阅读说明：本技术 治疗肿瘤的方法 (Method for treating tumors ) 是由 M·雷 N·O·西莫斯 D·潘迪亚 张汉 T·K·桑切斯 C·T·哈比森 P·M·萨伯 于 2020-03-27 设计创作，主要内容包括：本公开文本提供了一种用于治疗患有肿瘤的受试者的方法,所述方法包括向所述受试者施用治疗有效量的抗PD-1抗体或其抗原结合部分或抗PD-L1抗体或其抗原结合部分,其中所述受试者被鉴定为具有高炎症基因标签得分。在一些实施方案中,所述高炎症基因标签得分是通过测量从所述受试者获得的肿瘤样品中的一组炎症基因的表达来确定的,其中所述炎症基因组包含CD274(PD-L1)、CD8A、LAG3和STAT1。(The present disclosure provides a method for treating a subject having a tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding portion thereof or an anti-PD-L1 antibody or antigen-binding portion thereof, wherein the subject is identified as having a high inflammatory gene signature score. In some embodiments, the high inflammatory gene signature score is determined by measuring expression of a set of inflammatory genes in a tumor sample obtained from the subject, wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.)

1. A method for treating a human subject having a tumor, the method comprising (i) identifying a subject exhibiting a high inflammation signature score; and (ii) administering an anti-PD-1 antibody to the subject; wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

2. A method for treating a human subject having a tumor, the method comprising administering to the subject an anti-PD-1 antibody, wherein the subject was identified as exhibiting a high inflammation signature score prior to the administration;

wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

3.A method for identifying a human subject having a tumor suitable for treatment with an anti-PD-1 antibody, the method comprising: (i) measuring an inflammation signature score in a tumor sample obtained from the subject, and (ii) administering an anti-PD-1 antibody to the subject if the subject exhibits a high inflammation signature score;

wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

4. The method of any one of claims 1 to 3, wherein the inflammatory genome consists of less than about 20, less than about 18, less than about 15, less than about 13, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, or less than about 5 inflammatory genes.

5. The method of any one of claims 1 to 4, wherein the genome of inflammation consists essentially of: (i) CD274(PD-L1), CD8A, LAG3, and STAT1, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, or 15 additional inflammatory genes.

6. The method of claim 5, wherein the additional inflammatory gene is selected from the group consisting of: CCL2, CCL3, CCL4, CCL5, CCR5, CD27, CD274, CD276, CMKLR1, CXCL10, CXCL11, CXCL9, CXCR6, GZMA, GZMK, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DRA, HLA-DRB1, HLA-E, ICOS, IDO1, IFNG, IRF1, NKG7, PDCD1LG2, PRF1, PSMB10, TIGIT, and any combination thereof.

7. The method of any one of claims 1 to 4, wherein the genome of inflammation consists essentially of CD274(PD-L1), CD8A, LAG3, and STAT 1.

8. The method of any one of claims 1 to 4, wherein the genome of inflammation consists of CD274(PD-L1), CD8A, LAG3, and STAT 1.

9. The method of any one of claims 1 to 8, wherein the high inflammation signature score is characterized by an inflammation signature score that is greater than an average inflammation signature score, wherein the average inflammation signature score is determined by averaging the expression of the set of inflammation genes in tumor samples obtained from a population of subjects having the tumor.

10. The method of claim 9, wherein the average inflammation signature score is determined by averaging the expression of the set of inflammatory genes in tumor samples obtained from the population of subjects.

11. The method of claim 9 or 10, wherein the high inflammation tag score is characterized by an inflammation tag score at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the average inflammation tag score.

12. The method of any one of claims 9 to 11, wherein the high inflammation tag score is characterized by an inflammation tag score that is at least about 50% higher than the average inflammation tag score.

13. The method of any one of claims 9 to 12, wherein the high inflammation tag score is characterized by an inflammation tag score that is at least about 75% higher than the average inflammation tag score.

14. The method of any one of claims 1 to 13, wherein the tumor sample is a tumor tissue biopsy sample.

15. The method of any one of claims 1 to 14, wherein the tumor sample is formalin fixed paraffin embedded tumor tissue or freshly frozen tumor tissue.

16. The method of any one of claims 1 to 15, wherein the expression of the inflammatory gene in the inflammatory genome is determined by detecting the presence of inflammatory gene mRNA, the presence of a protein encoded by the inflammatory gene, or both.

17. The method of claim 16, wherein the presence of inflammatory gene mRNA is determined using reverse transcriptase PCR.

18. The method of claim 16 or 17, wherein the presence of a protein encoded by the inflammatory gene is determined using an IHC assay.

19. The method of claim 18, wherein the IHC assay is an automated IHC assay.

20. The method of any one of claims 1-19, wherein the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1.

21. The method of any one of claims 1-20, wherein the anti-PD-1 antibody binds the same epitope as nivolumab.

22. The method of any one of claims 1 to 21, wherein the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody or a portion thereof.

23. The method of any one of claims 1-22, wherein the anti-PD-1 antibody comprises a heavy chain constant region of human IgG1 or IgG4 isotype.

24. The method of any one of claims 1-23, wherein the anti-PD-1 antibody is nivolumab.

25. The method of any one of claims 1-23, wherein the anti-PD-1 antibody is pembrolizumab.

26. The method of any one of claims 1 to 25, wherein the anti-PD-1 antibody is administered about every 1,2, or 3 weeks at a dose in the range of from at least about 0.1mg/kg to at least about 10.0mg/kg body weight.

27. The method of claim 26, wherein the anti-PD-1 antibody is administered at a dose of at least about 3mg/kg body weight about once every 2 weeks.

28. The method of any one of claims 1 to 25, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in flat doses.

29. The method of any one of claims 1 to 25 and 28, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, or at least about 550 mg.

30. The method of any one of claims 1 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 240 mg.

31. The method of any one of claims 1 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 480 mg.

32. The method of any one of claims 1 to 25 and 28 to 31, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in flat doses about once every 1,2, 3, or 4 weeks.

33. The method of any one of claims 1 to 25, 28, 29, and 32, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose or about 240mg about once every two weeks.

34. The method of any one of claims 1 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480mg about once every four weeks.

35. The method of any one of claims 1 to 34, wherein the anti-PD-1 antibody is administered continuously for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

36. The method of any one of claims 1-35, wherein the anti-PD-1 antibody is formulated for intravenous administration.

37. The method of any one of claims 1 to 36, wherein the anti-PD-1 antibody is administered at a sub-therapeutic dose.

38. The method of any one of claims 1 to 37, further comprising administering an antibody or antigen-binding fragment thereof that specifically binds to cytotoxic T-lymphocyte-associated protein 4(CTLA-4) ("anti-CTLA-4 antibody").

39. The method of claim 38, wherein the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4.

40. The method of claim 38 or 39, wherein the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab.

41. The method of any one of claims 38 to 40, wherein the anti-CTLA-4 antibody is ipilimumab.

42. The method of any one of claims 38 to 40, wherein the anti-CTLA-4 antibody is tremelimumab.

43. The method of any one of claims 38 to 42, wherein the anti-CTLA-4 antibody is administered at a dose in the range of from 0.1mg/kg to 20.0mg/kg body weight once every 2, 3, 4,5, 6, 7 or 8 weeks.

44. The method of any one of claims 38 to 43, wherein the anti-CTLA-4 antibody is administered at a dose of 1mg/kg body weight once every 6 weeks.

45. The method of any one of claims 38 to 43, wherein the anti-CTLA-4 antibody is administered at a dose of 1mg/kg body weight once every 4 weeks.

46. The method of any one of claims 38 to 42, wherein the anti-CTLA-4 antibody is administered in flat doses.

47. The method of claim 46, wherein the anti-CTLA-4 antibody is administered in a flat dose of at least about 40mg, at least about 50mg, at least about 60mg, at least about 70mg, at least about 80mg, at least about 90mg, at least about 100mg, at least about 110mg, at least about 120mg, at least about 130mg, at least about 140mg, at least about 150mg, at least about 160mg, at least about 170mg, at least about 180mg, at least about 190mg, or at least about 200 mg.

48. The method of claim 46 or 47 wherein the anti-CLTA-4 antibody is administered at a flat dose about once every 2, 3, 4,5, 6, 7 or 8 weeks.

49. The method of any one of claims 1 to 48, wherein the tumor is derived from a cancer selected from the group consisting of: hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof.

50. The method of any one of claims 1-49, wherein the tumor is derived from hepatocellular carcinoma.

51. The method of any one of claims 1-49, wherein the tumor is derived from gastroesophageal cancer.

52. The method of any one of claims 1-49, wherein the tumor is derived from melanoma.

53. The method of any one of claims 1-52, wherein the tumor is recurrent.

54. The method of any one of claims 1-53, wherein the tumor is refractory.

55. The method of any one of claims 1 to 54, wherein the tumor is refractory following at least one prior therapy comprising administration of at least one anti-cancer agent.

56. The method of claim 55, wherein the at least one anti-cancer agent comprises standard of care therapy.

57. The method of claim 55 or 56, wherein the at least one anti-cancer agent comprises immunotherapy.

58. The method of any one of claims 1-57, wherein the tumor is locally advanced.

59. The method of any one of claims 1-58, wherein the tumor is metastatic.

60. The method of any one of claims 1-59, wherein the administering treats the tumor.

61. The method of any one of claims 1-60, wherein the administering reduces the size of the tumor.

62. The method of claim 61, wherein the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the size of the tumor prior to the administration.

63. The method of any one of claims 1 to 62, wherein the subject exhibits a progression-free survival of at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about one year, at least about eighteen months, at least about two years, at least about three years, at least about four years, or at least about five years after initial administration.

64. The method of any one of claims 1-63, wherein the subject exhibits stable disease after the administration.

65. The method of any one of claims 1-63, wherein the subject exhibits a partial response following the administration.

66. The method of any one of claims 1-63, wherein the subject exhibits a complete response after the administration.

67. A kit for treating a subject having a tumor, the kit comprising:

(a) an anti-PD-1 antibody at a dose ranging from about 4mg to about 500 mg; and

(b) instructions for using the anti-PD-1 antibody in the method according to any one of claims 1 to 66.

68. The kit of claim 67, further comprising an anti-CTLA-4 antibody.

69. The kit of claim 67 or 68, further comprising an anti-PD-L1 antibody.

Technical Field

The present disclosure provides a method for treating a subject having a tumor using immunotherapy.

Background

Human cancers have many genetic and epigenetic changes that produce novel antigens that are potentially recognized by the immune system (Sjoblom et al, Science (2006)314(5797): 268-. The adaptive immune system, composed of T and B lymphocytes, has a strong potential for cancer, has a broad capacity and precise specificity to respond to a wide variety of tumor antigens. In addition, the immune system exhibits considerable plasticity and memory components. The successful exploitation of all these attributes of the adaptive immune system will make immunotherapy unique among all cancer treatment modalities.

Until recently, cancer immunotherapy has focused a great deal of effort on approaches to enhance anti-tumor immune responses by adoptively metastasizing activated effector cells, immunizing against relevant antigens, or providing non-specific immune stimulators (such as cytokines). However, in the past decade, a great deal of effort to develop specific immune checkpoint pathway inhibitors has begun to provide new immunotherapeutic approaches for treating cancer, including the development of antibodies that specifically bind to the programmed death factor-1 (PD-1) receptor and block the inhibitory PD-1/PD-1 ligand pathway (such as nivolumab and pembrolizumab (previously pambrizumab); USAN committee Statement (USAN Council Statement), 2013)) (Topalian et al, 2012a, b; Topalian et al, 2014; Hamid et al, 2013; Hamid and Carvajal, 2013; McDermott and Atkins, 2013).

PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family (including CD28, CTLA-4, ICOS, PD-1, and BTLA). Two cell surface glycoprotein ligands of PD-1 have been identified, programmed death factor ligand-1 (PD-L1) and programmed death factor ligand-2 (PD-L2), which are expressed on antigen presenting cells as well as on many human cancers and have been shown to down regulate T cell activation and cytokine secretion upon binding to PD-1. In preclinical models, inhibition of the PD-1/PD-L1 interaction mediates potent antitumor activity (U.S. Pat. nos. 8,008,449 and 7,943,743), and treatment of cancer with antibody inhibitors of the PD-1/PD-L1 interaction has entered clinical trials (Brahmer et al, 2010; Topalian et al, 2012 a; Topalian et al, 2014; Hamid et al, 2013; Brahmer et al, 2012; Flies et al, 2011; pardol, 2012; Hamid and Carvajal, 2013).

Nivolumab (previously designated 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking down-regulation of anti-tumor T cell function (U.S. patent No. 8,008,449; Wang et al, 2014). Nivolumab has been shown to be active in a variety of advanced solid tumors including renal cell carcinoma (renal adenocarcinoma or suprarenal adenoids), melanoma, and non-small cell lung cancer (NSCLC) (topallian et al, 2012 a; topallian et al, 2014; Drake et al, 2013; WO 2013/173223).

The immune system and the response to immunotherapy are complex. In addition, the effectiveness of anticancer agents can vary according to unique patient characteristics. Thus, there is a need for targeted therapeutic strategies that identify patients more likely to respond to a particular anti-cancer agent, thereby improving the clinical outcome of patients diagnosed with cancer.

Disclosure of Invention

Certain aspects of the present disclosure relate to a method for treating a human subject having a tumor, the method comprising (i) identifying a subject exhibiting a high inflammation signature (signature) score; and (ii) administering an anti-PD-1 antibody to the subject; wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

Certain aspects of the present disclosure relate to a method for treating a human subject having a tumor, the method comprising administering to the subject an anti-PD-1 antibody, wherein the subject was identified as exhibiting a high inflammation signature score prior to the administration; wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

Certain aspects of the present disclosure relate to a method for identifying a human subject having a tumor suitable for treatment with an anti-PD-1 antibody, the method comprising: (i) measuring an inflammation signature score in a tumor sample obtained from the subject, and (ii) administering an anti-PD-1 antibody to the subject if the subject exhibits a high inflammation signature score; wherein the inflammation signature score is determined by measuring the expression of a set of inflammatory genes ("inflammation genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

In some embodiments, the inflammatory genome consists of less than about 20, less than about 18, less than about 15, less than about 13, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, or less than about 5 inflammatory genes. In some embodiments, the genome of inflammation consists essentially of: (i) CD274(PD-L1), CD8A, LAG3, and STAT1, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, or 15 additional inflammatory genes.

In some embodiments, the additional inflammatory gene is selected from the group consisting of: CCL2, CCL3, CCL4, CCL5, CCR5, CD27, CD274, CD276, CMKLR1, CXCL10, CXCL11, CXCL9, CXCR6, GZMA, GZMK, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DRA, HLA-DRB1, HLA-E, ICOS, IDO1, IFNG, IRF1, NKG7, PDCD1LG2, PRF1, PSMB10, TIGIT, and any combination thereof.

In some embodiments, the genome of inflammation consists essentially of CD274(PD-L1), CD8A, LAG3, and STAT 1. In some embodiments, the genome of inflammation consists of CD274(PD-L1), CD8A, LAG3, and STAT 1.

In some embodiments, the high inflammation signature score is characterized by an inflammation signature score that is greater than an average inflammation signature score, wherein the average inflammation signature score is determined by averaging the expression of the set of inflammation genes in tumor samples obtained from a population of subjects having the tumor.

In some embodiments, the average inflammation signature score is determined by averaging the expression of the set of inflammatory genes in tumor samples obtained from the population of subjects.

In some embodiments, the high inflammation tag score is characterized by an inflammation tag score that is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the average inflammation tag score. In some embodiments, the high inflammation tag score is characterized by an inflammation tag score that is at least about 50% higher than the average inflammation tag score. In some embodiments, the high inflammation tag score is characterized by an inflammation tag score that is at least about 75% higher than the average inflammation tag score.

In some embodiments, the tumor sample is a tumor tissue biopsy sample. In some embodiments, the tumor sample is formalin fixed paraffin embedded tumor tissue or freshly frozen tumor tissue. In some embodiments, the expression of the inflammatory gene in the genome of inflammation is determined by detecting the presence of inflammatory gene mRNA, the presence of a protein encoded by the inflammatory gene, or both. In some embodiments, the presence of inflammatory gene mRNA is determined using reverse transcriptase PCR. In some embodiments, the presence of a protein encoded by the inflammatory gene is determined using an IHC assay. In some embodiments, the IHC assay is an automated IHC assay.

In some embodiments, the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. In some embodiments, the anti-PD-1 antibody binds to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody, or a portion thereof. In some embodiments, the anti-PD-1 antibody comprises a heavy chain constant region of human IgG1 or IgG4 isotype. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

In some embodiments, the anti-PD-1 antibody is administered at a dose in the range of from at least about 0.1mg/kg to at least about 10.0mg/kg body weight about once every 1,2, or 3 weeks. In some embodiments, the anti-PD-1 antibody is administered at a dose of at least about 3mg/kg body weight about once every 2 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in flat doses. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, or at least about 550 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 240 mg. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 480 mg.

In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in flat doses about once every 1,2, 3, or 4 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose or about 240mg about once every two weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480mg about once every four weeks.

In some embodiments, the anti-PD-1 antibody is administered continuously as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In some embodiments, the anti-PD-1 antibody is formulated for intravenous administration. In some embodiments, the anti-PD-1 antibody is administered at a sub-therapeutic dose.

In some embodiments, the methods further comprise administering an antibody or antigen-binding fragment thereof that specifically binds to cytotoxic T lymphocyte-associated protein 4(CTLA-4) ("anti-CTLA-4 antibody"). In some embodiments, the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds the same epitope as ipilimumab or tremelimumab. In some embodiments, the anti-CTLA-4 antibody is ipilimumab. In some embodiments, the anti-CTLA-4 antibody is tremelimumab.

In some embodiments, the anti-CTLA-4 antibody is administered at a dose ranging from 0.1mg/kg to 20.0mg/kg body weight once every 2, 3, 4,5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of 1mg/kg body weight once every 6 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of 1mg/kg body weight once every 4 weeks.

In some embodiments, the anti-CTLA-4 antibody is administered in flat doses. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose of at least about 40mg, at least about 50mg, at least about 60mg, at least about 70mg, at least about 80mg, at least about 90mg, at least about 100mg, at least about 110mg, at least about 120mg, at least about 130mg, at least about 140mg, at least about 150mg, at least about 160mg, at least about 170mg, at least about 180mg, at least about 190mg, or at least about 200 mg. In some embodiments, the anti-CLTA-4 antibody is administered in a flat dose about once every 2, 3, 4,5, 6, 7, or 8 weeks.

In some embodiments, the tumor is derived from a cancer selected from the group consisting of: hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof. In some embodiments, the tumor is derived from hepatocellular carcinoma. In some embodiments, the tumor is derived from gastroesophageal cancer. In some embodiments, the tumor is derived from melanoma.

In some embodiments, the tumor is recurrent. In some embodiments, the tumor is refractory. In some embodiments, the tumor is refractory after at least one prior therapy comprising administration of at least one anti-cancer agent. In some embodiments, the at least one anti-cancer agent comprises a standard of care therapy. In some embodiments, the at least one anti-cancer agent comprises immunotherapy.

In some embodiments, the tumor is locally advanced. In some embodiments, the tumor is metastatic.

In some embodiments, the administering treats the tumor. In some embodiments, the administering reduces the size of the tumor. In some embodiments, the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the size of the tumor prior to the administration. In some embodiments, the subject exhibits progression-free survival of at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about one year, at least about eighteen months, at least about two years, at least about three years, at least about four years, or at least about five years after initial administration.

In some embodiments, the subject exhibits stable disease after the administration. In some embodiments, the subject exhibits a partial response after the administration. In some embodiments, the subject exhibits a complete response after the administration.

Certain aspects of the present disclosure relate to a kit for treating a subject having a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from about 4mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in any of the methods disclosed herein. In some embodiments, the kit further comprises an anti-CTLA-4 antibody. In some embodiments, the kit further comprises an anti-PD-L1 antibody.

Drawings

Figure 1 is a schematic of a study design for exploratory endpoint biomarker assessment of NIVO efficacy in patients with advanced hepatocellular carcinoma (HCC) and receiving prior Sorafenib (SOR) treatment ("SOR-experienced") and not receiving prior sorafenib treatment (SOR naive) in clinical trial NCT 01658878.

Fig.2A and 2B are waterfall plots showing the optimal reduction (%) of target lesions from baseline for all subjects in the total population (fig. 2A) and the population undergoing SOR (fig. 2B), where the subjects in each plot are labeled according to PD-L1 status. FIGS. 2C and 2D are schematic representations of the overall survival (month) of patients in the total population of patients with tumor cells PD-L1 ≧ 1% or < 1% (SOR naive and SOR experienced; FIG. 2C) and the population undergoing SOR alone (FIG. 2D), as indicated. The number of patients at risk for each PD-L1 group is indicated below the x-axis.

Figures 3A-3D are graphs showing the relationship between the best overall response and the percentage of cells expressing T cell markers selected from CD3 (figure 3A), CD4 (figure 3B), CD8 (figure 3C), and FOXP3 (figure 3D) relative to the overall population (SOR naive and SOR experienced).

Figures 4A-4D are schematic diagrams showing the overall survival of the total population (SOR naive and SOR experienced) stratified into three parts based on expression of the lowest, intermediate or highest level of T cell markers selected from CD3 (figure 4A), CD4 (figure 4B), CD8 (figure 4C) and FOXP3 (figure 4D). The number of patients at risk for each stratified group is indicated below the x-axis.

FIGS. 5A-5B are graphs showing the relationship between the optimal total response and the percentage of cells expressing macrophage markers selected from the group consisting of CD68 (FIG. 5A) and CD163 (FIG. 5B) relative to the total population (SOR naive and SOR experienced).

Figures 6A-6B are schematic diagrams showing the overall survival of the total population (SOR naive and SOR experienced) stratified into three based on expression of the lowest, intermediate or highest level T cell marker selected from CD68 (figure 6A) and CD163 (figure 6B). The number of patients at risk for each stratified group is indicated below the x-axis.

Figure 7A is a graph showing the relationship between the best overall response and the 4 gene signature score, as described herein. Figure 7B is a schematic showing the overall survival of the total population (SOR naive and SOR experienced) stratified into three based on expression of the lowest, intermediate or highest 4 gene inflammation signature scores. The number of patients at risk for each stratified group is indicated below the x-axis.

Figure 8 is a schematic diagram of a study design showing exploratory endpoint biomarker assessment for efficacy of nivolumab treatment with or without ipilimumab in patients with chemotherapy refractory gastroesophageal cancer in phase I/II clinical trial NCT 01928394.

Figures 9A-9B are graphs showing the relationship between the optimal total response and tumor PD-L1 expression for subjects treated with nivolumab 3mg/kg monotherapy or nivolumab 1mg/kg + ipilimumab 3mg/kg, nivolumab 3mg/kg + ipilimumab 1mg/kg, or nivolumab 1mg/kg + ipilimumab 1mg/kg (figure 9A) and the relationship between the optimal total response and PD-L1 combined positive score (CPS; figure 9B) (as defined herein).

FIGS. 10A-10F are graphs of the overall survival of patients in all treatment groups stratified by tumor PD-L1 expression ≧ 1% or < 1% (FIG. 10A),. gtoreq.5% or < 5% (FIG. 10B),. gtoreq.10% or < 10% (FIG. 10C) or stratified by PD-L1 CPS ≧ 1 or <1 (FIG. 10D),. gtoreq.5 or <5 (FIG. 10E),. gtoreq.10 or <10 (FIG. 10F), as indicated. The number of patients at risk for each PD-L1 group is indicated below the x-axis.

FIGS. 11A-10D are graphical representations of the overall survival of patients in the treatment groups of 1mg/kg of nivolumab + 3mg/kg of ipilimumab stratified by PD-L1 expression ≧ 1% or < 1% (FIG. 11A) or by PD-L1 CPS ≧ 1 or <1 (FIG. 11B),. gtoreq.5 or <5 (FIG. 11C),. gtoreq.10 or <10 (FIG. 11D), as indicated. The number of patients at risk for each PD-L1 group is indicated below the x-axis.

Fig. 12A-12D are graphs showing the relationship between the best overall response and the CD 8T cell signature (fig. 12A), PD-L1 transcript (fig. 12B), Ribas 10 gene signature (fig. 12C), and the 4 gene inflammation signature described herein (fig. 12D).

Figure 13 shows ROC analysis of the 4 gene immune tags and benefits.

Figure 14 is a schematic representation of the study design of exploratory endpoint biomarker assessment of efficacy of nivolumab monotherapy, ipilimumab monotherapy and nivolumab/ipilimumab combination therapy in patients with unresectable stage III or IV melanoma in NCT01721772 and NCT01844505 trials.

FIGS. 15A-15B are Kaplan-Meier plots of major findings, progression free survival (PFS; FIG. 15A) and overall survival (OS; FIG. 15B) for the intent-to-treat (ITT) population from NCT01844505 (FIGS. 15A-15B).

Figure 16 is a bar graph showing sample treatment of subjects treated with nivolumab + ipilimumab combination therapy, nivolumab monotherapy, or ipilimumab monotherapy in NCT01844505 and evaluated a4 gene signature score. The total number of each group is indicated above each column.

Figure 17 is a graph showing the relationship between the optimal overall response and the 4 gene inflammation signature score described herein in subjects administered nivolumab/ipilimumab combination therapy, nivolumab monotherapy or ipilimumab monotherapy in the NCT01844505 trial.

Figures 18A-18C are schematic diagrams showing progression free survival of subjects administered nivolumab/ipilimumab combination therapy (figure 18A), nivolumab monotherapy (figure 18B), or ipilimumab monotherapy (figure 18C), wherein subjects are stratified according to a high 4-gene inflammation signature score ("high ISS") or a low 4-gene inflammation signature score ("low ISS"). The number of patients at risk for each stratified group is indicated below the x-axis. Fig.18D shows the corresponding hazard ratio.

Figures 19A-19C are schematic diagrams showing the Overall Survival (OS) of subjects administered nivolumab/ipilimumab combination therapy (figure 19A), nivolumab monotherapy (figure 19B), or ipilimumab monotherapy (figure 19C), wherein the subjects are based on having a high 4 gene inflammation signature score ("high ISS") or a low 4 gene inflammation signature score ("low ISS"). The number of patients at risk for each stratified group is indicated below the x-axis. Fig.19D shows the corresponding hazard ratio.

Detailed Description

The present disclosure provides a method for treating a human subject having a tumor, the method comprising (i) identifying a subject exhibiting a high inflammation signature score; and (ii) administering to the subject a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. The present disclosure also provides a method for treating a human subject having a tumor, the method comprising administering a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the subject was identified as having a high inflammation signature score prior to the administration. The present disclosure also provides a method for identifying a human subject having a tumor suitable for treatment with a PD-1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody), the method comprising: (i) measuring an inflammation signature score in a tumor sample obtained from the subject, and (ii) administering a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, to the subject if the subject has a high inflammation signature score.

I. Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below, unless the context clearly provides otherwise. Additional definitions are set forth throughout this application.

By "administering" is meant physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for immunotherapy (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration, other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Other parenteral routes include oral, topical, epidermal or mucosal routes of administration, e.g. intranasally, vaginally, rectally, sublingually or topically. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

As used herein, an "adverse event" (AE) is any adverse and often unintentional or undesirable sign (including abnormal laboratory findings), symptom or disease associated with the use of medical treatment. For example, an adverse event may be associated with activation of the immune system or expansion of cells of the immune system (e.g., T cells) in response to a treatment. A medical treatment may have one or more related AEs, and each AE may have the same or a different level of severity. Reference to a method capable of "altering an adverse event" means a treatment regimen that reduces the incidence and/or severity of one or more AEs associated with the use of a different treatment regimen.

An "antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. The heavy chain constant region comprises three constant domains, i.e.C_H1、C_H2And C_H3. Each light chain comprises a light chain variable region (abbreviated herein as V)_L) And a light chain constant region. The light chain constant region comprises a constant domain, i.e.C_L。V_HAnd V_LRegions can be further subdivided into regions of high degeneracy, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V_HAnd V_LComprising three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). Thus, the term "anti-PD-1 antibody" includes whole antibodies and antigen-binding portions of whole antibodies having two heavy chains and two light chains that specifically bind to PD-1. Non-limiting examples of antigen-binding moieties are shown elsewhere herein.

The immunoglobulin may be derived from any well-known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG 4. "isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric antibodies and humanized antibodies; a human or non-human antibody; fully synthesizing an antibody; and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or antigen-binding portions of any of the above-described immunoglobulins, and includes monovalent and bivalent fragments or portions as well as single chain antibodies.

An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may be cross-reactive with other antigens (such as PD-1 molecules from different species). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules having a single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are examples of isolated antibodies. Monoclonal antibodies can be produced by hybridomas, recombinant, transgenic, or other techniques known to those skilled in the art.

"human antibodies" (HuMAb) refer to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

"humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDRs of a non-human antibody are replaced with corresponding amino acids derived from a human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDRs have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDRs are unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized antibodies" retain antigen specificity similar to the original antibody.

"chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

An "anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, an anti-PD-L1 antibody specifically binds to PD-L1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

An "antigen-binding portion" (also referred to as an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen to which the intact antibody binds. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody described herein) include (i) Fab fragments (fragments from papain cleavage) or (ii) fragments from V_L、V_HLC and CH1 domains; (ii) a F (ab')2 fragment (a fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) from V_HAnd the CH1 domain; (iv) v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) dAb fragments (Ward et al (1989) Nature 341:544-546) consisting of V_HDomain composition; (vi) separated from each other(vii) a combination of Complementarity Determining Regions (CDRs) and (vii) two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, despite the two domains V of the Fv fragment_LAnd V_HAre encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that can make them into a single protein chain in which V is present_LAnd V_HThe regions pair to form monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-58426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

"cancer" refers to a broad group of different diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and may also metastasize to distal parts of the body through the lymphatic system or blood stream.

The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of contracting a disease or suffering from a relapse of a disease by a method that includes inducing, enhancing, suppressing or otherwise modifying an immune response. "treatment" or "therapy" of a subject refers to any type of intervention or treatment performed on the subject, or administration of an active agent to the subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, severity or recurrence of a symptom, complication or condition, or biochemical indicator associated with the disease.

"programmed death factor-1" (PD-1) refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes variants, subtypes, and species homologs of human PD-1(hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession No. U64863.

"programmed death factor ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that down-regulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes variants, subtypes and species homologs of human PD-L1(hPD-L1), hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession No. Q9NZQ 7. The human PD-L1 protein is encoded by the human CD274 gene (NCBI gene ID: 29126).

As used herein, a PD-1 or PD-L1 "inhibitor" refers to any molecule that is capable of blocking, reducing, or otherwise limiting the interaction between PD-1 and PD-L1 and/or the activity of PD-1 and/or PD-L1. In some aspects, the inhibitor is an antibody or an antigen-binding fragment of an antibody. In other aspects, the inhibitor comprises a small molecule.

As used herein, "T cell surface glycoprotein CD8a chain" or "CD 8A" refers to an integral membrane glycoprotein that participates in an immune response and serves multiple functions in response to both external and internal attacks. In T cells, CD8a functions primarily as a co-receptor for MHC class I molecules/peptide complexes. CD8A interacts with both the T Cell Receptor (TCR) and MHC class I proteins presented by Antigen Presenting Cells (APC). In turn, CD8a recruits Src kinase LCK to the vicinity of the TCR-CD3 complex. LCK then initiates diverse intracellular signaling pathways by phosphorylating multiple substrates, ultimately leading to lymphokine production, motility, adhesion and activation of Cytotoxic T Lymphocytes (CTLs). This mechanism enables CTLs to recognize and eliminate infected and tumor cells. In NK cells, the presence of CD8A homodimers on the cell surface provides a survival mechanism that allows binding and lysis of multiple target cells. The CD8A homodimeric molecule also promoted survival and differentiation of activated lymphocytes into memory CD 8T cells. The complete CD8a amino acid sequence can be found under UniProtKB identification number P01732. The human CD8a protein is encoded by the human CD8a gene (NCBI gene ID: 925).

As used herein, "lymphocyte activation gene-3", "LAG 3", "LAG-3" or "CD 223" refers to type I transmembrane proteins expressed on the cell surface of activated CD4+ and CD8+ T cells, as well as NK and dendritic cell subsets. LAG-3 protein is closely related to CD4, a co-receptor for T helper cell activation. Both molecules have four extracellular Ig-like domains and need to bind to their ligands (major histocompatibility complex (MHC) class II) to exert their functional activity. LAG-3 protein is expressed only on the cell surface of activated T cells, and its lysis from the cell surface terminates LAG-3 signaling. LAG-3 may also be found as a soluble protein, which does not bind to MHC class II. LAG-3 also plays an important role in promoting regulatory T cell (Treg) activity and in down-regulating T cell activation and proliferation. Both natural and induced tregs express increased LAG-3, which is required for their maximal suppressive function. The complete human LAG-3 amino acid sequence can be found under UniProtKB identification number P18627. The human LAG3 protein is encoded by the human LAG3 gene (NCBI gene ID: 3902).

As used herein, "signal transducer and activator of transcription 1- α/β" or "STAT 1" refers to signal transducers and activators of transcription that mediate cellular responses to Interferon (IFN), the cytokine KITLG/SCF, and other cytokines and other growth factors. Upon binding of type I IFNs (IFN- α and IFN- β) to cell surface receptors, signaling via protein kinases results in activation of Jak kinases (TYK2 and Jak1) and results in tyrosine phosphorylation of STAT1 and STAT 2. Phosphorylated STATs dimerize and bind to ISGF3G/IRF-9 to form a complex called ISGF3 transcription factor, which enters the nucleus. ISGF3 binds to IFN-stimulated response elements (ISREs) to activate transcription of IFN-stimulated genes (ISGs), thereby driving the cell in an antiviral state. STAT1 is tyrosine-phosphorylated and serine-phosphorylated in response to type II IFN (IFN- γ). It then forms a homodimer called IFN- γ activating factor (GAF), migrates into the nucleus and binds to IFN- γ activating sequences (GAS) to drive expression of target genes, thereby inducing a cellular antiviral state. STAT1 is activated in response to KITLG/SCF and KIT signaling. STAT1 may also mediate cellular responses to activated FGFR1, FGFR2, FGFR3, and FGFR 4. The complete human STAT1 amino acid sequence can be found under UniProtKB identification number P42224. The human STAT1 protein is encoded by the human STAT1 gene (NCBI gene ID: 6772).

"cytotoxic T lymphocyte antigen-4" (CTLA-4) refers to an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is expressed in vivo only on T cells and binds to two ligands, namely CD80 and CD86 (also referred to as B7-1 and B7-2, respectively). The term "CTLA-4" as used herein includes human CTLA-4(hCTLA-4), variants, subtypes and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank accession number AAB 59385.

"subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents (such as mice, rats, and guinea pigs). In a preferred embodiment, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

The use of the term "flat dose" with respect to the methods and dosages of the present disclosure means a dose that is administered to a patient without regard to the patient's body weight or Body Surface Area (BSA). Thus, the flat dose is not provided at the mg/kg dose, but rather in the absolute amount of the agent (e.g., anti-PD-1 antibody). For example, a 60kg human and a 100kg human will receive the same dose of antibody (e.g., 240mg of anti-PD-1 antibody).

Use of the term "fixed dose" in reference to the methods of the present disclosure means that two or more different antibodies (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody or anti-PD-L1 antibody and anti-CTLA-4 antibody) in a single composition are present in the composition in a specific (fixed) ratio to each other. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg/ml). In some embodiments, the ratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 1:8, about 1:1, about 3:1, or about 1 (e.g.g.g.g.g.g.g.g.g.g.g.g.g., anti-PD-1 antibody or anti-PD-L1 antibody) over mg of the second antibody (e.g., anti-CTLA-4 antibody). For example, a 3:1 ratio of anti-PD-1 antibody to anti-CTLA-4 antibody can mean that the vial can contain about 240mg of anti-PD-1 antibody and 80mg of anti-CTLA-4 antibody or about 3mg/ml of anti-PD-1 antibody and 1mg/ml of anti-CTLA-4 antibody.

The term "weight-based dose" as referred to herein means a dose administered to a patient calculated based on the weight of the patient. For example, when a patient weighing 60kg requires 3mg/kg of anti-PD-1 antibody, one can calculate and use the appropriate amount of anti-PD-1 antibody (i.e., 180mg) for administration.

A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by measuring the activity of the agent in vitro assays.

For example, an "anti-cancer agent" promotes cancer regression in a subject. In a preferred embodiment, the therapeutically effective amount of the drug promotes regression of the cancer to the extent that the cancer is eliminated. By "promoting cancer regression" is meant that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptomatic periods, or prevention of injury or disability due to disease affliction. In addition, the terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or organism level resulting from administration of the drug.

For treatment of a tumor, for example, a therapeutically effective amount of an anti-cancer agent preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even more preferably by at least about 80%, relative to an untreated subject. In other preferred embodiments of the present disclosure, tumor regression may be observed and persist for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Despite these final measures of treatment effectiveness, the evaluation of immunotherapeutic drugs must also take into account immune-related response patterns.

An "immune response" is as understood in the art, and generally refers to a biological response in vertebrates to foreign factors (agents) or abnormalities, such as cancer cells, that protect the organism from these factors and the diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells or the liver, including antibodies, cytokines, and complements, that result in the selective targeting, binding, damage, destruction, and/or elimination of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells in the vertebrate body, or in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells (e.g., effector)T cells, Th cells, CD4⁺Cell, CD8⁺T cells or Treg cells), or any other cell of the immune system (e.g., NK cells).

An "immune-related response pattern" refers to the clinical response pattern typically observed in cancer patients treated with immunotherapeutic agents that produce an anti-tumor effect by inducing a cancer-specific immune response or by modifying the innate immune process. This response pattern is characterized by beneficial therapeutic effects after initial increase in tumor burden or appearance of new lesions, which would be classified as disease progression and would be synonymous with drug failure in the evaluation of traditional chemotherapeutic agents. Thus, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effect of these agents on the target disease.

As used herein, the terms "treatment" and "treatment" refer to any type of intervention or procedure performed on a subject with the purpose of reversing, alleviating, inhibiting, or slowing or preventing the progression, severity, or recurrence of symptoms, complications, disorders, or biochemical indicators associated with the disease, or improving overall survival. Treatment can be to a subject with a disease or a subject without a disease (e.g., for prophylaxis).

The term "effective dose" is defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from the date of diagnosis or the date of the beginning of treatment of a disease, such as cancer, that patients diagnosed as having the disease remain alive), or the prevention of injury or disability due to disease affliction. A therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or a "prophylactically effective dose" of any amount of a drug that inhibits the development or recurrence of a disease when administered to a subject at risk of developing a disease or the recurrence of a disease, either alone or in combination with another therapeutic agent. The ability of a therapeutic agent to promote disease regression or inhibit disease progression or recurrence can be evaluated using various methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

For example, an anti-cancer agent is a drug that promotes cancer regression in a subject. In some embodiments, the therapeutically effective amount of the drug promotes regression of the cancer to the extent that the cancer is eliminated. By "promoting cancer regression" is meant that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a reduction in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival, prevention of injury or disability due to disease affliction, or an improvement in disease symptoms otherwise in a patient. In addition, the terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or organism level resulting from administration of the drug.

For treatment of a tumor, for example, a therapeutically effective amount or dose of the drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated subject. In some embodiments, the therapeutically effective amount or dose of the drug completely inhibits cell growth or tumor growth, i.e., 100% inhibits cell growth or tumor growth. The ability of a compound to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, such properties of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to skilled practitioners. In some embodiments described herein, tumor regression may be observed and persist for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

As used herein, the term "biological sample" refers to biological material isolated from a subject. The biological sample may contain any biological material suitable for determining target gene expression, for example, by sequencing nucleic acids in a tumor (or circulating tumor cells) and identifying genomic changes in the sequenced nucleic acids. The biological sample may be any suitable biological tissue or fluid, such as, for example, tumor tissue, blood, plasma, and serum. In one embodiment, the sample is a tumor tissue biopsy sample, such as Formalin Fixed Paraffin Embedded (FFPE) tumor tissue or freshly frozen tumor tissue, and the like. In another embodiment, the biological sample is a liquid biopsy sample, which in some embodiments comprises one or more of blood, serum, plasma, circulating tumor cells, exosome RNA, ctDNA and cfDNA.

As used herein, the terms "about once per week", "about once per two weeks" or any other similar dosing interval term means an approximate number. "about once per week" may include every seven days ± one day, i.e. every six days to every eight days. "about once every two weeks" may include every fourteen days ± three days, i.e., every eleven days to every seventy-seven days. For example, similar approximations apply to about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, and about once every twelve weeks. In some embodiments, a dosing interval of about once every six weeks or about once every twelve weeks, respectively, means that a first dose may be administered on any day of the first week, and then the next dose may be administered on any day of the sixth or twelfth week. In other embodiments, a dosing interval of about once every six weeks or about once every twelve weeks means that a first dose is administered on a particular day of the first week (e.g., monday) and then the next dose is administered on the same day of the sixth or twelfth week (i.e., monday), respectively.

The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated component.

The term "about" or "consisting essentially of … …" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of … …" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" or "substantially comprising … …" may mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times a value. When a particular value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of … …" should be assumed to be within an acceptable error range for that particular value or composition.

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood as including the value of any integer within the range and the score for the value as appropriate (e.g., one tenth and one hundredth of an integer).

Abbreviations used herein are defined throughout this disclosure. Table 1 provides a list of additional abbreviations.

Table 1: list of abbreviations

Various aspects of the disclosure are described in further detail in the following subsections.

Methods of the present disclosure

The present disclosure relates to methods of treating a tumor in a human subject comprising administering to the subject a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the tumor exhibits a high inflammation signature score prior to the administration. In some embodiments, the inflammation signature score is determined by measuring the expression of a panel of inflammatory genes ("inflammatory genome") in a tumor sample obtained from the subject; and wherein the inflammatory genome comprises CD274(PD-L1), CD8A, LAG3, and STAT 1.

In some embodiments, the inflammatory genome consists of less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, or less than about 5 inflammatory genes. In some embodiments, the inflammatory genome consists of less than 20 genes. In some embodiments, the inflammatory genome consists of less than 19 genes. In some embodiments, the inflammatory genome consists of less than 18 genes. In some embodiments, the inflammatory genome consists of less than 17 genes. In some embodiments, the inflammatory genome consists of less than 16 genes. In some embodiments, the inflammatory genome consists of less than 15 genes. In some embodiments, the inflammatory genome consists of less than 14 genes. In some embodiments, the inflammatory genome consists of less than 13 genes. In some embodiments, the inflammatory genome consists of less than 12 genes. In some embodiments, the inflammatory genome consists of less than 11 genes. In some embodiments, the inflammatory genome consists of less than 10 genes. In some embodiments, the inflammatory genome consists of less than 9 genes. In some embodiments, the inflammatory genome consists of less than 8 genes. In some embodiments, the inflammatory genome consists of less than 7 genes. In some embodiments, the inflammatory genome consists of less than 6 genes. In some embodiments, the inflammatory genome consists of less than 5 genes. In certain embodiments, the inflammatory genome consists of 4 genes. In some embodiments, the genome of inflammation consists essentially of CD274(PD-L1), CD8A, LAG3, and STAT 1. In some embodiments, the genome of inflammation consists of CD274(PD-L1), CD8A, LAG3, and STAT 1.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1) and CD8A, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1) and LAG3, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1) and STAT1, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD8A and LAG3, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD8A and STAT1, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) LAG3 and STAT1, and (ii)2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, and LAG3, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, and STAT1, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1), LAG3, and STAT1, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274 CD8A, LAG3, and STAT1, and (ii)1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

In some embodiments, the genome of inflammation consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)1 additional inflammatory gene. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)2 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)3 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)4 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)5 additional inflammatory genes. In some inflammatory genomes, it consists mainly of: (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)6 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)7 additional inflammatory genes. In some inflammatory genomes, it consists mainly of: (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)8 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)9 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)10 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)11 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)12 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)13 additional inflammatory genes. In some inflammatory genomes, it consists essentially of (or consists of): (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)14 additional inflammatory genes. In some inflammatory genomes, it consists mainly of: (i) CD274(PD-L1), CD8A, LAG3 and STAT1, and (ii)15 additional inflammatory genes.

Various genes associated with inflammation are known in the art and may be included in the inflammatory genome disclosed herein. For example, the additional inflammatory gene may be selected from: CCL2, CCL3, CCL4, CCL5, CCR5, CD27, CD274, CD276, CMKLR1, CXCL10, CXCL11, CXCL9, CXCR6, GZMA, GZMK, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DRA, HLA-DRB1, HLA-E, ICOS, IDO1, IFNG, IRF1, NKG7, PDCD1LG2, PRF1, PSMB10, TIGIT, and any combination thereof.

In some embodiments, the genome of inflammation consists essentially of CD274(PD-L1), CD8A, LAG3, and STAT 1. In some embodiments, the genome of inflammation consists of CD274(PD-L1), CD8A, LAG3, and STAT 1.

Ii.a. inflammation signature score

As used herein, an inflammation signature score is a measure of the combined expression level of genes present in the genome of inflammation (e.g., comprising, consisting essentially of, or consisting of CD274(PD-L1), CD8A, LAG3, and STAT1) in a sample obtained from a subject. Any biological sample comprising one or more tumor cells can be used in the methods disclosed herein. In some embodiments, the sample is selected from a tumor biopsy sample, a blood sample, a serum sample, or any combination thereof. In certain embodiments, the sample is a tumor biopsy collected from the subject prior to administration of the anti-PD-1 antibody. In particular embodiments, the sample obtained from the subject is a formalin fixed tumor biopsy sample. In some embodiments, the sample obtained from the subject is a paraffin-embedded tumor biopsy. In some embodiments, the sample obtained from the subject is a freshly frozen tumor biopsy sample.

Any method known in the art for measuring the expression of a particular gene or set of genes may be used in the methods of the present disclosure. In some embodiments, expression of one or more inflammatory genes in the inflammatory genome is determined by detecting the presence of mRNA transcribed from an inflammatory gene, the presence of a protein encoded by the inflammatory gene, or both.

In some embodiments, expression of one or more inflammatory genes is determined by measuring the level of inflammatory gene mRNA in a sample obtained from the subject, e.g., by measuring the level of one or more of LAG3 mRNA, PD-L1 mRNA, CD8A mRNA, and STAT1 mRNA. In certain embodiments, the inflammatory gene score is determined by measuring LAG3 mRNA, PD-L1 mRNA, CD8A mRNA, and STAT1 mRNA levels in a sample obtained from the subject. Any method known in the art can be used to measure the level of inflammatory gene mRNA. In some embodiments, the inflammatory gene mRNA is measured using reverse transcriptase PCR. In some embodiments, the inflammatory gene mRNA is measured using RNA in situ hybridization.

In some embodiments, expression of one or more inflammatory genes is determined by measuring the level of an inflammatory gene protein in a sample obtained from the subject, e.g., by measuring the level of one or more of PD-L1, CD8A, LAG-3, and STAT 1. In certain embodiments, the inflammatory gene score is determined by measuring the levels of PD-L1, CD8A, LAG-3, and STAT1 in a sample obtained from the subject. Any method known in the art may be used to measure inflammatory gene protein levels. In some embodiments, the inflammatory gene protein is measured using an Immunohistochemistry (IHC) assay. In certain embodiments, the IHC assay is an automated IHC assay.

In some embodiments, the expression of one or more inflammatory genes of the inflammatory genome is normalized relative to the expression of one or more housekeeping genes. In some embodiments, the one or more housekeeping genes consists of genes that have relatively consistent expression across multiple tumor types in multiple subjects.

In some embodiments, the raw gene expression values are normalized according to a standard gene expression signature (GEP) protocol. In these embodiments, the gene expression signature score can be calculated as the median or average of the log 2-transformed normalized and scaled expression values for all target genes in the signature and presented on a linear scale. In certain embodiments, the score has a positive or negative value depending on whether gene expression is up-regulated or down-regulated under particular conditions.

In certain embodiments, a high inflammation signature score is characterized by an inflammation signature score that is greater than a reference inflammation signature score. In some embodiments, the reference inflammation signature score is a mean inflammation signature score. In some embodiments, the average inflammation signature score is determined by: the expression of genes present in the genome of inflammation in a tumor sample obtained from a population of subjects is measured and an average of the population of subjects is calculated. In some embodiments, each member of the population of subjects has the same tumor as the subject administered the anti-PD-1 antibody, the anti-PD-L1 antibody, the anti-CTLA-4 antibody, or any combination thereof. In particular embodiments, the average inflammation tag score is about-0.07, about-0.06, -0.05, about-0.04, about-0.03, or about-0.02. In a particular embodiment, the average inflammation signature score is about-0.04. In certain embodiments, the average inflammation signature score is about-0.0434.

In some embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 25% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 30% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 35% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 40% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 45% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 50% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 55% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 60% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 65% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 70% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 75% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 80% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 85% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 90% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 95% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 100% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 125% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 150% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 175% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 200% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 225% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 250% higher than the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 275% higher than the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 300% higher than the average inflammation signature score.

In some embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 1.25-fold, at least about 1.30-fold, at least about 1.35-fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold, at least about 2-fold, at least about 2.25-fold, at least about 2.50-fold, at least about 2.75-fold, at least about 3-fold, at least about 3.25-fold, at least about 3.50-fold, at least about 3.75-fold, or at least about 400-fold of the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.25 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 1.30 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.35 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 1.40 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.45 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.50 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.55 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.60 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 1.65 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.70 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.75 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.80 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.85 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.90 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 1.95 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 2 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 2.25 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 2.50 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 2.75 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 3 times the average inflammation signature score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 3.25 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 3.50 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation tag score that is at least about 3.75 times the average inflammation tag score. In certain embodiments, a high inflammation score is characterized by an inflammation signature score that is at least about 4 times the average inflammation signature score.

In certain embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 0.5, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 0.75, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 1.0, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 1.25, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 1.50, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 1.75, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 2.0, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 2.25, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 2.5, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 2.75, wherein the inflammation tag score is determined according to the methods disclosed herein. In some embodiments, a high inflammation tag score is characterized by an inflammation tag score of at least about 3.0, wherein the inflammation tag score is determined according to the methods disclosed herein.

II.B. antibodies

The present disclosure relates to a method for treating a human subject having cancer, the method comprising administering to the subject a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the subject is administered an anti-PD-1 monotherapy, e.g., wherein the subject is not administered one or more additional anti-cancer agents. In some embodiments, the subject is administered a combination therapy, e.g., wherein the subject is administered an anti-PD-1 antibody and one or more additional anti-cancer agents. In certain embodiments, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody.

In other aspects of the disclosure, an anti-PD-L1 antibody replaces the anti-PD-1 antibody. In certain embodiments, the method comprises administering to the subject an anti-PD-L1 antibody. In some embodiments, the subject is administered an anti-PD-L1 monotherapy. In some embodiments, the subject is administered a combination therapy comprising an anti-PD-L1 antibody and a second anti-cancer agent (e.g., an anti-CTLA-4 antibody).

Ii.b.1. anti-PD-1 antibodies useful in the present disclosure

anti-PD-1 antibodies known in the art can be used in the compositions and methods described herein. A variety of human monoclonal antibodies that specifically bind to PD-1 with high affinity have been disclosed in U.S. patent No. 8,008,449. anti-PD-1 human antibodies disclosed in U.S. patent No. 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) at1 × 10^-7K of M or less_DBinding to human PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; (b) (ii) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increasing T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (d) increasing interferon- γ production in an MLR assay; (e) increasing IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibit the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulating an antigen-specific memory response; (i) stimulation ofAntibody reaction; and (j) inhibiting tumor cell growth in vivo. anti-PD-1 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, in some embodiments at least five, of the foregoing characteristics.

Other anti-PD-1 monoclonal antibodies have been described, for example, in the following documents: U.S. patent nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, U.S. publication No. 2016/0272708, and PCT publication nos. WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2017/025016, WO 2017/106061, WO 2017/19846, WO 2017/024465, WO 2017/025016, WO 2017/132825 and WO 2017/133540, each of which is incorporated by reference in its entirety.

In some embodiments, the anti-PD-1 antibody is selected from nivolumab (also referred to as nivolumab)5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck; also known asPabolizumab and MK-3475; see WO 2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cimirapril mab (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001(TAIZHOU JUNSHI PHARMA; also known as teripril mab (tropipalimab); see Si-Yang Liu et al, j.hematol.oncol.10:136(2017), BGB-a317 (Beigene; also known as tirezumab; see WO 2015/35606 and US 2015/0079109), incsar 1210(Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et al, J.Hematol.Oncol.10:136(2017)), TSR-042(Tesaro Biopharmaceutical; also known as ANB 011; see WO 2014/179664), GLS-010(Wuxi/Harbin receptacle Pharmaceuticals; also known as WBP 3055; see Si-Yang Liu et al, J.Hematol.Oncol.10:136(2017)), AM-0001 (armor), STI-1110 (Sorrent's Therapeutics; see WO 2014/194302), age 2034 (Agenus; see WO 2017/040790), MGA012 (macrogenetics, see WO 2017/19846), BCD-100 (Biocad; kaplon et al, mAbs 10(2): 183-.

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking down-regulation of anti-tumor T cell function (U.S. Pat. No. 8,008,449; Wang et al, 2014Cancer immune res.2(9): 846-56).

In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4(S228P) antibody directed against human cell surface receptor PD-1 (programmed death factor-1 or programmed cell death factor-1). Pembrolizumab is described, for example, in U.S. patent nos. 8,354,509 and 8,900,587.

anti-PD-1 antibodies that can be used in the disclosed compositions and methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein (e.g., nivolumab) for binding to human PD-1 (see, e.g., U.S. patent nos. 8,008,449 and 8,779,105; WO 2013/173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as any anti-PD-1 antibody described herein (e.g., nivolumab). The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to this particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody (e.g., nivolumab) due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified in standard PD-1 binding assays (such as Biacore analysis, ELISA assays, or flow cytometry) based on their ability to cross-compete with nivolumab (see, e.g., WO 2013/173223).

In certain embodiments, an antibody that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of a human PD-1 antibody as nivolumab is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

anti-PD-1 antibodies useful in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It is well established that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes antigen-binding portions or fragments that bind to the PD-1 receptor and exhibit similar functional properties as those of whole antibodies in terms of inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

In some embodiments, the anti-PD-1 antibody is administered at a dose in the range of from 0.1mg/kg to 20.0mg/kg body weight once every 2, 3, 4,5, 6, 7, or 8 weeks, for example at 0.1mg/kg to 10.0mg/kg body weight once every 2, 3, or 4 weeks. In other embodiments, the anti-PD-1 antibody is administered at a dose of about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or 10mg/kg body weight once every 2 weeks. In other embodiments, the anti-PD-1 antibody is administered at a dose of about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or 10mg/kg body weight once every 3 weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 5mg/kg body weight about once every 3 weeks. In another embodiment, the anti-PD-1 antibody (e.g., nivolumab) is administered at a dose of about 3mg/kg body weight about once every 2 weeks. In other embodiments, the anti-PD-1 antibody (e.g., pembrolizumab) is administered at a dose of about 2mg/kg body weight about once every 3 weeks.

anti-PD-1 antibodies useful in the present disclosure can be administered in flat doses. In some embodiments, the anti-PD-1 antibody is administered in a flat dose of from about 100 to about 1000mg, from about 100mg to about 900mg, from about 100mg to about 800mg, from about 100mg to about 700mg, from about 100mg to about 600mg, from about 100mg to about 500mg, from about 200mg to about 1000mg, from about 200mg to about 900mg, from about 200mg to about 800mg, from about 200mg to about 700mg, from about 200mg to about 600mg, from about 200mg to about 500mg, from about 200mg to about 480mg, or from about 240mg to about 480 mg. In one embodiment, the anti-PD-1 antibody is administered in a flat dose amount of at least about 200mg, at least about 220mg, at least about 240mg, at least about 260mg, at least about 280mg, at least about 300mg, at least about 320mg, at least about 340mg, at least about 360mg, at least about 380mg, at least about 400mg, at least about 420mg, at least about 440mg, at least about 460mg, at least about 480mg, at least about 500mg, at least about 520mg, at least about 540mg, at least about 550mg, at least about 560mg, at least about 580mg, at least about 600mg, at least about 620mg, at least about 640mg, at least about 660mg, at least 680mg, at least about 700mg, or at least about 720mg at a dosing interval of about 1,2, 3, 4,5, 6, 7, 8,9, or 10 weeks. In another embodiment, the anti-PD-1 antibody is administered in flat doses of about 200mg to about 800mg, about 200mg to about 700mg, about 200mg to about 600mg, about 200mg to about 500mg at dosing intervals of about 1,2, 3, or 4 weeks.

In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about once every 3 weeks. In other embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about once every 2 weeks. In other embodiments, the anti-PD-1 antibody is administered at a flat dose of about 240mg about once every 2 weeks. In certain embodiments, the anti-PD-1 antibody is administered at a flat dose of about 480mg about once every 4 weeks.

In some embodiments, nivolumab is administered about once every 2 weeks in a flat dose of about 240 mg. In some embodiments, nivolumab is administered about once every 3 weeks in a flat dose of about 240 mg. In some embodiments, nivolumab is administered about once every 3 weeks in a flat dose of about 360 mg. In some embodiments, nivolumab is administered at a flat dose of about 480mg about once every 4 weeks.

In some embodiments, pembrolizumab is administered at a flat dose of about 200mg about every 2 weeks. In some embodiments, pembrolizumab is administered at a flat dose of about 200mg about every 3 weeks. In some embodiments, pembrolizumab is administered at a flat dose of about 400mg about every 4 weeks.

In some aspects, the PD-1 inhibitor is a small molecule. In some aspects, the PD-1 inhibitor comprises millamolece. In some aspects, the PD-1 inhibitor comprises a macrocyclic peptide. In certain aspects, the PD-1 inhibitor comprises BMS-986189. In some aspects, the PD-1 inhibitor comprises an inhibitor disclosed in international application No. WO 2014/151634, which is incorporated herein by reference in its entirety. In some aspects, the PD-1 inhibitor comprises INCMGA00012(Incyte Corporation). In some aspects, the PD-1 inhibitor comprises an anti-PD-1 antibody disclosed herein in combination with a PD-1 small molecule inhibitor.

Ii.b.2. anti-PD-L1 antibodies useful in the present disclosure

In certain embodiments, the anti-PD-1 antibody is replaced with an anti-PD-L1 antibody in any of the methods disclosed herein. anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the present disclosure. Examples of anti-PD-L1 antibodies that can be used in the compositions and methods of the present disclosure include the antibodies disclosed in U.S. patent No. 9,580,507. The anti-PD-L1 human monoclonal antibodies disclosed in U.S. patent No. 9,580,507 have been shown to exhibit one or more of the following characteristics: (a) at1 × 10^-7K of M or less_DIn combination with human PD-L1, as determined by surface plasmon resonance using a Biacore biosensor system; (b) increasing T cell proliferation in Mixed Lymphocyte Reaction (MLR) assay(ii) a (c) Increasing interferon- γ production in an MLR assay; (d) increasing IL-2 secretion in an MLR assay; (e) stimulating an antibody response; and (f) reversing the effects of T regulatory cells on T cell effector cells and/or dendritic cells. anti-PD-L1 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, in some embodiments at least five, of the foregoing characteristics.

In certain embodiments, the anti-PD-L1 antibody is selected from BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013/173223), Attributab (Roche; also known as Attributab)MPDL3280A, RG 7446; see US 8,217,149; see also Herbst et al (2013) J Clin Oncol 31 (suppl.: 3000), dutvacizumab (AstraZeneca; also known as IMFINZI^TMMEDI-4736; see WO 2011/066389), avizumab (Pfizer; also known asMSB-0010718C; see WO 2013/079174), STI-1014 (Sorrento; see WO 2013/181634), CX-072 (Cytomx; see WO 2016/149201), KN035(3D Med/Alphamab; see Zhang et al, Cell Discov.7:3 (3.2017), LY3300054(Eli Lilly Co.; see, e.g., WO 2017/034916), BGB-A333 (BeiGene; see Desai et al, JCO 36(15 suppl): TPS3113(2018)), and CK-301(Checkpoint Therapeutics; see Gorelik et al, AACR: Abstract 4606(2016 4.4)).

In certain embodiments, the PD-L1 antibody is atelizumabAtezumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

In certain embodiments, the PD-L1 antibody is dulvacizumab (IMFINZI)^TM). The dolvacizumab is human IgG1 kappa monoclonal antibody PD-L1.

In certain embodiments, the PD-L1 antibody is avilumabThe avilamumab is a human IgG1 lambda monoclonal antibody PD-L1.

anti-PD-L1 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein (e.g., atuzumab, dulzumab, and/or avizumab) for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibody binds the same epitope as any anti-PD-L1 antibody described herein (e.g., atelizumab, dulzumab, and/or avizumab). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to this particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibodies (e.g., atelizumab and/or avizumab) due to their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified in standard PD-L1 binding assays (such as Biacore analysis, ELISA assays, or flow cytometry) based on their ability to cross-compete with altuzumab and/or avizumab (see, e.g., WO 2013/173223).

In certain embodiments, an antibody that cross-competes with atuzumab, dulzumab, and/or avizumab for binding to human PD-L1 or binds to the same epitope region of human PD-L1 antibody as atuzumab, dulzumab, and/or avizumab is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

anti-PD-L1 antibodies useful in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It is well established that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

anti-PD-L1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes antigen-binding portions or fragments that bind to PD-L1 and exhibit similar functional properties as those of whole antibodies in terms of inhibiting receptor binding and upregulating the immune system. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atuzumab, dulzumab, and/or avizumab for binding to human PD-L1.

An anti-PD-L1 antibody useful in the present disclosure may be any PD-L1 antibody that specifically binds to PD-L1, such as an antibody that cross-competes with dolvacizumab, avizumab, or astuzumab for binding to human PD-1, such as an antibody that binds to the same epitope as dolvacizumab, avizumab, or astuzumab. In certain embodiments, the anti-PD-L1 antibody is dulvacizumab. In other embodiments, the anti-PD-L1 antibody is avizumab. In some embodiments, the anti-PD-L1 antibody is atelizumab.

In some embodiments, the anti-PD-L1 antibody is administered at a dose in the range of from about 0.1mg/kg to about 20.0mg/kg body weight, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg about once every 2, 3, 4,5, 6, 7, or 8 weeks.

In some embodiments, the anti-PD-L1 antibody is administered at a dose of about 15mg/kg body weight about once every 3 weeks. In other embodiments, the anti-PD-L1 antibody is administered at a dose of about 10mg/kg body weight about once every 2 weeks.

In other embodiments, the anti-PD-L1 antibodies useful in the present disclosure are flat doses. In some embodiments, the anti-PD-L1 antibody is administered in a flat dose amount of about 200mg to about 1600mg, about 200mg to about 1500mg, about 200mg to about 1400mg, about 200mg to about 1300mg, about 200mg to about 1200mg, about 200mg to about 1100mg, about 200mg to about 1000mg, about 200mg to about 900mg, about 200mg to about 800mg, about 200mg to about 700mg, about 200mg to about 600mg, about 700mg to about 1300mg, about 800mg to about 1200mg, about 700mg to about 900mg, or about 1100mg to about 1300 mg. In some embodiments, the anti-PD-L1 antibody is administered in a flat dose of at least about 240mg, at least about 300mg, at least about 320mg, at least about 400mg, at least about 480mg, at least about 500mg, at least about 560mg, at least about 600mg, at least about 640mg, at least about 700mg, at least 720mg, at least about 800mg, at least about 840mg, at least about 880mg, at least about 900mg, at least 960mg, at least about 1000mg, at least about 1040mg, at least about 1100mg, at least about 1120mg, at least about 1200mg, at least about 1280mg, at least about 1300mg, at least about 1360mg, or at least about 1400mg at dosing intervals of about 1,2, 3, or 4 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 1200mg about once every 3 weeks. In other embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 800mg about once every 2 weeks. In other embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 840mg about once every 2 weeks.

In some embodiments, the atezumab is administered at a flat dose of about 1200mg about once every 3 weeks. In some embodiments, the atezumab is administered at a flat dose of about 800mg about once every 2 weeks. In some embodiments, the atezumab is administered at a flat dose of about 840mg approximately once every 2 weeks.

In some embodiments, avitumumab is administered at a flat dose of about 800mg about once every 2 weeks.

In some embodiments, the doxoruzumab is administered at a dose of about 10mg/kg about once every 2 weeks. In some embodiments, the dulvacizumab is administered about once every 2 weeks in a flat dose of about 800 mg/kg. In some embodiments, the dulvacizumab is administered about once every 3 weeks in a flat dose of about 1200 mg/kg.

In some aspects, the PD-L1 inhibitor is a small molecule. In some aspects, the PD-L1 inhibitor comprises a millamole. In some aspects, the PD-L1 inhibitor comprises a macrocyclic peptide. In certain aspects, the PD-L1 inhibitor comprises BMS-986189.

In some aspects, the PD-L1 inhibitor comprises a millamole having the formula shown in formula (I):

wherein R is¹-R¹³Is an amino acid side chain, R^a-RⁿIs hydrogen, methyl, or forms a ring with an ortho-R group, and R¹⁴is-C (O) NHR¹⁵Wherein R is¹⁵Is hydrogen, or a glycine residue optionally substituted with another glycine residue and/or a tail which may improve pharmacokinetic properties. In some aspects, the PD-L1 inhibitor comprises a compound disclosed in international application No. WO 2014/151634, which is incorporated herein by reference in its entirety. In some aspects, the PD-L1 inhibitor comprises a compound disclosed in international application nos. WO 2016/039749, WO 2016/149351, WO 2016/077518, WO 2016/100285, WO 2016/100608, WO 2016/126646, WO 2016/057624, WO 2017/151830, WO 2017/176608, WO 2018/085750, WO 2018/237153, or WO 2019/070643, each of which is incorporated herein by reference in its entirety.

In certain aspects, the PD-L1 inhibitor comprises a small molecule PD-L1 inhibitor disclosed in international application nos. WO 2015/034820, WO 2015/160641, WO 2018/044963, WO 2017/066227, WO 2018/009505, WO 2018/183171, WO 2018/118848, WO 2019/147662, or WO 2019/169123, each of which is incorporated herein by reference in its entirety.

In some aspects, the PD-L1 inhibitor comprises a combination of an anti-PD-L1 antibody disclosed herein and a PD-L1 small molecule inhibitor disclosed herein.

anti-CTLA-4 antibodies

anti-CTLA-4 antibodies known in the art can be used in the compositions and methods of the present disclosure. The anti-CTLA-4 antibodies of the disclosure bind to human CTLA-4, thereby disrupting CTLA-4 interaction with the human B7 receptor. Since the interaction of CTLA-4 with B7 transduces signals that result in the inactivation of CTLA-4 receptor-bearing T cells, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity have been disclosed in U.S. patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in the following documents: U.S. patent nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121, and international publication nos. WO 2012/122444, WO 2007/113648, WO 2016/196237, and WO 2000/037504, each of which is incorporated herein by reference in its entirety. anti-CTLA-4 human monoclonal antibodies disclosed in U.S. patent No. 6,984,720 have been shown to exhibit one or more of the following characteristics: (a) at least about 10⁷M^-1Or about 10⁹M^-1Or about 10¹⁰M^-1To 10¹¹M^-1Or higher equilibrium association constant (K)_a) The reflected binding affinities bind specifically to human CTLA-4 as determined by Biacore analysis; (b) kinetic association constant (k)_a) Is at least about 10³About 10⁴Or about 10⁵m^-1s^-1(ii) a (c) Kinetic dissociation constant (k)_d) Is at least about 10³About 10⁴Or about 10⁵m^-1s^-1(ii) a And (d) inhibits binding of CTLA-4 to B7-1(CD80) and B7-2(CD 86). anti-CTLA-4 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the foregoing characteristics.

In certain embodiments, the CTLA-4 antibody is selected from ipilimumab (also referred to as ipilimumab)MDX-010, 10D 1; see U.S. Pat. No. 6,984,720), MK-1308(Merck), AGEN-1884(Agenus Inc.; see WO 2016/196237) and tremelimumab (AstraZeneca; also known as tiximumab (ticilimumab), CP-675,206; see WO 2000/037504 and Ribas, Update Cancer ther.2(3):133-39 (20)07)). In particular embodiments, the anti-CTLA-4 antibody is ipilimumab.

In particular embodiments, the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving Overall Survival (OS) in patients with advanced melanoma.

In particular embodiments, the CTLA-4 antibody is tremelimumab.

In particular embodiments, the CTLA-4 antibody is MK-1308.

In certain embodiments, the CTLA-4 antibody is AGEN-1884.

anti-CTLA-4 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with binding to human CTLA-4 with any of the anti-CTLA-4 antibodies disclosed herein (e.g., ipilimumab and/or tremelimumab). In some embodiments, the anti-CTLA-4 antibody binds the same epitope as any of the anti-CTLA-4 antibodies described herein (e.g., ipilimumab and/or tremelimumab). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to this particular epitope region. These cross-competing antibodies are expected to have functional properties that are very similar to those of the reference antibodies (e.g., ipilimumab and/or tremelimumab) due to their binding to the same epitope region of CTLA-4. Cross-competing antibodies can be readily identified in standard CTLA-4 binding assays (such as Biacore analysis, ELISA assays, or flow cytometry) based on their ability to cross-compete with ipilimumab and/or tremelimumab (see, e.g., WO 2013/173223).

In certain embodiments, the antibody that cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4 or binds to the same epitope region of a human CTLA-4 antibody as ipilimumab and/or tremelimumab is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

anti-CTLA-4 antibodies useful in the compositions and methods of the disclosed disclosures also include antigen-binding portions of the above antibodies. It is well established that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind with high specificity and affinity to CTLA-4, block CTLA-4 activity, and disrupt CTLA-4 interaction with the human B7 receptor. In any of the compositions or methods disclosed herein, an anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits similar functional properties as those of a whole antibody in inhibiting CTLA-4 interaction with the human B7 receptor and upregulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes for binding to human CTLA-4 with ipilimumab and/or tremelimumab.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered once every 2, 3, 4,5, 6, 7, or 8 weeks at a dose in the range of from 0.1mg/kg to 10.0mg/kg body weight. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1mg/kg or 3mg/kg body weight once every 3, 4,5, or 6 weeks. In one embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3mg/kg body weight once every 2 weeks. In another embodiment, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 1mg/kg body weight once every 6 weeks.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered in flat doses. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose amount of about 10 to about 1000mg, about 10mg to about 900mg, about 10mg to about 800mg, about 10mg to about 700mg, about 10mg to about 600mg, about 10mg to about 500mg, about 100mg to about 1000mg, about 100mg to about 900mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 100mg, about 100mg to about 500mg, about 100mg to about 480mg, or about 240mg to about 480 mg. In some embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered in an amount of a flat dose of at least about 60mg, at least about 80mg, at least about 100mg, at least about 120mg, at least about 140mg, at least about 160mg, at least about 180mg, at least about 200mg, at least about 220mg, at least about 240mg, at least about 260mg, at least about 280mg, at least about 300mg, at least about 320mg, at least about 340mg, at least about 360mg, at least about 380mg, at least about 400mg, at least about 420mg, at least about 440mg, at least about 460mg, at least about 480mg, at least about 500mg, at least about 520mg at least about 540mg, at least about 550mg, at least about 560mg, at least about 580mg, at least about 600mg, at least about 620mg, at least about 640mg, at least about 660mg, at least about 680mg, at least about 700mg, or at least about 720 mg. In another embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered in a flat dose about once every 1,2, 3, 4,5, 6, 7, or 8 weeks.

In some embodiments, ipilimumab is administered at a dose of about 3mg/kg about every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10mg/kg about every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10mg/kg about once every 12 weeks. In some embodiments, ipilimumab is administered in four doses.

Combination therapy of II.B.4

In certain embodiments, the anti-PD-1 antibody, the anti-PD-L1 antibody, and/or the anti-CTLA-4 antibody is administered in a therapeutically effective amount. In some embodiments, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody and an anti-CTLA-4 antibody. In other embodiments, the method comprises administering a therapeutically effective amount of an anti-PD-L1 antibody and an anti-CTLA-4 antibody. Any of the anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies disclosed herein can be used in the methods. In certain embodiments, the anti-PD-1 antibody comprises nivolumab. In some embodiments, the anti-PD-1 antibody comprises pembrolizumab. In some embodiments, the anti-PD-L1 antibody comprises atelizumab. In some embodiments, the anti-PD-L1 antibody comprises dolvacizumab. In some embodiments, the anti-PD-L1 antibody comprises avizumab. In some embodiments, the anti-CTLA-4 antibody comprises ipilimumab. In some embodiments, the anti-CTLA-4 antibody comprises ipilimumab anti-tremelimumab.

In some embodiments, each of (a) the anti-PD-1 antibody or the anti-PD-L1 antibody and (b) the anti-CTLA-4 antibody is administered about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, or about once every 6 weeks. In some embodiments, the anti-PD-1 antibody or the anti-PD-L1 antibody is administered about once every 2 weeks, about once every 3 weeks, or about once every 4 weeks, and the anti-CTLA-4 antibody is administered about once every 6 weeks. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered on the same day as the anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody or the anti-PD-L1 antibody and the anti-CTLA-4 antibody are administered on different days.

In some embodiments, the anti-CTLA-4 antibody is administered at a dose ranging from about 0.1mg/kg to about 20.0mg/kg body weight about once every 2, 3, 4,5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 0.1mg/kg, about 0.3mg/kg, about 0.6mg/kg, about 0.9mg/kg, about 1mg/kg, about 3mg/kg, about 6mg/kg, about 9mg/kg, about 10mg/kg, about 12mg/kg, about 15mg/kg, about 18mg/kg, or about 20 mg/kg. In certain embodiments, the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 4 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks.

In some embodiments, the anti-CTLA-4 antibody is administered in flat doses. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose ranging from at least about 40mg to at least about 1600 mg. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose of at least about 40mg, at least about 50mg, at least about 60mg, at least about 70mg, at least about 80mg, at least about 90mg, at least about 100mg, at least about 110mg, at least about 120mg, at least about 130mg, at least about 140mg, at least about 150mg, at least about 160mg, at least about 170mg, at least about 180mg, at least about 190mg, or at least about 200 mg. In some embodiments, the CTLA-4 antibody is administered in a flat dose of at least about 220mg, at least about 230mg, at least about 240mg, at least about 250mg, at least about 260mg, at least about 270mg, at least about 280mg, at least about 290mg, at least about 300mg, at least about 320mg, at least about 360mg, at least about 400mg, at least about 440mg, at least about 480mg, at least about 520mg, at least about 560mg, or at least about 600 mg. In some embodiments, the CTLA-4 antibody is administered in a flat dose of at least about 640mg, at least about 720mg, at least about 800mg, at least about 880mg, at least about 960mg, at least about 1040mg, at least about 1120mg, at least about 1200mg, at least about 1280mg, at least about 1360mg, at least about 1440mg, or at least about 1600 mg. In some embodiments, the anti-CTLA-4 antibody is administered at least once about every 2, 3, 4,5, 6, 7, or 8 weeks in flat doses.

In certain embodiments, the anti-PD-1 antibody is administered at a dose of about 2mg/kg about once every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a dose of about 3mg/kg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a dose of about 6mg/kg about once every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks.

In certain embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about once every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 240mg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 480mg about every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about every 6 weeks.

In certain embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about every 3 weeks and the anti-CTLA-4 antibody is administered at a flat dose of about 80mg about every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200mg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80mg about once every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 240mg about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80mg about every 6 weeks. In some embodiments, the anti-PD-1 antibody is administered at a flat dose of about 480mg about every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80mg about every 6 weeks.

In certain embodiments, the anti-PD-L1 antibody is administered at a dose of about 10mg/kg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a dose of about 15mg/kg about once every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks.

In certain embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 800mg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 1200mg about once every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg about once every 6 weeks.

In certain embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 800mg about once every 2 weeks and the anti-CTLA-4 antibody is administered at a flat dose of about 80mg about once every 6 weeks. In some embodiments, the anti-PD-L1 antibody is administered at a flat dose of about 1200mg about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80mg about every 6 weeks.

In some embodiments, the anti-PD-1 antibody (e.g., nivolumab) is administered at a dose of about 3mg/kg and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg on the same day, about every 3 weeks for 4 doses, and then the anti-PD-1 antibody (e.g., nivolumab) is administered at a flat dose of 240mg about every 2 weeks or at a flat dose of 480mg about every 4 weeks. In some embodiments, the anti-PD-1 antibody (e.g., nivolumab) is administered at a dose of about 1mg/kg and the anti-CTLA-4 antibody is administered at a dose of about 3mg/kg on the same day, about every 3 weeks for 4 doses, and then the anti-PD-1 antibody (e.g., nivolumab) is administered at a flat dose of 240mg about every 2 weeks or at a flat dose of 480mg about every 4 weeks.

II.B.5. additional anti-cancer therapies

In some aspects of the disclosure, the methods disclosed herein further comprise administering an anti-PD-1 antibody (or an anti-PD-L1 antibody) and an additional anti-cancer therapy. In certain embodiments, the methods comprise administering an anti-PD-1 antibody (or an anti-PD-L1 antibody), an anti-CTLA-4 antibody, and an additional anti-cancer therapy. The additional anti-cancer therapy can include any therapy known in the art for treating a tumor in a subject and/or any standard of care therapy as disclosed herein. In some embodiments, the additional anti-cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, including any of the chemotherapy disclosed herein. In some embodiments, the additional anti-cancer therapy comprises immunotherapy. In some embodiments, the additional anti-cancer therapy comprises administering an antibody, or antigen-binding portion thereof, that specifically binds to: LAG-3, TIGIT, TIM3, NKG2a, OX40, ICOS, MICA, CD137, KIR, TGF β, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof.

II.C. tumors

In some embodiments, the tumor is derived from a cancer selected from the group consisting of: hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof. In certain embodiments, the tumor is derived from hepatocellular carcinoma, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from gastroesophageal cancer, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from melanoma, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from bladder cancer, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from lung cancer, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from a renal cancer, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from a head and neck cancer, wherein the tumor has a high inflammation signature score. In certain embodiments, the tumor is derived from colon cancer, wherein the tumor has a high inflammation signature score.

In certain embodiments, the subject has received one, two, three, four, five or more prior cancer treatments. In other embodiments, the subject is treatment naive. In some embodiments, the subject has progressed on other cancer treatments. In certain embodiments, the prior cancer treatment comprises immunotherapy. In other embodiments, the prior cancer treatment comprises chemotherapy. In some embodiments, the tumor is recurrent. In some embodiments, the tumor is metastatic. In other embodiments, the tumor is not metastatic. In some embodiments, the tumor is locally advanced.

In some embodiments, the subject has received a prior therapy to treat the tumor, and the tumor is relapsed or refractory. In certain embodiments, the at least one prior therapy comprises a standard of care therapy. In some embodiments, the at least one prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, the at least one prior therapy comprises chemotherapy. In some embodiments, the subject has received prior immunooncology (I-O) therapy to treat the tumor, and the tumor is relapsed or refractory. In some embodiments, the subject has received more than one prior therapy to treat the tumor, and the subject is relapsed or refractory. In other embodiments, the subject has received anti-PD-1 or anti-PD-L1 antibody therapy.

In some embodiments, the prior treatment line comprises chemotherapy. In some embodiments, the chemotherapy comprises a platinum-based therapy. In some embodiments, the platinum-based therapy comprises a platinum-based antineoplastic agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthroline (phenanthriptin), picoplatin (picoplatin), satraplatin (satraplatin), and any combination thereof. In certain embodiments, the platinum-based therapy comprises cisplatin. In a particular embodiment, the platinum-based therapy comprises carboplatin.

In some embodiments, the at least one prior therapy is selected from therapies comprising administration of an anti-cancer agent selected from platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumabErlotinibCrizotinibCetuximabAnd any combination thereof. In certain embodiments, the at least one prior therapy comprises platinum-based dual drug chemotherapy.

In some embodiments, the subject has experienced disease progression after the at least one prior therapy. In certain embodiments, the subject has received at least two prior therapies, at least three prior therapies, at least four prior therapies, or at least five prior therapies. In certain embodiments, the subject has received at least two prior therapies. In one embodiment, the subject has experienced disease progression after the at least two prior therapies. In certain embodiments, the at least two prior therapies comprise a first prior therapy and a second prior therapy, wherein the subject has experienced disease progression after the first prior therapy and/or the second prior therapy, and wherein the first prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof; and wherein the second prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, the first prior therapy comprises platinum-based dual-drug chemotherapy and the second prior therapy comprises single-agent chemotherapy. In certain embodiments, the single agent chemotherapy comprises docetaxel.

Ii.e. pharmaceutical compositions and dosages

The therapeutic agents of the present disclosure may constitute compositions, such as pharmaceutical compositions, containing the antibody and/or cytokine and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), while the carrier for the antibody-and/or cytokine-containing composition is suitable for non-parenteral (e.g., oral) administration. In some embodiments, the subcutaneous injection is based on Halozyme TherapeuticsDrug delivery technology (see U.S. Pat. No. 7,767,429, which is incorporated herein by reference in its entirety).Co-formulation using antibodies with recombinant human hyaluronidase (rHuPH20), whichThe traditional limitation on the volume of subcutaneously deliverable biologies and drugs due to extracellular matrix is eliminated (see U.S. Pat. No. 7,767,429). The pharmaceutical compositions of the present disclosure may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Thus, in some embodiments, the pharmaceutical compositions for use in the present disclosure may further comprise a recombinant human hyaluronidase (e.g., rHuPH 20).

In some embodiments, the method comprises administering an anti-PD-1 antibody (or an anti-PD-L1 antibody) and an anti-CTLA-4 antibody, wherein the anti-PD-1 antibody (or the anti-PD-L1 antibody) is administered in a single composition with the anti-CTLA-4 antibody at a fixed dose. In some embodiments, the anti-PD-1 antibody is administered in a fixed dose with the anti-CTLA-4 antibody. In some embodiments, the anti-PD-L1 antibody is administered in a fixed dose with the anti-CTLA-4 antibody in a single composition. In some embodiments, the ratio of the anti-PD-1 antibody (or the anti-PD-L1 antibody) to the anti-CTLA-4 antibody is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 1:1, about 1:10, about 1:1, about 1:10, about 1:1, about 1:10, about 1:1, about 1:1, about 1:10, about 1:10, about 1:1, about 1:10, about 1:1, about 1:10, about 1:1, about 1:10, about 1:10, about 1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2: 1.

Although higher nivolumab monotherapy dosing of up to 10mg/kg once every two weeks has been achieved without reaching the Maximum Tolerated Dose (MTD), the significant toxicity reported in other trials of checkpoint inhibitor plus anti-angiogenic therapy (see, e.g., Johnson et al, 2013; Rini et al, 2011) supports the selection of nivolumab doses below 10 mg/kg.

Treatment is continued as long as clinical benefit is observed or until unacceptable toxicity or disease progression occurs. However, in certain embodiments, the dose of anti-PD-1, anti-PD-L1, and/or anti-CTLA-4 antibody administered is significantly lower than the approved dose, i.e., the sub-therapeutic dose, of the agent. The anti-PD-1, anti-PD-L1, and/or anti-CTLA-4 antibodies can be administered at doses that have been shown to produce the highest efficacy as monotherapy in clinical trials, e.g., about 3mg/kg nivolumab administered once every three weeks (topallian et al, 2012 a; topallian et al, 2012); or at significantly lower doses, i.e., at sub-therapeutic doses.

The dose and frequency will vary depending on the half-life of the antibody in the subject. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are typically administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of the life. In therapeutic applications, it is sometimes desirable to have relatively high doses at relatively short intervals until progression of the disease is reduced or terminated, and preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without undue toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and anamnesis of the patient being treated, and like factors well known in the medical arts. The compositions of the present disclosure can be administered by one or more routes of administration using one or more of a variety of methods well known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending on the desired result.

Kit III

Kits for therapeutic use comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody are also within the scope of the present disclosure. The kit typically includes a label indicating the intended use and instructions for use of the kit contents. The term label includes any writing or recording material provided on or with the kit or otherwise accompanying the kit. Accordingly, the present disclosure provides a kit for treating a subject having a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from 0.1 to 10mg/kg body weight or an anti-PD-L1 antibody at a dose ranging from 0.1 to 20mg/kg body weight; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. The present disclosure further provides a kit for treating a subject having a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from about 4mg to about 500mg or an anti-PD-L1 antibody at a dose ranging from about 4mg to about 2000 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. In some embodiments, the present disclosure provides a kit for treating a subject having a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from 200mg to 800mg or an anti-PD-L1 antibody at a dose ranging from 200mg to 1800 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein.

In certain embodiments for treating a human patient, the kit comprises an anti-human PD-1 antibody disclosed herein, e.g., nivolumab or pembrolizumab. In certain embodiments for treating a human patient, the kit comprises an anti-human PD-L1 antibody disclosed herein, e.g., atelizumab, dulvacizumab, or avizumab.

In some embodiments, the kit further comprises an anti-CTLA-4 antibody. In certain embodiments for treating a human patient, the kit comprises an anti-human CTLA-4 antibody disclosed herein, e.g., ipilimumab, tremelimumab, MK-1308, or AGEN-1884.

In some embodiments, the kit further comprises an inflammatory genomic assay disclosed herein. In some embodiments, the kit further comprises instructions for administering an anti-PD-1 antibody or an anti-PD-L1 antibody to a subject identified as having a high inflammation signature score according to the methods disclosed herein. In other embodiments, the kit further comprises an anti-CTLA-4 antibody and instructions for administering (a) the anti-PD-1 antibody or the anti-PD-L1 antibody and (b) the anti-CTLA-4 antibody to a subject identified as having a high inflammation signature score according to the methods disclosed herein.

All references cited above and all references cited herein are incorporated by reference in their entirety.

The following examples are provided by way of illustration and not by way of limitation.

Examples

Example 1: assessment of inflammatory biomarkers associated with clinical outcome of nivolumab-treated patients with advanced hepatocellular carcinoma

Liver cancer is the fourth leading cause of cancer-related mortality worldwide, with most liver cancers being hepatocellular carcinoma (HCC). Patients with advanced HCC have few effective treatment options, and agents that are able to achieve a robust and durable response remain an unmet need in hepatocytes. Clinical trials for approved first-and second-line targeted therapies reported median overall survival in the range of from 10.7-13.6 months and 10.2-10.6 months, respectively (see, Abou-Alfa et al, N Engl J med.379(1):54-63 (2018); Bruix et al, Lancet 389(10064):56-66 (2017); Llovet et al, N Engl J med.359(4):378-90 (2008); and Kudo et al, lancet.391(10126):1163-73 (2018)). Nivolumab ("NIVO") binds to PD-1 receptors expressed primarily on activated T cells and thus prevents the binding of PD-L1 and PD-L2 ligand expressed on tumor cells. In clinical trial NCT01658878, nivolumab demonstrated a durable response, manageable safety and long-term survival in patients with advanced HCC regardless of etiology with/without prior Sorafenib (SOR) treatment (see, El-Khoueiry et al, Lancet.389:2492-2502 (2017)). Based on the results from clinical trial NCT01658878, NIVO is approved in many countries (including the united states) for use in patients with HCC experiencing SOR.

This example relates to exploratory biomarker analysis from nivolumab treated patients with advanced HCC from clinical trial NCT 01658878.

Design of research

This data is associated with cohorts 1 and 2 of clinical trial NCT01658878, which had 262 subjects in total (fig. 1). Cohort 1 contained 80 SOR naive subjects, and cohort 2 contained 182 subjects undergoing SOR. Eleven subjects in cohort 1 and 37 subjects in cohort 2 were administered 0.1-10mg/kg nivolumab as part of a dose escalation analysis. As part of the dose expansion analysis, sixty-nine subjects in cohort 1 and 145 subjects in cohort 2 were administered 3mg/kg nivolumab. After the initial treatment, 154 subjects in cohort 2(9 subjects from the dose escalation study and 145 subjects from the dose extension study) were administered nivolumab maintenance at 3 mg/kg.

The main endpoints of the clinical trial NCT01658878 are safety and tolerability (dose escalation) and objective response rate (ORR; dose extension). Secondary endpoints included ORR (dose escalation), disease control rate, response time, duration of response, and overall survival. Exploratory endpoints include biomarker assessments, which are discussed herein.

Data generated from clinical trial NCT01658878, including an ORR of 14.3% in 50% of subjects and a duration of response (DOR) of at least 12 months, helped the USFDA approve nivolumab for treating patients with HCC experiencing SOR.

A qualified subject has (i) advanced HCC that is not suitable for histological confirmation of radical resection; (ii) the Child-Pugh score is less than or equal to 7 (incremental) or less than or equal to 6 (extended); (iii) progression on at least one previous systemic therapy line or intolerance or rejection of SOR; (iv) AST and ALT are less than or equal to 5 multiplied by the upper normal limit and bilirubin is less than or equal to 3 mg/dL; (v) for patients infected with HBV, a viral load of less than 100IU/mL and an effective antiviral therapy concomitant; and (vi) for patients infected with HCV, active infection or resolved infection as evidenced by detectable HCV RNA or antibodies. Subjects with any history of hepatic encephalopathy, previously or currently clinically significant ascites, or active HBV and HCV co-infection were excluded.

Pre-treated tumor samples (fresh or archived) were obtained from patients receiving increasing and expanding stages of 3mg/kg nivolumab (stored for IHC) or 0.1-10mg/kg nivolumab (stored for RNA sequencing).

Biomarker assessment

Analyzing the sample using (i) IHC to assess PD-L1, PD-1, T cell markers (CD3, CD4, CD8, FOXP3) and macrophage markers (CD68, CD 163); and (ii) RNA sequencing the sample to assess the tumor inflammation signature. Biomarkers were assessed to assess relevance to clinical outcome (including BOR) and overall survival by blinded independent review board (according to RECIST v 1.1). Analysis was performed using standard Limma and Cox regression frameworks.

Biomarker analysis

PD-L1

Of the total population, 195 subjects had evaluable PD-L1 data (SOR naive, n-58; SOR experienced, n-137; table 2). Clinically significant responses were observed in all subjects (including those with PD-L1< 1%), and 6 subjects had complete responses. In the total population, numerically higher objective response rates were observed in subjects with PD-L1 ≧ 1% and PD-L1< 1% overlapping 95% confidence intervals. Populations that experience SOR have ORR comparable to those of the total population.

In the total population, deep responses were observed regardless of the PD-L1 status (fig. 2A-2B). Tumor cell PD-L1 expression in at least 1% of tumor cells was significantly associated with overall survival (fig. 2C; P ═ 0.032). Typically, positive PD-L1 expression in at least 1% of tumor cells was associated with higher overall survival in subjects undergoing SOR, however this difference was not statistically significant (fig. 2D).

TABLE 2 optimal overall response of tumor cell PD-L1 status.

Tumor PD-L1 expression was not found to be significantly different when stratified by geographic region (asian vs non-asian; data not shown).

T cell markers

Prior to administration of nivolumab, expression profiles of T cell markers CD3, CD8, CD4, and FOX-3 were analyzed in tumor samples obtained from the subjects. The frequency of CD3 positive cells was observed to correlate with response (CR/PR compared to SD; P ═ 0.03; fig. 3A). No significant correlation was observed between CD4, CD8, or FOXP3 positive cell frequency and response (fig. 3B-3D). The frequency of CD3 positive cells was higher in the tumor microenvironment than the other T cell markers evaluated (data not shown). No significant differences in T cell marker distribution were found when stratified by viral etiology (HBV or HCV infection, or no infection; data not shown) or geographic region (asian versus non-asian; data not shown).

Tumor inflammation (as measured by CD3 or CD8 expression) had an insignificant trend towards improved overall survival (fig. 4A-4B; P ═ 0.08) and to a lesser extent CD4 or FOXP3 expression (fig. 4C-4D).

Macrophage markers

Prior to administration of nivolumab, the expression profile of macrophage markers CD68 and CD163 in tumor samples obtained from the subject was analyzed. No correlation between CD68 and CD163 expression and clinical outcome was observed (fig. 5A-5B and fig. 6A-6B). Furthermore, no significant differences in macrophage marker distribution were found when stratified by viral etiology (infected with HBV or HCV, or not; data not shown) or geographic region (Asian versus non-Asian; data not shown).

Tumor immunity gene label

For the subset of subjects for which data was available (n-37), gene expression profiling was performed using RNA sequencing to assess tumor immunoinfiltration and inflammatory signatures (table 3). In particular, several inflammatory signatures, such as the 4-gene inflammatory signature of the present disclosure (including CD274(PD-L1), CD8A, LAG3, and STAT1), Gajewski 13-gene inflammatory signature, the Merck 6-gene interferon gamma signature, the NanoString interferon gamma biological signature, and the NanoString T-cell depletion signature) were significantly associated with improved response and overall survival (table 3). In particular, significantly higher mean 4-gene inflammation signature scores as described herein were observed in patients experiencing partial response compared to stable disease (p ═ 0.05; fig. 7A). Furthermore, the mean median 4 gene inflammation score was significantly associated with improved overall survival (p ═ 0.01; fig. 7B).

TABLE 3 relationship of tumor immunogene signature to clinical response in the total population.

Danilova L, et al Proc Natl Acad sci.2016; 113E 7769-E7777; spranger S, et al nature.2015; 523: 231-; ayers M, et al J Clin invest.2017; 127:2930-2940.

No significant difference in the 4 gene inflammation signature scores was found when stratified by viral etiology (HBV or HCV infection, or no infection; data not shown) or geographic region (asian versus non-asian; data not shown).

In clinical trials NCT01658878 cohorts 1 and 2, a persistent response was observed in both SOR naive patients and patients undergoing SOR regardless of tumor cell PD-L1 status. In this retrospective analysis of pre-treated tumor samples from patients with advanced HCC, tumor cell PD-L1 expression correlated with OS; however, this correlation was not significant in patients experiencing SOR. CD3⁺T cell frequency correlates with response to nivolumab with a trend towards improved survival when CD3 and CD8 are positive. Higher scores for several inflammatory signatures, including the 4 gene inflammatory signature, were associated with improved response and overall survival.

Example 2: PD-L1 combination positive score and correlation of immunogene signature with efficacy of nivolumab + -ipilimumab in patients with metastatic gastroesophageal cancer

Combination therapy comprising Nivolumab (NIVO) and ipilimumab (IPI) demonstrated clinically meaningful anti-tumor activity and manageable safety profiles in patients with chemotherapy refractory gastroesophageal cancer in stage 1/2 (NCT 01928394; Janjigian YY, et al, J Clin oncol.2018; 36: 2836-. In the current exploratory analysis of clinical trial NCT01928394, the expression of selected immune gene signatures was evaluated to determine if there was a correlation with the efficacy of nivolumab monotherapy with combination therapy of ipilimumab.

Design of research

Patients with locally advanced or metastatic stomach/esophagus/GEJ cancers refractory to ≥ 1 prior chemotherapy are randomly assigned one of the following: nivolumab 3mg/kg (NIVO3) intravenously once every 2 weeks (n-59); nivolumab 1mg/kg plus ipilimumab 3mg/kg (NIVO1+ IPI3) once every 3 weeks for four cycles (n-49); or nivolumab 3mg/kg plus ipilimumab 1mg/kg (NIVO3+ IPI1) once every 3 weeks for four cycles (n ═ 52) (fig. 8). All combination regimens followed the receipt of NIVO3 every 2 weeks until disease progression or unacceptable Adverse Events (AEs).

The primary endpoint was the Objective Response Rate (ORR), defined as the best response of a complete or partial response divided by the number of patients treated, according to RECIST version 1.1. Secondary endpoints include Overall Survival (OS), Progression Free Survival (PFS), reaction time, duration of reaction (DOR), and safety. Tumor response was assessed every 6 weeks using imaging for 24 weeks, then every 12 weeks until disease progression or treatment discontinuation. Survival was monitored continuously during patient treatment and every 3 months after discontinuation of treatment. Exploratory endpoints included a correlation between tumor PD-L1 expression and efficacy and safety.

Key eligibility criteria for an esophageal gastric cancer cohort include diagnosis of locally advanced or metastatic gastric, esophageal or GEJ adenocarcinoma with disease progression while taking or intolerant of at least one chemotherapy regimen; measurable disease as assessed by the Solid tumor Response assessment Criteria (Response assessment in Solid Tumors, RECIST) version 1.118; the physical ability status of Eastern Cooperative Oncology Group (Eastern Cooperative Oncology Group) is 0 or 1; and adequate organ function. Patients with human epidermal growth factor receptor 2 positive tumors are eligible if they have previously been treated with trastuzumab. Key exclusion criteria included suspected autoimmune disease; hepatitis b virus or human immunodeficiency virus infection; a condition requiring a corticosteroid or other immunosuppressive drug; and prior immune checkpoint inhibitor therapies.

Biomarker analysis

PD-L1 expression

Biological samples were collected from subjects prior to immunotherapy, and a subset of the subject samples were available for PD-L1 expression analysis (table 4).

Table 4. baseline characteristics and responses: total population and population assessed by PD-L1.

^aThree patients in the dose escalation phase of NIVO1+ IPI1 were also included in the analysis. CR, complete reaction; ECOG, eastern cooperative group of tumors; NE, not evaluable; PD, progressive disease; PR, partial reaction; SD, stable disease.

PD-L1 Immunohistochemistry (IHC) was used to assess PD-L1 expression on tumors and tumor-associated immune cells. Tumor PD-L1 expression as used in this example represents the percentage of live tumor cells that show partial or complete membrane PD-L1 staining. Tumor PD-L1 expression was calculated according to formula II:

combined Positive Scores (CPS) include both tumor and tumor-associated immune cell PD-L1 expression. CPS is calculated according to formula III:

a better correlation of PD-L1 expression (fig. 9B) as expressed by CPS with the response was observed compared to PD-L1 expression (fig. 9A) on tumor cells. PD-L1 expression, expressed by CPS, had a higher prevalence (regardless of cutoff value) and had a better correlation with response at higher cutoff values than PD-L1 expression on tumor cells (table 5). At higher cut-off values, PD-L1 expression, expressed by CPS, showed a stronger correlation with overall survival compared to tumor PD-L1 expression (fig. 10A-10F).

Table 5 prevalence and response rates by PD-L1 expression on tumor cells and by CPS: all schemes

^aPD-L1 expression on tumor cells;^bPD-L1 expression by CPS;^cfor tumor PD-L1 expression, the cut-off values are presented as a percentage. For CPS, the cutoff value is presented as a score. NA, not applicable; ORR, objective response rate.

In the nivolumab 1mg/kg + ipilimumab 3mg/kg treatment group, PD-L1 expression, expressed by CPS, had a higher prevalence (regardless of cutoff value) and a better correlation with response at higher cutoff values compared to PD-L1 expression on tumor cells. Furthermore, PD-L1 expression, as expressed by CPS, showed a strong correlation with overall survival at higher cut-off values (fig. 11A-11D). This correlation was consistent and more evident in patients treated with nivolumab 1mg/kg + ipilimumab 3mg/kg in patients with all combination regimens (see fig. 10D-10F).

Table 6 prevalence and response rates by PD-L1 expression on tumor cells and by CPS: 1mg/kg of nivolumab + 3mg/kg of ipilimumab.

^aPD-L1 expression on tumor cells;^bPD-L1 expression by CPS;^cfor tumor PD-L1 expression, the cut-off values are presented as a percentage. For CPS, the cutoff value is presented in a score;^donly 1 patient had tumors PD-L1 ≥ 5% and ≥ 10%.

Genetic profiling

Biological samples were collected from subjects prior to immunotherapy, and a subset of the subject samples were available for gene expression profiling (table 7).

Table 7. baseline characteristics and responses: total population and gene expression profiling population.

Various gene expression signatures were analyzed on available samples (table 8). All gene expression signatures showed a trend associated with the response (table 8). Notably, significant correlations were observed between the 4-gene inflammation signature of the present disclosure (comprising CD274(PD-L1), CD8A, LAG3, and STAT 1; fig. 12D), CD 8T cell signature (fig. 12A), PD-L1 transcript (fig. 12B), and Ribas 10 gene interferon gamma signature (fig. 12C), where the 4-gene inflammation signature showed the strongest correlation with response (patients with CR/PR, n ═ 4; table 8). Although the number of responding patients was small for this analysis (n-4), a good differentiation of AUC was demonstrated (90% [ 95% CI, 77-100]) (fig. 13).

TABLE 8 Gene expression signatures and responses.

Gene signature/transcript	P valuea	Error discovery ratea,b
			4 gene inflammation label	0.00411	0.037
CD 8T cell tag1	0.0321	0.0862
			Gajewski 13 gene inflammation label2	0.127	0.164
Interferon gamma transcripts	0.0479	0.0862
			Ribas 10 gene interferon gamma label3	0.0416	0.0862
PD-L1 transcript	0.0621	0.0931
			T cell tag1	0.171	0.18

^aP-values and false discovery rates were derived from tests using 9 pre-assigned tags and genes. False discovery rate adjusted P value.^bEstimation of false discovery rate for a given number of tests/hypotheses.^cIn view of the small sample size, exploratory P-values are intended to describe the relative performance of different tags for correlation with the reaction. Siemers NO, et al PLoS one.2017; 12: e 0179726; spranger S, et al nature.2015; 523: 231-; ayers M, et al J Clin invest.2017; 127:2930-2940.

In this exploratory analysis, inflammatory gene signature expression was observed to correlate with response to nivolumab monotherapy and to combination therapy with ipilimumab. This correlation indicates the presence of an actionable biological factor that can be targeted by the immunooncology agent.

Example 3: genomic analysis and immunotherapy in advanced melanoma

Nivolumab (NIVO) and ipilimumab (IPI) are immune checkpoint inhibitors with unique but complementary activities. Combination therapy comprising nivolumab and ipilimumab and nivolumab and ipilimumab monotherapy are approved for the treatment of unresectable melanoma or metastatic melanoma.

In the study of various tumors, including melanoma, it was shown that the response to anti-PD-1 therapy correlates with the gene expression profile of T cell inflammation.

This example reports the results of an exploratory analysis of the correlation of novel inflammatory gene signatures with clinical outcome for nivolumab/ipilimumab combination therapy and nivolumab and ipilimumab monotherapy in melanoma.

Design of research

This example reports data collected from clinical trial NCT 01844505. In this trial 945 previously untreated patients with unresectable stage III or IV melanoma were randomly assigned at a 1:1:1 ratio to receive one of the following regimens: (i) 3mg nivolumab/kg body weight every 2 weeks (gaipilimumab-matched placebo) (n-316, 313 received treatment); (ii) 1mg nivolumab/kg every 3 weeks plus 3mg ipilimumab/kg every 3 weeks for 4 doses, then 3mg nivolumab/kg every 2 weeks for 3 cycles and more (n-314, 313 received treatment); or 3mg ipilimumab/kg every 3 weeks for 4 doses (garnivolumab matched placebo) (n ═ 315, 311 received treatment) (fig. 14). Both nivolumab and ipilimumab were administered by intravenous infusion.

Random stratification was performed according to tumor PD-L1 status (positive and negative or uncertain), BRAF mutation status (V600 mutation positive and wild type), and united states committee for cancer metastasis staging (M0, M1a or M1b and M1 c). Treatment is continued until disease progression (as defined by RECIST version 1.1), progression to an unacceptable toxic event, or withdrawal of consent.

Progression-free survival and overall survival are common primary endpoints. Secondary endpoints included objective response rates, tumor PD-L1 expression, and health-related quality of life. Exploratory endpoints include safety, pharmacokinetics, and biomarker analysis.

The 4-year follow-up of NCT01844505 demonstrated a sustained, sustained survival benefit of first-line nivolumab/ipilimumab combination therapy and nivolumab monotherapy in patients with advanced melanoma (ORRb,% (95% CI): 58% (52.6-63.8) NIVO + IPI; 45% (39.1-50.3) NIVO; 19% (14.9-23.8) IPI; median PFS, month (95% CI): 11.5(8.7-19.3) NIVO + IPI; 6.9(5.1-10.2) NIVO; 2.9(2.8-3.2) IPI; and median OS, month (95% CI): NR (38.2-NR) NIVO + IPI; 36.9(28.3-NR) NIVO; 19.9(16.9-24.6) IPI) (FIG. 15A-15B). NCT01844505 cannot be used for formal statistical comparisons between nivolumab/ipilimumab combination therapy and nivolumab monotherapy.

Purpose(s) to

The purpose of this analysis was to assess the correlation of inflammatory signatures with clinical response, PFS and OS with nivolumab-based immunooncology (I-O) therapy. For the inflammation signature analysis, tumor samples were pretreated using RNAseq analysis to estimate relative tumor inflammation using expression of 4 key genes-CD 274(PD-L1), CD8a, LAG3, and STAT1- (including the 4 gene inflammation signature score described herein). Relative median scores were used in NCT01844505 samples to assess the correlation of PFS and OS with the 4-gene inflammation signature score to define a high 4-gene inflammation signature score versus a low 4-gene inflammation signature score (median-0.0434). A summary of sample handling is provided in table 9 and fig. 16.

TABLE 9 sample handling NCT 01844505.

Results

The distribution of 4-gene inflammation signature scores was higher in patients responding to treatment with nivolumab/ipilimumab combination therapy, nivolumab monotherapy and ipilimumab monotherapy (figure 17). In all treatment groups, patients with high and low inflammation signature scores observed longer PFS (fig. 18A-18D). Longer OS was also observed in patients with high and low inflammation signature scores in all treatment groups (fig. 19A-19D).

In previously untreated metastatic melanoma, a high 4-gene inflammation signature score was observed to correlate with clinical response to immunooncology therapy and increased survival.