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 34 to 37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 38 to 40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a sequence identical to SEQ ID NO:15, the amino acid sequence of VH-FR1 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:19, the amino acid sequence of VH-FR2 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:20, the amino acid sequence of VH-FR3 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:24, the amino acid sequence of VH-FR4 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:27, or a VL-FR1 amino acid sequence identical in amino acid sequence as set forth in claim 27, and SEQ ID NO:31, VL-FR2 having the same amino acid sequence as set forth in SEQ ID NO, and SEQ ID NO:35, VL-FR3 which has the same amino acid sequence as set forth in SEQ ID NO, and to SEQ ID NO:38, and VL-FR4 amino acid sequence identical to the amino acid sequence set forth in seq id no. In certain embodiments, the antibody specifically binds human LIF.

The r5D8 antibody described herein was generated from rats immunized with DNA encoding human LIF. r5D8 was cloned and sequenced and included CDRs with the following amino acid sequences (using a combination of kabat and IMGT CDR numbering approaches): VH-CDR1 corresponding to SEQ ID NO. 1(GFTFSHAWMH), VH-CDR2 corresponding to SEQ ID NO. 4(QIKAKSDDYATYYAESVKG), VH-CDR3 corresponding to SEQ ID NO. 6(TCWEWDLDF), VL-CDR1 corresponding to SEQ ID NO. 9(RSSQSLLDSDGHTYLN), VL-CDR2 corresponding to SEQ ID NO. 11(SVSNLES) and VL-CDR3 corresponding to SEQ ID NO. 13 (MQATHAPPYT). The antibody has been humanized by CDR shifting of the plant, and this humanized version is referred to as h5D 8.

In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a VH-CDR1 that is at least 80% or 90% identical to that set forth in SEQ ID No. 1(GFTFSHAWMH), a VH-CDR2 that is at least 80%, 90% or 95% identical to that set forth in SEQ ID No. 4(QIKAKSDDYATYYAESVKG), and a VH-CDR3 that is at least 80% or 90% identical to that set forth in SEQ ID No. 6 (TCWEWDLDF). In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a VL-CDR1 that is at least 80% or 90% identical as set forth in SEQ ID No. 9(RSSQSLLDSDGHTYLN), a VL-CDR2 that is at least 80% identical as set forth in SEQ ID No. 11(SVSNLES), and a VL-CDR3 that is at least 80% or 90% identical as set forth in SEQ ID No. 13 (MQATHAPPYT). In certain embodiments, described herein are antibodies that specifically bind LIF, including VH-CDR1 set forth in SEQ ID NO:1(GFTFSHAWMH), VH-CDR2 set forth in SEQ ID NO:4(QIKAKSDDYATYYAESVKG), VH-CDR3 set forth in SEQ ID NO:6(TCWEWDLDF), VL-CDR1 set forth in SEQ ID NO:9(RSSQSLLDSDGHTYLN), VL-CDR2 set forth in SEQ ID NO:11(SVSNLES), and VL-CDR3 set forth in SEQ ID NO:13 (MQATHAPPYT). Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs of the disclosure. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 6, 9, 11, and 13. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any of SEQ ID NOs 1, 4, 6, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and does not affect binding affinity by more than 10%, 20%, or 30%. In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 14-17, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 18 or 19, a VH-FR3 amino acid sequence at least 90% identical to the amino acid sequence set forth in any of SEQ ID NOS 20-22, or a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 26-29, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 30-33, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 34-37, or a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 20, a VH-FR4 amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO. 24, a VL-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 27, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 31, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35, and a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In certain embodiments, the antibody specifically binds human LIF. In certain embodiments, the antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 26 to 29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 30 to 33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 34 to 37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 38 to 40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a sequence identical to SEQ ID NO:15, the amino acid sequence of VH-FR1 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:19, the amino acid sequence of VH-FR2 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:20, the amino acid sequence of VH-FR3 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:24, the amino acid sequence of VH-FR4 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:27, or a VL-FR1 amino acid sequence identical in amino acid sequence as set forth in claim 27, and SEQ ID NO:31, VL-FR2 having the same amino acid sequence as set forth in SEQ ID NO, and SEQ ID NO:35, VL-FR3 which has the same amino acid sequence as set forth in SEQ ID NO, and to SEQ ID NO:38, and VL-FR4 amino acid sequence identical to the amino acid sequence set forth in seq id no. In certain embodiments, the antibody specifically binds human LIF.

In certain embodiments, described herein are antibodies that specifically bind LIF comprising a VH-CDR1 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID No. 1(GFTFSHAWMH), a VH-CDR2 amino acid sequence at least 80%, 90% or 95% identical to that set forth in SEQ ID No. 4(QIKAKSDDYATYYAESVKG), and a VH-CDR3 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID No. 8 (TSWEWDLDF). In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a VL-CDR1 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID No. 9(RSSQSLLDSDGHTYLN), a VL-CDR2 amino acid sequence at least 80% identical to that set forth in SEQ ID No. 11(SVSNLES), and a VL-CDR3 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID No. 13 (MQATHAPPYT). In certain embodiments, described herein are antibodies that specifically bind LIF, comprising the VH-CDR1 amino acid sequence set forth in SEQ ID NO:1(GFTFSHAWMH), the VH-CDR2 amino acid sequence set forth in SEQ ID NO:4(QIKAKSDDYATYYAESVKG), the VH-CDR3 amino acid sequence set forth in SEQ ID NO:8(TSWEWDLDF), the VL-CDR1 amino acid sequence set forth in SEQ ID NO:9(RSSQSLLDSDGHTYLN), the VL-CDR2 amino acid sequence set forth in SEQ ID NO:11(SVSNLES), and the VL-CDR3 amino acid sequence set forth in SEQ ID NO:13 (MQATHAPPYT). Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs of the disclosure. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 8, 9, 11, and 13. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any of SEQ ID NOs 1, 4, 8, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and does not affect binding affinity by more than 10%, 20%, or 30%. In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 14-17, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 18 or 19, a VH-FR3 amino acid sequence at least 90% identical to the amino acid sequence set forth in any of SEQ ID NOS 20-22, or a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 26-29, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 30-33, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 34-37, or a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOS 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a VH-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 15, a VH-FR2 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 19, a VH-FR3 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 20, a VH-FR4 amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO. 24, a VL-FR1 amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 27, a VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 31, a VL-FR3 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 35, and a VL-FR4 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 38. In certain embodiments, the antibody specifically binds human LIF. In certain embodiments, the antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 26 to 29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 30 to 33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 34 to 37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs 38 to 40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID No. 38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions comprise a sequence identical to SEQ ID NO:15, the amino acid sequence of VH-FR1 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:19, the amino acid sequence of VH-FR2 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:20, the amino acid sequence of VH-FR3 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:24, the amino acid sequence of VH-FR4 having the same amino acid sequence as set forth in seq id no, and SEQ ID NO:27, or a VL-FR1 amino acid sequence identical in amino acid sequence as set forth in claim 27, and SEQ ID NO:31, VL-FR2 having the same amino acid sequence as set forth in SEQ ID NO, and SEQ ID NO:35, VL-FR3 which has the same amino acid sequence as set forth in SEQ ID NO, and to SEQ ID NO:38, and VL-FR4 amino acid sequence identical to the amino acid sequence set forth in seq id no. In certain embodiments, the antibody specifically binds human LIF.

In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs 41, 42, and 44. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs 41, 42, and 44. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized light chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs 45-48. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs 45-48. In certain embodiments, the antibody specifically binds human LIF.

In certain embodiments, described herein is an antibody that specifically binds LIF, comprising a humanized heavy chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and a humanized light chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; and a humanized light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46.

In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 66; and a humanized light chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; and a humanized light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46.

In certain embodiments, described herein is an antibody that specifically binds LIF, comprising a humanized heavy chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs 57-60; and a humanized light chain variable region comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs 61-64. In certain embodiments, described herein is an antibody that specifically binds LIF, comprising a humanized heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 57-60; and a humanized light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 61-64.

In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 58; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 67; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 62.

In certain embodiments, described herein are recombinant antibodies that specifically bind Leukemia Inhibitory Factor (LIF), comprising: heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

In certain embodiments, described herein are recombinant antibodies that specifically bind Leukemia Inhibitory Factor (LIF), comprising: heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5; heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6; light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10; light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs of the disclosure. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 2, 5, 6, 10, 12, and 13. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any of SEQ ID NOs 2, 5, 6, 10, 12, and 13 by 1, 2, 3, or 4 amino acids and does not affect binding affinity by more than 10%, 20%, or 30%.

In certain embodiments, described herein are recombinant antibodies that specifically bind Leukemia Inhibitory Factor (LIF), comprising: heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; heavy chain complementarity determining region 2(VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs of the disclosure. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 3, 4, 7, 9, 11, and 13. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any of SEQ ID NOs 3, 4, 7, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and does not affect binding affinity by more than 10%, 20%, or 30%.

In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs 49-52; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS 53-56. In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a humanized heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 49-52; and a humanized light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 53-56.

In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 50; and a humanized light chain comprising an amino acid sequence at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, described herein are antibodies that specifically bind LIF, comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 54.

Epitopes bound by therapeutically useful LIF antibodies

Described herein are unique epitopes of human LIF that, when bound, inhibit the biological activity of LIF (e.g., STAT3 phosphorylation) and inhibit tumor growth in vivo. The epitopes described herein consist of two discontinuous amino acid fragments (from residue 13 to residue 32 and residues 120 to 138 of human LIF) that are present in two different topological domains (alpha helices a and C) of the human LIF protein. This binding is a combination of weak (van der waals attraction), moderate (hydrogen bonding) and strong (salt bridge) interactions. In certain embodiments, the contact residue is a residue on LIF that forms a hydrogen bond with a residue on the anti-LIF antibody. In certain embodiments, the contact residues are residues on LIF that form salt bridges with residues on the anti-LIF antibody. In certain embodiments, the contact residue is a residue on LIF that forms van der waals forces with a residue on the anti-LIF antibody and is within at least 5, 4, or 3 angstroms.

In certain embodiments, the methods and compositions described herein comprising a LIF binding antibody and a PD-1 axis inhibitor comprise an isolated antibody that binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are isolated antibodies that bind all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are isolated antibodies that bind all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody. In a certain embodiment, the antibody interacts with helices a and C of LIF. In a certain embodiment, the antibody blocks the interaction of LIF with gp 130.

In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 6, 9, 11, and 13, which antibody binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 6, 9, 11, and 13, which antibody binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 8, 9, 11, and 13, which antibody binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 8, 9, 11, and 13, which antibody binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, described herein is an antibody comprising a CDR that differs by 1, 2, 3, 4, or 5 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 6, 9, 11, and 13 and binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein is an antibody comprising a CDR that differs by 1, 2, 3, 4, or 5 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 6, 9, 11, and 13 and binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, described herein is an antibody comprising a CDR that differs by 1, 2, 3, 4, or 5 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 8, 9, 11, and 13 and binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein is an antibody comprising a CDR that differs by 1, 2, 3, 4, or 5 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs 1, 4, 8, 9, 11, and 13 and binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, described herein are antibodies that specifically bind LIF comprising a humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and a humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO 46 and binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are antibodies that specifically bind LIF comprising a humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and a humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO. 46, and binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, described herein are antibodies that specifically bind LIF comprising a humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 66; and a humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO 46 and binds to any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are antibodies that specifically bind LIF comprising a humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 66; and a humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO. 46, and binds to all of the following residues: 68 of SEQ ID NO: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, the antibody binds only residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in a strong interaction with the antibody.

In certain embodiments, the antibodies disclosed herein inhibit LIF signaling in a cell. In certain embodiments, the IC for biologically inhibiting the antibody in U-251 cells under serum starvation conditions₅₀Less than or equal to about 100, 75, 50, 40, 30, 20, 10, 5, or 1 nanomolar. In certain embodiments, the IC for biologically inhibiting the antibody in U-251 cells under serum starvation conditions₅₀Less than or equal to about 900, 800, 700, 600, 500, 400, 300, 200, or 100 nanomolar.

In certain embodiments, the antibodies disclosed herein are useful for treating tumors and cancers that express LIF. In certain embodiments, an individual treated with an antibody of the disclosure has been selected as having an LIF-positive tumor/cancer for treatment. In certain embodiments, the tumor is LIF positive or produces elevated levels of LIF. In certain embodiments, LIF positivity is determined as compared to a reference value or a set of pathological criteria. In certain embodiments, a LIF-positive tumor expresses 2-fold, 3-fold, 5-fold, 10-fold, 100-fold or more LIF over the non-transformed cells from which it is derived. In certain embodiments, the tumor has acquired ectopic expression of LIF. LIF-positive tumors can be determined histologically using, for example, immunohistochemistry with anti-LIF antibodies; mRNA quantification by commonly used molecular biological methods, such as, for example, by real-time PCR or RNA-seq; or, for example, by western blot, flow cytometry, ELISA, or homogeneous protein quantification (e.g., ) Protein quantification was performed. In some embodiments of the present invention, the,the antibodies are useful for treating patients diagnosed with cancer. In certain embodiments, the cancer comprises, or is, one or more cancer stem cells.

In certain embodiments, the antibodies disclosed herein are useful for treating tumors in cancers that express the LIF receptor (CD 118). LIF receptor positive tumors can be determined by histopathology or flow cytometry, and in certain embodiments, include cells that bind LIF receptor antibodies that are 2x, 3x, 4x, 5x, 10x or more greater than isotype controls. In certain embodiments, the tumor has acquired ectopic expression of the LIF receptor. In a certain embodiment, the cancer is a cancer stem cell. In a certain embodiment, LIF-positive tumors or cancers can be determined by immunohistochemistry using anti-LIF and anti-LIF antibodies. In a certain embodiment, LIF-positive tumors are determined by IHC analysis, wherein LIF levels are in the top 10%, 20%, 30%, 40% or top 50% of the tumors.

The antibodies described herein affect many of the results. In a certain embodiment, an antibody described herein can reduce the presence of M2 macrophages in a tumor model tumor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control antibody (e.g., an isotype control). M2 macrophages can be identified by staining CCL22 and CD206 in IHC sections or by flow cytometry of tumor infiltrating immune cells or bone marrow cells. In a certain embodiment, an antibody described herein can reduce binding of LIF to gp130 tumor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to a control antibody (e.g., an isotype control). In a certain embodiment, an antibody described herein can reduce LIF signaling in a LIF-reactive cell line by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control antibody (e.g., an isotype control). LIF signaling can be measured, for example, by western blotting of phosphorylated STAT3 (downstream target of LIF signaling). The antibodies herein are also highly specific for LIF, as compared to other IL-6 family member cytokines. In certain embodiments, the antibody binds human LIF with an affinity of about 10x, about 50x, or about 100x greater than the affinity of any other IL-6 family member cytokine. In certain embodiments, the LIF antibody does not bind to other IL-6 family member cytokines produced in mammalian systems. In certain embodiments, the antibody does not bind to oncostatin M produced in a mammalian system.

In certain embodiments, the LIF-binding polypeptides and antibodies can be administered by any route suitable for administration of antibody-containing pharmaceutical compositions, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered on a suitable dosage schedule, e.g., weekly, twice weekly, monthly, twice monthly, etc. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1mg/kg and about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5mg/kg and about 30 mg/kg. The LIF-binding polypeptide or antibody can be administered intravenously over a period of at least about 60 minutes; however, this time period may vary somewhat depending on the conditions associated with each individual administration.

PD-1 axis inhibitors

The PD-1 axis is the signaling pathway by which PD-1 inhibits T cell responses, including the interaction of PD-1 with PDL-1 or PDL-2. The LIF-binding polypeptides and antibodies described herein can be combined with PD-1 axis inhibitors and used in methods of treating tumors, cancers, or other neoplasms. In certain embodiments, the LIF-binding polypeptides and antibodies described herein can be combined with PD-1 axis inhibitors in pharmaceutical compositions useful for treating cancer, tumors, or other neoplasms. The h5D8 antibodies described herein can be combined with PD-1 axis inhibitors and used in methods of treating tumors, cancers, or other neoplasms. In certain embodiments, the h5D8 antibodies described herein can be combined with PD-1 axis inhibitors in pharmaceutical compositions useful for the treatment of cancer, tumors, or other neoplasms.

The PD-1 axis inhibitors utilized in the compositions and methods herein can inhibit signaling through PD-1(CD279), PDL-1(CD274), or PDL-2(CD 273). The inhibitor may be an antibody or antibody fragment, a soluble ligand-Fc fusion construct, or a small molecule inhibitor. In certain embodiments, the PD-1 axis inhibitor comprises an antibody or a PD-1 binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment that specifically binds PD-1(CD279) comprises pabulizumab, nivolumab, AMP-514, sibatuzumab, tirezlizumab (BGB-a317), pembrolizumab, or a PD-1(CD279) binding fragment thereof. In certain embodiments, the PD-1 axis inhibitor is a PD-L2 Fc fusion protein (e.g., AMP-224). In certain embodiments, the PD-1 axis inhibitor comprises an antibody or PDL-1 binding fragment that specifically binds PDL-1(CD 274). In certain embodiments, the antibody or antigen-binding fragment that specifically binds to PDL-1(CD274) comprises de vacizumab (MEDI4376), alemtuzumab, avizumab-936559, or FAZ053, or a PDL-1(CD274) binding fragment thereof. In certain embodiments, the PD-1 axis inhibitor comprises an antibody or PDL-2 binding fragment thereof that specifically binds PDL-2(CD 273).

In certain embodiments, the PD-1 axis inhibitor includes one or more small molecule inhibitors, such as N- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

In certain embodiments, the PD-1 axis inhibitor can be administered by any route suitable for administration of small molecule polypeptides or antibody-containing pharmaceutical compositions, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, intracerebrally, or orally. In certain embodiments, the PD-1 axis inhibitory antibody is administered intravenously. In certain embodiments, the PD-1 axis inhibitory antibody is administered on a suitable dosage schedule, e.g., weekly, twice weekly, monthly, twice monthly, bi-weekly, tri-weekly, or once every four weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1mg/kg and about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5mg/kg and about 30 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5mg/kg and about 20 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5mg/kg and about 15 mg/kg. In certain embodiments, the therapeutically acceptable amount is about 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, or 20 mg/kg. In one example, Devolumab may be administered biweekly at a dose of about 10 mg/kg.

In certain embodiments, the PD-1 axis inhibitor may be administered to the subject at a flat dosage level of between about 100 milligrams and about 1000 milligrams. In certain embodiments, the administration of the PD-1 axis inhibitor to an individual may be at a flat dosage level of between about 200 mg and about 800 mg, between about 200 mg and about 600 mg, between about 200 mg and about 500 mg, between about 300 mg and about 500 mg. In certain embodiments, the PD-1 axis inhibitor may be administered to a subject at a flat dosage level of about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 milligrams. In certain embodiments, administration of the PD-1 axis inhibitor to the individual may be at a level suitable for monotherapy. For example, nivolumab may be administered at a dose of about 240 mg every two weeks or about 480 mg every four weeks. In another example, the palbociclumab may be administered at about 200 mg once every three weeks.

Dose of h5D8

In certain embodiments, the h5D8 antibody can be administered by any route suitable for administration of a pharmaceutical composition comprising the antibody, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, h5D8 is administered intravenously. In certain embodiments, h5D8 is administered on a suitable dosage schedule, e.g., weekly, twice weekly, monthly, twice monthly, etc. In certain embodiments, h5D8 is administered once every three weeks. H5D8 may be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1mg/kg and about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5mg/kg and about 30 mg/kg. The h5D8 antibody can be administered in flat doses regardless of the weight or mass of the individual to whom the h5D8 antibody is administered. The h5D8 antibody may be administered in flat doses regardless of the weight or mass of the individual to whom the h5D8 antibody is administered, provided that the individual has a mass of at least about 37.5 kilograms. Flat doses of h5D8 may be administered from about 75 mg to about 2000 mg. Flat doses of h5D8 may be administered from about 225 mg to about 2000 mg, about 750 mg to about 2000 mg, about 1125 mg to about 2000 mg, or about 1500 mg to about 2000 mg. A flat dose of h5D8 may be administered at about 75 mg. A flat dose of h5D8 may be administered at about 225 mg. A flat dose of h5D8 may be administered at about 750 mg. A flat dose of h5D8 can be administered at about 1125 milligrams. A flat dose of h5D8 may be administered at about 1500 mg. A flat dose of h5D8 may be administered at about 2000 mg.

Other doses of h5D8 are contemplated. Flat doses of h5D8 may be administered at about 50, 100, 150, 175, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2025, 2050, 2075, or 2100 milligrams. Any of these doses can be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

Flat doses of h5D8 may be administered once per week from about 75 mg to about 2000 mg. Flat doses of h5D8 may be administered once per week from about 75 mg to about 1500 mg. Flat doses of h5D8 may be administered once per week from about 225 mg to about 1500 mg, from about 750 mg to about 1500 mg, from about 1125 mg to about 1500 mg. A flat dose of h5D8 may be administered once a week at about 75 mg. A flat dose of h5D8 may be administered once a week at about 225 mg. A flat dose of h5D8 may be administered once a week at about 750 mg. A flat dose of h5D8 can be administered once a week at about 1125 milligrams. A flat dose of h5D8 may be administered once a week at about 1500 mg. A flat dose of h5D8 may be administered once per week at about 2000 mg.

Flat doses of h5D8 may be administered from about 75 mg to about 2000 mg once every two weeks. Flat doses of h5D8 may be administered from about 75 mg to about 1500 mg once every two weeks. Flat doses of h5D8 may be administered once every two weeks from about 225 mg to about 1500 mg, from about 750 mg to about 1500 mg, from about 1125 mg to about 1500 mg. A flat dose of h5D8 may be administered at about 75 mg once every two weeks. A flat dose of h5D8 may be administered at about 225 mg once every two weeks. A flat dose of h5D8 may be administered at about 750 mg once every two weeks. A flat dose of h5D8 can be administered at about 1125 milligrams once every two weeks. A flat dose of h5D8 may be administered at about 1500 mg once every two weeks. A flat dose of h5D8 may be administered at about 2000 milligrams once every two weeks.

Flat doses of h5D8 may be administered from about 75 milligrams to about 2000 milligrams once every three weeks. Flat doses of h5D8 may be administered once every three weeks from about 75 mg to about 1500 mg. Flat doses of h5D8 may be administered once every three weeks from about 225 mg to about 1500 mg, from about 750 mg to about 1500 mg, from about 1125 mg to about 1500 mg. A flat dose of h5D8 may be administered at about 75 mg once every three weeks. A flat dose of h5D8 may be administered at about 225 milligrams once every three weeks. A flat dose of h5D8 may be administered at about 750 milligrams once every three weeks. A flat dose of h5D8 may be administered once every three weeks at about 1125 milligrams. A flat dose of h5D8 may be administered at about 1500 milligrams once every three weeks. A flat dose of h5D8 may be administered at about 2000 milligrams once every three weeks.

Flat doses of h5D8 may be administered from about 75 milligrams to about 2000 milligrams once every four weeks. Flat doses of h5D8 may be administered from about 75 milligrams to about 1500 milligrams once every four weeks. Flat doses of h5D8 may be administered once every four weeks from about 225 mg to about 1500 mg, about 750 mg to about 1500 mg, about 1125 mg to about 1500 mg. A flat dose of h5D8 may be administered at about 75 milligrams once every four weeks. A flat dose of h5D8 may be administered at about 225 milligrams once every four weeks. A flat dose of h5D8 may be administered at about 750 milligrams once every four weeks. A flat dose of h5D8 can be administered at about 1125 milligrams once every four weeks. A flat dose of h5D8 may be administered at about 1500 milligrams once every four weeks. A flat dose of h5D8 may be administered at about 2000 milligrams once every four weeks.

The h5D8 antibody may be administered at a dose based on the weight or mass of the individual to whom the h5D8 antibody is administered. Body weight-modifying doses of h5D8 can be administered from about 1mg/kg to about 25 mg/kg. A weight adjusted dose of h5D8 may be administered from about 3mg/kg to about 25mg/kg, from about 10mg/kg to about 25mg/kg, from about 15mg/kg to about 25mg/kg, or from about 20mg/kg to about 25 mg/kg. A weight adjusted dose of h5D8 can be administered at about 1 mg/kg. A weight adjusted dose of h5D8 can be administered at about 3 mg/kg. A weight adjusted dose of h5D8 may be administered at about 10 mg/kg. A weight adjusted dose of h5D8 may be administered at about 15 mg/kg. A weight adjusted dose of h5D8 may be administered at about 20 mg/kg. A weight adjusted dose of h5D8 may be administered at about 25 mg/kg.

A weight adjusted dose of h5D8 can be administered once every three weeks from about 1mg/kg to about 25 mg/kg. The weight-adjusted dose of h5D8 may be administered once per one, two, three, or four weeks at about 3mg/kg to about 25mg/kg, about 10mg/kg to about 20mg/kg, about 15mg/kg to about 25mg/kg, about 20mg/kg to about 25 mg/kg.

Other weight adjusted doses of h5D8 are contemplated. A weight-adjusted dose of h5D8 may be administered at about 2mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, or 30 mg/kg. Any of these doses can be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

A weight adjusted dose of h5D8 can be administered once a week at about 1 mg/kg. A weight adjusted dose of h5D8 can be administered once a week at about 3 mg/kg. A weight adjusted dose of h5D8 may be administered once a week at about 10 mg/kg. A weight adjusted dose of h5D8 may be administered once a week at about 15 mg/kg. A weight adjusted dose of h5D8 can be administered once a week at about 20 mg/kg. A weight adjusted dose of h5D8 can be administered once a week at about 25 mg/kg.

A weight adjusted dose of h5D8 may be administered at about 1mg/kg once every two weeks. A weight adjusted dose of h5D8 may be administered at about 3mg/kg once every two weeks. A weight adjusted dose of h5D8 may be administered at about 10mg/kg once every two weeks. A weight adjusted dose of h5D8 may be administered at about 15mg/kg once every two weeks. A weight adjusted dose of h5D8 may be administered at about 20mg/kg once every two weeks. A weight adjusted dose of h5D8 may be administered at about 25mg/kg once every two weeks.

A weight adjusted dose of h5D8 may be administered at about 1mg/kg once every three weeks. A weight adjusted dose of h5D8 may be administered at about 3mg/kg once every three weeks. A weight adjusted dose of h5D8 may be administered at about 10mg/kg once every three weeks. A weight adjusted dose of h5D8 may be administered at about 15mg/kg once every three weeks. A weight adjusted dose of h5D8 may be administered at about 20mg/kg once every three weeks. A weight adjusted dose of h5D8 may be administered at about 25mg/kg once every three weeks.

A weight adjusted dose of h5D8 may be administered at about 1mg/kg once every four weeks. A weight adjusted dose of h5D8 may be administered at about 3mg/kg once every four weeks. A weight adjusted dose of h5D8 may be administered at about 10mg/kg once every four weeks. A weight adjusted dose of h5D8 may be administered at about 15mg/kg once every four weeks. A weight adjusted dose of h5D8 may be administered at about 20mg/kg once every four weeks. A weight adjusted dose of h5D8 may be administered at about 25mg/kg once every four weeks.

Any of the doses detailed herein can be administered intravenously over a period of at least about 60 minutes; however, this time period may vary somewhat depending on the conditions associated with each individual administration.

Dosage schedules for combination therapy

Combination therapy comprising a LIF-binding polypeptide and a PD-1 axis inhibitor can be administered in a variety of ways. The LIF-binding polypeptide and the PD-1 axis inhibitor can be administered simultaneously on the same schedule or at different times on different schedules. When administered simultaneously, administration can be by separate formulations or a single formulation comprising the LIF-binding polypeptide and the PD-1 axis inhibitor. The administration may be mixed, e.g., LIF-binding polypeptide may be administered intravenously, while PD-1 axis inhibitor may be administered orally or parenterally by injection. In certain embodiments, the LIF-binding polypeptide is administered intravenously, parenterally, subcutaneously, intratumorally, or orally. In certain embodiments, the PD-1 axis inhibitor is administered intravenously, parenterally, subcutaneously, intratumorally, or orally.

When the individual is treated with the combination therapy according to the same regimen, the LIF-binding polypeptide and the PD-1 axis inhibitor can be administered weekly, biweekly, every three weeks, or every four weeks. The LIF-binding polypeptide and the PD-1 axis inhibitor can be administered separately or as a single formulation. The H5D8 and PD-1 axis inhibitor can be applied separately or as a single formulation.

LIF-binding polypeptides and PD-1 axis inhibitors can be used interchangeably when the individual is subjected to combination therapy on different schedules. In certain embodiments, the subject may be administered one or more PD-1 axis inhibitors prior to administration of the LIF-binding polypeptide. The LIF-binding polypeptide can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of the PD-1 axis inhibitor. The LIF-binding polypeptide can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of the PD-1 axis inhibitor. The h5D8 antibody can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of the PD-1 axis inhibitor. The h5D8 antibody can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of the PD-1 axis inhibitor.

The LIF-binding polypeptide can be administered to the individual one or more times prior to administration of the PD-1 axis inhibitor. In certain embodiments, the PD-1 axis inhibitor can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of the LIF binding polypeptide. In certain embodiments, the PD-1 axis inhibitor can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of the LIF binding polypeptide. In certain embodiments, the PD-1 axis inhibitor may be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of the h5D8 antibody. In certain embodiments, the PD-1 axis inhibitor may be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of the h5D8 antibody.

In certain embodiments, the LIF-binding polypeptide can be administered to the individual once per week, and the PD1 axis inhibitor can be administered to the individual every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the LIF-binding polypeptide can be administered to the individual once every two weeks, and the PD1 axis inhibitor can be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the LIF-binding polypeptide can be administered to the individual once every three weeks, and the PD1 axis inhibitor can be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the LIF-binding polypeptide can be administered to the individual once every four weeks, and the PD1 axis inhibitor can be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the PD1 axis inhibitor may be administered to the individual once per week, and the LIF binding polypeptide may be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the PD1 axis inhibitor can be administered to the individual once every two weeks, and the LIF binding polypeptide can be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the PD1 axis inhibitor may be administered to the individual once every three weeks, and the LIF binding polypeptide may be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the PD1 axis inhibitor may be administered to the individual once every four weeks, and the LIF binding polypeptide may be administered to the individual weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the h5D8 may be administered to the subject one or more times prior to administration of the PD-1 axis inhibitor. In certain embodiments, the h5D8 may be administered to the subject once per week, and the PD1 axis inhibitor may be administered to the subject weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the h5D8 may be administered to the subject once every two weeks, and the PD1 axis inhibitor may be administered to the subject weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the h5D8 may be administered to the subject once every three weeks, and the PD1 axis inhibitor may be administered to the subject weekly, biweekly, every three weeks, or every four weeks. In certain embodiments, the h5D8 may be administered to the subject once every four weeks, and the PD1 axis inhibitor may be administered to the subject weekly, biweekly, every three weeks, or every four weeks.

Combination therapies according to the present disclosure may include combinations in which one or both of the activating components (e.g., LIF binding polypeptide and PD-1 inhibitor) are not effective by themselves, but are effective when administered as part of a combination therapy. In certain embodiments, the PD-1 inhibitor is administered at a level that is ineffective for monotherapy but effective in combination with the LIF binding polypeptide. In certain embodiments, the PD-1 inhibitor is administered at a level that is ineffective for monotherapy but effective in combination with the h5D8 antibody. In certain embodiments, the LIF-binding polypeptide is administered at a level that is ineffective for monotherapy but effective in combination with a PD-1 inhibitor. In certain embodiments, h5D8 is administered at a level that is not effective for monotherapy but is effective in combination with a PD-1 inhibitor. In certain embodiments, both the LIF-binding polypeptide and the PD-1 inhibitor are administered at levels that are ineffective for monotherapy but effective in combination. In certain embodiments, both the h5D8 and the PD-1 inhibitor are administered at levels that are ineffective for monotherapy but effective in combination.

Treatment indications

In certain embodiments, disclosed herein are methods and compositions useful for treating cancer or tumors. In certain embodiments, the cancer comprises breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone marrow, blood, thymus, uterus, testis, and liver tumors. In certain embodiments, tumors that can be treated with the antibodies of the invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioblastoma, glioma, endovascular carcinoma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and/or teratoma. In certain embodiments, the tumor/cancer is selected from the group consisting of: lentigo-like melanoma on extremities, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, babbitt adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, intradermal sinus tumor, endometrial hyperplasia, endometrial interstitial sarcoma, endometrioid adenocarcinoma, ventricular septal sarcoma, ewing sarcoma, focal nodular hyperplasia, gastric adenoma, germ cell line tumor, glioblastoma, glucagonoma, hemangioblastoma, angioendothelioma, hemangioma, hepatic adenoma, hepatic adenocarcinoma, hepatocellular carcinoma, insulinoma (insulinite), intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, human immunodeficiency virus, human, Leiomyosarcoma, melanoma, malignant mesothelioma, schwannoma, medulloblastoma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, serous adenoma of the nipple, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pneumocblastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal/vulvar carcinoma, vasoactive intestinal peptide tumor (Ppoma), and Wilm's tumor (Wilm's tulor). In certain embodiments, the tumors/cancers to be treated with one or more antibodies of the invention include brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma (preferably grade II, III or IV astrocytoma), glioblastoma multiforme, small cell carcinoma and non-small cell carcinoma (preferably non-small cell lung cancer), lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer (preferably ductal breast cancer and/or breast cancer). In certain embodiments, the cancer treated with an antibody of the disclosure comprises glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises lung cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises colon cancer. In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In a certain embodiment, the cancer is refractory to other therapies. In a certain embodiment, the cancer treated is recurrent. In a certain embodiment, the cancer is relapsed/refractory glioblastoma, pancreatic, ovarian, colon, prostate, or lung cancer. In certain embodiments, the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer. In certain embodiments, the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer comprises an advanced solid tumor. In certain embodiments, the individual is refractory to prior treatment with a LIF-binding antibody as monotherapy. In certain embodiments, the subject is refractory to prior treatment with a PD-1 axis inhibitor as monotherapy.

Pharmaceutically acceptable excipients, carriers and diluents

In certain embodiments, the PD-1 axis inhibitor and LIF binding polypeptide of the present disclosure are components of a pharmaceutical composition. In certain embodiments, the PD-1 axis inhibitor and LIF binding polypeptide of the present disclosure are components of the same pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the pharmaceutical composition comprises between about 0.6% and 1.2% NaCl. In certain embodiments, the pharmaceutical composition comprises between about 0.7% and 1.1% NaCl. In certain embodiments, the pharmaceutical composition comprises between about 0.8% and 1.0% NaCl. In certain embodiments, the pharmaceutical composition comprises about 5% dextrose. In certain embodiments, the pharmaceutical composition further comprises one or more of: buffers such as acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethyl aminomethane (Tris); surfactants, for example, polysorbate 80 (tween 80), polysorbate 20 (tween 20), polysorbate, and poloxamer 188; polyols/disaccharides/polysaccharides, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, histidine, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; and chelating agents, such as EDTA or EGTA.

In certain embodiments, a PD-1 axis inhibitor, LIF-binding polypeptide, or both a PD-1 axis inhibitor and LIF-binding polypeptide of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the PD-1 axis inhibitor and the LIF binding polypeptide are administered from the same solution. In certain embodiments, the solution comprises a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the solution comprises between about 0.6% and 1.2% NaCl. In certain embodiments, the solution comprises between about 0.7% and 1.1% NaCl. In certain embodiments, the solution comprises between about 0.8% and 1.0% NaCl. In certain embodiments, the highly concentrated stock solution of antibodies can be diluted in about 0.9% NaCl. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution further comprises one or more of: buffers such as acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethyl aminomethane (Tris); surfactants, for example, polysorbate 80 (tween 80), polysorbate 20 (tween 20), polysorbate, and poloxamer 188; polyols/disaccharides/polysaccharides such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40 and combinations thereof; amino acids, for example, histidine, glycine or arginine; antioxidants, for example, ascorbic acid, methionine, and combinations thereof; and chelating agents, such as EDTA or EGTA. In certain embodiments, the PD-1 axis inhibitor and LIF binding polypeptide of the present disclosure are lyophilized for transport/storage and reconstitution prior to administration. In certain embodiments, the lyophilized antibody formulation includes bulking agents such as mannitol, sorbitol, sucrose, trehalose, and dextran 40 and combinations thereof. In a certain embodiment, the anti-LIF antibodies of the present disclosure can be shipped and stored as a concentrated stock solution that is diluted for use at the treatment site. In certain embodiments, the stock solution comprises about 25mM histidine, about 6% sucrose, about 0.01% polysorbate, and about 20mg/mL of anti-LIF antibody. In certain embodiments, the pH of the solution is about 6.0. In certain embodiments, the form administered to the subject is an aqueous solution comprising about 25mM histidine, about 6% sucrose, about 0.01% polysorbate 80, and about 20mg/mL h5D8 antibody. In certain embodiments, the pH of the solution is about 6.0.

In certain embodiments, the PD-1 axis inhibitor and LIF binding polypeptide of the present disclosure are administered in suspension in a sterile solution. In certain embodiments, the PD-1 axis inhibitor and the LIF binding polypeptide are administered from the same solution. In certain embodiments, the PD-1 axis inhibitor and the LIF binding polypeptide are administered from separate solutions. In certain embodiments, the solution comprises a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the solution comprises between about 0.6% and 1.2% NaCl. In certain embodiments, the solution comprises between about 0.7% and 1.1% NaCl. In certain embodiments, the solution comprises between about 0.8% and 1.0% NaCl. In certain embodiments, the highly concentrated stock solution of antibodies can be diluted in about 0.9% NaCl. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution further comprises one or more of: buffers such as acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethyl aminomethane (Tris); surfactants, for example, polysorbate 80 (tween 80), polysorbate 20 (tween 20), polysorbate, and poloxamer 188; polyols/disaccharides/polysaccharides such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40 and combinations thereof; amino acids, for example, histidine, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; and chelating agents, such as EDTA or EGTA. In certain embodiments, the PD-1 axis inhibitor and LIF binding polypeptide of the present disclosure are lyophilized for transport/storage and reconstitution prior to administration. In certain embodiments, the lyophilized antibody formulation includes bulking agents such as mannitol, sorbitol, sucrose, trehalose, and dextran 40 and combinations thereof. In a certain embodiment, the anti-LIF antibodies of the present disclosure can be shipped and stored as a concentrated stock solution that is diluted for use at the treatment site. In certain embodiments, the stock solution comprises about 25mM histidine, about 6% sucrose, about 0.01% polysorbate, and about 20mg/mL of anti-LIF antibody. In certain embodiments, the pH of the solution is about 6.0. In certain embodiments, the form administered to the subject is an aqueous solution comprising about 25mM histidine, about 6% sucrose, about 0.01% polysorbate 80, and about 20mg/mL h5D8 antibody. In certain embodiments, the pH of the solution is about 6.0.

Also described herein are kits for performing the combination therapies described herein. In certain embodiments, the kit comprises a LIF-binding polypeptide and a PD-1 axis inhibitor. In certain embodiments, the kit comprises h5D8 and a PD-1 axis inhibitor. One or both of the two components may be contained in a vial of glass or other suitable material in lyophilized or liquid form.

The h5D8 antibody described herein can be included in a kit comprising a vial containing a sterile solution comprising the h5D8 antibody at a concentration of about 20mg/mL, about 25mM histidine, about 6% sucrose, and about 0.01% polysorbate 80. The vial may be a disposable glass vial. The disposable glass vial may be filled with about 10 milliliters of 5D8 antibody at a concentration of about 20mg/mL h5D8 antibody, about 25mM histidine, about 6% sucrose, and about 0.01% polysorbate 80. In certain embodiments, the pH of the solution is about 6.0. The h5D8 antibody described herein can be included in a kit comprising a vial containing a lyophilized composition comprising the h5D8 antibody that, when reconstituted in an appropriate amount of sterile diluent, produces a concentration of about 20mg/mL of the h5D8 antibody, about 25mM histidine, about 6% sucrose, and about 0.01% polysorbate 80. The vial may be a disposable glass vial.

The antibodies described herein may be administered or prepared or diluted for administration in different ways depending on the dosage level ultimately to be delivered to the patient. This may be done, for example, to optimize the drug properties of the patient dose, e.g., to reduce particulate matter. H5D8 may be prepared at a concentration of about 8mg/mL, regardless of the final dose delivered to the patient. In certain embodiments, h5D8 can be prepared at a level of no more than about 10, 9, 8, 7, 6, 5, or 4 mg/mL. In certain embodiments, h5D8 can be prepared at a level greater than about 1, 2, 3, 4, 5, 6, or 7 mg/mL.

Examples of the invention

The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

Example 1-Generation of rat antibodies specific for LIF

The cDNA encoding amino acids 23-202 of human LIF was cloned into an expression plasmid (Aldefron GmbH, Flisburg, Germany). Groups of experimental rats (Wistar) were immunized by intradermal administration of DNA-coated gold particles using a hand-held particle bombardment device ("gene gun"). Cell surface expression on transiently transfected HEK cells was confirmed with an anti-tag antibody recognizing a tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations and HEK cells transiently transfected with the above expression plasmids were tested in flow cytometry. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas that produce antibodies specific for LIF are identified by screening in flow cytometry assays, as described above. Cell pellets of positive hybridoma cells were prepared using RNA protective agents (RNAlater, catalog No. AM7020, zemer feishel Scientific) and further processed for sequencing of antibody variable domains.

Example 2 Generation of mouse antibodies specific for LIF

The cDNA encoding amino acids 23-202 of human LIF was cloned into an expression plasmid (Aldefron GmbH, Flisburg, Germany). Groups of experimental mice (NMRI) were immunized by intradermal administration of DNA-coated gold particles using a hand-held particle bombardment device ("gene gun"). Cell surface expression on transiently transfected HEK cells was confirmed with an anti-tag antibody recognizing a tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations and HEK cells transiently transfected with the above expression plasmids were tested in flow cytometry. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas that produce antibodies specific for LIF are identified by screening in flow cytometry assays, as described above. Cell pellets of positive hybridoma cells were prepared using an RNA protective agent (RNAlater, catalog No. AM7020, zemer feishell scientific) and further processed for sequencing of antibody variable domains.

Example 3 humanization of rat antibodies specific for LIF

One clone (5D8) was selected from the rat immunization for subsequent humanization. Humanization was performed using standard CDR grafting methods. The heavy and light chain regions were cloned from the 5D8 hybridoma using standard molecular cloning techniques and sequenced by the Sanger method. BLAST searches were then performed against the human heavy and light chain variable sequences and 4 sequences were selected from each variable sequence as acceptor frameworks for humanization. These acceptor frameworks were deimmunized to remove T cell reactive epitopes. The heavy and light chain CDRs 1, CDR2 and CDR3 of 5D8 were cloned into 4 different heavy chain acceptor frameworks (H1 to H4) and 4 different light chain frameworks (L1 to L4). All 16 different antibodies were then tested: expression in CHO-S cells (Selexis); inhibition of LIF-induced STAT3 phosphorylation; and binding affinity by Surface Plasmon Resonance (SPR). These experiments are summarized in table 1.

After 10 days of cell culture, in flasks in fed-batch culture (3X 10 inoculum)⁵Individual cells/mL, 200mL culture volume) were compared for expression performance of transfected cells. At this point, cells were harvested and secreted antibodies were purified using a protein a column and then quantified. All humanized antibodies were expressed, except for the antibody using the H3 heavy chain (SEQ ID NO: 43). The H2 and L2 variable regions performed well compared to the other variable regions (SEQ ID NO:42 and SEQ ID NO: 46).

Inhibition of LIF-induced STAT3 phosphorylation at tyrosine 705 was determined by western blotting. U251 glioma cells were plated at a density of 100.000 cells/well in 6-well plates. Cells were cultured in complete medium for 24 hours before any treatment, after which the serum was starved for 8 hours. Thereafter, the cells containing the indicator antibody were allowed to stand overnight at a concentration of 10. mu.g/ml. After treatment, proteins were obtained in phosphatase and protease inhibitors containing radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, seimer feishell scientific) and used for western blotting. For western blotting, membranes were incubated in 5% skim milk powder-TBST for 1 hour and with primary antibody overnight (p-STAT3, cat # blockade 9145, Cell Signaling or STAT3, cat # 9132, Cell Signaling) or 30 minutes (β -actin peroxidase, cat # a3854, Sigma Aldrich). The membrane was then washed with TBST, incubated with secondary antibody and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog No. 34076, seimer feishell scientific). These results are shown in figure 1. The darker the pSTAT3 band, the less inhibitory effect. Inhibition was higher in lanes labeled 5D8 (non-humanized rat), a (H0L0), C (H1L2), D (H1L3) and G (H2L 2); moderate inhibition in H (H2L3), O (H4L2) and P (H4L 3); there was no inhibition in B (H1L1), E (H1L4), F (H2L1), I (H2L4), N (H4L1) and Q (H4L 4).

Antibodies exhibiting inhibition of LIF-induced STAT3 phosphorylation were then analyzed by SPR to determine binding affinity. Briefly, Biacore was used^TMThe 2002 instrument observed binding of a (H0L0), C (H1L2), D (H1L3), and G (H2L2), H (H2L3), and O (H4L2) humanized antibodies to amine-coupled hLIF. Mathematical sensorgram fitting (langmuir interaction model [ a + B ═ AB) through all sensorgrams generated on all sensor chip surfaces at six ligand concentrations]) Kinetic constants and affinities were determined. The best fit curve (min Chi2) for each concentration was used to calculate kinetic constants and affinities. See table 1.

Since the experimental setup used a bivalent antibody as analyte, the best-fit sensor maps were also based on a bivalent analyte fitting model [ a + B ═ AB; AB + B ═ AB2] were analyzed to understand in more detail the targeted binding mechanism of the humanized antibodies. Using a bivalent fitting model [ a + B ═ AB; kinetic sensorgram analysis of AB + B ═ AB2] confirmed the relative affinity ordering of mAb samples.

Humanized 5D8, including H2 and L2, was chosen for more in-depth analysis due to its high binding affinity and high batch culture yield.

Example 4 humanization of clone 5D8 improved binding to LIF

The H2L2 clone (H5D8) was selected for further analysis and compared for binding to parental rat 5D8(r5D8) and mouse clone 1B2 by SPR. The 1B2 antibody was a previously disclosed mouse anti-LIF antibody previously deposited in Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM ACC3054) and used for comparative purposes. Recombinant human LIF purified from E.coli and HEK-293 cells, respectively, was used as ligand. LIF from human or e.coli was covalently coupled to the surface of the Biacore optical sensor chip using amine coupling chemistry and binding affinity was calculated from kinetic constants.

Materials and methods

Human LIF from e.coli was obtained from Millipore, reference LIF 1010; human LIF from HEK-293 cells was obtained from ACRO Biosystems (ACRO Biosystems) reference LIF-H521 b. LIF was coupled to the sensor chip using a Biacore amine coupling kit (BR-1000-50; GE Healthcare, Uppsala). Using CM5 optical sensor chip (BR-1000-12; GE healthcare group, Uppsala) in Biacore^TMThe 2002 instrument runs the sample. Biacore HBS-EP buffer was used during machine runs (BR-1001-88; GE healthcare group, Uppsala). Kinetic analysis of binding sensorgrams was performed using BIAevaluation 4.1 software. With increasing analyte concentration, fitting of the mathematical sensorgrams through all sensorgrams generated on all sensor chip surfaces (langmuir interaction model [ a + B ═ AB |) ]) Kinetic constants and affinities were determined. The sensor map was also fitted to the model [ a + B ═ AB; AB + B ═ AB₂]Analysis, including compositional analysis, is performed to generate an estimate of the bivalent contribution to the determined langmuir antibody-target affinity (e.g., affinity contribution). Best fit curve (min Chi) for each concentration²) For calculation of kinetic constants and affinities. A summary of these affinity experiments is shown in Table 2 (human LIF made in E.coli) and Table 3 (human LIF made in HEK293 cells).

The langmuir 1:1 sensorgram fit model from this set of experiments indicated that the humanized 5D8(h5D8) antibody had approximately 10-25 times higher affinity for human LIF than mouse 1B2 and r5D 8.

Next, the h5D8 antibody was tested against LIF of multiple species by SPR. The h5D8 SPR binding kinetics were performed on recombinant LIF analytes from different species and expression systems: human LIF (e.coli, HEK293 cells); mouse LIF (e.coli, CHO cells); rat LIF (e.coli); cynomolgus monkey LIF (yeast, HEK293 cells).

Materials and methods

The h5D8 antibody was immobilized on the sensor chip surface by non-covalent Fc specific capture. Recombinant ig (fc) -specific staphylococcus aureus protein a/G was used as a capture agent, allowing spatially uniform and flexible presentation of anti-LIF antibodies to LIF analytes. The sources of LIF analytes are as follows: human LIF (from E.coli; Michibo reference LIF 1050); human LIF (from HEK cells, ACRO biosystems LIF-H521); mouse LIF (E.coli; catalog number NF-LIF2010, Mirabbo); mouse LIF (from CHO cells; Repurogold, catalog number RCP 09056); monkey LIF (yeast, Kingfisher Biotech catalog No. RP 1074Y); monkey LIF produced in HEK-293 cells. Overall, h5D8 exhibited binding to LIF of several species. An overview of the affinity experiments is shown in table 4.

EXAMPLE 5 humanized clone 5D8 inhibition of LIF-induced STAT3 phosphorylation in vitro

To determine the biological activity of h5D8, humanized and parental forms were tested in the LIF-activated cell culture model. Figure 2A shows that humanized clones exhibited increased inhibition of STAT3 phosphorylation (Tyr 705) when glioma cell lines were incubated with human LIF. Figure 2B shows an experiment of the same setup of figure 2A repeated with different dilutions of the h5D8 antibody.

Materials and methods

U251 glioma cells were plated at a density of 150,000 cells/well in 6-well plates. Cells were cultured in complete medium for 24 hours prior to any treatment. Thereafter, r5D8 anti-LIF antibody or h5D8 anti-LIF antibody was used at a concentration of 10. mu.g/ml overnight treatment or no treatment (control cells).

After treatment, proteins were obtained in phosphatase and protease inhibitors containing radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, seimer feishell scientific) and used for western blotting. For western blotting, membranes were incubated in 5% non-fat milk-TBST for 1 hour and with primary antibody overnight (p-STAT3, cat # blockade 9145, cell signaling or STAT3, cat # 9132, cell signaling) or 30 minutes (β -actin peroxidase, cat # a3854, sigma aldrich). The membrane was then washed with TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog No. 34076, seimer feishell scientific).

Example 6-endogenous levels of LIF in U-251 cells IC for h5D8 antibody treatment₅₀The value is obtained.

The IC of the biological inhibition of h5D8 in U-251 cells under serum starvation conditions was also determined₅₀As low as 490 picomoles (FIG. 3A). See fig. 3A and 3B and representative results of table 5.

Materials and methods

The U-251 cells were seeded at 600,000 cells per 6cm plate (each condition). Cells were treated with h5D8 at the corresponding concentration (titration) overnight at 37 ℃ under serum starvation (0.1% FBS). As a positive control for pSTAT3, recombinant LIF (R & D #7734-LF/CF) was used to stimulate cells at 1.79nM for 10 min at 37 ℃. As a negative control for pSTAT3, a JAK I inhibitor (Calbiochem #420099) was used for 30 minutes at 1uM at 37 ℃. Cells were then harvested as lysates on ice following the protocol of the Meso Scale Discovery multi-point detection system total STAT3 (cat # K150SND-2) and Phospho-STAT3(Tyr705) (cat # K150SVD-2) kits to measure protein levels detectable by MSD Meso Sector S600.

Example 7-other antibodies that specifically bind to human LIF

Additional rat antibody clones that specifically bind human LIF were identified (10G7 and 6B5), an overview of their binding characteristics is shown in table 6 below, using clone 1B2 as a comparison.

Materials and methods

Recombinant LIF target protein [ human LIF (e.coli); millibo catalog number LIF 1010 and human LIF (HEK293 cells); ACRO biosystems Catalogue number LIF-H521B ] kinetic real-time binding assays were performed on anti-LIF mAbs 1B2, 10G7 and 6B5 immobilized on the surface of a CM5 optical sensor chip as analytes.

Kinetic constants and affinities were obtained by mathematical sensor map fitting using the langmuir 1:1 binding model applying global (simultaneous fitting of sensor atlas) and single curve fitting algorithms. Through k_obsThe analysis assesses the rationality of the global fit.

Example 8-other anti-LIF antibodies inhibit LIF-induced STAT3 phosphorylation in vitro

Other clones were tested for their ability to inhibit LIF-induced STAT3 phosphorylation in cell culture. As shown in figure 4, clone 10G7 and r5D8, previously detailed, exhibited a high inhibition of LIF-induced STAT3 phosphorylation compared to clone 1B 2. anti-LIF polyclonal antiserum (positive) was included as a positive control. Although 6B5 exhibited no inhibitory effect, this may be due to the possible lack of binding of 6B5 to the unglycosylated LIF used in this experiment.

Materials and methods

Patient-derived glioma cells were seeded at a density of 150,000 cells/well in 6-well plates. Prior to any treatment, cells were cultured for 24 hours in GBM medium consisting of neuronal basal medium (Life Technologies) supplemented with B27 (Life Technologies), penicillin/streptomycin and growth factors (20ng/ml EGF and 20ng/ml FGF-2 (pepro Technologies) ]). The following day, whether cells were treated with recombinant LIF produced in E.coli or a mixture of recombinant LIF and the indicator antibody for 15 minutes (final concentration of antibody 10. mu.g/ml, final concentration of recombinant LIF 20 ng/ml). After treatment, proteins were obtained in phosphatase and protease inhibitors containing radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, seimer feishell scientific) and used for western blotting. For western blotting, membranes were incubated in 5% non-fat milk-TBST for 1 hour and with primary antibody overnight (p-STAT3, cat # block 9145, cell signaling) or 30 minutes (β -actin peroxidase, cat # a3854, sigma aldrich). The membrane was then washed with TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog No. 34076, seimer feishell scientific).

Example 9-LIF is highly overexpressed in various tumor types

Immunohistochemistry experiments were performed on a variety of human tumor types to determine the extent of LIF expression. As shown in fig. 5, LIF is highly expressed in glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer (CRC), and pancreatic cancer.

EXAMPLE 10 humanized clone h5D8 inhibits tumor growth in a mouse model of non-Small cell Lung cancer

To determine the ability of the humanized 5D8 clone to inhibit LIF-positive cancer in vivo, the antibody was tested in a non-small cell lung cancer (NSCLC) mouse model. Figure 6A shows that tumor growth was reduced in mice treated with this antibody compared to vehicle negative control. Fig. 6B shows data generated using the r5D8 version.

Materials and methods

Murine non-small cell lung carcinoma (NSCLC) cell line KLN205 with high LIF levels was stably infected with a lentivirus expressing the firefly luciferase gene for in vivo bioluminescence monitoring. To establish the mouse model, 5x 10 was placed by intercostal puncture⁵One KLN205 non-small cell lung cancer (NSCLC) cell was implanted in situ into the left lung of an 8-week-old immunoreceptive DBA/2 mouse. Mice were treated intraperitoneally twice weekly with either control vehicle or with 15mg/kg or 30mg/kg of h5D8 antibody, and tumor growth was monitored by bioluminescence. For bioluminescence imaging, mice received an intraperitoneal injection of 0.2mL of 15mg/mL D-luciferin under 1% -2% inhaled isoflurane anesthesia. Bioluminescence signals were monitored using the IVIS system 2000 series (Xenogen Corp.), alamida, ca, usa, which consisted of a high sensitivity cooled CCD camera. Imaging data were gridded using live imaging software (hinoko corporation) and the total bioluminescence signal was integrated in each box-like region. The data was analyzed using total photon flux emission (photons/sec) in the region of interest (ROI). The results demonstrate that treatment with the h5D8 antibody promotes tumor regression. Data are presented as mean ± SEM.

Example 11-h5D8 inhibition of tumor growth in a mouse model of glioblastoma multiforme

In an in situ GBM tumor model using the luciferase-expressing human cell line U251, r5D8 significantly reduced the tumor volume in mice administered twice weekly Intraperitoneal (IP) injections with 300 μ g r5D8 and h5D 8. The results of this study are shown in fig. 7A (quantification at day 26 post-treatment). This experiment was also performed using either 200 μ g or 300 μ g of humanized h5D8 treated mice, showing a statistically significant tumor reduction 7 days after treatment.

Materials and methodsMethod of

U251 cells stably expressing luciferase were harvested, washed in PBS, centrifuged at 400g for 5 minutes, resuspended in PBS, and counted using an automated cell counter (Countess, Invitrogen). Cells were kept on ice to maintain optimal viability. Intraperitoneal injection of ketamineXylose amineMice were anesthetized (75 mg/kg and 10mg/kg, respectively). Each mouse was carefully placed in a stereotactic apparatus and fixed. The hair of the head was removed with depilatory cream and the skin of the head was incised with a scalpel to expose the skull. A small cut was made carefully with the drill at a position 1.8mm lateral to the lambda surface and 1mm anterior to the lambda surface. mu.L of cells were seeded into right tattoos 2.5mm deep using a Hamilton 30G syringe. The head incision was closed with Hystoacryl tissue adhesive (Brann) and mice were injected with the subcutaneous analgesic meloxicam (1 mg/kg). The final number of cells implanted per mouse was 3x 10⁵。

Mice were treated with h5D8 intraperitoneally twice weekly. Treatment was started on day 0 immediately after tumor cell inoculation. Mice received 2 doses of h5D8 or vehicle control altogether.

Body weight and tumor volume: body weight was measured 2 times per week and tumor growth was quantified by bioluminescence on day 7 (genistein IVIS spectra). To quantify the bioluminescent activity in vivo, mice were anesthetized with isoflurane and injected intraperitoneally with a luciferin substrate (PerkinElmer) (167 μ g/kg).

Tumor size determined by bioluminescence (IVIS spectra, jinomori) was assessed on day 7. Individual tumor measurements and mean ± SEM were calculated for each treatment group. Statistical significance was determined by unpaired nonparametric Mann-Whitney U test.

Example 12-h5D8 inhibition of tumor growth in a mouse model of ovarian cancer

The efficacy of r5D8 was evaluated in two other syngeneic tumor models. In ovarian orthotopic tumor model ID8, intraperitoneal administration of 300 μ g r5D8 twice weekly significantly inhibited tumor growth as measured by abdominal volume (fig. 8A and 8B). The results in fig. 8C show that h5D8 also reduced tumor volume at doses of 200 μ g and above.

Materials and methods

ID8 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Invitrogen), 40U/mL penicillin and 40. mu.g/mL streptomycin (PenStrep) (Gibco, Invitrogen) and 0.25. mu.g/mL plasmacyclin (Plasmocin) (Invivogen)).

ID8 cells were harvested, washed in PBS, centrifuged at 400g for 5 minutes, and resuspended in PBS. Cells were placed on ice to maintain optimal viability and 200 μ L of cell suspension was injected intraperitoneally with a 27G needle. The final number of cells implanted in mice was 5x 10⁶。

Mice were treated twice weekly with h5D8 administered by intraperitoneal injection at different doses as indicated. Body weight was measured 2 times per week and tumor progression was monitored by measuring the abdominal circumference using a caliper (Fisher Scientific).

Example 13-r5D8 inhibition of tumor growth in a mouse model of colorectal cancer

In mice with subcutaneous colon CT26 tumor, r5D8 (administered 300 μ g intraperitoneally twice weekly) significantly inhibited tumor growth (fig. 9A and 9B).

Materials and methods

CT26 cells were cultured in Roseviv park memorial Institute (Roswell park mechanical Institute) medium (RPMI [ Gibco, Invitrogen ]) supplemented with 10% Fetal Bovine Serum (FBS), 40U/mL penicillin, and 40. mu.g/mL streptomycin (PenStrep) and 0.25. mu.g/mL plasmacyclin.

Digestion of CT26 cells (8X 10) with Trypsin⁵) Washed with PBS, centrifuged at 400g for 5 min, and resuspended in 100 μ LPBS. Will be provided withCells were placed on ice to avoid cell death. The CT26 cells were administered to mice by subcutaneous injection using a 27G needle.

Mice were administered 300 μ g r5D8 or vehicle control by intraperitoneal Injection (IP) twice weekly starting on day 3 post-CT 26 cell implantation.

Body weight and tumor volume were measured three times per week. Tumor volume was measured using calipers (feishell technologies).

Example 14-r5D8 reduced inflammatory infiltration in tumor models

As shown in figure 10A, the expression of CCL22 (marker for M2 polarized macrophages) was significantly reduced in tumors treated with r5D8 in situ model of U251 GBM. This finding was also confirmed in a physiologically relevant organotypic tissue slice culture model using r5D8, as shown in figure 10B, where three patient samples had significantly reduced CCL22 and CD206(MRC1) expression (also a marker for M2 macrophages) after treatment (compare the upper (control) and lower (treated) limits of MRC1 and CCL 22). In addition, r5D8 also reduced CCL22 in syngeneic ID8 (fig. 10C) and CT26 (fig. 10D) tumors in immune competent mice⁺M2 macrophages. H5D8 treatment also programmed macrophages towards an immunostimulatory phenotype in the syngeneic CT26 tumor model (fig. 10E). h5D8 treatment increased macrophages with the M1 phenotype as shown by the increased CD206 negative/MHCII positive fraction, while macrophages with the M2 phenotype were decreased as shown by the decreased CD206 positive/MHCII negative fraction. Fig. 10F shows gene expression data from monocytes cultured in conditioned medium of LIF-knockdown U251 cells. MRC1, CCL2, CCL1, and CTSK (indicated by triangles) all showed significant reduction in expression.

Example 15-r5D8 increase non-myeloid Effector cells

To investigate other immune mechanisms, the effect of r5D8 on T cells and other non-myeloid immune effector cells within the tumor microenvironment was evaluated. As shown in FIG. 11A, r5D8 treatment resulted in an increase in NK cells in tumors and total and activated CD4 in the ovarian orthotopic ID8 syngeneic model⁺And CD8⁺T cells are increased. Similarly, r5D8 increased intratumoral NK cells, increased CD4+ and CD in the colon syngeneic CT26 tumor model, as shown in fig. 11B8+ T cells, and tends to decrease CD4⁺CD25⁺FoxP3⁺T-reg cells. As shown in FIG. 11C, CD4 was also observed in the syngeneic orthotopic KLN205 tumor model following r5D8 treatment⁺CD25⁺FoxP3⁺Tendency of T-reg cells to decrease. As shown in FIG. 12, consistent with the requirement for T-cell mediated efficacy, CD4 in the CT26 model⁺And CD8⁺Depletion of T cells inhibited the anti-tumor efficacy of r5D 8.

Materials and methods for T cell depletion

In the presence of 10% fetal bovine serum (FBS [ Gibco, Invitrogen Co.)]) 40U/mL penicillin and 40. mu.g/mL streptomycin (PenStrep [ Gibco, Invitrogen Co.)]) And 0.25. mu.g/mL plasmacycline (Nevay) in RPMI medium (Gibco, Invitrogen) to culture CT26 cells. CT26 cells (5X 10) were collected⁵) Washed with PBS, centrifuged at 400g for 5 min, and resuspended in 100 μ LPBS. Cells were placed on ice to avoid cell death. CT26 cells were administered to mice in both ribs by subcutaneous injection using a 27G syringe. R5D8 was administered intraperitoneally twice weekly to treat mice as shown in the study design. Mice were administered vehicle control (PBS), rat r5D8 and/or anti-CD 4 and anti-CD 8 by intraperitoneal Injection (IP) twice weekly as described in the study design. All antibody treatments were administered concomitantly.

EXAMPLE 16 Crystal Structure of h5D8 complexed with human LIF

The crystal structure of h5D8 was resolved to a resolution of 3.1 angstroms in order to determine the epitope on LIF to which h5D8 binds and to determine the h5D8 residues involved in binding. The co-crystal structure revealed that the N-terminal loop of LIF was centered between the h5D8 light and heavy chain variable regions (fig. 13A). In addition, h5D8 interacts with residues on helices a and C of LIF, forming discrete and conformational epitopes. Binding was driven by several salt bridges, H-bonds and van der waals interactions (table 7, fig. 13B). The h5D8 epitope of LIF spans the region of interaction with gp 130. See boularger, m.j., Bankovich, a.j., korteme, t., Baker, D. & Garcia, k.c. conversion mechanisms for recognition of different cytokines by the shared signaling receptor gp 130. Molecular cell [ Molecular cell ]12, 577-cell 589 (2003). The results are summarized in table 7 below and depicted in fig. 13.

Materials and methods

LIF was maintained in HEK293S (GntI)^-/-) Transient expression in cells and purification using Ni-NTA affinity chromatography followed by gel filtration chromatography in 20mM Tris pH 8.0 and 150mM NaCl. The recombinant h5D8 Fab was transiently expressed in HEK293F cells and purified using KappaSelect affinity chromatography followed by cation exchange chromatography. Purified h5D8 Fab and LIF were mixed at a molar ratio of 1:2.5 and incubated at room temperature for 30 minutes before deglycosylation using EndoH. The complex was subsequently purified using gel filtration chromatography. The complex was concentrated to 20mg/mL and crystallization experiments were performed using sparse matrix screening. Crystals were formed at 4 ℃ under conditions comprising 19% (v/v) isopropanol, 19% (w/v) PEG4000, 5% (v/v) glycerol, 0.095M sodium citrate pH 5.6. The crystal diffracts on the 08ID-1 beam line of a Canadian Light Source (CLS) The resolution of (2). Section D [ Crystal bulletin: D Vol.D. ] according to Kabsch et al xds]Biological crystallography]66, 125-. According to McCoy et al Phaser crystalline software]JAppl crystallogor [ journal of applied crystallography ]]40,658-The phase machine determines the structure by molecular exchange. Several iterations of model construction and refinement were performed using Coot and phenix_{Work by}And R_{Freedom of movement}. See Emsley et al, Features and developments of Features and evaluation of Coot [ Coot]Section D [ Crystal science: D bundling ]]Biological crystallography]66, 486-; and Adams et al, PHENIX: a comprehensive Python-based system for macromolecular structural solution]Section D [ Crystal science: D bundling ]]Biological crystallography]66,213-221(2010). These numbers are reported in PyMOL (PyMOL molecular graphics System, version 2.0, Schrodinger Limited liability company ( LLC)).

Example 17-h5D8 high specificity for LIF

Binding of h5D8 to other LIF family members was tested to determine binding specificity. When both proteins were produced in E.coli, using Octet96 analysis, h5D8 bound human LIF approximately 100-fold more than the most homologous IL-6 family member oncostatin M (OSM) to LIF. When both proteins were produced in mammalian systems, h5D8 exhibited no binding to OSM. The data are summarized in table 8.

Materials and methods

Octet binding experiments: the reagents were used and prepared according to the manufacturer's manual. The basic kinetics experiments were performed using Octet data acquisition software version 9.0.0.26 as follows: setting of sensors/programs: i) equilibration (60 seconds); ii) load (15 seconds); iii) baseline (60 seconds); iv) association (180 seconds); and v) dissociation (600 seconds)

Octet affinity of h5D8 for cytokines: the basic kinetics experiments were performed using Octet data acquisition software version 9.0.0.26 as follows: the amine reactive second generation biosensor (AR2G) was hydrated in water for a minimum of 15 minutes. Conjugation of h5D8 to the biosensor was performed using an amine coupled second generation kit according to ForteBio technical instructions 26 (see reference). The immersion step was carried out at 30 ℃ 1000rpm as follows: i) equilibrating in water for 60 seconds; ii) activation in 20mM ECD, 10mM sulfo-NHS in water for 300 sec; iii) 10. mu.g/ml h5D8 was fixed in 10mM sodium acetate pH 6.0 for 600 seconds; iv) quenching in 1M ethanolamine (pH 8.5) for 300 seconds; v) baseline in water for 120 seconds. Kinetics experiments were then carried out at 30 ℃, 1000rpm by the following immersion and reading steps: vi) baseline 60 seconds in 1X kinetic buffer; vii) associate the appropriate cytokine train dilutions for 180 seconds in 1X kinetic buffer; viii) dissociation in 1X kinetic buffer for 300 seconds; ix) three regeneration/neutralization cycles were alternated between 10mM glycine pH 2.0 and 1X kinetic buffer, respectively (5 seconds each, 3 cycles). After regeneration, the biosensor is reused for subsequent binding assays.

Human recombinant LIF produced by mammalian cells was from ACRO biosystems (LIF-H521 b); human recombinant OSM produced in mammalian cells is from R & D (8475-OM/CF); and human recombinant OSM produced in E.coli cells from R & D (295-OM-050/CF).

Example 18-h5D8 fab Crystal Structure

Five crystal structures of h5D8 Fab under broad spectrum chemical conditions were determined. The high resolution of these structures indicates that the conformation of the CDR residues is associated with less flexibility and is highly similar in different chemical environments. The unique feature of this antibody is the presence of an atypical cysteine at position 100 of the variable heavy region. Structural analysis indicated that cysteine was unpaired and largely inaccessible to solvents.

The H5D8 Fab was obtained by papain digestion of its IgG followed by purification using standard affinity, ion exchange and size chromatography techniques. Obtaining crystals by gas phase diffusion method and determining resolution inToFive crystal structures ranging in between. Although the crystallization conditions varied across five different pH levels: 5.6, 6.0, 6.5, 7.5 and 8.5, all structures resolved in the same crystallographic space group and with similar unit cell dimensions (P212121, ). As such, these crystal structures allow for a three-dimensional arrangement of h5D8 Fab that is less hindered by crystal stacking artifacts and spans a broad spectrum of chemical conditions.

The electron density of all Complementarity Determining Region (CDR) residues was observed and subsequently modeled. Notably, the LCDR1 and HCDR2 adopt an elongated configuration that forms a binding groove in the center of the paratope with the shallow LCDR3 and HCDR3 regions (fig. 14A). The five structures are very similar at all residues, with the root mean square deviation of all atoms at Andin the same manner (fig. 14A). These results indicate that the conformation of the CDR residues is maintained in various chemical environments, including pH levels ranging between 5.6 and 8.5 and ionic strengths ranging between 150mM and 1M. Analysis of the electrostatic surface of the h5D8 paratope revealed that positively and negatively charged regions also contribute to hydrophilicity, without widespread hydrophobic patches. h5D8 has the rare feature of an atypical cysteine at the base of HCDR3(Cys 100). In all five structures, the free cysteines are ordered and do not form any disulfide mismatches. Furthermore, it is not acidified by Cys (cysteine) or glutathione (glutamate)Cystatinization) and van der waals interactions with the backbone and side chain atoms of Leu4, Phe27, Trp33, Met34, Glu102, and Leu105 of the heavy chain: ( Distance) (fig. 14B). Finally, Cys100 is a largely buried structural residue that appears to be involved in mediating the conformation of CDR1 and HCDR 3. Thus, as observed by the uniform distribution of this region in our five crystal structures, it is less likely to be reactive with other cysteines.

Materials and methods

H5D8-1 IgG was obtained from Corntaile Biologics (Catalent Biologics) and formulated in 25mM histidine, 6% sucrose, 0.01% polysorbate 80 (at pH 6.0). The formulated IgG was extensively buffer exchanged to PBS using a 10K MWCO concentrator (millipore) and then digested at 1:100 micrograms papain (sigma) in PBS, 1.25mM EDTA, 10mM cysteine for 1 hour at 37 ℃. Papain-digested IgG was passed through a protein a column (GE healthcare) using an AKTA Start chromatography system (GE healthcare). The protein a flow-through containing the h5D8 Fab was recovered and its buffer exchanged for 20mM sodium acetate at pH 5.6 using a 10K MWCO concentrator (millipore). The resulting samples were loaded onto Mono S cation exchange columns (GE healthcare) using an AKTA Pure chromatography system (GE healthcare). Elution with a 1M potassium chloride gradient produced a major h5D8 Fab peak which was recovered, concentrated and purified to size homogeneity in 20mM Tris-HCl, 150mM sodium chloride (pH 8.0) using a Superdex 200 Incase gel filtration column (GE healthcare group). The high purity of the h5D8 Fab was confirmed by SDS-PAGE under reducing and non-reducing conditions.

The purified h5D8 Fab was concentrated to 25mg/mL using a 10K MWCO concentrator (Millipore). Vapor diffusion crystallization experiments were set up using an Oryx 4 dispenser (Douglas Instruments) at 20 ℃ using sparse matrix 96 condition commercial sieves JCSG TOP96(Rigaku reagent) and MCSG-1 (Anatasce). After four days under the following five crystallization conditions, crystals were obtained and harvested: 1)0.085M sodium citrate, 25.5% (w/v) PEG 4000, 0.17M ammonium acetate, 15% (v/v) glycerol, pH 5.6; 2)0.1M MES, 20% (w/v) PEG 6000, 1M lithium chloride, pH 6.0; 3)0.1M MES, 20% (w/v) PEG 4000, 0.6M sodium chloride, pH 6.5; 4)0.085M HEPES sodium, 17% (w/v) PEG 4000, 8.5% (v/v) 2-propanol, 15% (v/v) glycerol, pH 7.5; 5)0.08M Tris, 24% (w/v) PEG 4000, 0.16M magnesium chloride, 20% (v/v) glycerol, pH 8.5. The mother liquor containing crystals was supplemented with 5% -15% (v/v) glycerol or 10% (v/v) ethylene glycol as required before rapid freezing in liquid nitrogen. The crystal was subjected to X-ray synchrotron radiation with an advanced photon source beam line 23-ID-D (Chicago, Ill.) and a diffraction pattern was recorded on a Pilatus36M detector. Data were processed using XDS and the structure was determined by molecular replacement using phase shifters. Perfected in PHENIX, an iterative model was built in Coot. Pictures were generated in PyMOL. All software is accessed via SBGrid.

Example 19-h5D8 mutation at cysteine 100 maintained binding

Analysis of h5D8 revealed a free cysteine residue at position 100 (C100) of the heavy chain variable region. The H5D8 variant was generated by substituting C100 with each naturally occurring amino acid to characterize binding to and affinity for human and mouse LIF. Binding was characterized using ELISA and Octet assays. The results are summarized in Table 9. The ELISA EC50 curve is shown in fig. 15 (fig. 15A human LIF and fig. 15B mouse LIF).

Materials and methods

ELISA: binding of the h5D 8C 100 variant to human and mouse LIF was determined by ELISA. Recombinant human or mouse LIF protein was plated at 1ug/mL onto Maxisorp 384-well plates overnight at 4 ℃. Plates were blocked with 1x blocking buffer for 2 hours at room temperature. Titrations of each h5D 8C 100 variant were added and combined for 1 hour at room temperature. Plates were washed three times with PBS + 0.05% Tween-20. HRP-conjugated anti-human IgG was added and bound for 30 min at room temperature. Plates were washed three times with PBS + 0.05% tween-20 and developed using 1x TMB substrate. The reaction was stopped with 1M HCl and the absorbance at 450nm was measured. Graph generation and nonlinear regression analysis were performed using Graphpad Prism.

Octet RED 96: the affinity of the h5D 8C 100 variant for human and mouse LIF was determined by BLI using the Octet RED96 system. After a baseline of 30 seconds in 1x kinetic buffer, the h5D 8C 100 variant was loaded onto an anti-human Fc biosensor at 7.5 ug/mL. Titration of human or mouse LIF protein was correlated to the loaded biosensor for 90 seconds and dissociated in 1x kinetic buffer for 300 seconds. KD was calculated by data analysis software using a 1:1 global fitting model.

Example 20-h5D8 blocking LIF binding to gp130 in vitro

To determine whether h5D8 prevented LIF from binding to LIFR, a molecular binding assay was performed using the Octet RED96 platform. H5D8 was loaded onto AHC biosensors by anti-human Fc capture. The biosensor was then immersed in LIF and association was observed as expected (fig. 16A, middle third). Subsequently, the biosensor was immersed in different concentrations of LIFR. Dose-dependent association was observed (fig. 16A, right third). Control experiments demonstrated that this association was LIF specific (not shown) and not due to non-specific interaction of LIFR with h5D8 or with the biosensor.

To further characterize the binding of h5D8 and LIF, a series of ELISA binding experiments were performed. H5D8 and LIF were preincubated and then introduced onto plates coated with recombinant human LIFR (hLIFR) or gp 130. The lack of binding between the h5D8/LIF complex and the coated substrate indicates that h5D8 somehow disrupts the binding of LIF to the receptor. In addition, a control antibody that does not bind LIF (isotype control, indicated by (-) or binds LIF at a known binding site (B09 does not compete with gp130 or LIFR for LIF binding; r5D8 is the rat parental version of h5D 8) was also used. The ELISA results demonstrated that h5D8/LIF complex was able to bind hLIFR (as was the r5D8/LIF complex), indicating that these antibodies were unable to prevent LIF/LIFR association (fig. 16A). In contrast, the h5D8/LIF complex (and r5D8/LIF complex) was unable to bind recombinant human gp130 (FIG. 16B). This indicates that when LIF binds to h5D8, the gp130 binding site of LIF is affected.

Example 21 LIF and LIFR expression in human tissues

To determine the expression levels of LIF and LIFR, real-time quantitative PCR was performed on many different types of human tissues. The average expression levels shown in FIGS. 17A and 17B are given as copy number per 100ng total RNA. Most tissues express at least 100 copies per 100ng total RNA. LIF mRNA expression was highest in human adipose tissue (mesenteric ileum [1]), vascular tissue (choroid plexus [6] and mesenteric [8]), and umbilical cord [68] tissues; lowest in brain tissue (cortex [20] and substantia nigra [28 ]). LIFRmRNA is expressed most strongly in human adipose tissue (mesenteric ileum [1]), vascular tissue (lung [9]), brain tissue [11-28] and thyroid [66] tissue; lowest among PBMCs [31 ]. LIF and LIFR mRNA expression levels in cynomolgus monkey tissues were similar to those observed in human tissues, with high LIF expression in adipose tissues, high LIFR expression in adipose tissues and low in PBMCs (data not shown).

The organization numbers of fig. 17A and 17B are: 1-fat (mesenteric ileum); 2-adrenal gland; 3-bladder; 4-bladder (trigone vesicae); 5-blood vessels (brain: middle cerebral artery); 6-blood vessels (choroid plexus); 7-blood vessels (coronary arteries); 8-blood vessels (mesentery (colon)); 9-blood vessels (lungs); 10-blood vessels (kidneys); 11-brain (amygdala); 12-brain (tail nucleus); 13-brain (cerebellum); 14 brain- (cortex: anterior cingulate); 15-brain (cortex: posterior cingulate region); 16-brain (cortex: lateral to frontal lobe); 17-brain (cortex: medial frontal lobe); 18-brain (cortex: occipital bone); 19-brain (cortex: parietal lobe); 20-brain (cortex: temporal lobe); 21-brain (dorsal-raphe-nucleus); 22-brain (hippocampus); 23-brain (hypothalamus: anterior zone); 24-brain (hypothalamus: posterior region); 25-brain (locus coeruleus); 26-brain (medulla oblongata); 27-brain (nucleus accumbens); 28-brain (substantia nigra); 29-mammary gland; 30-cecum; 31-Peripheral Blood Mononuclear Cells (PBMCs); 32-colon; 33-Dorsal Root Ganglion (DRG); 34-duodenum; 35-fallopian tube; 36-gallbladder; 37-heart (left atrium); 38-heart (left ventricle); 39-ileum; 40-jejunum; 41-kidney (cortex); 42-kidney (medulla oblongata); 43-kidney (pelvis); 44-liver (parenchyma); 45-liver (bronchi: first order); 46-liver (bronchi: three stages); 47-lung (parenchyma); 48-lymph glands (tonsils); 49-muscle (bone); 50-esophagus; 51-ovary; 52-pancreas; 53-pineal body; 54-pituitary gland; 55-placenta; 56-prostate; 57-rectum; 58-skin (foreskin); 69-spinal cord; 60-spleen (parenchyma); 61-stomach (antrum); 62-stomach (corpus gastri); 63-stomach (fundus); 64-stomach (pyloric canal); 65-testis; 66-thyroid gland; 67-trachea; 68-umbilical cord; 69-ureter; 70-uterus (cervix); 71-uterus (myometrium); and 72-vas deferens.

Example 22-h5D8 and anti-PD-1 antibodies inhibit tumor growth in a mouse model of colorectal cancer

The therapeutic efficacy of h5D8 was evaluated in combination with PD-1 inhibitors in syngeneic CT26 and MC38 models. Mice treated with the combination of PD-1 inhibitor and h5D8 exhibited reduced CT26 tumor growth compared to mice treated with PD-1 inhibitor or h5D8 alone, as shown in fig. 18A and 18B. While the long-lasting survival benefit of h5D8 monotherapy was not observed (fig. 18C), but was only rarely observed with anti-PD 1 therapy (fig. 18C), the combination of h5D8 and anti-PD 1 brought about 40% and 30% long-term survival benefit, respectively, in treated CT26 and MC38 tumor-bearing mice (fig. 18C). Importantly, long-term tumor-free CT26 survivors were resistant to tumor re-implantation, consistent with obtaining durable adaptive immunity (fig. 18D).

To investigate other immune mechanisms that the combination of h5D8 and PD-1 inhibitor had an effect on tumor growth, the effect of the combination on the functionality of infiltrating CD 8T cells was evaluated as shown in fig. 19A and 19B. No effect or nominal increase in CD8 TIL was observed in CT26 and MC38 tumors, respectively, using monotherapy h5D8 treatment. Although the effect on CD8 TIL was small due to anti-PD 1 monotherapy, the combination with h5D8 significantly increased CD8 TIL in both CT26 and MC38 tumors, suggesting that the increase in efficacy observed with this combination may be driven by an increase in CD8 TIL (fig. 19C and 19D).

Tumors harvested from mice treated with the combination of h5D8 and anti-PD 1 showed not only an increase in CD8 TIL, but also an increase in cytolytic, proliferative and antigen-experienced subpopulations identified by GZMB, Ki67 and CD44, respectively, relative to the control and monotherapy-treated groups. The combination treated tumors also showed an increase in the ratio of CD8/Treg, M1/M2 and CD8/CD11b, demonstrating that effective modulation of the Tumor Microenvironment (TME) is beneficial for anti-tumor immunity. These data support that LIF drives inhibition of TME at least in part by the inhibitory polarization of macrophages, which subsequently blunts host anti-tumor immunity. Inhibition of LIF with h5D8 reversed this effect and in combination with T cell promoting therapies (such as anti-PD 1 therapies) driven strong anti-tumor immunity, achieving a long-lasting survival benefit in a cancer mouse model.

To examine whether CD8 TIL differs on a per cell basis in tumors harvested in mice treated with either anti-PD 1 monotherapy or the combination of h5D8 and anti-PD 1, the ability of CD8 TIL to produce IFN γ in response to tumor-specific antigens was examined. CD8 TIL isolated from CT26 tumors treated with a combination of anti-PD 1 or h5D8 and anti-PD 1 showed no functional differences including the immunodominant rejection antigen of CT26 when stimulated ex vivo with AH1 peptide (gp 70; 423-431a.a.) (FIG. 19C). Similarly, CD8 TIL isolated from MC38 tumors treated with a combination of anti-PD 1 or h5D8 and anti-PD 1 also showed no functional difference when stimulated ex vivo with an immunodominant tumor antigen peptide (p15 e; 604-611a.a.), indicating that the increase in combined efficacy was driven by an overall increase in CD8 TIL frequency rather than a functional increase in CD8 TIL per cell.

Materials and methods

H5D8 and PD-1 inhibitor antibody clone RMP1-14 (BioXCell) were administered twice weekly at doses of 15mg/kg and 10mg/kg, respectively, and tumor volume was monitored by caliper-based measurements.

Example 23-correlation between LIF, TAM and Treg

The effect of LIF on the cancer immune system was determined by measuring the relative abundance of tumor-associated macrophages (TAMs) and regulatory T cells (tregs) in 28 types of solid tumors from the cancer genomic map (TCGA). In several tumor types, a significant correlation between LIF and TAM and Treg was observed (fig. 20A and 20B). Glioblastoma (GBM), prostate adenocarcinoma, thyroid carcinoma and ovarian carcinoma are 4 tumor types that showed the highest correlation between LIF, TAM and Treg, while high LIF expression was shown in each sample (fig. 20A and 20B). Extensive LIF expression was observed in GBM tumors expressed by tumor cells and immune cell infiltrates (fig. 24).

In both human GBM and the analysis of the TCGA dataset for ovarian cancer (OV), a significant positive correlation was found between LIF and CCL2, CD163, and CD206 (fig. 28). No correlation was observed between LIF and CXCL9 (data not shown), but relatively low levels of CXCL9 mRNA were observed in each tumor. These results were verified at the protein level by analyzing a cohort of 20 GBM patients and tumor-bearing LIF, CXCL9, CCL2, CD163, and CD206 IHC. A strong positive correlation between LIF and CCL2, CD163, and CD206 was observed (fig. 21E). CXCL9 was expressed in isolated cell clusters, accounting for the low level of CXCL9 mRNA present in tumors. Notably, CXCL9 showed a negative correlation with LIF in human GBM (fig. 21E).

The examples described herein further illustrate that LIF is in CD8⁺T cell depletion plays a key role and promotes the presence of pre-neoplastic TAMs. It was observed that blockade of LIF in tumors expressing high levels of LIF reduced CD206, CD163, and CCL2 in TAMs and induced CXCL9 expression. Blockade of LIF releases epigenetic silencing of CXCL9, triggering CD8⁺T cell tumor infiltration. LIF neutralizing antibodies in combination with inhibition of the PD1 immune checkpoint promote tumor regression and increased overall survival.

Materials and methods

RNA-seq data for 9,403 patients with 28 different solid tumors was downloaded from a cancer genome map (TCGA) of a fiber server (fiber. org, version 2016 — 01 — 28). For all downstream analyses, expression data (RSEM) were log2 transformed. Next, genetic signatures were obtained for four immune populations of interest: TAM, Treg, CD4⁺T cells and CD8⁺T cells. Then calculating the correlation between LIF expression and the gene signatures of four immune populations, and LIF and a set of objectivesCorrelation between genes.

Example 24-repression of LIF function in GL261N, RCAS and ID8 models

The potential immunomodulatory role of LIF in cancer was studied in immune competent mouse GBM and ovarian cancer (tumor types in which LIF is highly associated with TAM and Treg) models. GBM cell lines, GL261N (a derivative of the GL261 cell line), GFAP-tv-a RCAS-PDGFA, shp53, shNF1(RCAS) transgenic model and ovarian cancer cell line ID8, which produce tumors in the brain (GL261N and RCAS) and peritoneum (ID8) of mice were identified as expressing high levels of LIF (fig. 25).

LIF function in the GL261N, RCAS and ID8 models was blocked using neutralizing antibodies. A reduction in tumor growth and an increase in survival was observed in these models (fig. 20C, 20G, 20H, 20K, 20P). Blockade of LIF in the GL261 tumor model (tumor that did not express LIF) did not inhibit tumor growth (fig. 25E). Neutralizing antibodies against LIF induced a significant reduction in p-STAT3 levels, suggesting that LIF is the major cytokine inducing the JAK-STAT3 pathway in these animal models (selected based on high LIF expression) (fig. 20D and 20L). Furthermore, although no significant reduction in Ki67 positive cells was observed, an increase in cleaved caspase 3(CC3) was observed, indicating that blockade of LIF induced tumor cell death (fig. 20D and 20L).

Materials and methods

All animal experiments were approved by the institutional animal care committee of the wald hilbertron (vald' Hebron) institute and conducted according to their guidelines, in compliance with the european union and national directives. Female C57BL/6 and NOD SCID were purchased from Janvier, Inc. (Janvier). For brain tumor models, 3x 10⁵Individual GL261N, GL261 or RCAS cells, all with luciferase expression, were stereotactically inoculated into the striatum (1 mm anterior and 1.8mm lateral; 2.5mm within parenchyma) of the right hemisphere of the 8-week-old C57BL/6 mouse. For ovarian tumor models, 5x 10 ⁶Individual ID8 ovarian cancer cells were injected intraperitoneally into 8-week-old C57BL/6 mice. anti-LIF or control IgG was administered intraperitoneally twice weekly at doses of 300 μ g (ID8) or 600 μ g (GL261N, GL261 and RCAS). In addition, 200 μ g rat anti-mouse was injected intraperitoneally twice weeklyMurine PD1 blocking antibody (anti-PD 1, Beehrlich cell Co.), anti-mouse/human/rat CCL2 antibody (MCP-1, Beehrlich cell Co.) or 3 μ g anti-mouse CXCL9 antibody (R)&D) In that respect By weight and abdominal circumference (ID8) or using the Profenox corporationSpectra (GL261N, GL261 and RCAS) were measured for bioluminescence to monitor tumor progression. Mice were euthanized when they exhibited clinical signs of illness or adverse stress (i.e. cachexia, anorexia or increased respiratory rate) or tumors began to interfere with normal bodily functions.

EXAMPLE 25 anti-LIF treatment of GL261N tumors

The role of the immune system in response to anti-LIF therapy was evaluated using immunodeficient animals. In RAG^-/-Or NOD SCID mice (all mouse strains lacking adaptive immune response), treatment with anti-LIF showed no significant effect on tumor growth (fig. 25F). The results indicate that the anti-tumor response to LIF blockade is mainly mediated by an adaptive immune response.

Materials and methods

Using the method of example 24, except that the mouse used was RAG from Jackson Laboratories (Jackson Laboratories)^-/-、CCL2^-/-And CXCL9^-/-And NOD SCID γ (NSG) from Charles River (Charles River).

Example 26 anti-LIF treatment decreases the number of pre-tumor TAMs and increases CD8⁺T cell tumor infiltration

By observing pre-tumor TAM (CD11 b)⁺Ly6G^-Ly6C^-CD206⁺CD163⁺MHCII^{Is low in}) To further investigate the molecular mechanisms involved in the immune response to anti-LIF treatment (fig. 20E, 20I, 20M). Importantly, CD8 following anti-LIF treatment⁺Tumor infiltration of T cells was accompanied by an increase (fig. 20D, 20F, 20J, 20L, 20N). Treg and NK cell numbers decreased and increased, respectively, after treatment with anti-LIF (fig. 25G-25J). Infiltrative CD8⁺T cells express GZMA, indicating that they are mediating cellsToxic effects (fig. 26A). Furthermore, CD8⁺The compartment of the T cells expressed PD1 (fig. 26B, 26C). Monocytes derived from recruitment (CD11 b)⁺Ly6G^-Ly6C^-CD49d⁺)¹²Decreased in response to anti-LIF (fig. 26D), no control of dendritic cell population (CD11 b) was observed (fig. 26D)⁺、CD11c⁺、MHCII⁺) (FIG. 26E) or the level of IL-10 or IL-12 in the tissue (FIG. 26F).

Materials and methods

Mice were euthanized and tumors were isolated. GL261N and the RCAS tumor were enzymatically digested with a brain tumor dissociation kit, and myelin was removed with myelin removal bead II, both from santana whirlpool biotechnology (Miltenyi Biotec). The ID8 tumor was treated with a mouse tumor dissociation kit (american and whirlpool biotechnology) and ascites fluid was collected. Human GBM samples of the organotypic model and patient-derived xenografts were enzymatically digested with a human tumor dissociation kit (american and whirlpool biotechnology).

Separation of CD11b from GL261N cell suspensions using anti-Ly 6C-APC and anti-APC microbeads and anti-Ly 6G microbeads⁺Cells to deplete Ly6G⁺And Ly6C⁺Population, then magnetic beads with CD11 b. CD45 with anti-mouse CD45 magnetic beads⁺And (5) separating the cells. Separation of CD11b from ID8 cell suspensions using anti-CD 11b magnetic beads⁺A cell. Finally, CD45 was isolated from organotypic slices using anti-human CD45 magnetic beads⁺A cell. All separation steps were performed using a MultiMACS Cell24 separator Plus, magnetic beads purchased from whirlpool biotechnology, inc.

Murine anti-CD 3, CD4, CD335, CD163, MHC class II, CXCR3 (eBioscience), CD45, CD8, F4/80, CD11b, CD11c, CD206, CD49d (BD Bioscience)), LIFR (novius Biologicals) Ly6G, Ly6C, CCR2, and PD1 (biogegenerated) antibodies were used for flow cytometry. Intracellular staining was performed using a specific staining kit (e biosciences) for FoxP3, granzyme a (gzma), CXCL9(e biosciences) and CCL2 (bio-legend). To go toFlow studies (flow human study) used antibodies against CD11b, CD14(BD biosciences), CD45, CD3, CXCR3 (bioglass) and CD8(BD pharmaceuticals (BD Pharmigen)). In some cases, the samples were previously incubated with LIVE/DEAD fixable yellow DEAD stain kit (seimer feishell science) to determine viability. As a positive control for human GBM organic and patient-derived xenografts, 10 ⁶Individual PBMCs (referred to as "spiked PBMCs") were added to the control samples to establish the leukocyte population.

Samples were collected on a BD lsrfortessa cell analyzer or Navios (Beckman Coulter) and the data analyzed using Flow Jo software.

Example 27-results of LIF mediated modulation of tumor immune infiltrates whether LIF mediated modulation of tumor immune infiltrates was the cause or outcome of an anti-tumor response to LIF blockade was assessed by performing an acute treatment experiment in which mice were treated with anti-LIF for 4 days after tumor establishment. Treatment for 4 days did not affect tumor growth (fig. 26G), but was sufficient to engage CD8⁺T cell tumor infiltration (fig. 26H). This indicates CD8⁺T cell infiltration is not the result of an anti-tumor response to LIF blockade.

Example 28-Gene response verification

By isolating CD11b from the ID8 mouse model⁺Cells treated with anti-LIF antibodies and subjected to transcriptome analysis to determine genes associated with down-regulated oncogenic phenotypes. The genes identified were CCL2, CCL3, CCL7, PF4, CTSK, CD206 and CD 163. Also, interestingly, CXCL9 was up-regulated (fig. 21A). The aforementioned gene reaction was verified by qRT-PCR in ID8 and GL261N models (fig. 21B).

CXCL9 and CCL2 stand out as CD8 respectively⁺T cell tumor infiltration and key chemokines for TAM and Treg recruitment. Neutralization of LIF in TAM by neutralization (CD11 b)⁺Ly6G^-Ly6C^-) Modulation of CXCL9 and CCL2 (fig. 21C). Immunostaining and isolation of TAMs showed that CXCL9, CCL2, CD206 and CD163 were predominantly expressed in TAMs (fig. 21D), and treatment with anti-LIF could modulate their expression (fig. 21C, 21D). CXCR3(CXCL9 receptor), CCR2(CCL 2)Receptor) and LIFR in TAM and CD8⁺Expression in T cells (fig. 27A). qRT-PCR analysis quantified CD11b from GL261N tumors⁺Ly6G^-Ly6C^-And CD11b^-Ly6G^-Ly6C^-Presence of CD11b and CXCL9 mRNA in cells. (FIG. 27B)

Materials and methods

Cells were lysed for mRNA extraction (RNeasy Mini or Micro kit, Qiagen (Qiagen)), Reverse transcribed (iScript Reverse Supermix from burle (BioRad) for mRNA), and qRT-PCR was performed using Taqman probes from Applied Biosystems according to the manufacturer's recommendations. For paraffin-embedded sections, RNA was obtained by using a High Pure FFPET RNA isolation kit (Roche) and following the manufacturer's instructions. The reaction was performed in a CFX384 Touch (TM) real-time PCR detection system (Bio-Rad), and the results were expressed as fold-changes relative to the control samples calculated by the Ct method. Murine or human ACTB or GAPDH was used as an internal normalization control.

RNA was determined on the Affymetrix microarray platform using mouse Gene 2.1 ST. Next, it was normalized based on the Robust Microarray mean (RMA). Genes differentially expressed in anti-LIF treated mice were determined by bayesian linear regression using limma Bioconductor software package, considering paired samples.

Example 29 anti-LIF response in CXCL9 and CCL2 knockout mice

Knockout of CXCL9 and CCL2 genes (CXCL 9)^-/-，CCL2^-/-) Mouse models were tested for the relevance of CXCL9 and CCL2 modulation in LIF oncogenic function. Tumors in these mouse models were treated with blocking antibodies against CXCL9 and CCL 2. Interestingly, at CXCL9^-/-The antitumor response to LIF inhibition was attenuated in mice, but in CCL2^-/-There was no attenuation in the mice (fig. 21F). Similarly, this CXCL9 neutralizing antibody, but not the CCL2 antibody, attenuated the anti-cancer response against LIF (fig. 21F). These results indicate that the primary mediator of the anti-LIF response is CXCL 9. As expected, blockade of CXCL9 reduced response to anti-LCD8 of IF⁺T cell tumor infiltration (fig. 21G).

Materials and methods

The slides were dewaxed and hydrated. 10 min 10% peroxidase (H) at room temperature using citrate antigen extraction solution (DAKO) pH 6 or pH 9 ₂O₂) And blocking solution (2% BSA) for 1h antigen extraction. EnVision FLEX + (DAKO) was used as the detection system, followed by counter staining with hematoxylin, dehydration and fixation (DPX) according to the manufacturer's instructions. Quantification of LIF, CCL2, CD163, CD206, and CXCL9 staining in patient GBM tumors was expressed as H-score (3 x percent strong staining +2 x percent moderate staining + percent weak staining) ranging from 0 to 300. Quantification of p-STAT3, Ki67, CC3 and CD8 was performed with ImageJ, the total number of cells in three different fields per mouse was counted, five mice per group, and the percentage of positive cells was calculated. Data are presented as mean ± SEM.

Immunohistochemical antibodies: human LIF (Atlas; 1:200), murine LIF (Abcam; 1:200), murine p-STAT3 (cell signaling; 1:50), murine Ki67 (Ebos; 1:200), murine cleaved caspase 3(CC3) (cell signaling; 1:500), murine CD8 (Poolss; Bioss; 1:200), human/murine CCL2 (Nowessox biologics; 1:200), human CXCL9 (Muraseisel technologies; 1:100) and human CD163 (Leica Novacastra; 1: 200).

Nuclei were counterstained with DAPI and images were captured using a laser scanning confocal NIKON Eclipse Ti microscope. Quantitation of immunofluorescence was performed with ImageJ, counting all or up to 100 cells positive for CD11b, Iba1 or CD3 in 2-3 different fields per mouse of 3-5 mice/group, and calculating the percentage of these cells positive for CCL2, CD206 and CD163 in Iba1 (for GL 261N) or CD68/CD11b (for ID 8) positive populations. For CXCL9, the percentage of cells within the total cell population surrounded by this cytokine signal was calculated. For organotypic slices, 3-4 fields (n-3) per patient were quantified. For organotypic tissue immunofluorescence, five different subjects were treated with Fiji-Image J software for each condition Z-stack image of (a). For CD8⁺T cells, CD8 calculated⁺Percentage of T cells in the total population. Data are presented as mean ± SEM.

Immunofluorescence antibodies: human/murine CCL2 (Nuoweisi biologics 1:200), human/murine CD11b (Ebosh 1:2000), human/murine Iba1 (Wako 1:1000), murine CD68 (Ebosh 1:200), human/murine CD206 (Ebosh 1:500), murine CD163 (Ebosh 1:200), CXCL9 (murine, Nuoweisi biologics 1: 200; human, Saimeri Seishehell science 1:200), and human CD8(DAKO 1: 200).

Example 30 Effect of LIF on Primary culture of mouse bone marrow-derived macrophages (BMDM)

The effect of LIF on primary culture of mouse BMDM was studied to explore the molecular mechanisms by which LIF modulates CXCL9 and CCL2 in macrophages. LIF modulated IFN γ or IL 4-induced expression of several M1-like and M2-like markers in BMDM (fig. 22A). Except when BMDM was treated with IFN γ, no CXCL9 expression was detected. Recombinant LIF repressed induction of CXCL9 by IFN γ at both mRNA and protein levels (fig. 22B and 22C). Patient-derived TAM obtained from fresh human GBM tumor (CD11 b)⁺CD14⁺) CXCL9 was also regulated by IFN γ and LIF (fig. 22D and 29A). These results were further verified after recombinant LIF was observed to repress induction of CXCL9 by LPS at both mRNA and protein levels. (FIG. 29B). Thus, LIF acts as a repressor of CXCL9 induction. No binding of the CXCL9 promoter of p-STAT3 was observed after LIF treatment (data not shown). Consistent with this, LIF treatment was found to increase the level of trimethylated H3 lysine 27(H3K27me3), decrease the level of acetylated H4(H4ac), and increase the binding of EZH2 to the CXCL9 promoter region, indicating that LIF modulates CXCL9 expression through epigenetic silencing (fig. 22E).

Materials and methods

Bone marrow-derived macrophages (BMDM) were obtained from six to ten week old C57BL/6 mice. Briefly, bone marrow precursors were cultured in DMEM (Life technologies) supplemented with 20% heat-inactivated FBS and 30% L cell conditioned media (cm) as macrophage colony stimulationThe source of the factor. Differentiated macrophages were obtained after 6 days of culture. L cell cm was obtained from L929 cells grown in DMEM (Life technologies) supplemented with 10% heat-inactivated FBS. Human macrophages were isolated from human GBM samples. Briefly, tumor tissue was enzymatically digested with a tumor dissociation kit, and CD11b was isolated using CD11b magnetic beads and MultiMACS Cell24 separator Plus (both from american whirlpool biotechnology corporation)⁺A cell. CD11b to be obtained⁺Cells were cultured in RPMI medium (life technologies) supplemented with 10% heat-inactivated FBS. Recombinant LIF, IFN γ, LPS and IL4 were purchased from Michibo, Inc., R, respectively&D systems Co Ltd (R)&D Systems), sigma and Creative, baiomatt (Creative BioMart).

Chromatin immunoprecipitation was performed according to the Upstate (millipore) standard protocol. Briefly, 1.2X 10⁷Individual BMDMs were fixed using 1% formaldehyde at 37 ℃ for 10 minutes, harvested and sonicated to generate 200-fold 500bp chromatin fragments. The 20j.tg sheared chromatin was then immunoprecipitated with 2j.tg anti p-STAT3(Tyr705) antibody, anti trimethyl-histone H3(Lys27) (cell signaling), anti acetyl H4 (millipore) or anti EZH2 (millipore) overnight. The immune complexes were recovered using 20j.tl protein G magnetic beads, washed and eluted. The cross-linking was reversed at 65 ℃ for 4h and the immunoprecipitated DNA was recovered using the PCR purification kit from Qiagen. Genomic regions of interest were identified by real-time quantitative pcr (qpcr) using SYBR Green premix (invitrogen).

Example 3 modulation of immune cell tumor infiltration by LIF in patients

To demonstrate that LIF regulates immune cell tumor infiltration by suppressing CXCL9 in the actual cancer patient tumor, organotypic tissue cultures were generated from freshly obtained GBM samples from the patients. These organotypic models allow for the short-term culture of tumor sections that maintain the patient's tumor tissue architecture and stroma, including immune cells. Organotypic tissue cultures from 3 patients with high levels of LIF expressed by tumor cells (fig. 22H). There was a large infiltration of TAMs in all 3 cultures as detected by Iba1 marker, most of which expressed CCL2, CD163, and CD 206. Interestingly, treatment of organotypic cultures with neutralizing antibodies against LIF for 3 days promoted a decrease in CCL2, CD163, and CD206, and an increase in CXCL9 expression (fig. 22H).

Materials and methods

Human GBM samples were obtained from the university hospital wald hilberuron and the clinic hospital. The clinical protocol was approved by the institutional review board of wald hilbert and office hospital (CEIC), with informed consent from all subjects.

GBM neurospheres were generated as follows. Briefly, tumor samples were processed 30 minutes after surgical resection. The minced meat pieces of the human GBM sample were digested with 200U/ml collagenase I (Sigma) and 500U/ml DNase I (Sigma) in PBS under vigorous stirring at 37 ℃ for 1 h. The single cell suspension was filtered through a 70 μm cell filter (BD Falcon) and washed with PBS. Finally, the cells were resuspended and subsequently cultured in GBM medium consisting of neuronal basal medium supplemented with B27, penicillin/streptomycin (all from life technologies) and growth factors (20ng/ml EGF and 20ng/ml FGF-2 (petabte).

GBM organotypic slice cultures were generated as follows. After excision, the surgical specimens were cut into rectangular pieces 5-10mm long and 1-2mm wide with a scalpel and transferred to 0.4 μm membrane culture inserts (Millipore) in 6-well plates, respectively. 1.2ml of neuronal basal medium (life technologies) supplemented with B27 (life technologies), penicillin/streptomycin (life technologies) and growth factors (20ng/ml EGF and 20ng/ml FGF-2) (PeproTech) were placed into each well before placing the inserts into the 6-well plates. The culture was maintained at 37 ℃, constant humidity, 95% air and 5% CO²The following steps. One day later, sections were treated with rat anti-LIF blocking antibody or its corresponding normal IgG (10. mu.g/ml) for 3 days. For blockade of CXCL9 studies, human CXCL9 (R) will be targeted&System D) was added to the culture at 1.5 μ g/ml. In some cases, 0.1ng/ml human rIFN γ (R) was added&D systems company) 24 h. In parallel, Peripheral Blood Mononuclear Cells (PBMCs) were obtained from whole blood of the same patient by centrifugation density separation using lymphosep (biowest)). PBMCs were cryopreserved in RPMI medium supplemented with 10% inactivated FBS and 10% DMSO until use. For immune cell infiltration assays, control or anti-LIF sections were embedded in Matrigel (Matrigel) (Corning), followed by 1 × 10 ⁶Individual PBMCs were added to complete RPMI medium in 24-well plates. In addition, the supernatant was collected and organotypic slices were recovered from the matrigel and further processed for IF and flow cytometry. Under some conditions, a concentration of 10 is used⁶Individual cells/ml PBS resuspended PBMCs and incubated with 5 μ M cell tracking CFSE (invitrogen) for 20 minutes. After incubation, cells were washed with RPMI and added to the sections embedded in matrigel. After 24h, the fluorescent PBMCs invading the matrigel were evaluated under a microscope by counting migrating cells in five different regions under each condition.

Example 32-treatment with anti-LIF increased CXCL9 and decreased CCL2 expression in tumors expressing high levels of LIF

The effect of LIF on immune cell tumor infiltration was evaluated. Organotypic slices from 3 patients expressing high levels of LIF were incubated with Peripheral Blood Mononuclear Cells (PBMCs) from the same patient following anti-LIF treatment (fig. 23A). Treatment with anti-LIF increased CXCL9 and decreased CCL2 expression (fig. 23B), and induced infiltration of immune cells into the matrigel surrounding the tumor sample (fig. 23B). Notably, CD8⁺T cells were recruited into tumor tissue following LIF blockade (fig. 23B, 23C), and this effect was dependent on CXCL9, as neutralization of CXCL9 prevented CD8 ⁺T cell infiltration (fig. 23D).

Similar results were confirmed in the context of in vivo models. NSG mice were inoculated with tumor fragments from 4 patients whose tumors express high LIF levels and these mice were treated with LIF neutralizing antibodies for 5 days. Next, PBMCs of each patient were inoculated in mice. Interestingly, mice treated with anti-LIF showed CD8⁺Increased T cell tumor infiltration, and most infiltrating CD8⁺T cells expressed CXCL9 receptor CXCR3 (fig. 23E).

Example 33-blockade by PD1 increases antitumor response

A mouse model with overt tumors was treated with anti-LIF and anti-PD 1 antibodies, and further reduction in tumor growth was observed with combinations of LIF and PD1 blockade compared to each of GL261N, RCAS, and ID8 tumors treated alone (fig. 30). Also importantly, the combination treatment of anti-LIF and anti-PD 1 increased overall survival and induced tumor regression (fig. 23F and 23G).

Mice exhibiting complete tumor regression were collected and re-inoculated with 3x 10⁵And (4) tumor cells. No tumors were visualized in these mice, whereas tumors grew rapidly in naive mice inoculated with the same number of cells in parallel (fig. 23H). The results of this restimulation experiment indicate that the combination treatment of anti-LIF and anti-PD 1 resulted in immunological memory.

Embodiments of the claimed invention

Specific embodiments of the invention described herein are described.

1. Use of a Leukemia Inhibitory Factor (LIF) -binding polypeptide in combination with a PD-1, PDL-1 or PDL-2 signaling inhibitor to treat cancer in an individual.

2. The use of example 1, wherein the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in separate formulations.

3. The use of example 1, wherein the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered to the individual in the same formulation.

4. The use of embodiment 1 or 2, wherein the LIF binding polypeptide is administered to the individual prior to administering the PD-1, PDL-1 or PDL-2 signaling inhibitor to the individual.

5. The use of example 1 or 2, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual prior to administering the LIF binding polypeptide to the individual.

6. The use of example 1 or 2, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling is administered to the individual at the same time as the LIF-binding polypeptide is administered to the individual.

7. The use of any one of embodiments 1-6, wherein the LIF-binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region.

8. The use of any one of embodiments 1-7, wherein the LIF-binding polypeptide comprises an antibody that specifically binds LIF.

9. The use of example 8, wherein the antibody that specifically binds LIF comprises at least one framework region derived from the framework regions of a human antibody.

10. The use of embodiment 8, wherein the antibody that specifically binds to LIF is humanized.

11. The use of example 8, wherein the antibody that specifically binds to LIF is deimmunized.

12. The use of example 8, wherein the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains.

13. The use of example 8, wherein the antibody that specifically binds to LIF is an IgG antibody.

14. The use of example 8, wherein the antibody that specifically binds to LIF is Fab, F (ab)₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies.

15. The use of any one of embodiments 8-14, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

b) Immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

16. The use of any one of embodiments 8-15, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and

b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 45-48.

17. The use of embodiment 16, wherein the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 46.

18. The use as described in example 17 wherein the VH sequence is identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

19. The use of any one of embodiments 8-15, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and

b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64.

20. The use of any one of embodiments 8-19, wherein the antibody that specifically binds to LIF is administered at less than about 200 picomolar K_DAnd (4) combining.

21. The use of any one of embodiments 8-19, wherein the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining.

22. The use of any one of embodiments 8 to 21, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof.

23. The use of embodiment 22, wherein the antibody specifically binds to PD-1.

24. The use of embodiment 22, wherein the antibody comprises palivizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

25. The use of embodiment 22, wherein the antibody specifically binds to PDL-1 or PDL-2.

26. The use of embodiment 25, wherein the antibody comprises Devolumab, Attributab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

27. The use of any one of embodiments 8 to 16, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

28. The use of embodiment 27, wherein the Fc fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

29. The use of any one of embodiments 1 to 28, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

30. The use of embodiment 29, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

31. The use of any one of embodiments 1-30, wherein the cancer comprises advanced solid tumors, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

32. The use of embodiment 31, wherein the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic ductal adenocarcinoma.

33. The use of any one of embodiments 1-32, wherein the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a PD-1, PDL-1 or PDL-2 signaling inhibitor administered as a monotherapy.

34. A method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) leukemia Inhibitory Factor (LIF) binding polypeptides; and

b) PD-1(CD279), PDL-1(CD274) or PDL-2(CD273) signaling inhibitors.

35. The method of embodiment 34, wherein the LIF-binding polypeptide and the PD-1(CD279), PDL-1(CD274), or PDL-2(CD273) signaling inhibitor are administered separately.

36. The method of embodiment 34, comprising administering to the individual having cancer an effective amount of the LIF binding polypeptide.

37. The method of embodiment 34, comprising administering to the individual having cancer an effective amount of the PD-1 inhibitor.

38. The method of any one of embodiments 34-37, wherein the LIF binding polypeptide comprises an immunoglobulin variable region or a fragment of an immunoglobulin heavy chain constant region.

39. The method of any one of embodiments 34-37, wherein the LIF-binding polypeptide comprises an antibody that specifically binds to LIF.

40. The method of example 39, wherein the antibody that specifically binds LIF comprises at least one framework region derived from the framework regions of a human antibody.

41. The method of embodiment 39, wherein the antibody that specifically binds LIF is humanized.

42. The method of embodiment 39, wherein the antibody that specifically binds to LIF is deimmunized.

43. The method of embodiment 39, wherein the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains.

44. The method of embodiment 39, wherein the antibody that specifically binds LIF is an IgG antibody.

45. The method of embodiment 39, wherein the antibody that specifically binds to LIF is Fab, F (ab)₂Single domain antibodies, single chain variable fragments (scFv) or nanobodies.

46. The method of any one of embodiments 39-45, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

47. The method of any one of embodiments 39-46, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and

b) An immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOS 45-48.

48. The method of embodiment 47, wherein the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 46.

49. The method of embodiment 48, wherein the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO. 46.

50. The method of any one of embodiments 39-46, wherein the antibody that specifically binds LIF comprises:

a) an immunoglobulin heavy chain sequence having an amino acid sequence at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs 57-60 or 67; and

b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOS 61-64.

51. The method of any one of embodiments 39-50, wherein the antibody that specifically binds to LIF is present at a K of less than about 200 picomolar_DAnd (4) combining.

52. The method of any one of embodiments 39 to 50, wherein the antibody that specifically binds to LIF is administered at a K of less than about 100 picomolar_DAnd (4) combining.

53. The method of any one of embodiments 34 to 52, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises an antibody or a PD-1, PDL-1 or PDL-2 binding fragment thereof.

54. The method of embodiment 53, wherein the antibody specifically binds PD-1.

55. The method of embodiment 54, wherein the antibody comprises palivizumab, nivolumab, AMP-514, tirezumab, sibatuzumab, or a PD-1 binding fragment thereof.

56. The method of embodiment 53, wherein the antibody specifically binds to PDL-1 or PDL-2.

57. The method of embodiment 56, wherein the antibody comprises Devolumab, Attributab, Avermemab, BMS-936559, or FAZ053, or a PDL-1 or PDL-2 binding fragment thereof.

58. The method of any one of embodiments 34-52, wherein the inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises an Fc fusion protein that binds PD-1, PDL-1 or PDL-2.

59. The method of embodiment 58, wherein the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof.

60. The method of any one of embodiments 34 to 52, wherein the PD-1, PDL-1 or PDL-2 signaling inhibitor comprises a PD-1, PDL-1 or PDL-2 small molecule inhibitor.

61. The method of embodiment 60, wherein the small molecule inhibitor of PD-1, PDL-1 or PDL-2 signaling comprises: n- {2- [ ({ 2-methoxy-6- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] pyridin-3-yl } methyl) amino ] ethyl } acetamide (BMS 202); (2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) -5-methylbenzyl) -D-serine hydrochloride; (2R,4R) -1- (5-chloro-2- ((3-cyanobenzyl) oxy) -4- ((3- (2, 3-dihydrobenzo [ b ] [1,4] dioxin-6-yl) -2-methylbenzyl) oxy) benzyl) -4-hydroxypyrrolidine-2-carboxylic acid; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenylindole; 3- (4, 6-dichloro-1, 3, 5-triazin-2-yl) -1-phenyl-1 h-indole; l- α -glutamine, N2, N6-bis (L-seryl-L-aspartyl-L-threonyl-L-seryl-L- α -glutamyl-L-seryl-L-phenylalanyl) -L-lysyl-L-phenylalanyl-L-arginyl-L-pentyl-L-threonyl-L-glutamyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutamyl-L-isoleucyl-L-lysyl; (2S) -1- [ [2, 6-dimethoxy-4- [ (2-methyl [1,1' -biphenyl ] -3-yl) methoxy ] phenyl ] methyl ] -2-piperidinecarboxylic acid; glycinamide, N- (2-mercaptoacetyl) -L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylmannosyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1 → 14) -thioether; or a derivative or analogue thereof.

62. The method of any one of embodiments 34 to 61, wherein the cancer comprises advanced solid tumor, glioblastoma, gastric cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, soft tissue cancer, or any combination thereof.

63. The method of embodiment 62, wherein the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic ductal adenocarcinoma.

64. The method of any one of embodiments 34-63, wherein the cancer is refractory to treatment with a therapeutic amount of an LIF-binding polypeptide inhibitor.

65. The method of any one of embodiments 34 to 63, wherein the cancer is refractory to treatment with a therapeutic amount of a PD-1, PDL-1 or PDL-2 signaling inhibitor.

66. The method of any one of embodiments 34-65, wherein the Leukemia Inhibitory Factor (LIF) binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered separately.

67. The method of any one of embodiments 34 to 65, wherein the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered simultaneously.

68. The method of any one of embodiments 34-65, wherein the LIF-binding polypeptide and the PD-1, PDL-1 or PDL-2 signaling inhibitor are administered in a single composition.

69. Use of an antibody that specifically binds Leukemia Inhibitory Factor (LIF) in combination with a PD-1 or PDL-1 binding antibody for treating cancer in an individual, wherein the LIF binding antibody comprises:

a) an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

b) immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

c) an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

d) an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs 9 or 10;

e) immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and

f) immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

70. A method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) An antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

an immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 9 or 10;

immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 11 or 12; and

an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and

b) PD-1 or PDL-1 binding antibodies.

71. The method of embodiment 70, comprising administering to the individual having cancer an effective amount of the antibody that specifically binds LIF.

72. The method of embodiment 70, comprising administering an effective amount of the PD-1 or PDL-1 binding antibody to the individual having cancer.

73. The method of any one of embodiments 70 to 72, wherein the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately.

74. The method of any one of embodiments 70-73, wherein the cancer is glioblastoma multiforme (GBM), NSCLC (non-small cell lung cancer), ovarian cancer, colorectal cancer, thyroid cancer, pancreatic cancer, or a combination thereof.

75. A method of reducing pre-Tumor Associated Macrophages (TAMs) in a tumor of an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

an immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 9 or 10;

Immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 11 or 12; and

an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and

b) PD-1 or PDL-1 binding antibodies.

76. The method of embodiment 75, comprising administering to the individual having cancer an effective amount of the antibody that specifically binds LIF.

77. The method of embodiment 75, comprising administering to the individual having cancer an effective amount of the PD-1 or PDL-1 binding antibody.

78. The method of any one of embodiments 75 to 77, wherein the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately.

79. The method of any one of embodiments 75 to 78, wherein the TAM exhibits cell surface expression of any 1, 2 or 3 molecules selected from the list consisting of CD11b, CD206, and CD 163.

80. The method of any one of embodiments 75-78, wherein the tumor comprises a lung tumor, a brain tumor, a pancreatic tumor, a breast tumor, a kidney tumor, a colorectal tumor, an ovarian tumor, or a combination thereof.

81. A method of generating immunological memory in an individual having cancer comprising administering to the individual having cancer an effective amount of a combination of:

a) An antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

an immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 9 or 10;

immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 11 or 12; and

an immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and

b) PD-1 or PDL-1 binding antibodies.

82. The method of embodiment 81, comprising administering to the individual having cancer an effective amount of the antibody that specifically binds LIF.

83. The method of embodiment 81, comprising administering to the individual having cancer an effective amount of the PD-1 or PDL-1 binding antibody.

84. The method of any one of embodiments 81 to 83, wherein the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately.

85. The method of any one of embodiments 81 to 84, wherein immunological memory is mediated by CD8+ T cells.

86. The method of any one of embodiments 81 to 84, wherein immunological memory is mediated by CD4+ T cells.

87. A method of increasing the amount of T lymphocytes in a tumor in an individual comprising administering to the individual with the tumor an effective amount of a combination of:

a) an antibody that specifically binds Leukemia Inhibitory Factor (LIF), the antibody comprising:

i. an immunoglobulin heavy chain complementarity determining region 1(VH-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-3;

an immunoglobulin heavy chain complementarity determining region 2(VH-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 4 or 5;

an immunoglobulin heavy chain complementarity determining region 3(VH-CDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 6-8;

an immunoglobulin light chain complementarity determining region 1(VL-CDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 9 or 10;

immunoglobulin light chain complementarity determining region 2(VL-CDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOS: 11 or 12; and

An immunoglobulin light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and

b) PD-1 or PDL-1 binding antibodies.

88. The method of embodiment 87, comprising administering to the individual having cancer an effective amount of the antibody that specifically binds LIF.

89. The method of embodiment 87, comprising administering to the individual having cancer an effective amount of the PD-1 or PDL-1 binding antibody.

90. The method of embodiment 87, wherein the antibody that specifically binds LIF and the PD-1 or PDL-1 binding antibody are administered separately.

91. The method of any one of embodiments 87 to 90, wherein the T lymphocytes comprise CD8+ T cells.

92. The method of any one of embodiments 87 to 90, wherein the T lymphocytes comprise CD4+ T cells.

93. The method of any one of embodiments 87 to 90, wherein the tumor comprises a lung tumor, a brain tumor, a pancreatic tumor, a breast tumor, a kidney tumor, a colorectal tumor, an ovarian tumor, or a combination thereof.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

All publications, patent applications, issued patents, and other documents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference. To the extent that a definition in this disclosure is contradictory, a definition contained in the text incorporated by reference is excluded.

Sequence of