Tumor treatment methods using anti-PD-1 antibodies

Abstract

This invention provides a therapeutic method using an antibody or its antigen-binding portion that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. [Solution] This disclosure provides a method for treating a subject suffering from a tumor, characterized by administering to the subject an antibody or its antigen-binding portion that specifically binds to the PD-1 receptor and inhibits PD-1 activity. In one embodiment, the tumor is derived from non-small cell lung cancer (NSCLC). In one embodiment, the tumor expresses programmed death ligand-1. In one embodiment, the subject has the wild-type STK11 gene.

Description

[Technical Field]

[0001] This disclosure relates to a method for treating a tumor, characterized by administering an anti-programmed death-1 (PD-1) antibody to a target, wherein the target has wild-type STK11. [Background technology]

[0002] Human cancers possess numerous genetic and epigenetic alterations, giving rise to neoantigens that can potentially be recognized by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T and B lymphocytes, has a broad capacity to respond to various tumor antigens and possesses potent anti-cancer capabilities due to its sophisticated specificity. Furthermore, this immune system exhibits high flexibility and memory structure. If all of these characteristics of the adaptive immune system are successfully utilized, immunotherapy will become unique among all cancer treatment modalities.

[0003] PD-1 is an important immune checkpoint receptor expressed by T and B cells that mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 (programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2)), expressed in antigen-presenting cells and many human cancers, have been identified and shown to downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0004] Nivolumab (formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby preventing downregulation of antitumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0005] NSCLC is the leading cause of cancer death in the United States and worldwide (NCCN GUIDELINES®, version 3.2014 - Non-Small Cell Lactation Cancer, most recently accessed May 14, 2014: available at www.nccn.org / professionals / physician_gls / pdf / nscl.pdf). Patients with stage IV disease who are relatively less sensitive to chemotherapy but show good activity indicators (PS) can benefit from treatment with chemotherapy agents including platinum-based drugs (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine, as well as various combinations of these drugs. [Overview of the project]

[0006] This disclosure provides a method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering to the subject an antibody or its antigen-binding portion ("anti-PD-1 antibody") that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity, if the STK11 gene is wild-type. In another embodiment, this disclosure provides a method for treating a subject suffering from a tumor, characterized by administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a wild-type STK11 gene. In another embodiment, this disclosure provides a method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody therapy, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering to the subject an anti-PD-1 antibody if the STK11 gene is wild-type. In one embodiment, the method further includes detecting the mutation status of a marker gene selected from the group consisting of KRAS, TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0007] In another embodiment, the disclosure relates to a method for treating a subject suffering from a tumor, characterized by (i) determining a mutation in a marker gene in the subject; then (ii) administering an anti-PD-1 antibody to the subject if the marker gene is mutated; and a method for selecting the marker gene from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In another embodiment, the disclosure relates to a method for treating a subject suffering from a tumor, characterized by administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a mutated marker gene, and a method for selecting the marker gene from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In other embodiments, the Disclosure relates to a method for identifying subjects suffering from tumors suitable for anti-PD-1 antibody therapy, characterized by (i) determining the mutation status of a marker gene in the subject; and (ii) administering an anti-PD-1 antibody to the subject if the marker gene is mutated; and the method for selecting the marker gene from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In one embodiment, TP53 is mutated. In one embodiment, CDKN2A is mutated. In one embodiment, PTPND, CUBN, and HERC1 are mutated.

[0008] In one embodiment, the marker gene includes nonsensical mutations. In a particular embodiment, the marker gene includes nonsense, frameshift, or splicing mutations.

[0009] In one embodiment, the tumor is derived from lung cancer. In a particular embodiment, the tumor is derived from small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In one embodiment, the tumor is derived from NSCLC. In one embodiment, the tumor is derived from non-squamous NSCLC. In another embodiment, the tumor is derived from squamous NSCLC.

[0010] In one embodiment, the method further includes detecting PD-L1 expression in the tumor before administration. In one embodiment, the tumor expresses PD-L1 in a scattered pattern. In one embodiment, the tumor expresses PD-L1 in a heterogeneous pattern.

[0011] In one embodiment, the mutation status of the STK11 gene is investigated by sequencing the STK11 gene.

[0012] In one embodiment, the tumor exhibits a TMB state, which is a high tumor mutational load (TMB). In one embodiment, the TMB state of the tumor is determined by sequencing the nucleic acids in the tumor and identifying genomic changes in the sequenced nucleic acids.

[0013] In one embodiment, the tumor exhibits high levels of inflammation. In one embodiment, the inflammation is measured by the expression of STK11.

[0014] In one embodiment, the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. In one embodiment, the anti-PD-1 antibody binds to the same epitope as nivolumab. In one embodiment, the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody or a portion thereof. In one embodiment, the anti-PD-1 antibody contains a heavy chain constant region of a human IgG1 or IgG4 isotype. In one embodiment, the anti-PD-1 antibody is nivolumab.

[0015] In one embodiment, the anti-PD-1 antibody is administered once every 1, 2, or 3 weeks in a dose ranging from at least about 0.1 mg / kg to at least about 10.0 mg / kg body weight. In one embodiment, the anti-PD-1 antibody or its antigen-binding moiety is administered in a flat dose. In one embodiment, the anti-PD-1 antibody or its antigen-binding moiety is administered in a flat dose or at about 240 mg.

[0016] In one embodiment, the administration treats a tumor. In one embodiment, the administration reduces tumor size. In one embodiment, the subject exhibits progression-free survival for at least about 1 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 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the first administration. In one embodiment, the subject exhibits partial remission after administration. In one embodiment, the subject exhibits complete remission after administration.

[0017] In other embodiments, the Disclosure provides a kit for treating a subject affected by a tumor, comprising (a) an anti-PD-1 antibody in a dose ranging from about 4 mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in the method described herein. In one embodiment, the kit further comprises an anti-PD-L1 antibody.

[0018] Embodiment E1. A method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) if the STK11 gene is wild-type, administering to the subject an antibody or its antigen-binding portion ("anti-PD-1 antibody") that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity.

[0019] E2. A method for treating a subject suffering from a tumor, comprising administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a wild-type STK11 gene.

[0020] E3. A method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody treatment, comprising: (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering an anti-PD-1 antibody to the subject if the STK11 gene is wild-type.

[0021] E4. The method according to any one of E1 to E3, further comprising detecting the mutation status of a marker gene selected from the group consisting of KRAS, TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0022] E5. A method for treating a subject suffering from a tumor, comprising: (i) determining the mutation status of a marker gene in the subject; and then (ii) administering to the subject an antibody or an antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity (an "anti-PD-1 antibody") if the marker gene has a mutation; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0023] E6. A method for treating a subject suffering from a tumor, comprising administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a marker gene with a mutation, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0024] A method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody treatment, comprising: (i) determining the mutation status of a marker gene in the subject; and then (ii) administering an anti-PD-1 antibody to the subject if the marker gene has a mutation, wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0025] E8. The method according to any one of E4 to E7, wherein TP53 has a mutation.

[0026] E9. The method according to any one of E4 to E8, wherein CDKN2A has a mutation.

[0027] E10. The method according to any one of E4 to E9, wherein PTPND, CUBN, and HERC1 have mutations.

[0028] E11. The method according to any one of E4 to E10, wherein the marker gene contains a non-synonymous mutation.

[0029] [[ID=A]]E12. The method according to any one of E4 to EA, wherein the marker gene contains a nonsense, frameshift, or splicing mutation.

[0030] E13. The method according to any one of E1 to E12, wherein the tumor is derived from lung cancer.

[0031] E14. The method according to E13, wherein the tumor is derived from small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0032] E15. The method according to E14, wherein the tumor is derived from NSCLC.

[0033] E16. The method according to E15, wherein the tumor is derived from non-squamous NSCLC.

[0034] It should be noted that there is an error in the original text where "E12" is followed by "E1~E12" in "E13. The method according to any one of E1~E12, wherein the tumor is derived from lung cancer.", which may cause confusion. I have translated it as accurately as possible based on the existing content. Also, there is an "ID=A" in the translated text which is not in the original, this is likely a result of an error in the original text's numbering system and has been translated as is to maintain consistency with the original's format. If this is a specific requirement, please provide more context for a more accurate translation.E17. The method according to E15, wherein the tumor is derived from squamous cell NSCLC.

[0035] E18. The method according to any one of E1 to E17, further comprising detecting PD-L1 expression in a tumor before administration.

[0036] E19. The method according to E18, wherein the tumor expresses PD-L1 in a scattered pattern.

[0037] The method according to E19, wherein the scattered pattern of PD-L1 expression is characterized by PD-L1 H-scores of approximately 60-500, 80-480, 100-460, 120-440, 140-420, 160-400, 180-380, 200-360, 200-340, 200-320, or 200-300.

[0038] E21. The method according to E19, wherein the scattered pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, at least approximately 110, at least approximately 120, at least approximately 130, at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 225, at least approximately 250, at least approximately 275, or at least approximately 300.

[0039] E22. The method described in E21, wherein the scattered pattern of PD-L1 expression is characterized by at least approximately 200 PD-L1 H-scores.

[0040] E23. The method according to E18, wherein the tumor expresses PD-L1 in a heterogeneous pattern.

[0041] E24. The method according to E23, wherein the heterogeneous pattern of PD-L1 expression is characterized by PD-L1 H-scores of approximately 1 to approximately 50, approximately 5 to approximately 45, approximately 10 to approximately 40, or approximately 15 to approximately 35, and the PD-L1 expression is limited to one or more different parts of the tumor.

[0042] E25. The method according to E23, wherein the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40.

[0043] E26. The method according to E25, wherein the heterogeneous pattern of PD-L1 expression is characterized by at least approximately 15 PD-L1 H-scores.

[0044] E27. The method according to any one of E1, 3, 4, and 13-26, wherein the mutational state of the STK11 gene is determined by sequencing the STK11 gene.

[0045] E28. The method according to any one of E18-E27, wherein PD-L1 expression is detected by an immunohistochemistry (IHC) assay.

[0046] E29. The method according to E28, wherein the IHC assay is an automated IHC assay.

[0047] E30. The method according to E28 or E29, wherein the IHC assay is performed using an anti-PD-L1 monoclonal antibody that specifically binds to PD-L1, and the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

[0048] E31. The method according to any one of E18-E30, wherein at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of tumor cells express PD-L1.

[0049] E32. The method according to any one of E1 to E31, wherein the tumor exhibits a tumor mutational burden (TMB) state of high TMB.

[0050] E33. The method according to E32, wherein the tumor TMB state is determined by sequencing nucleic acids in the tumor and identifying genomic changes in the sequenced nucleic acids.

[0051] E34. The method according to E33, wherein the genomic alteration comprises one or more somatic mutations.

[0052] E35. The method according to E33 or E34, wherein the genomic alteration comprises one or more nonsynonymous mutations.

[0053] E36. The method according to any one of E33 to E35, wherein the genomic alteration comprises one or more missense mutations.

[0054] E37. The method according to any one of E33 to E36, wherein the genomic alteration comprises one or more alterations selected from the group consisting of base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and any combination thereof.

[0055] E38. The above high TMB is at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, and at least The method according to any one of E32-E37, which is a score of 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, or at least 500.

[0056] E39. The method according to any one of E32 to E38, wherein the high TMB is a score of at least 215, at least 220, at least 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, at least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249, or at least 250.

[0057] E40. The method according to any one of E32 to E39, wherein the high TMB is a score of at least 243.

[0058] E41. The method according to any one of E32 to E40, further comprising comparing the TMB state of the subject with a reference TMB value.

[0059] E42. The method according to E41, wherein the TMB state of the subject is within the highest quantile of the reference TMB value.

[0060] E43. The method according to E41, wherein the TMB state of the subject is within the highest tertile of the reference TMB value.

[0061] E44. The method according to any one of E32 to E43, wherein the TMB state is determined by genome sequencing.

[0062] E45. The method according to any one of E32 to E43, wherein the TMB state is determined by exome sequencing.

[0063] E46. The method according to any one of E32 to E45, wherein the TMB state is determined by genome profiling.

[0064] E47. The method according to any one of E1 to E46, wherein the tumor exhibits high inflammation.

[0065] E48. The method according to E47, wherein the inflammation is measured by the expression of STK11.

[0066] E49. The method according to any one of E1 to E48, wherein the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1.

[0067] E50. The method according to any one of E1 to E49, wherein the anti-PD-1 antibody binds to the same epitope as nivolumab.

[0068] E51. The method according to any one of E1 to E50, wherein the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody, or a part thereof.

[0069] E52. The method according to any one of E1 to E51, wherein the anti-PD-1 antibody comprises a heavy chain constant region of a human IgG1 or IgG4 isotype.

[0070] E53. The method according to any one of E1 to E52, wherein the anti-PD-1 antibody is nivolumab.

[0071] E54. The method according to any one of E1 to E53, wherein the anti-PD-1 antibody is pembrolizumab.

[0072] E55. The method according to any one of E1 to E54, wherein the anti-PD-1 antibody is administered once every 1, 2, or 3 weeks at a dose in the range of at least about 0.1 mg / kg to at least about 10.0 mg / kg body weight.

[0073] E56. The method according to E55, wherein the anti-PD-1 antibody is administered once every two weeks at a dose of at least about 3 mg / kg body weight.

[0074] E57. The method according to any one of E1 to E56, wherein the anti-PD-1 antibody or its antigen-binding portion is administered in a flat dose.

[0075] E58. The method according to any one of E1 to E54 and 57, wherein the anti-PD-1 antibody or its antigen-binding moiety 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.

[0076] E59. The method according to any one of E1 to E54, 57, and 58, wherein the anti-PD-1 antibody or its antigen-binding portion is administered in a flat dose of approximately 240 mg.

[0077] E60. The method according to any one of E1-E54 and 57-E59, wherein the anti-PD-1 antibody or its antigen-binding moiety is administered in a flat dose once every 1, 2, 3, or 4 weeks.

[0078] E61. The method according to any one of E1 to E60, wherein the anti-PD-1 antibody is administered for as long as a clinical benefit is observed or until uncontrollable toxicity or disease progression occurs.

[0079] E62. The anti-PD-1 antibody is formulated for intravenous administration. The method according to any one of E1 to E61.

[0080] E63. The method according to any one of E1 to E62, wherein the anti-PD-1 antibody is administered in a dose less than or equal to a therapeutic dose.

[0081] E64. The method according to any one of E1 to E63, wherein the administration treats the tumor.

[0082] E65. The method according to any one of E1 to E64, wherein the administration reduces tumor size.

[0083] E66. The method according to E65, wherein the tumor size is reduced to at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size before administration.

[0084] E67. The method according to any one of E1 to E66, wherein the subject exhibits progression-free survival for at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after the first dose.

[0085] E68. The method according to any one of E1 to E67, wherein the subject exhibits a stable condition after the administration.

[0086] E69. The method according to any one of E1 to E67, wherein the subject exhibits a partial response after the administration.

[0087] E70. The method according to any one of E1 to E67, wherein the subject shows a complete response after the administration.

[0088] E71. A kit for treating subjects suffering from tumors, (a) Anti-PD-1 antibody in doses ranging from approximately 4 mg to approximately 500 mg; and (b) Instructions for using the anti-PD-1 antibody in any one of the methods described in E1 to E70. A kit that includes this.

[0089] E72. The kit described in E71, further comprising an anti-PD-L1 antibody. [Brief explanation of the drawing]

[0090] [Figure 1] Figures 1A-1D show immunohistochemical (IHC) diagrams illustrating different patterns of PD-L1 expression in commercially available NSCLC tumors. PD-L1 expression patterns can be classified as scattered (Figure 1A), heterogeneous (Figure 1B), tumor-stromal junction (Figure 1C), and negative (Figure 1D).

[0091] [Figure 2] Figures 2A-2B show the distribution of PD-L1 H-scores in each PD-L1 pattern (i.e., scattered (D), heterogeneous (H), negative (N), and tumor-stromal junction (T)) as shown in Figures 1A-1D (Figure 2A), and the distribution of PD-L1 H-scores in two NSCLC subtypes (i.e., adenocarcinoma and squamous cell carcinoma) (Figure 2D).

[0092] [Figure 3] Figures 3A-3C show IHC diagrams corresponding to scattered (Figure 3A), tumor-stromal junction (Figure 3B), and negative (Figure 3C) PD-L1 expression patterns from biopsies derived from patients treated with nivolumab monotherapy.

[0093] [Figure 4] Figure 4 shows the PD-L1 H-scores of patients who received nivolumab monotherapy. The dominant PD-L1 pattern in the majority of complete responders (CR) and partial responders (PR) is a scattered pattern.

[0094] [Figure 5] Figures 5A and 5B show the overall CI score (Figure 5A) and PD-L1 CI score (Figure 5B) based on the dominant pattern of PD-L1 tumors.

[0095] [Figure 6] Figure 6 shows multiplexed IHC images stained for PD-L1, CD68, and CD3.

[0096] [Figure 7] Figures 7A and 7B show PD-L1 expression in NSCLC tumors as measured by RNA sequencing (Figure 7A), and mutational load in NSCLC tumors as measured by exome sequencing (Figure 7B).

[0097] [Figure 8]Figures 8A and 8B show the correlation between the number of missense mutations in NSCLC tumors, as measured by the CI score, and overall inflammation (Figure 8A), and the correlation between the number of missense mutations in NSCLC tumors, as measured by the PDLP1pos CI score, and PD-L1+ inflammation (Figure 8B).

[0098] [Figure 9] Figures 9A and 9B show the mutation frequencies in the PD-L1 expression patterns observed for different biomarkers (TP53, STK11, KEAP1, KRAS, EGFR, and MET). Figure 9A shows the mutation frequencies in the PD-L1 expression patterns observed for different biomarkers (TP53, STK11, KEAP1, KRAS, EGFR, and MET). D = scattered, H = heterogeneous, I = tumor-stromal junction, N = negative. Figure 9B shows the presence ("y") or absence ("n") of STK11 mutations in PD-L1 expression as measured by RNA sequencing (RNAseq).

[0099] [Figure 10] Figures 10A-10C show the correlation between the presence ("y") or absence ("n") of the STK11 mutation and the PD-L1+ CI score (Figure 10A). Figure 10B shows the numerical data corresponding to the information shown in Figure 10A. Figure 10C shows the numerical data corresponding to the overall inflammation score in NSCLC tumors, depending on the presence ("STK11-MUT") or absence ("STK11-WT") of the STK11 mutation.

[0100] [Figure 11]Figure 11 shows immunoprint analysis of 24 NSCLC tumor samples, classifying them according to their inflammatory pattern (sigClass) by analyzing the levels of FOLR2, VSIG4, CD163, CLEC4D, CSF1R, CD86, MS4A1, CD79B, CD19, KIR2DS4, CD3E, CCR4, CCR8, and CD8A. Samples were classified into low ("sigClass low"), intermediate ("sigClass med"), and high ("sigClass hi") inflammation. Samples were also classified by the presence ("STK11 mut") or absence ("STK11 wt") of the STK11 mutation. In addition, the samples were classified according to their PD-L1 expression pattern into negative ("PDL1_Pattern 2 Negative"), scattered ("PDL1_Pattern 2 Scattered"), heterogeneous ("PDL1_Pattern 2 Heterogeneous"), and tumor-stromal junction ("PDL1_Pattern 2TS").

[0101] [Figure 12A] Figures 12A and 12B are graphical representations of survival probabilities for subjects with advanced NSCLC having wild-type STK11 (data labels 2 and 4) or mutant STK11 (data labels 1 and 3) treated with physician-selected chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). Figure 12A includes mutant STK11 subjects with all nonsynonymous mutations for STK11, while Figure 12B includes only mutant STK11 subjects with nonsense, frameshift, or splicing mutations for STK11. The number of subjects at risk at each time point for each group is shown below the X-axis. [Figure 12B] Same as above

[0102] [Figure 13]Figure 13 is a graphical representation of survival probabilities for subjects with progressive NSCLC who have KRAS mutations and wild-type STK11 (data labels 2 and 4) or mutant STK11 (data labels 1 and 3) (the subjects were treated with physician-selected chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4)). The number of subjects at risk at each time point for each group is shown below the X axis.

[0103] [Figure 14A] Figures 14A and 14B are graphical representations of survival probabilities for non-squamous NSCLC patients with wild-type STK11 (data labels 2 and 4) or STK11 (data labels 1 and 3) with non-synonymous mutations, treated with physician-selected chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). Figure 14A includes all patients meeting this criterion, while Figure 14B includes only patients with additional KRAS mutations. The number of patients at risk at each time point for each group is shown below the X-axis. [Figure 14B] Same as above

[0104] [Figure 15-1]Figures 15A-15E are graphical representations of the correlation between PD-L1 expression levels and STK11 mutation status. Figures 15A and 15B show the distribution of WT or mutant STK11 subjects who achieved complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) after treatment with physician-selected chemotherapy (Figure 15A) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; Figure 15B). Figure 15C is a graphical representation of the distribution of WT or mutant STK11 subjects who achieved complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) after treatment with physician-selected chemotherapy or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab) in relation to PD-L1 expression levels. Figures 15D and 15E show the distribution of PDL1 expression in WT or mutant STK11 subjects treated with physician-selected chemotherapy (Figure 15D) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; Figure 15E). The Y-axis shows the percentage of tumor cells expressing PD-L1 (Figures 15A-15E). Figure 15F is a table showing the status of specific mutations for a subset of subjects with STK11 mutations. [Figure 15-2] Same as above [Figure 15-3] Same as above [Figure 15-4] Same as above

[0105] [Figure 16A]Figure 16A is a graphical representation of survival probabilities for subjects with progressive NSCLC who have KRAS mutations and wild-type TP53 (data labels 2 and 4) or mutant TP53 (data labels 1 and 3) (the subjects were treated with physician-selected chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4)). Figures 16B-16C are graphical representations of survival probabilities for subjects with progressive NSCLC who have wild-type (data labels 2 and 4) or mutant (data labels 1 and 3) CDKN2A (Figure 16B) or PTPND / CUBN / HERC1 (Figure 16C) treated with physician-selected chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). The number of subjects at risk at each time point for each group is shown below the X axis (Figures 16A-16C). Figure 16D is a screenshot showing the distribution of HERC1, CUBNM, and PTPRD mutations in the 1144 subjects analyzed. [Figure 16B] Same as above [Figure 16C] Same as above [Figure 16D] Same as above

[0106] [Figure 17-1] Figures 17A-17B are distribution maps showing the association between tumor mutational load and STK11 mutation status in all analyzed progressive NSCLC subjects (Figure 17A) or subpopulations with additional KRAS mutations (Figure 17B). Figure 17C is a graph showing the distribution of WT or mutant STK11 subjects exhibiting complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) after treatment with physician-selected chemotherapy or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab) in relation to tumor mutational load (TMB; Figure 17C). [Figure 17-2] Same as above

[0107] [Figure 18A]Figures 18A–18D are graphical representations of survival probabilities for non-squamous NSCLC subjects with wild-type STK11 (data labels 2 and 4) or mutations in STK11 (data labels 1 and 3) treated with docetaxel (data labels 1 and 2) or a second-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). Figure 18A includes all subjects with either non-synonymous STK11 mutations; Figure 18B includes subjects with either non-synonymous STK11 mutations or KRAS mutations; Figure 18C includes all subjects with either nonsense, frameshift, or splicing mutations in STK11; and Figure 18D includes subjects with either nonsense, frameshift, or splicing mutations in either STK11 or KRAS. The number of subjects at risk at each time point for each group is shown below the X axis (Figures 18A–18D). [Figure 18B] Same as above [Figure 18C] Same as above [Figure 18D] Same as above

[0108] [Figure 19] Figure 19 is a graphical representation of survival probabilities for squamous cell NSCLC patients with wild-type STK11 (data labels 2 and 4) or STK11 mutations (data labels 1 and 3) treated with docetaxel (data labels 1 and 2) or a second-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). The number of at-risk patients at each time point for each group is shown below the X axis (Figure 19). [Modes for carrying out the invention]

[0109] This disclosure relates to a method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of the STK11 gene in the subject; and (ii) if the STK11 gene is wild-type, administering to the subject an antibody or its antigen-binding portion that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity ("anti-PD-1 antibody") or an antibody or its antigen-binding portion that specifically binds to programmed death-ligand 1 (PD-L1) and inhibits PD-1 activity ("anti-PD-L1 antibody"). In one embodiment, the tumor is derived from NSCLC.

[0110] term To make this disclosure more easily understandable, certain terms are first defined. Unless otherwise specifically provided herein, each of the following terms has the meaning set forth below, as used in this application. Further definitions are provided throughout this application.

[0111] "Administer" means the physical introduction of a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for anti-PD-1 antibodies include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration by injection or infusion. As used herein, "parenteral administration" usually means a mode of administration other than intestinal and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the combination is administered by a non-parenteral route, and in some embodiments, orally. Other non-parenteral routes include, for example, topical, epithelial, or mucosal routes of administration, such as intranasal, intravaginal, transrectal, sublingual, or topical. The administration can also be done, for example, once, multiple times, and / or once or more times over a long period of time.

[0112] As used herein, "adverse event" (AE) refers to an undesirable, generally unintended or unwanted sign (including abnormal findings on examination), symptom, or disease associated with the application of a medical treatment. For example, an adverse event may be associated with activation of the immune system or proliferation of immune system cells (e.g., T cells) in response to a treatment. A medical treatment may exhibit one or more associated AEs, and each AE may exhibit the same or different severity levels. A method that can "alter an adverse event" means a treatment plan that reduces the occurrence and / or severity of one or more AEs associated with the application of a different treatment plan.

[0113] "Antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen and contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or antigen-binding portions thereof. Each H chain includes a heavy chain variable region (abbreviated herein as V H and a heavy chain constant region. The heavy chain constant region includes at least three constant domains C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (abbreviated herein as V L and a light chain constant region. The light chain constant region includes one constant domain, C L . The V H and V L regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs) that incorporate highly conserved regions called framework regions (FRs). Each V H and V L includes three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains include a binding domain that interacts with an antigen. The constant region of an antibody can mediate binding to host tissues or factors of the immunoglobulin, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0114] Immunoglobulins may originate from any of the generally known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG subclass is also well known to those skilled in the art and includes, but not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" means an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, as an example, natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; totally synthetic Abs; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise indicated and the context indicates, the term "antibody" also includes any antigen-binding fragment or antigen-binding moiety of the immunoglobulins, including monovalent and bivalent fragments or parts, and single-chain antibodies.

[0115] "Isolated antibody" means an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PD-1 substantially does not contain antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may cross-react to other antigens, such as PD-1 molecules from different species. Furthermore, an isolated antibody may substantially not contain other cellular material and / or chemical substances.

[0116] The term “monoclonal antibody” (“mAb”) refers to an antibody molecule in a single molecular composition, i.e., a non-naturally occurring preparation of an antibody molecule whose major sequence is essentially identical and which exhibits binding specificity and affinity to a particular epitope. Monoclonal antibodies are an example of isolated antibodies. MAbs can be prepared by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0117] A “human” antibody (HuMAb) means an antibody having a variable region in which both the framework and CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this disclosure may include amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutagenesis in vivo). However, as used herein, the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted onto a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0118] A “humanized antibody” refers to an antibody in which any, almost, or all of the non-human antibody's CDR domain is replaced with a corresponding amino acid derived from human immunoglobulin. In one embodiment of the humanized form of an antibody, any, almost, or all of the non-human antibody's CDR domain is replaced with an amino acid derived from human immunoglobulin, while any, almost, or all of one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not cause the antibody to lose its ability to bind to a particular antigen. A “humanized” antibody retains antigenic properties similar to the antigen specificity of the original antibody.

[0119] A "chimeric antibody" is an antibody in which the variable region originates from one species and the constant region originates from another species; for example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0120] An "anti-antigen" antibody is an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1.

[0121] The "antigen-binding portion" (also called the "antigen-binding fragment") of an antibody refers to one or more fragments of the antibody that possess the ability to specifically bind to the antigen to which the entire antibody binds.

[0122] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells within the body. Uncontrolled cell division and proliferation can form malignant tumors that invade adjacent tissues and can even metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0123] "Serine / threonine kinase 11" or "STK11" (also known as "polarity-associated protein LKB1," "renal cancer antigen NY-REN-19," "hepatic kinase B1," "EC2.7.11.1," and "HLKB1") refers to a member of the serine / threonine kinase family that modulates cell polarity and functions as a tumor suppressor. STK11 regulates the activity of AMP-activated protein kinase (AMPK) family members, thereby playing a role in various processes such as cellular metabolism, cell polarity, apoptosis, and DNA damage response. STK11 is ubiquitously expressed and is most highly expressed in the testes and fetal liver. STK11 is generally inactivated in NSCLC, particularly tumors harboring KRAS mutations. As described herein, loss of mutant STK11, e.g., wild-type expression of STK11, correlates with decreased or abnormal PD-L1 expression in tumors derived from NSCLC. In one embodiment, loss of expression of a mutant STK11, for example, wild-type STK11, occurs in tumors derived from NSCLC, and the tumors either express or do not express wild-type KRAS (for example, the tumors either have or do not have a KRAS mutation). In one embodiment, the STK11 variant is an STK11 variant previously described, for example, in Koyama et al., Cancer Res. 76(5):999-1008 (2016), Skoulidis et al., Cancer Discov. 5(8):860-77 (2015), and / or Skoulidis et al., Cancer Disclov., May 17, 2018, DOI: 10.1158 / 2159-8290.CD-18-0099 (each of which is incorporated herein in its entirety with proper attribution).

[0124] "KRAS" refers to the gene encoding the GTPase KRAS protein, a member of the ras subfamily of small GTPases. Approximately 15–25% of patients with lung adenocarcinoma have tumor-associated KRAS mutations, the majority of which result in constitutive activity of KRAS signaling. As used herein, "TP53" refers to the gene encoding the tumor suppressor protein p53. p53 acts to regulate cell division, and loss-of-function mutations cause abnormal cell division and proliferation. Approximately half of all cancers contain somatic mutations in TP53. As used herein, "cyclin-dependent kinase inhibitor 2A" or "CDKN2A" refers to the gene encoding cyclin-dependent kinase inhibitor 2A, which acts as a tumor suppressor by inducing cell cycle arrest during G1 and G2 phases. Loss-of-function mutations in CDKN2A are common in lung cancer. As used herein, "CUBN" refers to the gene encoding cubin, the receptor for the intrinsic factor-vitamin B12 complex. As used herein, "HERC1" refers to the gene encoding member 1 (HERC1), a member of the HERC family containing HECT and RLD domains in the E3 ubiquitin protein ligase family. HERC1 can activate guanine nucleotide exchange in ARF1 and Rab proteins and may be involved in membrane transport processes.

[0125] The term “immunotherapy” means the treatment of a subject who is suffering from a disease, or who is at risk of suffering from a disease or relapse of a disease, by means of inducing, enhancing, suppressing, or modulating an immune response. “Treatment” or “therapy” of a subject means an intervention or method performed on a subject to alleviate, improve, suppress, delay, or prevent the onset, exacerbation, progression, severity, or relapse of disease-related symptoms, complications, conditions, or biochemical signs, or the administration of an active agent to such subject.

[0126] As used herein, “PD-L1 positive” can be used interchangeably with “at least about 1% PD-L1 expression.” In one embodiment, the PD-L1 expression can be used by any method known in the art. In another embodiment, the PD-L1 expression is measured by automated IHC. Thus, a PD-L1 positive tumor may represent at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of tumor cells expressing PD-L1 as measured by automated IHC. In one embodiment, “PD-L1 positive” means that there are at least 100 cells expressing PD-L1 on the cell surface.

[0127] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on T cells that have been activated in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants of hPD-1, isoforms, and species homologs, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank acceptance number U64863.

[0128] Programmed death ligand 1 (PD-L1) is one of two cell surface glycoprotein ligands to PD-1 that downregulate T cell activation and cytokine secretion by binding to PD-1 (the other being PD-L2). As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank acceptance number Q9NZQ7.

[0129] The term "subject" includes either humans or non-human animals. The term "non-human animals" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In one embodiment, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0130] The “therapeutic dose” or “therapeutic amount” of a drug or therapeutic agent is the amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or promotes disease regression, as indicated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or impairment due to the distress of the disease. The efficacy of a therapeutic agent that promotes disease regression can be evaluated using various methods known to those skilled in the art, for example, in human subjects in clinical trials, in animal model systems where efficacy in humans is predicted, or by measuring the activity of the drug in an in vitro assay.

[0131] As used herein, “sub-therapeutic dose” means a dose lower than the usual or typical dose of a therapeutic compound (e.g., an antibody) when administered alone for the treatment of a hyperproliferative disorder (e.g., cancer).

[0132] For example, an "anti-cancer agent" promotes the regression of cancer in a subject or inhibits further tumor growth. In one embodiment, a therapeutically effective dose of the drug promotes cancer regression until the cancer is eliminated. "Promotes cancer regression" means that administration of a therapeutically effective dose of the drug alone or in combination with an anti-cancer agent results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, or prevention of disability or impairment due to the distress of the disease. In addition, the therapeutic terms "effective" and "efficacy" include both pharmacological efficacy and physiological safety. Pharmacological efficacy means the ability of a drug to promote cancer regression in a patient. Physiological safety means toxicity levels or other adverse physiological effects (side effects) at the cellular, organ, and / or biological levels resulting from the administration of the drug.

[0133] As an example for the treatment of tumors, a therapeutically effective dose of an anticancer drug can inhibit cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to an untreated subject. In other embodiments of this disclosure, tumor regression may be observed and continued for at least about 20 days, at least about 40 days, or at least about 60 days. Despite these final measurements of therapeutic effect, the evaluation of immunotherapeutic drugs must also take into account “immune-related” response patterns.

[0134] The “immune-related” response pattern refers to a common clinical response pattern observed in cancer patients treated with immunotherapies that produce antitumor effects by inducing cancer-specific immune responses or by modulating endogenous immune processes. This response pattern is characterized by beneficial therapeutic effects following an initial increase in tumor tissue volume or the appearance of new lesions, which in conventional chemotherapy evaluations would be classified as disease progression and synonymous with drug failure. Therefore, proper evaluation of immunotherapies may require long-term monitoring of their effects on the targeted disease.

[0135] A therapeutically effective dose of a drug includes a “preventive effective dose,” which is the amount of drug administered alone or in combination with an antitumor agent to a subject at risk of developing cancer (e.g., a subject exhibiting a pre-malignant condition) or a subject at risk of cancer recurrence, that suppresses the development or recurrence of said cancer. In some embodiments, the preventive effective dose inhibits the development or recurrence of cancer as a whole. To “suppress” the development or recurrence of cancer means to reduce the likelihood of cancer progression or recurrence, or to inhibit the development or recurrence of cancer as a whole.

[0136] As used herein, the term “wild-type” means a gene having a nucleotide sequence that codes for a protein exhibiting an amino acid sequence known in the art as a common sequence. For example, in one embodiment, “wild-type” STK11 has a nucleotide sequence that codes for the STK11 protein having the same amino acid sequence as the amino acid sequence of the protein with UniProt identifier Q15831-1. In one embodiment, a “wild-type” gene may have a mutation in the conventional nucleotide sequence of the gene, as long as the mutation is synonymous (e.g., a nucleotide mutation that does not change the amino acid sequence of the resulting protein). Conversely, as used herein, a “mutant” or “mutant” gene means a gene having one or more nucleotide substitutions, insertions, or deletions that alter the amino acid sequence of the resulting protein (e.g., a nonsynonymous mutation). A mutant gene may or may not be expressible. In one embodiment, a nonsynonymous mutation is a “nonsense mutation” in which a nucleotide substitution or deletion results in an immature stop codon. In one embodiment, a non-synonymous mutation is a “frameshift mutation” in which a nucleotide substitution or deletion results in an insertion or deletion of many nucleotides that are not divisible by three, causing a shift in the translation of the sequence. In one embodiment, a non-synonymous mutation is a “splicing mutation” in which a nucleotide substitution or deletion interferes with or creates a splicing site.

[0137] As used herein, the term “tumor mutational load” (TMB) refers to the number of somatic mutations in the tumor genome and / or the number of somatic mutations per region of the tumor genome. Germline (genetic) variants are excluded when measuring TMB because their immune system is likely to recognize them as its own. TMB is a genetic analysis of the tumor genome and can be measured using sequencing methods well known to those skilled in the art. In one embodiment, TMB is measured using the total number of missense mutations in the tumor, identified by standardizing the tumor being compared with a germline sample to exclude heritable germline genetic variations. A sufficient amount of sample is required to measure TMB. In one embodiment, a tissue sample (e.g., a minimum of 10 sections) is used for evaluation.

[0138] The TMB state can be a numerical or relative value (e.g., high, medium, or low) within the highest quantile or tertile of the reference set.

[0139] As used herein, the term "high TMB" refers to the number of somatic mutations in the tumor genome that exceed the number of somatic mutations that are normal or average. In one embodiment, TMB is at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, and at least In one embodiment, high TMB scores of at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, or at least 500; in another embodiment, high TMB scores of at least 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, at least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249, or at least 250; in one embodiment, high TMB scores of at least 243. In another embodiment, "high TMB" means the TMB within the highest quantile of the reference TMB value.For example, the “reference TMB value” can be determined by grouping the values ​​of all subjects by evaluable TMB data according to the quantile distribution of TMB; that is, by ranking subjects from the highest to the lowest number of gene changes and classifying them into a specific number of groups. In one embodiment, all subjects with evaluable TMB data are ranked and classified into three categories, where “high TMB” is within the highest quantile of the reference TMB value. In one embodiment, the quartile boundaries are 0 < 100 gene changes; 100 to 243 gene changes; and > 243 gene changes. Once ranked, subjects with evaluable TMB data should be understood to be classifiable into one of the following number of groups (e.g., quartiles, quintiles, etc.).

[0140] As used herein, the term “intermediate TMB” means a number of somatic mutations in the tumor genome that is normal or average, and the term “low TMB” means a number of somatic mutations in the tumor genome that is less than normal or average. In one embodiment, “high TMB” is a score of at least 243, “intermediate TMB” is a score of 100 to 242, and “low TMB” is a score of 100 or less (or 0 to 100).

[0141] In one embodiment, TMB status may correlate with smoking status. In particular, subjects who currently smoke or have smoked in the past (d) often show more genetic changes, such as missense mutations, than subjects who do not smoke (d).

[0142] Tumors exhibiting high TMB may also exhibit high neoantigen levels. As used herein, the term “neoantigen” refers to a newly formed antigen that was not previously recognized by the immune system. Neoantigens can be proteins or peptides recognized by the immune system as foreign (or non-self). Transcription of a gene in the genome of a tumor carrying a somatic mutation produces mutant mRNA, which, upon translation, generates a mutant protein, which is subsequently processed, transported into the ER lumen, and then binds to the MHC class I complex, thereby facilitating T cell recognition of the neoantigen. Recognition of the neoantigen can promote T cell activation, clonal proliferation, and differentiation into effector and memory T cells.

[0143] Tumor TMB status can be used alone or in combination with other factors when determining whether a patient is likely to benefit from a particular anticancer agent or type of treatment or therapy, such as an immunotherapy agent, such as an anti-PD-1 antibody or its antigen-binding moiety, or an anti-PD-L1 antibody or its antigen-binding moiety. In one embodiment, a high TMB status (or high TMB) indicates a high likelihood of benefiting from immunotherapy, and can thus be used to identify patients who are more likely to benefit from treatment with an anti-PD-1 antibody or its antigen-binding moiety. As used herein, the term “benefiting from treatment” means improvement in one or more of the following: overall survival, progression-free survival, partial response, complete response, and overall response rate, and may include reduction in tumor growth or size, reduction in the severity of disease symptoms, increased frequency and duration of disease-free periods, or prevention of disability or impairment due to disease-related distress.

[0144] The terms “measure,” “measured,” or “measured” mean examining the measurable amount of somatic variation in the biological sample of interest, when referring to the TMB state or mutational state of a gene. Measurements are evaluated as being performed by sequencing nucleic acids in the sample, e.g., cDNA, mRNA, exoRNA, ctDNA, and cfDNA. Measurements are performed on the sample of interest and / or a reference sample, and may, for example, be newly detected or correspond to previous measurements. Measurements can be performed using, for example, PCR, qPCR, Sanger sequencing, genome profiling (including comprehensive gene panels), exome sequencing, genome sequencing, and / or other methods described herein, as are well known to those skilled in the art. In one embodiment, the measurement identifies genomic variations in sequenced nucleic acids. Genomic (or gene) profiling may involve a panel of a given set of genes, e.g., 150–500 genes, and in one example, the genomic variations evaluated by the gene panel correlate with the total somatic variation being evaluated.

[0145] As used herein, the term “genomic alteration” means a change (or mutation) in the nucleotide sequence of the tumor genome (such change is not present in the germline nucleotide sequence) and, in some embodiments, includes, but is not limited to, base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and any combination thereof. In some embodiments, the genomic alteration measured in a biological sample is a missense mutation.

[0146] As used herein, the term “biological sample” means a biological substance isolated from a subject. A biological sample may include, for example, a biological substance suitable for investigating TMB by sequencing nucleic acids in a tumor (or circulating tumor cells) and identifying genomic variations in the sequenced nucleic acids. The biological sample may be a suitable biological tissue or liquid, such as tumor tissue, blood, plasma, and serum. In one embodiment, the sample is a tumor tissue biopsy, such as formalin-fixed and paraffin-embedded tumor tissue or fresh-frozen tumor tissue. In another embodiment, the biological sample is, in one embodiment, a liquid biopsy containing one or more blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA, and cfDNA.

[0147] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of the listed or enumerated components.

[0148] The terms “about” or “essentially include” mean a numerical value or composition that is within the acceptable margin of error of a particular numerical value or composition as determined by a person skilled in the art, and in part by the method by which the numerical value or composition is measured or determined (i.e., the limits of the measurement system). For example, “about” or “essentially include” may mean within one or more standard deviations per single implementation in the art. Alternatively, “about” or “essentially include” may mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg may include any numerical value between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). Furthermore, particularly with respect to biological systems or processes, the term may mean up to one decimal place or up to five times the numerical value. Where a particular numerical value or composition is provided in this application and claims, unless otherwise specified, the meaning of “about” or “essentially include” should be within the acceptable margin of error of the particular numerical value or composition.

[0149] The terms “approximately once every week,” “approximately once every two weeks,” or other similar terms for dosing intervals as used herein mean approximate numbers. “Approximately once every week” may include every 7 days ± 1 day, i.e., every 6 days to every 8 days. “Approximately once every two weeks” may include every 14 days ± 3 days, i.e., every 11 days to every 17 days. Similar approximations apply, for example, to every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, and every 12 weeks. In one embodiment, a dosing interval of approximately every 6 weeks or every 12 weeks means that the first dose may be administered on any day in week 1, followed by the next dose on any day in week 6 or week 12, respectively. In other embodiments, an interval of approximately every 6 weeks or approximately every 12 weeks means that the first dose is administered on a specific day in the first week (e.g., Monday), followed by subsequent doses on the same day of the week in the sixth or twelfth week, respectively (i.e., Monday).

[0150] As used herein, the term "weight-based dose" means that the dose administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-PD-1 antibody, an appropriate amount of anti-PD-1 antibody (i.e., 180 mg) can be calculated and used for administration.

[0151] The use of the term “fixed dose” in the Methods of the Disclosure means that two or more different antibodies (e.g., anti-PD-1 antibody and a second antibody) in a single composition are present in the composition in particular in (fixed) ratios relative to each other. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In some 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 1:18 The ratios are approximately 1:200, 200:1, 180:1, 160:1, 140:1, 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or approximately 2:1 mg of the first antibody (e.g., anti-PD-1 antibody). For example, a 3:1 ratio of anti-PD-1 antibody to a second antibody may mean that the vial contains approximately 240 mg of anti-PD-1 antibody and 80 mg of the second antibody, or approximately 3 mg / ml of anti-PD-1 antibody and 1 mg / ml of the second antibody.

[0152] The use of the term "flat dose" in relation to the methods and dosages in this disclosure means a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Therefore, flat doses are provided as absolute amounts, not as doses of mg / kg of the drug (e.g., anti-PD-1 antibody). For example, a 60 kg person and a 100 kg person would receive the same dose of antibody (e.g., 240 mg of anti-PD-1 antibody).

[0153] As described herein, ranges of concentration, ranges of percentages, ranges of ratios, or ranges of integers should be understood to include integers, and where appropriate fractions thereof (e.g., one-tenth and one-hundredth of an integer), within the range described, unless otherwise specified.

[0154] Various aspects of this disclosure are described in further detail in the following sections.

[0155] Method of Disclosure This disclosure provides a method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering to the subject an antibody or its antigen-binding portion ("anti-PD-1 antibody") that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In one embodiment, if the subject suffering from the tumor (e.g., NSCLC, e.g., non-squamous NSCLC) has a wild-type STK11 gene, the anti-PD-1 antibody is administered. In one embodiment, this disclosure relates to a method for treating a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC), characterized by administering an anti-PD-1 antibody to the subject, wherein the subject has been identified as having a wild-type STK11 gene. In one embodiment, the present disclosure relates to a method for identifying subjects suffering from a tumor suitable for anti-PD-1 antibody therapy (e.g., NSCLC, e.g., non-squamous NSCLC), comprising: (i) determining the mutational status of the STK11 gene in the subject; and (ii) administering an anti-PD-1 antibody to the subject if the STK11 gene is wild-type.

[0156] Furthermore, the present disclosure provides a method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering to the subject an antibody or its antigen-binding portion ("anti-PD-L1 antibody") that specifically binds to programmed death-ligand 1 (PD-L1) and inhibits PD-1 activity. In one embodiment, if the subject suffering from the tumor (e.g., NSCLC, e.g., non-squamous NSCLC) has a wild-type STK11 gene, the anti-PD-L1 antibody is administered. In one embodiment, the present disclosure relates to a method for treating a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC), characterized by administering an anti-PD-L1 antibody to the subject, wherein the subject has been identified as having a wild-type STK11 gene. In one embodiment, the present disclosure relates to a method for identifying a subject suffering from a tumor suitable for anti-PD-L1 antibody therapy (e.g., NSCLC, e.g., non-squamous NSCLC), characterized by (i) determining the mutational status of the STK11 gene in the subject; and (ii) administering an anti-PD-L1 antibody to the subject if the STK11 gene is wild-type.

[0157] In another embodiment, the Disclosure relates to a method for treating a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC), the method comprising (i) determining the mutational status of the STK11 gene in the subject; and then (ii) identifying the subject, for example, not administering an anti-PD-1 antibody to the subject, or terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist, if the STK11 gene contains non-synonymous mutations. Another aspect of the present disclosure is a method for identifying subjects suffering from tumors unsuitable for anti-PD-1 antibody therapy (e.g., NSCLC, e.g., non-squamous NSCLC), comprising: (i) determining the mutation status of the STK11 gene in the subject; and then (ii) identifying the subject, if the STK11 gene contains non-synonymous mutations, by determining that the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody therapy, for example, by administering an anticancer agent that is not a PD-1 antagonist.

[0158] In another embodiment, the present disclosure relates to a method for treating a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC), characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) identifying the subject, for example, by not administering anti-PD-L1 antibodies to the subject, or by terminating or enhancing anti-PD-L1 antibody treatment, for example, by administering an anticancer agent that is not a PD-L1 antagonist, if the STK11 gene contains non-synonymous mutations. Another aspect of the present disclosure is a method for identifying subjects suffering from tumors unsuitable for anti-PD-L1 antibody therapy (e.g., NSCLC, e.g., non-squamous NSCLC), comprising: (i) determining the mutation status of the STK11 gene in the subject; and then (ii) identifying the subject, if the STK11 gene contains non-synonymous mutations, by determining that the subject is unsuitable for administration of anti-PD-L1 antibodies, for example, by not administering anti-PD-L1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody therapy, for example, by administering an anticancer agent that is not a PD-L1 antagonist.

[0159] In another aspect, the Disclosure relates to a method for treating a subject suffering from a tumor, characterized by (i) determining the mutation status of a marker gene in the subject; and then (ii) administering an anti-PD-1 antibody to the subject if the marker gene is mutated, wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Another aspect of the Disclosure relates to a method for treating a subject suffering from a tumor, characterized by administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a mutant marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Further other aspects of the present disclosure relate to a method for identifying subjects with tumors suitable for anti-PD-1 antibody therapy, comprising: (i) determining the mutation status of a marker gene in the subject; and (ii) administering an anti-PD-1 antibody to the subject if the marker gene is mutated; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0160] In another aspect, the Disclosure relates to a method for treating a subject with a tumor, characterized by (i) determining the mutation status of a marker gene in the subject; and (ii) administering an anti-PD-L1 antibody to the subject if the marker gene is mutated, wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Another aspect of the Disclosure relates to a method for treating a subject with a tumor, characterized by administering an anti-PD-L1 antibody to the subject, wherein the subject is identified as having a mutant marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Further other aspects of the present disclosure relate to a method for identifying subjects with tumors suitable for anti-PD-L1 antibody therapy, comprising: (i) determining mutations in a marker gene in the subject; and (ii) administering an anti-PD-L1 antibody to the subject if the marker gene is mutated; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0161] In one embodiment, the subject possesses a variant of TP53. In one embodiment, the subject possesses a variant of CDKN2A. In one embodiment, the subject possesses a variant of PTPND. In one embodiment, the subject possesses a variant of CUBN. In one embodiment, the subject possesses a variant of HERC1. In one embodiment, the subject possesses variants of PTPND and CUBN. In one embodiment, the subject possesses variants of PTPND and HERC1. In one embodiment, the subject possesses variants of CUBN and HERC1. In one embodiment, the subject possesses variants of PTPND, CUBN, and HERC1.

[0162] In one embodiment, the wild-type STK11 contains one or more synonymous mutations, the mutations in the genomic sequence do not affect the sequence of the expressed protein. In one embodiment, the mutant STK11 contains non-synonymous mutations. In one embodiment, the mutant STK11 contains nonsense mutations. In one embodiment, the mutant STK11 contains frameshift mutations. In one embodiment, the mutant STK11 contains splicing mutations. In one embodiment, the mutant STK11 is expressed as mRNA and protein. In one embodiment, the mutant STK11 protein is functional. In another embodiment, the mutant STK11 protein has reduced activity. In another embodiment, the mutant STK11 protein is nonfunctional.

[0163] In one embodiment, mutant TP53 includes nonsynonymous mutations. In one embodiment, mutant TP53 includes nonsense mutations. In one embodiment, mutant TP53 includes frameshift mutations. In one embodiment, mutant TP53 includes splicing mutations. In one embodiment, mutant TP53 is expressed as mRNA and protein. In one embodiment, the mutant TP53 protein is functional. In another embodiment, the mutant TP53 protein has reduced activity. In yet another embodiment, the mutant TP53 protein is nonfunctional.

[0164] In one embodiment, mutant CDKN2A contains nonsensical mutations. In another embodiment, mutant CDKN2A contains nonsense mutations. In another embodiment, mutant CDKN2A contains frameshift mutations. In another embodiment, mutant CDKN2A contains splicing mutations. In one embodiment, mutant CDKN2A is expressed as mRNA and protein. In one embodiment, the mutant CDKN2A protein is functional. In yet another embodiment, the mutant CDKN2A protein has reduced activity. In yet another embodiment, the mutant CDKN2A protein is nonfunctional.

[0165] In one embodiment, mutant PTPND includes nonsynonymous mutations. In one embodiment, mutant PTPND includes nonsense mutations. In one embodiment, mutant PTPND includes frameshift mutations. In one embodiment, mutant PTPND includes splicing mutations. In one embodiment, mutant PTPND is expressed as mRNA and protein. In one embodiment, the mutant PTPND protein is functional. In another embodiment, the mutant PTPND protein has reduced activity. In yet another embodiment, the mutant PTPND protein is nonfunctional.

[0166] In one embodiment, mutant CUBN includes nonsensical mutations. In another embodiment, mutant CUBN includes nonsense mutations. In another embodiment, mutant CUBN includes frameshift mutations. In another embodiment, mutant CUBN includes splicing mutations. In one embodiment, mutant CUBN is expressed as mRNA and protein. In one embodiment, the mutant CUBN protein is functional. In another embodiment, the mutant CUBN protein has reduced activity. In yet another embodiment, the mutant CUBN protein is nonfunctional.

[0167] In one embodiment, mutant HERC1 contains nonsensical mutations. In one embodiment, mutant HERC1 contains nonsense mutations. In one embodiment, mutant HERC1 contains frameshift mutations. In one embodiment, mutant HERC1 contains splicing mutations. In one embodiment, mutant HERC1 is expressed as mRNA and protein. In one embodiment, the mutant HERC1 protein is functional. In another embodiment, the mutant HERC1 protein has reduced activity. In another embodiment, the mutant HERC1 protein is nonfunctional.

[0168] In one embodiment, the tumor originates from lung cancer. In another embodiment, the tumor originates from NSCLC. In one embodiment, the subject is a human patient. In another embodiment, the subject is a chemotherapy-naive patient (e.g., a patient who has not received any chemotherapy). In yet another embodiment, the subject of the combination therapy of the present invention is a patient who has received another cancer treatment (e.g., chemotherapy) but is resistant to or refractory to such another cancer treatment.

[0169] In one embodiment, the disclosure provides a method for treating a subject with squamous cell NSCLC, characterized by (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering an anti-PD-1 antibody (or anti-PD-L1 antibody) to the subject if the STK11 gene is mutated. In one embodiment, the disclosure provides a method for treating a subject with squamous cell NSCLC, characterized by administering an anti-PD-1 antibody to the subject, wherein the subject has been identified as having a mutant STK11 gene. In one embodiment, the disclosure provides a method for identifying a subject with squamous cell NSCLC suitable for anti-PD-1 antibody therapy, comprising (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering an anti-PD-1 antibody to the subject if the STK11 gene is mutated.

[0170] In another embodiment, the present disclosure relates to a method for treating a subject suffering from squamous cellulosic NSCLC, characterized by (i) determining the mutational status of the STK11 gene in the subject; and (ii) if the STK11 gene contains nonsynonymous mutations, administering an anti-PD-1 antibody (or anti-PD-L1 antibody) to the subject, or terminating or enhancing anti-PD-1 antibody (or anti-PD-L1 antibody) treatment.

[0171] In some embodiments, the treatment of the Disclosure (e.g., administration of an anti-PD-1 antibody or an anti-PD-L1 antibody) effectively increases the survival of the subject. In some embodiments, the anti-PD-1 antibody treatment of the Disclosure increases the progression-free survival of the subject. In some embodiments, the anti-PD-1 antibody treatment of the Disclosure increases the progression-free survival of the subject compared to standard treatment. After administration of anti-PD-1 antibody treatment, subjects with tumors may exhibit an overall survival of at least approximately 10 months, at least approximately 11 months, at least approximately 12 months, at least approximately 13 months, at least approximately 14 months, at least approximately 15 months, at least approximately 16 months, at least approximately 17 months, at least approximately 18 months, at least approximately 19 months, at least approximately 20 months, at least approximately 21 months, at least approximately 22 months, at least approximately 23 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years.

[0172] In other embodiments, the survival or overall survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, or at least about 1 year compared to another subject treated with standard treatment (e.g., docetaxel) alone or with a different dosing schedule of said treatment. For example, the survival or overall survival of a subject treated with an anti-PD-1 antibody described herein is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 75% compared to another subject treated with standard treatment (e.g., docetaxel) alone or with a different dosing schedule of combination therapy.

[0173] In one embodiment, the treatments described herein effectively increase the progression-free survival of the subject. In one embodiment, the subject exhibits a progression-free survival of at least about 1 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 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0174] In one embodiment, administration of an anti-PD-1 antibody treats a tumor. In one embodiment, the administration reduces the tumor size. In one embodiment, the tumor size is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% compared to the tumor size before administration. In another embodiment, the subject exhibits a progression-free survival period of at least about 1 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 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the first administration. In one embodiment, the subject exhibits a stable disease state after administration. In one embodiment, the subject exhibits a partial response after administration. In another embodiment, the subject exhibits a complete response after administration. In yet another embodiment, the subject exhibits an improved objective response rate (ORR) after administration compared to a subject treated with standard care.

[0175] PD-L1 expression In one embodiment, the subject has tumor cells that are PD-L1+. In one embodiment, the subject has cancer cells that are PD-L1-. In one embodiment, the subject does not smoke at all. In one embodiment, the subject used to smoke. In one embodiment, the subject currently smokes. In one embodiment, the subject has squamous cancer cells. In one embodiment, the subject has non-squamous cancer cells.

[0176] In one embodiment, the tumor exhibits a scattered pattern of PD-L1 expression. In one embodiment, the scattered pattern of PD-L1 expression is characterized by H-scores of PD-L1 in the following ranges: approximately 60–500, approximately 70–490, approximately 80–480, approximately 90–470, approximately 100–460, approximately 110–450, approximately 120–440, approximately 130–430, approximately 140–420, approximately 150–410, approximately 160–400, approximately 170–390, approximately 180–380, approximately 190–370, approximately 200–360, approximately 20–350, approximately 200–340, approximately 200–330, approximately 200–320, approximately 200–310, or approximately 200–300. In one embodiment, the scattered pattern of PD-L1 expression is characterized by H-scores of at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, at least approximately 110, at least approximately 120, at least approximately 130, at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 225, at least approximately 230, at least approximately 240, at least approximately 250, at least approximately 260, at least approximately 270, at least approximately 275, at least approximately 280, at least approximately 290, or at least approximately 300 for PD-L1. In one embodiment, the scattered pattern of PD-L1 expression is characterized by H-scores of at least approximately 200 for PD-L1. In another embodiment, the scattered pattern of PD-L1 expression is characterized by the H-scores of at least approximately 240 PD-L1 cells. In one embodiment, the scattered pattern of PD-L1 expression is characterized by the H-scores of at least approximately 260 PD-L1 cells.

[0177] In one embodiment, the tumor exhibits a heterogeneous pattern of PD-L1 expression. In one embodiment, the heterogeneous pattern of PD-L1 expression is characterized by PD-L1 H-scores of approximately 1 to approximately 50, approximately 5 to approximately 45, approximately 10 to approximately 40, or approximately 15 to approximately 35, and the PD-L1 expression is limited to one or more different parts of the tumor. In one embodiment, the heterogeneous pattern of PD-L1 expression is characterized by PD-L1 H-scores of at least approximately 5, at least approximately 10, at least approximately 15, at least approximately 20, at least approximately 25, at least approximately 30, at least approximately 35, or at least approximately 40. In one embodiment, the heterogeneous pattern of PD-L1 expression is characterized by PD-L1 H-scores of at least approximately 15. In another embodiment, the heterogeneous pattern of PD-L1 expression is characterized by PD-L1 H-scores of at least approximately 20. In one embodiment, heterogeneous patterns of PD-L1 expression are characterized by a subset of tumors containing at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, or at least 150 PD-L1 cells expressing PD-L1. In one embodiment, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the tumor cells express PD-L1.

[0178] In one embodiment, the tumor exhibits PD-L1 expression at the tumor-stromal junction. In one embodiment, PD-L1 expression at the tumor-stromal junction is characterized by PD-L1 expression due to the proximity of tumor cells to the junction (e.g., approximately 1 cell diameter, 2 cell diameter, 3 cell diameter, 4 cell diameter, 5 cell diameter, 6 cell diameter, 7 cell diameter, 8 cell diameter, 9 cell diameter, or 10 cell diameter). In one embodiment, PD-L1 expression at the tumor-stromal junction is characterized by PD-L1 expression on the tumor surface.

[0179] The PD-L1 expression status of tumors in a subject can be measured before administering the compositions described herein or using the methods described herein. In one embodiment, the PD-L1 expression level of the tumor is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In another embodiment, the PD-L1 expression status of the tumor is at least about 1%. In another embodiment, the PD-L1 expression status of the tumor is at least about 5%. In one embodiment, the PD-L1 expression status of the tumor is at least about 10%. In another embodiment, the PD-L1 expression status of the tumor is at least about 25%. In a particular embodiment, the PD-L1 expression status of the tumor is at least about 50%.

[0180] In one embodiment, the method of the present disclosure (i) determines the mutation status of the STK11 gene in a subject affected by a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detects PD-L1 expression in the tumor; and then (iii) determines if the STK11 gene is wild-type and PD-L1 expression is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%. The present invention is characterized by administering an anti-PD-1 antibody to the subject if the percentage is at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In one embodiment, the present invention is characterized by (i) determining the mutation status of the STK11 gene in a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detecting PD-L1 expression in the tumor; and then (iii) administering an anti-PD-1 antibody to the subject if the STK11 gene is wild-type and the PD-L1 expression is at least about 15%. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detecting PD-L1 expression in the tumor; and then (iii) administering an anti-PD-1 antibody to the subject if the STK11 gene is wild-type and the PD-L1 expression is at least about 25%. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detecting PD-L1 expression in the tumor if the STK11 gene is wild-type and the PD-L1 expression is at least about 50%; and then (iii) administering an anti-PD-1 antibody to the subject.

[0181] In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject by determining that if the STK11 gene contains non-synonymous mutations and the PD-L1 expression in the tumor is about 50% or less, then the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); (ii) detecting PD-L1 expression in the tumor; and (iii) identifying a subject in which the STK11 gene contains non-synonymous mutations and the PD-L1 expression in the tumor is approximately 40% or less, and therefore the subject is deemed unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); and then (iii) identifying the subject by determining that if the STK11 gene contains non-synonymous mutations and the PD-L1 expression of the tumor is 30% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist.In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 25% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 20% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 15% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 10% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist.In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 5% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 3% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 2% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist. In one embodiment, the method of the present disclosure is characterized by (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and then (iii) identifying the subject by determining that if the STK11 gene contains nonsynonymous mutations and the PD-L1 expression in the tumor is about 1% or less, the subject is unsuitable for administration of anti-PD-1 antibodies, for example, by not administering anti-PD-1 antibodies to the subject, or by terminating or enhancing anti-PD-1 antibody treatment, for example, by administering an anticancer agent that is not a PD-1 antagonist.

[0182] In one embodiment, the tumor may exhibit a high level of inflammation. Increased inflammation can serve as an indicator of the scattered PD-L1 expression pattern. Therefore, high tumor inflammation can serve as an indicator of the response to anti-PD-1 antibody therapy.

[0183] Detection method One embodiment of this disclosure relates to determining the mutation status of one or more marker genes (e.g., STK11) in a subject. Methods known in the art can be used to determine whether the subject possesses a wild-type or mutant version of the marker gene. In one embodiment, the target marker gene is sequenced using a method available in the art, and the sequence of the marker gene is compared to known sequences of the same marker gene in the art. In one embodiment, the marker gene has a nonsensical mutation. In one embodiment, the marker gene has a nonsense mutation. In one embodiment, the marker gene has a frameshift mutation. In one embodiment, the marker gene has a splicing mutation. In one embodiment, the mutant marker gene is expressed. In another embodiment, the mutant marker gene is not expressed.

[0184] In one embodiment, the mutation status of a marker gene is determined by detecting the expression of the marker gene. In another embodiment, the mutant form of the target gene is not expressed, and the resulting deletion of mRNA and / or protein indicates the presence of the mutant. In yet another embodiment, the mutation status of a marker gene is determined by sequencing the resulting mRNA and / or protein. In yet another embodiment, the mutation status of a marker gene is determined by immunohistochemistry of the resulting protein.

[0185] In one embodiment, the subject has one wild-type copy and one mutant copy of the marker gene. In another embodiment, the subject has two wild-type copies of the marker gene and no mutant copies of the marker gene. In yet another embodiment, the subject has two mutant copies of the marker gene and no wild-type marker gene. In yet another embodiment, the two mutant copies of the marker gene are identical. In yet another embodiment, the subject has two different mutant copies of the marker gene.

[0186] One embodiment of this disclosure relates to determining and / or measuring the expression levels of one or more marker genes in a tumor of interest. To evaluate PD-L1 expression, in one embodiment, a test tissue sample may be obtained from a patient in need of treatment. In another embodiment, the evaluation of PD-L1 expression can be carried out without obtaining a test tissue sample. In one embodiment, the selection of a suitable patient includes (i) providing a test tissue sample obtained from a patient with cancer in the tissue, the test tissue sample containing tumor cells and / or tumor-infiltrating inflammatory cells; and then (ii) evaluating the percentage of cells in the test tissue sample expressing PD-L1 on the cell surface, based on the evaluation that the percentage of cells in the test tissue sample expressing PD-L1 on the cell surface is higher than a predetermined threshold.

[0187] However, in a method that includes measuring PD-L1 expression in a test tissue sample, the step of providing a test tissue sample obtained from a patient should be understood to be an optional step. Also, in some embodiments, the “measurement” or “evaluation” step for identifying or determining the number or percentage of cells in a test tissue sample that express PD-L1 (e.g., PD-L1 expression on the cell surface) is performed by a modified method for measuring PD-L1 expression, for example, by performing a reverse transcriptase polymerase chain reaction (RT-PCR) assay or an IHC assay. In other embodiments, without a modified method, PD-L1 expression is measured, for example, by reviewing a report of test results from a laboratory. In some embodiments, the steps of the method, including those up to and including the evaluation of PD-L1 expression, provide intermediate results that can be made available to a physician or other healthcare provider for use in selecting a suitable candidate for an anti-PD-1 antibody or anti-PD-L1 antibody therapy. In some embodiments, the step of providing such intermediate results is performed by a physician or another person performing the procedure under the direction of a physician. In other embodiments, these steps are performed by an independent laboratory or an independent person, for example, a laboratory technician.

[0188] In one embodiment of this method, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In a further embodiment, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection. In another embodiment, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In a further embodiment, the presence of PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In one embodiment, PD-L1 expression is assayed by IHC. In all other embodiments of these methods, cell surface expression of PD-L1 is assayed, for example, using IHC or in vivo imaging.

[0189] Imaging technologies provide essential tools in cancer research and treatment. Recent developments in molecular imaging systems, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence molecular tomography (FMT), bioluminescence imaging (BLI), laser scanning confocal microscopy (LSCM), and multiphoton microscopy (MPM), foreshadow further applications of these technologies in cancer research. Some of these molecular imaging systems enable clinicians to visualize not only the location of tumors within the body, but also the expression and activity of specific molecules, cells, and biological processes that influence tumor behavior and / or responsiveness to therapeutic agents (Condeelis and Weissleder, "In vivo imaging in cancer," Cold Spring Harb. Perspect. Biol. 2(12):a003848 (2010)). Regarding antibody specificity, the combination of PET sensitivity and resolution allows immunoPET imaging to monitor and assay antigen expression, particularly in tissue samples (McCabe and Wu, "Positive progress in immunoPET—not just a coincidence," Cancer Biother. Radiopharm. 25(3):253-61 (2010); Olafsen et al., "ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)," Protein Eng. Des. Sel. 23(4):243-9 (2010)). In any embodiment of this method, PD-L1 expression is assayed by immunoPET imaging. In any embodiment of this method, the percentage of cells in a test tissue sample expressing PD-L1 is evaluated by performing an assay to determine the presence of PD-L1 polypeptides on the cell surface of the test tissue sample. In some embodiments, the test tissue sample is an FFPE tissue sample.In another embodiment, the presence of the PD-L1 polypeptide is determined by an IHC assay. In a further embodiment, the IHC assay is performed using an automated method. In one embodiment, the IHC assay is performed using an anti-PD-L1 monoclonal antibody that binds to the PD-L1 polypeptide.

[0190] In one embodiment of this method, an automated IHC method is used to assay the expression of PD-L1 on the cell surface in FFPE tissue samples. This disclosure provides a method for detecting the presence of human PD-L1 antigen in a test tissue sample or for quantifying the level or percentage of human PD-L1 antigen in a sample expressing the antigen, the method comprising contacting the test sample and a negative control sample with a monoclonal antibody that specifically binds to human PD-L1 under conditions that allow for the formation of a complex between the antibody or a portion thereof and human PD-L1. In one embodiment, the test and control tissue samples are FFPE samples. The formation of the complex is then detected, and any difference in the formation of the complex between the test sample and the negative control sample indicates the presence of human PD-L1 antigen in the sample. Various methods are used to quantify PD-L1 expression.

[0191] In certain embodiments, an automated IHC method includes activating an automated staining apparatus which comprises the steps of (a) deparaffinizing and rehydrating embedded tissue sections in an automated staining apparatus; (b) activating the antigen using a decloaking chamber and pH 6 buffer heated to 110°C for 10 minutes; (c) placing reagents in the automated staining apparatus; then (d) neutralizing endogenous peroxidase in the tissue sample; blocking nonspecific protein binding sites on the slide; incubating the slide with primary Ab; incubating with a postprimary blocking agent; incubating with NovoLink polymer; adding a chromogen substrate and developing color; and then counterstaining with hematoxylin.

[0192] To assess PD-L1 expression in tumor tissue samples, pathologists count the number of membrane PD-L1+ tumor cells in each field of view under a microscope, estimate the percentage of positive cells mentally, and then average them to a final percentage. Different staining intensities are defined as 0 / negative, 1+ / weak, 2+ / intermediate, and 3+ / strong. Typically, percentage values ​​are assigned first to the 0 and 3+ buckets, then to the intermediate 1+ and 2+ intensities. For highly heterogeneous tissues, the sample is divided into sections, each section is scored separately, and then combined into a single set of percentage values. The percentages of negative and positive cells for each staining intensity are determined from each section, and the median is assigned to each section. Final percentage values ​​are assigned to the tissue for each staining intensity category (negative, 1+, 2+, and 3+). The sum of all staining intensities must be 100%. In one embodiment, the threshold number of cells required to be PD-L1 positive is at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200 cells. In one embodiment, the threshold number of cells required to be PD-L1 positive is at least about 100 cells.

[0193] Staining is also evaluated in tumor-infiltrating inflammatory cells (e.g., macrophages and lymphocytes). Macrophages serve as internal positive controls, as staining is often observed in the majority of macrophages. While staining with a 3+ intensity is not required, the absence of macrophage staining should not be considered a rule-of-failure. Macrophages and lymphocytes are evaluated for cell membrane staining and recorded only as positive or negative for each cell category for all samples. Staining is also characterized according to the naming convention of intratumoral / extratumoral immune cells. "Intratumoral" means that immune cells are located on the boundary of the tumor region without being physically inserted within the tumor tissue and / or between tumor cells. "Extratumoral" means that there is no physical association with the tumor, and immune cells are found in peripheral or adjacent tissues associated with connective tissue.

[0194] In one embodiment of the method for assigning these scores, the sample is scored by two separate pathologists, and these scores are later combined. In another embodiment, the identification of positive and negative cells is scored using appropriate software.

[0195] The organization score (histoscore) or "H-score" is used as a more quantitative measure of IHC data. The organization score is calculated as follows: Tissue score = [(% tumor x 1 (low intensity)) + (% tumor x 2 (intermediate intensity)) + (% tumor x 3 (high intensity)].

[0196] To determine the tissue score, pathologists estimate the percentage of stained cells in each intensity category within the sample. Because the expression of most biomarkers is heterogeneous, the tissue score is a more accurate representative of overall expression. The final tissue score ranges from 0 (no expression) to 300 (maximum expression).

[0197] Another method for quantifying PD-L1 expression in IHC test tissue samples is to determine the modified inflammation score (AIS), which is defined as the intensity of inflammation by multiplying it by the percentage of PD-L1 expression by tumor-infiltrating inflammatory cells (Taube et al., "Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape," Sci. Transl. Med. 4(127):127ra37 (2012)).

[0198] Tumor Mutation Bounds (TMB) Other aspects of this disclosure relate to measuring the TMB of tumor tissue obtained from a subject. As a tumor grows, somatic mutations that are not present in germline DNA accumulate. Tumor mutational load (TMB) means the number of somatic mutations in the tumor genome (after taking into account germline mutant DNA) and / or the number of somatic mutations per region of the tumor genome. The acquisition of somatic mutations, i.e., higher TMB, can be influenced by different mechanisms, such as exposure to exogenous mutation inducers (e.g., smoking or UV light exposure) and DNA mismatch repair mutations (e.g., MSI in colorectal and esophageal cancer). In solid tumors, approximately 95% of mutations are single nucleotide substitutions (Vogelstein et al., Science (2013) 339:1546-1558). As used herein, “non-synonymous mutation” means a nucleotide mutation that alters the amino acid sequence of a protein. Missense and nonsense mutations can both be non-synonymous mutations. In this specification, “missense mutation” means a non-synonymous point mutation in which a single nucleotide change results in a codon encoding a different amino acid. In this specification, “nonsense mutation” means a non-synonymous point mutation in which a codon is changed to an immature stop codon resulting in the cleavage of the resulting protein.

[0199] In one embodiment, somatic mutations may be expressed at the RNA and / or protein level, giving rise to neoantigens (also called neoepitopes). Neoantigens may influence immune-mediated antitumor responses. For example, recognition of a neoantigen may promote T cell activation, clonal proliferation, and differentiation into effector and memory T cells.

[0200] As a tumor grows, early clonal mutations (or "trunk mutations") can be carried to almost all or all tumor cells, while later mutations (or "branch mutations") can occur only in some tumor cells or regions (Yap et al., Sci Tranl Med (2012) 4:1-5; Jamai-Hanjani et al., (2015) Clin Cancer Res 21:1258-1266). As a result, neoantigens derived from clonal "trunk" mutations spread more extensively in the tumor genome than those from "branch" mutations, and therefore can generate many T cells that are reactive to clonal neoantigens (McGranahan et al., (2016) 351:1463-1469). In general, tumors with high TMB also have high neoantigen levels and can result in high oncoimmunogenicity and high T cell reactivity and antitumor response. Therefore, cancers with high TMB can respond well to immunotherapy, such as treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0201] Advances in sequencing technology enable the assessment of tumor genomic mutation status. Sequencing methods known to those skilled in the art can be used to sequence nucleic acids derived from tumor genomes (e.g., obtained from biological samples from subjects affected by tumors). In one embodiment, PCR or qPCR, Sanger sequencing, or next-generation sequencing (e.g., genomic profiling, exome sequencing, or genomic sequencing) can be used to measure TMB. In one embodiment, TMB status is measured using genomic profiling. Genomic profiling involves analyzing nucleic acids (including coding and non-coding regions) derived from tumor samples and can be performed using methods including selection of optimized nucleic acids to be incorporated, read alignment, and mutation calling. In one embodiment, gene profiling provides next-generation sequencing (NGS) based analysis of tumors, which can be optimized on a per-cancer, per-gene, and / or per-site basis. Genome profiling can incorporate the use of multiple individually modified alignment methods or algorithms to optimize performance in sequencing methods, particularly those involving massively parallel sequencing of numerous diverse genetic events across many different genes. Genome profiling provides comprehensive analysis of target cancer genomes at clinical quality, and the results of the genetic analysis can be relevant to the relevant scientific and medical knowledge for improving the quality and efficiency of cancer treatment.

[0202] Genome profiling relates to a predetermined set of gene panels containing only 5 genes, 1000 genes, approximately 25 to 750 genes, approximately 100 to 800 genes, approximately 150 to 500 genes, approximately 200 to 400 genes, or approximately 250 to 350 genes. In one embodiment, the genome profile includes at least 300 genes, at least 305 genes, at least 310 genes, at least 315 genes, at least 320 genes, at least 325 genes, at least 330 genes, at least 335 genes, at least 340 genes, at least 345 genes, at least 350 genes, at least 355 genes, at least 360 genes, at least 365 genes, at least 370 genes, at least 375 genes, at least 380 genes, at least 385 genes, at least 390 genes, at least 395 genes, or at least 400 genes. In another embodiment, the genome profile includes at least 325 genes. In certain embodiments, the genome profile includes at least 315 cancer-related genes and 28 gene introns (FOUNDATIONONE®), or the whole DNA coding sequences of 406 genes, 31 rearranged gene introns, and 265 gene RNA sequences (cDNA) (FOUNDATIONONE® Heme). In another embodiment, the genome profile includes 26 genes and 1000 associated mutations (EXODX® Solid Tumor). In yet another embodiment, the genome profile includes 76 genes (Guardant360). In yet another embodiment, the genome profile includes 73 genes (Guardant360). In yet another embodiment, the genome profile includes 354 genes and 28 gene introns for rearrangement (FOUNDATIONONE® CDX™). In one embodiment, the genome profile is FOUNDATIONONE® F1CDx.In another embodiment, the genome profile includes 468 genes (MSK-IMPACT™). One or more genes may be added to the genome profile as further genes identified as being related to oncology.

[0203] In yet another specific embodiment, the genome profiling detects all variants, i.e., single-nucleotide variants, insertions / deletions (indels), copy number variations, and rearrangements, such as translocations, expression, and epigenetic markers.

[0204] A comprehensive gene panel may include predetermined genes selected based on the type of tumor being analyzed. Therefore, the genomic profile used to measure TMB status can be selected based on the type of tumor the subject is affected by. In one embodiment, the genomic profile may include a set of genes specifically for solid tumors. In another embodiment, the genomic profile may include a set of genes specifically for hematological malignancies and sarcomas.

[0205] ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, and 1 of the 1-year-old manufacturer are ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF. PDCD1LG2, RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1 DGFRA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RI CTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf39), KAT6A(MYST3), MR E11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLI1, KDM5A, MSH2, PIK3 C2B, ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PIK3CA, RPT OR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, A MER1(FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RU NX1T1, TERT(APC, CARD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PI K3R1, SDHA, TET2, AR, CBFB, CTNNB1, FGF10, GPR124, KEL, MYCL(MYCL1), PIK 3R2, SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC TNFAIP3, ARFRP1, CCND1, CYLD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNF RSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1, SETD2 TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, T OP2A, ARID2, CCNE1, DICER1, FGF4, HGF, KMT2D(MLL2), NFE2L2, PPP2R1A, SL IT2, TP53, ASXL1, CD274, DNMT3A, FGF6, HNF1A, KRAS, NFKBIA, PRDM1, SMAD2.TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKAR1A, SMAD4, T SHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMA RCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1, NRAS, PTCH1, SNCAIP, WI SP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2, NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO1, BARD 1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1 , CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2 C, ESR1, FUBP1, IRF2, MED12, PALB2, RAF1, SPTA1, BCOR, CEBPA, EZH2, GABRA6, IRF4, MEF2B, PARK2, RANBP2, SRC, BCORL1, CHD2, FAM46C, GATA1, IRS2, MEN1, PAX5, RARA, STAG2, BLM, CHD4, FANCA, GATA2, JAK1, MET, PBRM1, RB1, STAT3, and one or more genes selected from the group consisting of any combination thereof. In other embodiments, the TMB analysis includes ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6,and further include identifying genomic alterations in one or more of the MYBs. In other embodiments, the mutational status of the SKT11 gene can be assessed as part of the TMB analysis or as described above.

[0206] In this embodiment, the TMB status based on genome profiling correlates strongly with the TMB status based on whole exome sequencing or whole genome sequencing.

[0207] TMB can be measured using tissue biopsy samples or ctDNA and / or liquid biopsy samples. ctDNA can be used to measure TMB status according to whole exome sequencing, whole genome sequencing, or genomic profiling using available methods (e.g., GRAIL).

[0208] TMB status can be used alone or in combination with other factors as a means of predicting tumor responsiveness to treatment, particularly treatment with cancer immunotherapy agents (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies). In one embodiment, tumor TMB status alone is used to identify patients with tumors that are more likely to respond to treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies. In another embodiment, PD-L1 status and TMB status are used to identify patients with tumors that are more likely to respond to treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0209] In one embodiment, the method of the present disclosure further includes measuring the TMB status of a subject before administering an anti-PD-1 antibody. In one embodiment, the method includes administering the anti-PD-1 antibody to a subject that harbors wild-type STK11 and exhibits a high TMB status. In another embodiment, the method includes refraining from administering the anti-PD-1 antibody to the subject, or discontinuing or enhancing anti-PD-1 antibody therapy, if the subject harbors a mutant form of STK11 and exhibits a high TMB status. In one embodiment, the method of the present disclosure further includes measuring the TMB status of a subject before administering an anti-PD-1 antibody. In one embodiment, the method includes administering the anti-PD-1 antibody to a subject that harbors wild-type STK11 and exhibits an intermediate TMB status. In another embodiment, the method includes refraining from administering the anti-PD-1 antibody to the subject, or discontinuing or enhancing anti-PD-1 antibody therapy, if the subject harbors a mutant form of STK11 and exhibits an intermediate TMB status. In other embodiments, the method includes refraining from administering an anti-PD-1 antibody to a subject if the subject harbors a variant of STK11 and exhibits a low TMB state, or terminating or enhancing anti-PD-1 antibody therapy.

[0210] FOUNDATIONONE® Assay The FOUNDATIONONE® assay is a comprehensive genomic profiling assay for solid tumors, including solid tumors of the lung, colon, and breast, melanoma, and ovarian cancer, but is not limited to those listed below. The FOUNDATIONONE® assay uses a hybrid capture next-generation sequencing method to identify genomic changes (base substitutions, insertions and deletions, copy number variations, and rearrangements) and select genomic characteristics (e.g., TMB and microsatellite instability). The assay covers 322 unique genes (including the full coding regions of 315 cancer-related genes and selected introns derived from 28 genes). A complete list of FOUNDATIONONE® assay genes is provided in Tables 1 and 2. See FOUNDATIONONE:Technical Specification (Foundation Medicine, Inc.), available at FoundationMedicine.com, last accessed March 16, 2018 (its entirety is incorporated herein by attribution). Table 1: List of genes whose entire coding sequence is assayed using the FOUNDATIONONE® assay. [Table 1] TIFF2026053613000002.tif56125 Table 2: List of genes whose selected introns are assayed in the FOUNDATIONONE® assay. [Table 2]

[0211] FOUNDATIONONE® Heme Assay The FOUNDATIONONE® Heme assay is a comprehensive genomic profiling assay for hematological malignancies and sarcomas. The FOUNDATIONONE® Heme assay uses hybrid capture next-generation sequencing to identify genomic alterations (base substitutions, insertions and deletions, copy number variations, and rearrangements) and select genomic characteristics (e.g., TMB and microsatellite instability). The assay analyzes the coding regions of 406 genes, selected introns of 31 genes, and RNA sequences of 265 genes commonly rearranged in cancer. A complete list of FOUNDATIONONE® Heme assay genes is provided in Tables 3, 4, and 5. See also the FOUNDATIONONE® HEME:Technical Specification (Foundation Medicine, Inc.), available at FoundationMedicine.com (last accessed March 16, 2018) (its entirety is incorporated herein by attribution). Table 3: List of genes whose entire coding sequence is assayed using the FOUNDATIONONE® Heme assay. [Table 3] TIFF2026053613000005.tif188133 TIFF2026053613000006.tif11133 Table 4: List of genes in which selected introns are assayed using the FOUNDATIONONE® Heme assay. [Table 4] Table 5: List of genes whose RNA sequences are assayed using the FOUNDATIONONE® Heme assay. [Table 5] TIFF2026053613000009.tif26128

[0212] EXODX® Solid Tumor Assay In one embodiment, TMB is measured using the EXODX® Solid Tumor Assay. The EXODX® Solid Tumor Assay is an exoRNA and cfDNA-based assay that detects actionable mutations in the cancer pathway. The EXODX® Solid Tumor Assay is a plasma-based assay that does not require tissue samples. The EXODX® Solid Tumor Assay covers 26 genes and 1000 mutations. The specific genes covered by the EXODX® Solid Tumor Assay are shown in Table 6. See the plasma-based solid tumor mutation panel available at exosomedx.com, last visited March 16, 2018 (Exosome Diagnostics, Inc.). Table 6: Genes covered by the EXODX® solid tumor assay. [Table 6]

[0213] Guardant360 assay In one embodiment, TMB status is determined using the Guardant360 assay. The Guardant360 assay measures mutations in at least 73 genes (Table 7), 23 indels (Table 8), 18 CNVs (Table 9), and 6 fusion genes (Table 10). See GuardantHealth.com, last accessed March 16, 2018. Table 7: Guardant360 assay genes. [Table 7] Table 8: Indels from the Guardant360 assay. [Table 8] Table 9: Guardant360 assay amplification (CNV). [Table 9] Table 10: Guardant360 assay fusion. [Table 10]

[0214] ILLUMINA® TruSight assay In one embodiment, TMB is determined using the TruSight Tumor 170 assay (ILLUMINA®). The TruSight Tumor 170 assay is a next-generation sequencing assay that covers 170 genes associated with common solid tumors, simultaneously analyzing DNA and RNA. The TruSight Tumor 170 assay evaluates fusions, splice variants, insertions / deletions, single-nucleotide variants (SNVs), and amplifications. The list of genes for the TruSight Tumor 170 assay is shown in Tables 11-13. Table 11: TruSight tumor 170 assay genes (amplification). [Table 11] Table 12: TruSight tumor 170 assay genes (fusions). [Table 12] Table 13: TruSight tumor 170 assay genes (minor mutations). [Table 13]

[0215] FOUNDATIONONE® F1CDx Assay FOUNDATIONONE® CDX™ ("F1CDx") is a next-generation sequencing-based in vitro diagnostic device for detecting 324 genes and selected gene rearrangements, as well as substitution, insertion, and deletion changes (indels) and copy number changes (CNAs) in genomic characteristics (including microsatellite instability (MSI) and tumor mutational burden (TMB)), using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. F1CDx is approved by the U.S. Food and Drug Administration (FDA) for use in several tumors, including NSCLC, melanoma, breast cancer, colorectal cancer, and ovarian cancer.

[0216] The F1CDx assay uses a single-pass DNA extraction method from routine FFPE biopsy or surgical excision specimens, of which 50–1000 ng are captured based on the construction and hybridization of a whole-genome shotgun library of selected intron regions from 309 cancer-related genes (all coding exons, one promoter region, one non-coding RNA), and 34 commonly rearranged genes (21 of which contain coding exons). Tables 14 and 15 provide a complete list of genes included in F1CDx. In total, the assay detects alterations in a total of 324 genes. Using the ILLUMINA® HiSeq4000 platform, the libraries selected by hybrid capture are sequenced with high uniform depth (targeting a central coverage of >500X with >99% of exons at coverage >100X). The sequence data is then processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number changes (amplification and homozygous gene deletions), and selected genomic rearrangements (e.g., gene fusions). Furthermore, genomic characteristics (including microsatellite instability (MSI) and tumor mutational burden (TMB)) are reported. Table 14: Genes containing the entire coding exon region included in FOUNDATIONONE® CDX® for the detection of substitutions, insertions and deletions (indels), and copy number changes (CNAs). [Table 14] Table 15: Selected genes with intron regions for detection of gene rearrangements, one gene with a 3'UTR, one gene with a promoter region, and one ncRNA gene. [Table 15]

[0217] The F1CDx assay identifies various alterations in gene and / or intron sequences, including substitutions, insertions / deletions, and CNAs. The F1CDx assay has been identified as consistent with previously externally validated NGS assays and FOUNDATIONONE®(F1 LDT) assays. See FOUNDATIONONE®CDX®: Technical Information (Foundation Medicine, Inc.) (in its entirety incorporated herein with due attribution), available on FoundationMedicine.com, last accessed March 16, 2018.

[0218] MSK-IMPACT™ In one embodiment, TMB status is assayed using the MSK-IMPACT® assay. The MSK-IMPACT® assay uses next-generation sequencing to analyze the mutation status of 468 genes. Target genes are captured and sequenced using an ILLUMINA® HISEQ® instrument. The MSK-IMPACT® assay is approved by the U.S. FDA for the detection of somatic mutations and microsatellite instability in solid malignancies. A complete list of the 468 genes analyzed by the MSK-IMPACT® assay is shown in Table 16. See Evaluation of Automatic Class III Designation for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets): Decision Summary (U.S. Food and Drug Administration), available November 15, 2017, at accessdata.fda.gov. Table 16: Genes analyzed by the MSK-IMPACT® assay. [Table 16]

[0219] NEOGENICS® NEOTYPE® Assay In one embodiment, the TMB is determined using the NEOGENICS® NEOTYOPE® assay. In another embodiment, the TMB is determined using the NEOTYPE® discovery profile. In yet another embodiment, the TMB is determined using the NEOTYPE® solid tumor profile. The NEOGENICS® assay measures the number of non-synonymous DNA coding sequence changes per megabase of sequenced DNA.

[0220] ONCOMINE® Tumor Mutagenesis Assay In one embodiment, TMB is determined using the THERMOPISHER SCIENTIFIC® ONCOMINE® tumor mutation assay. The THERMOPISHER SCIENTIFIC® ION TORRENT® ONCOMINE® tumor mutation assay is a targeted NGS assay that quantifies somatic mutations to determine tumor mutation levels. The assay covers 1.7 Mb of DNA.

[0221] NOVOGENE® NOVOPM® ASA In one embodiment, TMB is determined using the NOVOGENE® NOVOPM® assay. In another embodiment, TMB is determined using the NOVOGENE® NOVOPM® cancer panel assay. The NOVOGENE® NOVOPM® cancer panel assay analyzes the entire coding regions of 548 genes and the introns (representing approximately 1.5 Mb of DNA) of 21 genes, and is a comprehensive NGS cancer panel suitable for the diagnosis and / or treatment of solid tumors according to the National Comprehensive Cancer Information Network (NCCN) guidelines and medical literature. The assay detects genomic abnormalities such as SNVs, indels, fusions, and copy number variations (CNVs).

[0222] Other TMB assays In one embodiment, TMB is determined using a TMB assay provided by CARIS® Life Sciences. In another embodiment, TMB is determined using a PESONALIS® ACEIMMUNOID assay. In yet another embodiment, TMB is determined using a PGDX® CANCERXOME®-R assay.

[0223] In yet another specific embodiment, the genome profiling detects all variants, i.e., single-nucleotide variants, insertions / deletions (indels), copy number variations, and rearrangements, such as translocations, expression, and epigenetic markers.

[0224] A comprehensive gene panel often includes predetermined genes selected based on the tumor type being analyzed. Therefore, the genomic profile used to measure TMB status can be selected based on the tumor type the subject is affected by. In one embodiment, the genomic profile may include a set of genes specifically for solid tumors. In other embodiments, the genomic profile may include a set of genes specifically for hematological malignancies and sarcomas.

[0225] ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, and 1 of the 1-year-old manufacturer are ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF. PDCD1LG2, RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1 DGFRA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RI CTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf39), KAT6A(MYST3), MR E11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLI1, KDM5A, MSH2, PIK3 C2B, ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PIK3CA, RPT OR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, A MER1(FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RU NX1T1, TERT(APC, CARD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PI K3R1, SDHA, TET2, AR, CBFB, CTNNB1, FGF10, GPR124, KEL, MYCL(MYCL1), PIK 3R2, SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC TNFAIP3, ARFRP1, CCND1, CYLD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNF RSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1, SETD2 TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, T OP2A, ARID2, CCNE1, DICER1, FGF4, HGF, KMT2D(MLL2), NFE2L2, PPP2R1A, SL IT2, TP53, ASXL1, CD274, DNMT3A, FGF6, HNF1A, KRAS, NFKBIA, PRDM1, SMAD2.TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKAR1A, SMAD4, T SHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMA RCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1, NRAS, PTCH1, SNCAIP, WI SP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2, NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO1, BARD 1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1 , CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2 C, ESR1, FUBP1, IRF2, MED12, PALB2, RAF1, SPTA1, BCOR, CEBPA, EZH2, GABRA6, IRF4, MEF2B, PARK2, RANBP2, SRC, BCORL1, CHD2, FAM46C, GATA1, IRS2, MEN1, PAX5, RARA, STAG2, BLM, CHD4, FANCA, GATA2, JAK1, MET, PBRM1, RB1, STAT3, and one or more genes selected from the group consisting of any combination thereof. In other embodiments, TMB analysis includes ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6,and further include identifying genomic alterations in one or more of the MYBs.

[0226] In another embodiment, the genome profile includes ABL1, 12B, ABL2, ACTB, ACVR1, ACVR1B, AGO2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, AMER1(FAM123B or WTX), AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1, ARHGAP26(GRAF), ARID1A, ARID1B, ARID2, ARID5B, ARv7, ASMTL, ASXL1, ASXL2, ATM, ATR, ATRX, AURK A, AURKB, AXIN1, AXIN2, AXL, B2M, BABAM1, BAP1, BARD1, BBC3, BCL10, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, B MPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BRIP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD1 1, CARM1, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC42, CDC73, CDH 1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBPA, CENPA, CHD2, CHD4, CHEK1, CHEK2, CIC, C IITA, CKS1B, CPS1, CREBBP, CRKL, CRLF2, CSDE1, CSF1R, CSF3R, CTCF, CTLA-4, CTNNB1, CTNNA1, CTNNB1, CUL3, CUL4A, CUX1, CXCR4, CYLD, CYP17A1, CYSLT R2, DAXX, DCUN1D1, DDR1, DDR2, DDX3X, DH2, DICER1, DIS3, DNAJB1, DNM2, DNMT1, DNMT3A, DNMT3B, DOT1L, DROSHA, DTX1, DUSP2, DUSP4, DUSP9, E2F3, EBF1,ECT2L, EED, EGFL7, EGFR, EIF1AX, EIF4A2, EIF4E, ELF3, ELP2, EML4, EML4-A LK, EP300, EPAS1, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2, ERBB3 ERBB4, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERF, ERG, ERRFI1, ERRFl1, ESR 1, ETS1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXOSC6, EZH1, EZH2, FAF1, FAM175A FAM46C, FAM58A, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL FAS, FAS(TNFRSF6), FAT1, FBXO11, FBXO31, FBXW7, FGF1, FGF10, FGF12, FG F14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGFR1 FGFR2, FGFR3, FGFR4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH1, FOXA 1. FOXL2, FOXO1, FOXO3, FOXP1, FRS2, FUBP1, FYN, GABRA6, GADD45B, GATA1 GATA2、GATA3、GATA4、GATA6、GEN1、GID4(C17orf39)、GID4(C17orf39)、GLI 1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRIN2A, GR M3, GSK3B, GTSE1, H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, Hedgehog, HER -2 / NEU;ERBB2、HGF、HIST1H1C、HIST1H1D、HIST1H1E、HIST1H2AC、HIST1H2A G, HIST1H2AL, HIST1H2AM, HIST1H2BC, HIST1H2BD, HIST1H2BJ, HIST1H2BK, H IST1H2BO、HIST1H3A、HIST1H3B、HIST1H3C、HIST1H3D、HIST1H3E、HIST1H3F HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HIST2H3C, HIST2H3D, HIST3H3HLA-A, HLA-B, HNF1A, HOXB13, HRAS, HSD3B1, HSP90AA1, IK, ICOSLG, ID3, IDH1, IDH2, IFNGR1, IGF1, IGF1R, IGF2, IKBKE, IKZF1, IKZF2, IKZF3, IL10, I L7R、INHA、INHBA、INPP4A、INPP4B、INPP5D(SHIP)、INPPL1、INSR、IRF1、IRF 2、IRF4、IRF8、IRS1、IRS2、JAK1、JAK2、JAK3、JARID2、JUN、K14、KAT6A(MYST3) )、KAT6A(MYST3)、KDM2B、KDM4C、KDM5A、KDM5C、KDM6A、KDR、KEAP1、KEL、KIF5B、KIT、KLF4、KLHL6、KMT2A、KMT2A(MLL)、KMT2B、KMT2C、KMT2C(MLL3)、KMT2 D、KMT2D(MLL2)、KNSTRN、KRAS、LAMP1、LATS1、LATS2、LEF1、LMO1、LRP1B、LR RK2、LTK、LYN、LZTR1、MAF、MAFB、MAGED1、MAGI2、MALT1、MAP2K1、MAP2K1(MEK) 1)、MAP2K2、MAP2K2(MEK2)、MAP2K4、MAP3、MAP3K1、MAP3K13、MAP3K14、MAP3 K6、MAP3K7、MAPK1、MAPK3、MAPKAP1、MAX、MCL1、MDC1、MDM2、MDM4、MED12、MEF 2B、MEF2C、MEK1、MEN1、MERTK、MET、MGA、MIB1、MITF、MKI67、MKNK1、MLH1、ML LT3、MPL、MRE11A、MRE11A、MSH2、MSH3、MSH6、MSI1、MSI2、MST1、MST1R、MTAP、 MTOR、MUTYH、MYC、MYCL、MYCL(MYCL1)、MYCL(MYCL1)、MYCL1、MYCN、MYD88、M YO18A、MYOD1、NBN、NCOA3、NCOR1、NCOR2、NCSTN、NEGR1、NF1、NF2、NFE2L2、NF KBIA、NKX2-1、NKX3-1、NOD1、NOTCH1、NOTCH2、NOTCH3、NOTCH4、NPM1、NRAS、 NRG1、NSD1、NT5C2、NTHL1、NTRK1、NTRK2、NTRK3、NUF2、NUP93、NUP98、P2RY8、PAG1、PAK1、PAK3、PAK7、PALB2、PARK2、PARP1、PARP2、PARP3、PASK、PAX3、PAX5、PAX7、PBRM1、PC、PCBP1、PCLO、PDCD1、PDCD1(PD-1)、PDCD11、PDCD1LG2、PDCD1LG2(PD-L2)、PDGFRA、PDGFRB、PDK1、PDPK1、PGR、PHF6、PHOX2B、PIK3C2B、PIK3C2G、PIK3C3、PIK3CA、PIK3CB、PIK3CD、PIK3CG、PIK3R1、PIK3R2、PIK3R3、PIM1、PLCG2、PLK2、PMAIP1、PMS1、PMS2、PNRC1、POLD1、POLE、POT1、PPARG、PPM1D、PPP2、PPP2R1A、PPP2R2A、PPP4R2、PPP6C、PRDM1、PRDM14、PREX2、PRKAR1A、PRKCI、PRKD1、PRKDC、PRSS8、PTCH1、PTEN、PTP4A1、PTPN11、PTPN2、PTPN6(SHP-1)、PTPRD、PTPRO、PTPRS、PTPRT、QKI、R1A、RAB35、RAC1、RAC2、RAD21、RAD50、RAD51、RAD51B、RAD51C、RAD51D、RAD52、RAD54L、RAF1、RANBP2、RARA、RASA1、RASGEF1A、RB1、RBM10、RECQL、RECQL4、REL、RELN、RET、RFWD2、RHEB、RHOA、RICTOR、RIT1、RNF43、ROS1、RPS6KA4、RPS6KB1、RPS6KB2、RPTOR、RRAGC、RRAS、RRAS2、RTEL1、RUNX1、RUNX1T1、RXRA、RYBP、S1PR2、SDHA、SDHAF2、SDHB、SDHC、SDHD、SERP2、SESN1、SESN2、SESN3、SETBP1、SETD2、SETD8、SF3B1、SGK1、SH2B3、SH2D1A、SHOC2、SHQ1、SLIT2、SLX4、SMAD2、SMAD3、SMAD4、SMARCA1、SMARCA4、SMARCB1、SMARCD1、SMC1A、SMC3、SMO、SMYD3、SNCAIP、SOCS1、SOCS2、SOCS3、SOS1、SOX10、SOX17、SOX2、SOX9、SPEN、SPOP、SPRED1、SPTA1、SRC, SRSF2, STAG2, STAT3, STAT4, STAT5A, STAT5B, STAT6, STK11, STK19, STK40, SUFU, SUZ12, SYK, TAF1, TAP1, TAP2, TBL1XR1, TBX3, TCEB1, TCF3, TCF3(E2A), TCF7L2, TCL1A(TCL1), T EK, TERC, TERT, TERT promoter, TET1, TET2, TFRC, TGFBR1, TGFBR2, TIPARP, TLL2, TMEM127, TMEM30A, TMPRSS2, TMSB4XP8(TMSL3), TNFAIP3, TNFRSF11A, TNFRSF14, TNFRSF17, TOP1, TOP2 It includes one or more genes selected from the group consisting of A, TP53, TP53BP1, TP63, TRAF2, TRAF3, TRAF5, TRAF7, TSC1, TSC2, TSHR, TUSC3, TYK2, TYRO3, U2AF1, U2AF2, UPF1, VEGFA, VHL, VTCN1, WDR90, WHSC1, WHSC1(MMSETorNSD2), WHSC1L1, WISP3, WT1, WWTR1, XBP1, XIAP, XPO1, XRCC2, YAP1, YES1, YY1AP1, ZBTB2, ZFHX3, ZMYM3, ZNF217, ZNF24(ZSCAN3), ZNF703, ZRSR2, and any combination thereof.

[0227] In another embodiment, the genome profiling assay includes ABL1, 12B, ABL2, ACTB, ACVR1, ACVR1B, AGO2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, AMER1(FAM123BorWTX), AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1, ARHGAP26(GRAF), ARID1A, ARID1B, ARID2, ARID5B, ARv7, ASMTL, ASXL1, ASXL2, ATM, ATR, and ATRX. , AURKA, AURKB, AXIN1, AXIN2, AXL, B2M, BABAM1, BAP1, BARD1, BBC3, BCL10, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BRIP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD11, CARM1, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC42, CDC7 3, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBPA, CENPA, CHD2, CHD4, CHEK1, CHEK2, CIC, CIITA, CKS1B, CPS1, CREBBP, CRKL, CRLF2, CSDE1, CSF1R, CSF3R, CTCF, CTLA-4, CTNNB1, CTNNA1, CTNNB1, CUL3, CUL4A, CUX1, CXCR4, CYLD, CYP17A1, CYSLTR2, DAXX, DCUN1D1, DDR1, DDR2, DDX3X, DH2, DICER1, DIS3, DNAJB1, DNM2, DNMT1, DNMT3A, DNMT3B, DOT1L, DROSHA, DTX1, DUSP2, DUSP4, DUSP9, E2F3,EBF1, ECT2L, EED, EGFL7, EGFR, EIF1AX, EIF4A2, EIF4E, ELF3, ELP2, EML4, E ML4-ALK, EP300, EPAS1, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2 ERBB3, ERBB4, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERF, ERG, ERRFI1, ERRFl 1, ESR1, ETS1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXOSC6, EZH1, EZH2, FAF1, FA M175A, FAM46C, FAM58A, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI FANCL, FAS, FAS(TNFRSF6), FAT1, FBXO11, FBXO31, FBXW7, FGF1, FGF10, FG F12, FGF14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9 FGFR1, FGFR2, FGFR3, FGFR4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH 1, FOXA1, FOXL2, FOXO1, FOXO3, FOXP1, FRS2, FUBP1, FYN, GABRA6, GADD45B. STEP1、STEP2、STEP3、STEP4、STEP6、GEN1、GID4(C17orf39)、GID4(C17orf3 9) GLI1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRI N2A, GRM3, GSK3B, GTSE1, H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, Hedge hog、HER-2 / NEU;ERBB2、HGF、HIST1H1C、HIST1H1D、HIST1H1E、HIST1H2AC、H IST1H2AG, HIST1H2AL, HIST1H2AM, HIST1H2BC, HIST1H2BD, HIST1H2BJ, HIS T1H2BK, HIST1H2BO, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HIST2H3C, HIST2H3DHIST3H3, HLA-A, HLA-B, HNF1A, HOXB13, HRAS, HSD3B1, HSP90AA1, ICK, ICOSLG, ID3, IDH1, IDH2, IFNGR1, IGF1, IGF1R, IGF2, IKBKE, IKZF1, IKZF2, IKZF3 、IL10、IL7R、INHA、INHBA、INPP4A、INPP4B、INPP5D(SHIP)、INPPL1、INSR、I RF1、IRF2、IRF4、IRF8、IRS1、IRS2、JAK1、JAK2、JAK3、JARID2、JUN、K14、KAT6 A(MYST3), KAT6A(MYST3), KDM2B, KDM4C, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIF5B, KIT, KLF4, KLHL6, KMT2A, KMT2A(MLL), KMT2B, KMT2C, KMT2C(MLL) 3)、KMT2D、KMT2D(MLL2)、KNSTRN、KRAS、LAMP1、LATS1、LATS2、LEF1、LMO1、L RP1B、LRRK2、LTK、LYN、LZTR1、MAF、MAFB、MAGED1、MAGI2、MALT1、MAP2K1、MAP 2K1(MEK1)、MAP2K2、MAP2K2(MEK2)、MAP2K4、MAP3、MAP3K1、MAP3K13、MAP3K 14、MAP3K6、MAP3K7、MAPK1、MAPK3、MAPKAP1、MAX、MCL1、MDC1、MDM2、MDM4、ME D12, MEF2B, MEF2C, MEK1, MEN1, MERTK, MET, MGA, MIB1, MITF, MKI67, MKNK1, MLH1, MLLT3, MPL, MRE11A, MRE11A, MSH2, MSH3, MSH6, MSI1, MSI2, MST1, MST1 R、MTAP、MTOR、MUTYH、MYC、MYCL、MYCL(MYCL1)、MYCL(MYCL1)、MYCL1、MYCN、 MYD88、MYO18A、MYOD1、NBN、NCOA3、NCOR1、NCOR2、NCSTN、NEGR1、NF1、NF2、NF E2L2、NFKBIA、NKX2-1、NKX3-1、NOD1、NOTCH1、NOTCH2、NOTCH3、NOTCH4、NPM1 、NRAS、NRG1、NSD1、NT5C2、NTHL1、NTRK1、NTRK2、NTRK3、NUF2、NUP93、NUP98、P2RY8、PAG1、PAK1、PAK3、PAK7、PALB2、PARK2、PARP1、PARP2、PARP3、PASK、PAX3、PAX5、PAX7、PBRM1、PC、PCBP1、PCLO、PDCD1、PDCD1(PD-1)、PDCD11、PDCD1LG2、PDCD1LG2(PD-L2)、PDGFRA、PDGFRB、PDK1、PDPK1、PGR、PHF6、PHOX2B、PIK3C2B、PIK3C2G、PIK3C3、PIK3CA、PIK3CB、PIK3CD、PIK3CG、PIK3R1、PIK3R2、PIK3R3、PIM1、PLCG2、PLK2、PMAIP1、PMS1、PMS2、PNRC1、POLD1、POLE、POT1、PPARG、PPM1D、PPP2、PPP2R1A、PPP2R2A、PPP4R2、PPP6C、PRDM1、PRDM14、PREX2、PRKAR1A、PRKCI、PRKD1、PRKDC、PRSS8、PTCH1、PTEN、PTP4A1、PTPN11、PTPN2、PTPN6(SHP-1)、PTPRD、PTPRO、PTPRS、PTPRT、QKI、R1A、RAB35、RAC1、RAC2、RAD21、RAD50、RAD51、RAD51B、RAD51C、RAD51D、RAD52、RAD54L、RAF1、RANBP2、RARA、RASA1、RASGEF1A、RB1、RBM10、RECQL、RECQL4、REL、RELN、RET、RFWD2、RHEB、RHOA、RICTOR、RIT1、RNF43、ROS1、RPS6KA4、RPS6KB1、RPS6KB2、RPTOR、RRAGC、RRAS、RRAS2、RTEL1、RUNX1、RUNX1T1、RXRA、RYBP、S1PR2、SDHA、SDHAF2、SDHB、SDHC、SDHD、SERP2、SESN1、SESN2、SESN3、SETBP1、SETD2、SETD8、SF3B1、SGK1、SH2B3、SH2D1A、SHOC2、SHQ1、SLIT2、SLX4、SMAD2、SMAD3、SMAD4、SMARCA1、SMARCA4、SMARCB1、SMARCD1、SMC1A、SMC3、SMO、SMYD3、SNCAIP、SOCS1、SOCS2、SOCS3、SOS1、SOX10、SOX17、SOX2、SOX9、SPEN、SPOP、SPRED1、SPTA1, SRC, SRSF2, STAG2, STAT3, STAT4, STAT5A, STAT5B, STAT6, STK11, STK19, STK40, SUFU, SUZ12, SYK, TAF1, TAP1, TAP2, TBL1XR1, TBX 3, TCEB1, TCF3, TCF3(E2A), TCF7L2, TCL1A(TCL1), TEK, TERC, TERT, TERT promoter, TET1, TET2, TFRC, TGFBR1, TGFBR2, TIPARP, TLL2, TMEM12 7, TMEM30A, TMPRSS2, TMSB4XP8 (TMSL3), TNFAIP3, TNFRSF11A, TNFRSF14, TNFRSF17, TOP1, TOP2A, TP53, TP53BP1, TP63, TRAF2, TRAF3, TRAF5, TRAF7, TSC1, TSC2, TSHR, TUSC3, TYK2, TYRO3, U2AF1, U2AF2, UPF1, VEGFA, VHL, VTCN1, WDR90, WHSC1, WHSC1 (MMSET or NSD2), WHSC1L1, At least approximately 20, at least approximately 30, at least approximately 40, at least approximately 50, at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, and fewer than 20 selected from the group consisting of WISP3, WT1, WWTR1, XBP1, XIAP, XPO1, XRCC2, YAP1, YES1, YY1AP1, ZBTB2, ZFHX3, ZMYM3, ZNF217, ZNF24 (ZSCAN3), ZNF703, ZRSR2, and any combination thereof. Each gene contains approximately 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes.

[0228] In another embodiment, the genome profile includes one or more genes selected from the genes listed in Tables 1-16.

[0229] In one embodiment, TMB status based on genomic profiling correlates strongly with TMB status based on whole exome or whole genome sequencing. The evidence provided herein shows that the use of genomic profiling assays, such as the F1CDx assay, is consistent with whole exome and / or whole genome sequencing assays. These data support the use of genomic profiling assays as a more effective method for measuring TMB status without compromising the quality of TMB status prediction.

[0230] TMB can be measured using tissue biopsy samples, or circulating tumor DNA (ctDNA), cfDNA (cell-free DNA), and / or liquid biopsy samples. ctDNA can be used to measure TMB status according to whole exome sequencing, whole genome sequencing, or genomic profiling using available methods (e.g., GRAIL).

[0231] Subjects are identified as suitable for immunotherapy (e.g., using an anti-PD-1 antibody or its antigen-binding moiety, or an anti-PD-L1 antibody or its antigen-binding moiety) based on the measurement of TMB status and the identification of high TMB. In one embodiment, the TMB score is calculated as the total number of non-synonymous missense mutations in the tumor, as measured by whole exome sequencing or whole-genome sequencing. In one embodiment, high TMB is defined as at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, and low TMB. Having a score of at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, or at least 500.In another embodiment, a high TMB has a score of at least 215, at least 220, at least 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, at least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249, or at least 250. In a particular embodiment, a high TMB has a score of at least 243. In another embodiment, a high TMB has a score of at least 244. In one embodiment, a high TMB has a score of at least 245. In another embodiment, a high TMB has a score of at least 246. In another embodiment, the high TMB has a score of at least 247. In another embodiment, the high TMB has a score of at least 248. In another embodiment, the high TMB has a score of at least 249. In another embodiment, the high TMB has a score of at least 250. In another embodiment, the high TMB has an integer score of 200 to 300 or higher. In another embodiment, the high TMB has an integer score of 210 to 290 or higher. In another embodiment, the high TMB has an integer score of 220 to 280 or higher. In another embodiment, the high TMB has an integer score of 230 to 270 or higher. In another embodiment, the high TMB has an integer score of 235 to 265 or higher.

[0232] Alternatively, high TMB can be a relative value rather than an absolute value. In one embodiment, the TMB state of the subject is compared to a reference TMB value. In one embodiment, the TMB state of the subject is within the highest quantile of the reference TMB value. In another embodiment, the TMB state of the subject is within the highest tertile of the reference TMB value.

[0233] In certain embodiments, the TMB state is expressed as the number of mutations per sample, per cell, per exome, or per length of DNA (e.g., Mb). In certain embodiments, a tumor exhibits a high TMB state if it has at least about 50 mutations / tumor, at least about 55 mutations / tumor, at least about 60 mutations / tumor, at least about 65 mutations / tumor, at least about 70 mutations / tumor, at least about 75 mutations / tumor, at least about 80 mutations / tumor, at least about 85 mutations / tumor, at least about 90 mutations / tumor, at least about 95 mutations / tumor, at least about 100 mutations / tumor, at least about 105 mutations / tumor, at least about 110 mutations / tumor, at least about 115 mutations / tumor, or at least about 120 mutations / tumor. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 125 mutations / tumor, at least about 150 mutations / tumor, at least about 175 mutations / tumor, at least about 200 mutations / tumor, at least about 225 mutations / tumor, at least about 250 mutations / tumor, at least about 275 mutations / tumor, at least about 300 mutations / tumor, at least about 350 mutations / tumor, at least about 400 mutations / tumor, or at least about 500 mutations / tumor. In one particular embodiment, a tumor exhibits a high TMB state if it has at least about 100 mutations / tumor.

[0234] In certain embodiments, a tumor exhibits a high TMB state if it has at least about 5 mutations per megabase (mutations / Mb), at least about 6 mutations / Mb, at least about 7 mutations / Mb, at least about 8 mutations / Mb, at least about 9 mutations / Mb, at least about 10 mutations / Mb, at least about 11 mutations / Mb, at least about 12 mutations / Mb, at least about 13 mutations / Mb, at least about 14 mutations / Mb, at least about 15 mutations / Mb, at least about 20 mutations / Mb, at least about 25 mutations / Mb, at least about 30 mutations / Mb, at least about 35 mutations / Mb, at least about 40 mutations / Mb, at least about 45 mutations / Mb, at least about 50 mutations / Mb, at least about 75 mutations / Mb, or at least about 100 mutations / Mb, for example, genome sequenced according to, for example, the FOUNDATIONONE® CDX® assay. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 5 mutations / Mb. In one embodiment, a tumor exhibits a high TMB state if it has at least about 10 mutations / Mb. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 11 mutations / Mb. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 12 mutations / Mb. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 13 mutations / Mb. In certain embodiments, a tumor exhibits a high TMB state if it has at least about 14 mutations / Mb. In one embodiment, a tumor exhibits a high TMB state if it has at least about 15 mutations / Mb.

[0235] Because the number of mutations can vary by tumor type and other factors (see Q4 and Q5), the numerical values associated with "high TMB" and "low TMB" can differ by tumor type.

[0236] <> Non-small cell lung cancer This method can treat tumors at any stage. In one embodiment, the tumor originates from NSCLC at any stage. There are at least seven stages used for NSCLC (latent stage, stage 0 (in vivo carcinoma), stage I, stage II, stage IIIA, stage IIIB, and stage IV). In the latent stage, the cancer cannot be observed by imaging or bronchoscopy. In stage 0, cancer cells are found in the airway lining.

[0237] In one embodiment, the method treats stage I non-squamous NSCLC. Stage I NSCLC is further divided into stages IA and IB. In stage IA, the tumor is located only in the lung and is 3 centimeters or less in size. In stage IB, the cancer has not spread to the lymph nodes and is true in one or more of the following: 1) the tumor is 3 centimeters or larger but not larger than 5 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchus; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) a portion of the lung is collapsed or has pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchus.

[0238] In another embodiment, the method of the present disclosure treats stage II non-squamous NSCLC. Stage II NSCLC is classified into stages IIA and IIB. In stage IIA, the cancer may or may not have spread to the lymph nodes. If the cancer has spread to the lymph nodes, the cancer has spread only to the ipsilateral thoracic lymph nodes (lungs or lymph nodes within the lungs or near the trachea) and one or more of the following are true: 1) the tumor is no larger than 5 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchus; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) a portion of the lung is collapsed or has pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchus. The tumor is also considered to be stage IIA if the cancer has not spread to the lymph nodes, and one or more of the following are true: 1) the tumor is larger than 5 centimeters but not larger than 7 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchus; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) part of the lung is collapsed or has pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchus. In stage IIB, the cancer may or may not have spread to the lymph nodes. If the cancer has spread to the lymph nodes, the cancer has spread only to the lymph nodes in the chest on the same side as the tumor (lung, or lymph nodes within the lung or near the trachea), and one or more of the following are true: 1) the tumor is larger than 5 centimeters but not larger than 7 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchus; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) part of the lung is collapsed or has pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchus.The tumor is also considered to be stage IIB if the cancer has not spread to the lymph nodes, and one or more of the following are true: 1) the tumor is larger than 7 centimeters; 2) the cancer has spread to the main bronchus, chest wall, diaphragm, or nerves controlling the diaphragm (furthermore, at least 2 centimeters below where the trachea connects to the bronchus); 3) the cancer has spread to the membrane surrounding the heart or the membrane covering the chest wall; 4) the entire lung is collapsed or has developed pneumonia (inflammation of the lung); or 5) one or more other tumors are present in the same lobe of the lung.

[0239] In other embodiments, any method of this disclosure treats stage III non-squamous NSCLC. Stage IIIA is classified into three sub-sections. These three sub-sections are based on 1) tumor size; 2) location of the tumor; and 3) whether (if any) lymph nodes are cancerous. In the first type of stage IIIA NSCLC, the cancer has spread only to lymph nodes in the chest on the same side as the tumor, and the cancerous lymph nodes are near the sternum or where the bronchi enter the lungs. Furthermore: 1) the tumor may be of any size; 2) part of the lung (where the trachea connects to the bronchi) or the entire lung may collapse or develop pneumonia (inflammation of the lung); 3) one or more other tumors may be present in the same lobe of the lung; and 4) the cancer may spread to any of the following: a) the main bronchi (not the area where the trachea connects to the bronchi), b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lung or the membranes covering the chest wall, e) the membranes surrounding the heart. In the second type of NSCLC of stage IIIA, the cancer has spread to lymph nodes in the lung on the same side as the tumor, and the cancerous lymph nodes are either within the lung or near the bronchi. Furthermore: 1) the tumor may be of any size; 2) the entire lung may be collapsed or have pneumonia (inflammation of the lung); 3) there may be one or more other tumors in any of the lung lobes affected by cancer; and 4) the cancer may spread to any of the following: a) the main bronchi (not the area where the trachea connects to the bronchi), b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lungs or the membranes covering the chest wall, e) the heart or the membranes surrounding it, f) major blood vessels that lead to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal organs), j) the sternum or skeleton, or k) the tracheal bifurcation (where the trachea connects to the bronchi). In the third type of stage IIIA NSCLC, the cancer has not spread to the lymph nodes, the tumor may be of any size, and the cancer has spread to any one of the following: a) the heart, b) major blood vessels leading to or from the heart, c) the trachea, d) the esophagus, e) nerves controlling the larynx (vocal organs), f) the sternum or skeleton, or g) the tracheal bifurcation (where the trachea connects to the bronchi).Stage IIIB is classified into two sub-types based on 1) tumor size, 2) location of the tumor, and 3) whether lymph nodes are affected by cancer. In the first type of Stage IIIB NSCLC, the cancer has spread to the lymph nodes on the opposite side of the chest from the tumor. Furthermore, 1) the tumor may be of any size; 2) part of the lung (where the trachea connects to the bronchi) or the entire lung may be collapsed or have pneumonia (inflammation of the lung); 3) one or more other tumors may be present in any of the lobes of the lung affected by cancer; and 4) the cancer may have spread to any of the following: a) the main bronchi, b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lung or the membranes covering the chest wall, e) the heart or the membranes surrounding it, f) major blood vessels that lead to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal organs), j) the sternum or skeleton, or k) the tracheal bifurcation (where the trachea connects to the bronchi). In the second type of stage IIIB NSCLC, the cancer has spread to lymph nodes in the chest on the same side as the tumor. The cancerous lymph nodes are near the sternum or where the bronchi enter the lungs. Furthermore, 1) the tumor may be of any size; 2) another tumor may be present in a different lobe of the same lung; and 3) the cancer may have spread to any of the following: a) the heart, b) major blood vessels leading to or from the heart, c) the trachea, d) the esophagus, e) the nerves controlling the larynx (vocal organs), f) the sternum or skeleton, or g) the tracheal bifurcation (where the trachea connects to the bronchi).

[0240] In one embodiment, the method of the present disclosure treats stage IV non-squamous NSCLC. In stage IV NSCLC, the tumor may be of any size, and the cancer may have spread to the lymph nodes. One or more of the following are true in stage IV NSCLC: 1) one or more tumors are present in both lungs; 2) the cancer is observed in the fluid surrounding the lungs or heart; and 3) the cancer has spread to other parts of the body (e.g., the brain, liver, adrenal glands, kidneys, or bones).

[0241] In another embodiment, NSCLC treatable by this method is squamous cell carcinoma (epidermoid carcinoma) (squamous NSCLC). It is known that approximately 25% to 30% of all lung cancers are squamous cell carcinomas. These cancers begin in the squamous epithelium (flat cells that line the inside of the airways in the lungs). They are often associated with a history of smoking and tend to be found in the central part of the lung (bronchi) near the major airways.

[0242] In one embodiment of this method, the anti-PD-1 antibody is nivolumab. In another embodiment, it is pembrolizumab. Generally, the anti-PD-1 antibody is formulated for intravenous administration. In one embodiment, the anti-PD-1 antibody is administered by intravenous infusion over 60 minutes. In one embodiment, the anti-PD-1 antibody is administered as a pharmaceutically acceptable formulation. In one embodiment, the anti-PD-1 antibody or its antigen-binding moiety is administered in a dose below the therapeutic dose.

[0243] Anti-PD-1 antibodies or anti-PD-L1 antibodies useful in this disclosure Anti-PD-1 antibodies known in the art can be used in the compositions and methods described herein. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity are described in U.S. Patent No. 8,008,449. Anti-PD-1 human antibodies are shown to exhibit one or more of the following characteristics as described in U.S. Patent No. 8,008,449: (a) 1 x 10⁻¹⁶ as can be examined by surface plasmon resonance using a Biacore biosensor system. -7(b) binds to human PD-1 with a KD of M or less; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) activates antigen-specific memory responses; (i) activates antibody responses; and (j) inhibits tumor cell proliferation in vivo. Anti-PD-1 antibodies that can be used in this disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments, at least five, of the above features.

[0244] Other anti-PD-1 monoclonal antibodies include, for example, 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. WO2012 / 145493, WO2008 / 156712, WO2015 / No. 112900, No. WO2012 / 145493, No. WO2015 / 112800, No. WO2014 / 206107, No. WO2015 / 35606, No. WO No. 2015 / 085847, No. WO2014 / 179664, No. WO2017 / 020291, No. WO2017 / 020858, No. WO2016 / 19736 No. 7, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 1 No. 94302, No. WO2017 / 040790, No. WO2017 / 133540, No. WO2017 / 132827, No. WO2017 / 024465, No. WO2 This information is found in issues 017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540 (each of which is incorporated in its entirety with proper attribution).

[0245] In one embodiment, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), semiprimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al., J. Hematol. See Oncol. 10:136 (2017), BGB-A317 (Beigene; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO2015 / 085847; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136) The group is selected from the following: (see 2017), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics, see WO2017 / 19846), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540).

[0246] In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a complete human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively inhibits interaction with PD-1 ligands (PD-L1 and PD-L2), thereby preventing downregulation of antitumor T cell function (see U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0247] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patents 8,354,509 and 8,900,587.

[0248] The anti-PD-1 antibodies that can be used in the compositions and methods of this disclosure also include isolated antibodies, such as nivolumab (e.g., U.S. Patents 8,008,449 and 8,779,105; see WO2013 / 173223), that specifically bind to human PD-1 and cross-compete for the binding of human PD-1 to the anti-PD-1 antibodies described herein. In one embodiment, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies 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 interfere with the binding of other cross-competing antibodies to a particular epitope region. These cross-competing antibodies are expected to have very similar functional properties to 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 based on their ability to cross-compete with nivolumab in standard PD-1 binding assays (e.g., Biacore analysis, ELISA assay, or flow cytometry) (see, for example, WO2013 / 173223).

[0249] In one embodiment, an antibody (nivolumab) that cross-competes for the binding of human PD-1 to a human PD-1 antibody, or that binds to the same epitope region of a human PD-1 antibody, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0250] The anti-PD-1 antibodies used in the compositions and methods of this disclosure also include an antigen-binding moiety of the antibody. It has been shown that the antigen-binding function of the antibody may be performed by a fragment of the full-length antibody.

[0251] An anti-PD-1 antibody suitable for use in the compositions and methods of this disclosure is an antibody that binds to PD-1 with high specificity and affinity, inhibits the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods described herein, the anti-PD-1 "antibody" includes an antigen-binding moiety or fragment that binds to the PD-1 receptor, inhibits ligand binding, and exhibits functional properties similar to the whole antibody in upstreamly regulating the immune system. In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety cross-competes with nivolumab for binding to human PD-1.

[0252] Anti-PD-1 antibodies or anti-PD-L1 antibodies useful in this disclosure In some embodiments, the anti-PD-1 antibody used in the present method can be replaced with another PD-1 or anti-PD-L1 antagonist. For example, since anti-PD-L1 antibodies inhibit the interaction between PD-1 and PD-L1, thereby exhibiting a similar effect on the PD-1 signaling pathway, anti-PD-L1 antibodies can be used in the methods described herein to replace the use of anti-PD-1 antibodies. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the present disclosure. An example of an anti-PD-L1 antibody useful in the compositions and methods of the present disclosure is the antibody described in U.S. Patent No. 9,580,507. Anti-PD-L1 human monoclonal antibodies are shown to exhibit one or more of the following properties as described in U.S. Patent No. 9,580,507: (a) 1 x 10⁻¹⁶ so as to be examined by surface plasmon resonance using a Biacore biosensor system. -7 (b) binds to human PD-L1 with a KD of M or less; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) activates the antibody response; and (g) reverses the effect of T regulatory cells in T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in this disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments, at least five of the above features.

[0253] In one embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent No. 7,943,743 and WO2013 / 173223), and atezolizumab (Roche; TECENTRIQ®; also known as MPDL3280A, RG7446; see U.S. 8,217,149; Herbst et al. (2013) J Clin Oncol). See also 31(suppl):3000), durvalumab (AstraZeneca; IMFINZI®, also known as MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; BAVENCIO®, also known as MSB-0010718C; see WO2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly The group is selected from the following: Co. (see, for example, WO2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0254] In one embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0255] In one embodiment, the PD-L1 antibody is durvalumab (IMFINZI®). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0256] In one embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0257] In other embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

[0258] Anti-PD-L1 antibodies that can be used in the compositions and methods of the present disclosure are also isolated antibodies that specifically bind to human PD-L1 and cross-compete with the binding of human PD-L1 and the anti-PD-L1 antibodies described herein, for example, atezolizumab, durvalumab, and / or avelumab. In certain embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein (for example, atezolizumab, durvalumab, and / or avelumab). 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 a particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of a reference antibody (for example, atezolizumab and / or avelumab) due to their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays (for example, Biacore analysis, ELISA assay or flow cytometry) (see, for example, WO2013 / 173223).

[0259] In certain embodiments, an antibody (atezolizumab, durvalumab, and / or avelumab) that cross-competes with the binding of human PD-L1 and a human PD-L1 antibody or binds to the same epitope region of a human PD-L1 antibody is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0260] The anti-PD-L1 antibodies used in the compositions and methods of this disclosure also include an antigen-binding moiety of the antibody. It has been shown that the antigen-binding function of the antibody may be performed by a fragment of the full-length antibody.

[0261] An anti-PD-L1 antibody suitable for use in the compositions and methods of this disclosure is an antibody that binds to PD-1 with high specificity and affinity, inhibits PD-1 binding, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods described herein, the anti-PD-L1 "antibody" includes an antigen-binding moiety or fragment that binds to the PD-L1 receptor, inhibits ligand binding, and exhibits functional properties similar to the whole antibody in upstreamly regulating the immune system. In some embodiments, the anti-PD-L1 antibody or its antigen-binding moiety cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0262] Standard treatment for lung cancer Standard treatments for different types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a coalition of 21 major cancer centers in the United States, publishes the NCCN Guidelines® in Oncology, which provide detailed and up-to-date information on standard treatments for a wide variety of cancers (see NCCN Guidelines® (2014) (most recently accessed May 14, 2014: available at www.nccn.org / professionals / physician_gls / pdf / nscl.pdf)).

[0263] NSCLC is the leading cause of cancer death in the United States and worldwide, exceeding the combined death rate from breast, colon, and prostate cancers. In the United States, an estimated 228,190 new cases of lung and bronchial NSCLC are diagnosed annually, and 159,480 people die from the disease ((Siegel et al. (2014) CA Cancer J Clin 64(1):9-29). The majority of patients (approximately 78%) are diagnosed with advanced / recurrent or metastatic disease. Metastasis from lung cancer to the adrenal glands is common in approximately 33% of patients with such metastases. NSCLC treatment has shown gradually improved overall survival (OS), but the benefit is steady state (median OS for late-stage patients is only 1 year). Progression after 1L treatment occurs in almost all of these patients, and the 5-year survival rate is only 3.6% for refractory conditions. From 2005 to 2009, the overall 5-year relative survival rate for lung cancer in the United States was 15.9% (NCCN GUIDELINES®, version 3.2014 - Non-Small Cell Carcinoma, most recently accessed May 14, 2014 (available at www.nccn.org / professionals / physician_gls / pdf / nscl.pdf).

[0264] Surgery, radiotherapy (RT), and chemotherapy are three common modalities used to treat patients with NSCLC. As a class, NSCLC is relatively less sensitive to chemotherapy and RT compared to small cell carcinoma. Generally, for patients with stage I or II disease, surgical resection offers the best treatment opportunity, often with chemotherapy used both before and after surgery. RT can also be used as adjuvant therapy (initial local treatment) for patients with resectable NSCLC, or as palliative therapy for patients with incurable NSCLC.

[0265] Patients with stage IV disease showing a good performance status (PS) can benefit from chemotherapy. Many drugs, including platinum-based drugs (e.g., cisplatin, carboplatin), taxane-based drugs (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine, are useful for stage IV NSCLC. Combinations of many of these drugs result in a 30-40% one-year survival rate, which is superior to single-agent therapies. Specific targeted therapies have been developed for the treatment of advanced lung cancer. For example, bevacizumab (AVASTIN®) is a monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A). Erlotinib (Tarceva®) is a small molecule TKI that targets the epidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is a small molecule TKI that targets ALK and MET and is used to treat NSCLC in patients carrying mutant ALK fusion genes. Cetuximab (Erbitux®) is a monoclonal antibody that targets EGFR.

[0266] There is a need for treatment options in certain patients with squamous cell NSCLC (representing up to 25% of all NSCLC cases) due to the complete lack of treatment options after first-line (1L) treatment. Monotherapy is the standard treatment after progression with platinum-based double chemotherapy (Pt-doublet), resulting in a median overall survival (OS) of approximately 7 months. Erlotinib can be used less frequently, but docetaxel remains the standard treatment in this elective. Pemetrexed also produces clinically comparable efficacy but has been shown to have significantly fewer side effects compared to docetaxel in second-line (2L) treatment for patients with advanced NSCLC (Hanna et al. (2004) J Clin Oncol 22:1589-97). There are currently no approved treatments for use in lung cancer beyond third-line (3L) treatment. Pemetrexed and bevacizumab are not approved for squamous cell NSCLC, and molecular targeted therapies have limited applicability. These unmet needs in advanced lung cancer are exacerbated by the recent failure of STIMUVAX® to improve OS in the Phase 3 trial of Oncothyreon and Merck KgaA, the failure of the c-Met kinase inhibitor (tivantinib) to meet the survival endpoint in the ArQule and Daiichi Sankyo trial, the failure of Eli Lilly's ALIMTA® to improve OS in the late-stage trial of Roche's AVASTIN®, and the failure of the small molecule VEGF-R antagonist motesanib to meet the clinical endpoint in the late-stage trial of Amgen and Takeda Pharmaceutical.

[0267] Immunotherapy for lung cancer There is a clear need for effective drugs to treat patients whose disease has progressed despite multiple targeted therapies, and for treatments that extend survival beyond current standard care. Recent approaches to immunotherapy, particularly blocking immune checkpoints (including CTLA-4, PD-1, and PD-L1 inhibitory pathways), have shown promise (Creelan et al. (2014) Cancer Control 21(1):80-89). However, there remains a need to identify patients who may be more responsive to immunotherapy, especially those who may be more responsive to anti-PD-1 or anti-PD-L1 antibody treatment.

[0268] Pharmaceutical compositions and dosages The therapeutic agents of the Disclosure may comprise a composition, for example, a pharmaceutical composition comprising one or more antibodies and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic and absorption retardant agents, and the like. In one embodiment, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion). The pharmaceutical compositions of the Disclosure may comprise one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants (e.g., preservatives, wetting agents, emulsifiers, and dispersants).

[0269] This disclosure provides a dosing schedule that can deliver a desirable response, e.g., the maximum therapeutic response and / or the minimum side effects. For the administration of anti-PD-1 antibodies, the dose may range from approximately 0.01 to approximately 10 mg / kg, approximately 1 to approximately 9 mg / kg, approximately 2 to approximately 8 mg / kg, approximately 3 to approximately 7 mg / kg, approximately 3 to approximately 6 mg / kg, approximately 0.01 to approximately 5 mg / kg, or approximately 1 to approximately 3 mg / kg of the subject's body weight. For example, the dose may be approximately 0.1, approximately 0.3, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 mg / kg body weight. The dosing schedule is generally designed to achieve exposure that results in sustained receptor occupancy (RO), based on the typical pharmacokinetic properties of the antibody. An exemplary treatment plan may include administrations approximately once every week, once every two weeks, once every three weeks, once every four weeks, once every month, and once every three to six months or longer. In one embodiment, the anti-PD-1 antibody (e.g., nivolumab) is administered to the subject approximately every two weeks. The anti-PD-1 antibody may be administered in at least two doses, each dose being in an amount of approximately 0.01 mg / kg to approximately 5 mg / kg, for example, 3 mg / kg, with a two-week interval between the two doses. In one embodiment, the anti-PD-1 antibody may be administered in at least three, four, five, six, or seven doses (i.e., multiple doses), each dose being in an amount of approximately 0.01 mg / kg to approximately 10 mg / kg, for example, 1 mg / kg, 3 mg / kg, or 6 mg / kg, with a two-week interval between two adjacent doses. The aforementioned dosage and schedule may be modified during the course of treatment. In one embodiment, a dosing regimen for the anti-PD-1 antibody of this disclosure includes intravenous administration of approximately 0.1 to approximately 5 mg / kg body weight, approximately 1 to approximately 5 mg / kg body weight, or approximately 1 to approximately 3 mg / kg body weight (the antibody is administered every approximately 14 to 21 days for up to approximately 6 weeks or approximately 12 weeks until complete response or progressive disease is confirmed).In one embodiment, the antibody therapy, or the combination therapy described herein, is continued for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 month, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or at least about 10 years.

[0270] For the administration of anti-PD-L1 antibodies, the dose may range from approximately 1 to 20 mg / kg, 1 to 19 mg / kg, 2 to 18 mg / kg, 3 to 17 mg / kg, 3 to 16 mg / kg, 4 to 15 mg / kg, 5 to 14 mg / kg, 6 to 13 mg / kg, 7 to 12 mg / kg, or 8 to 12 mg / kg of the subject's body weight. For example, the dose may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg / kg of body weight. The administration schedule is generally designed to achieve exposure that results in sustained receptor occupancy (RO), based on the typical pharmacokinetic properties of the antibody. An exemplary treatment plan may include administrations approximately once every week, once every two weeks, once every three weeks, once every four weeks, once every month, and once every three to six months or longer. In one embodiment, the anti-PD-L1 antibody is administered to the subject approximately every two weeks. The anti-PD-L1 antibody may be administered in at least two doses, each dose being approximately 6 mg / kg to approximately 18 mg / kg, for example, 10 mg / kg, with a two-week interval between the two doses. In one embodiment, the anti-PD-L1 antibody may be administered in at least three, four, five, six, or seven doses (i.e., multiple doses), each dose being approximately 6 mg / kg to approximately 18 mg / kg, for example, 10 mg / kg or 15 mg / kg, with a two-week interval between two adjacent doses. The doses and schedule may be modified during the course of treatment. In one embodiment, a dosing regimen for the anti-PD-L1 antibody of the Disclosure includes intravenous administration of approximately 1 to approximately 18 mg / kg body weight, approximately 6 to approximately 15 mg / kg body weight, or approximately 10 to approximately 15 mg / kg body weight (the antibody is administered every approximately 14 to 21 days for up to approximately 6 weeks or approximately 12 weeks until complete response or progressive disease is confirmed).In one embodiment, the antibody therapy, or the combination therapy described herein, is continued for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 month, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or at least about 10 years.

[0271] When used in combination with other treatments (e.g., other immunotherapies), the dose of the anti-PD-1 antibody can be reduced compared to the dose used as monotherapy. Typically, it is 3 mg / kg or less, but doses of nivolumab of 0.001 mg / kg or more are sub-therapeutic doses. Sub-therapeutic doses of the anti-PD-1 antibody used in the method described herein are 0.001 mg / kg or more and 3 mg / kg or less. In some embodiments, sub-therapeutic doses are approximately 0.001 mg / kg to approximately 1 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, approximately 0.1 mg / kg to approximately 1 mg / kg, or approximately 0.001 mg / kg to approximately 0.1 mg / kg body weight. In one embodiment, doses below the therapeutic dose are at least about 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, or at least about 1.0 mg / kg body weight. Receptor occupancy data from 15 subjects who received nivolumab at doses of 0.3 mg / kg to 10 mg / kg suggest that PD-1 occupancy may be dose-dependent within this dose range. Considering all doses, the mean occupancy was 85% (ranging from 70% to 97%), and the mean steady-state occupancy was 72% (ranging from 59% to 81%) (Brahmer et al. (2010) J Clin Oncol 28:3167-75). Therefore, a dose of 0.3 mg / kg may allow sufficient exposure to produce maximum biological activity.

[0272] In one embodiment of the present disclosure, the anti-PD-1 antibody is administered at a dose of 3 mg / kg. In another embodiment of the present disclosure, the anti-PD-1 antibody is administered at a dose of 1 mg / kg.

[0273] In one embodiment, the dose of the anti-PD-1 antibody (or anti-PD-L1 antibody) is a flat dose in the pharmaceutical composition. In another embodiment, the method of the present disclosure can be used in a flat dose (a dose administered to the patient regardless of the patient's weight). In one embodiment, the flat dose of the anti-PD-1 antibody or its antigen-binding moiety is at least about 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 600 mg, 640 mg, 680 mg, 720 mg, 760 mg, 800 mg, 840 mg, 880 mg, 920 mg, 960 mg, 1000 mg, 1040 mg, 1080 mg, 1120 mg, 1160 mg, or 1200 mg. For example, the flat dose of nivolumab may be about 240 mg. For example, the flat dose of pembrolizumab may be approximately 200 mg. In one embodiment, the anti-PD-1 antibody or its antigen-binding portion is administered in a dose of approximately 240 mg. In another embodiment, the anti-PD-1 antibody or its antigen-binding portion is administered in a dose of approximately 360 mg. In another embodiment, the anti-PD-1 antibody or its antigen-binding portion is administered in a dose of approximately 480 mg. In one embodiment, the flat dose of the anti-PD-1 antibody or its antigen-binding portion is administered once every approximately one week, two weeks, three weeks, four weeks, five weeks, or six weeks. In one embodiment, 240 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every two weeks. In another embodiment, 360 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every three weeks. In yet another embodiment, 480 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every four weeks.

[0274] In one embodiment, the flat dose of the anti-PD-L1 antibody or its antigen-binding portion is at least about 600 mg, 620 mg, 640 mg, 660 mg, 680 mg, 700 mg, 720 mg, 740 mg, 760 mg, 780 mg, 800 mg, 820 mg, 840 mg, 860 mg, 880 mg, 900 mg, 920 mg, 940 mg, 960 mg, 1000 mg, 1040 mg, 1080 mg, 1120 mg, 1160 mg, 1200 mg, 1240 mg, 1280 mg, 1320 mg, 1360 mg, 1400 mg, 1440 mg, 1480 mg, 1520 mg, 1560 mg, 1600 mg, 1640 mg, 1680 mg, 1720 mg, 1760 mg, or 1800 mg. For example, the flat dose of atezolizumab (TECENTRIQ®) may be approximately 1200 mg. For example, the flat dose of durvalumab (IMFINZI®) may be approximately 800 mg. For example, the flat dose of avelumab (BAVENCIO®) may be approximately 800 mg. In one embodiment, the anti-PD-L1 antibody or its antigen-binding moiety is administered in a dose of approximately 800 mg. In one embodiment, the anti-PD-L1 antibody or its antigen-binding moiety is administered in a dose of approximately 1200 mg. In one embodiment, the anti-PD-L1 antibody or its antigen-binding moiety is administered in a dose of approximately 1600 mg. In one embodiment, the flat dose of the anti-PD-L1 antibody or its antigen-binding moiety is administered once every approximately one week, two weeks, three weeks, four weeks, five weeks, or six weeks. In one embodiment, approximately 800 mg of the anti-PD-L1 antibody or antigen-binding fragment is administered once every two weeks. In another embodiment, approximately 1200 mg of the anti-PD-L1 antibody or antigen-binding fragment is administered once every four weeks.

[0275] Dosage and frequency vary depending on the half-life of the antibody in the subject. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and then non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, typically, relatively low doses are administered over a long period at relatively long intervals. Some patients continue treatment for life. In therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression slows or stops, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, the patient may be administered under a prophylactic plan.

[0276] The actual dose levels of the active ingredients in the pharmaceutical compositions of this disclosure can be varied to obtain an amount of active ingredient that is not highly toxic to the patient and is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of this disclosure used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular composition used, the age, biometrics, weight, condition, overall health, and medical history of the patient being treated, and other factors well known in the pharmaceutical field. The compositions of this disclosure can be administered by one or more routes of administration using one or more of the various methods well known in the art. As will be understood by those skilled in the art, the route of administration and / or mode of administration will vary depending on the desired outcome.

[0277] kit Kits comprising an anti-PD-1 antibody or an anti-PD-L1 antibody also fall within the scope of the present invention. Kits generally include labeling and instructions for use indicating the intended use of the kit's contents. The term “labeling” includes descriptions or records of substances contained in, provided with, or accompanying the kit. Thus, this disclosure provides a kit for treating a subject affected by a tumor, comprising (a) an anti-PD-1 antibody in a dose ranging from about 4 mg to about 500 mg; and (b) instructions for use of the anti-PD-1 antibody in the manner described herein. In one embodiment for treating a human patient, the kit comprises an anti-human PD-1 antibody as described herein, e.g., nivolumab or pembrolizumab. In one embodiment, the kit further comprises an anti-PD-L1 antibody. In one embodiment, the kit further comprises instructions for detecting the mutational status of STK11 in a tumor sample. In another embodiment, the kit further comprises instructions for detecting PD-L1 expression in a tumor sample.

[0278] For example, this disclosure provides the following embodiments. [1] An antibody or its antigen-binding portion ("anti-PD-1 antibody") for treating a subject suffering from a tumor, wherein the subject has been identified as having a wild-type STK11 gene. [2] The anti-PD-1 antibody according to claim 1, wherein the subject has been identified as having a mutant marker gene selected from the group consisting of KRAS, TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. [3] An antibody or its antigen-binding portion ("anti-PD-1 antibody") for treating a subject suffering from a tumor, wherein the subject has been identified as having a mutant marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. [4] The anti-PD-1 antibody according to claim 2 or 3, wherein the marker gene includes a nonsynonymous mutation, a nonsense mutation, a frameshift mutation, or a splicing mutation. [5] The tumor is derived from lung cancer, and is an anti-PD-1 antibody according to any one of items 1 to 4 above. [6] The anti-PD-1 antibody according to item 5, wherein the tumor is derived from small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). [7] An anti-PD-1 antibody according to any one of 1 to 6, wherein PD-L1 expression is detected in the tumor before administration of the anti-PD-1 antibody. [8] The anti-PD-1 antibody according to 7, wherein the tumor expresses PD-L1 in a scattered or heterogeneous pattern. [9] The anti-PD-1 antibody according to any one of 1 to 8, wherein the tumor has a tumor mutational burden (TMB) state of high TMB.

[10] The tumor TMB status is determined by sequencing nucleic acids in the tumor and then identifying genomic changes in the sequenced nucleic acids, wherein the genomic changes include one or more changes selected from the group consisting of somatic mutations, nonsynonymous mutations, missense mutations, base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and any combination thereof. The anti-PD-1 antibody as described in 9 above.

[11] The tumor exhibits high levels of inflammation. An anti-PD-1 antibody as described in any of items 1 to 10 above.

[12] The anti-PD-1 antibody according to any one of claims 1 to 11, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

[13] The anti-PD-1 antibody according to any one of claims 1 to 12, wherein the anti-PD-1 antibody is administered once every 1, 2, or 3 weeks at a dose in the range of at least about 0.1 mg / kg to at least about 10.0 mg / kg body weight.

[14] The anti-PD-1 antibody or its antigen-binding portion is administered once every 1, 2, 3, or 4 weeks in a flat dose of approximately 240 mg or approximately 480 mg. The anti-PD-1 antibody as described in any of items 1 to 13 above.

[15] A kit for treating subjects suffering from tumors, (a) Anti-PD-1 antibody in doses ranging from approximately 4 mg to approximately 500 mg; and (b) A kit comprising instructions for administering the anti-PD-1 antibody to subjects identified as having the wild-type STK11 gene. This disclosure is further illustrated by the following embodiments, which should not be construed as further limiting. All references cited throughout this application are incorporated herein by attribution. [Examples]

[0279] STK11 mutation as a biomarker for nivolumab response PD-L1 is expressed in NSCLC tumors (e.g., commercially available NSCLC tumors) with different expression patterns (Figure 1). These patterns are described as scattered, heterogeneous, tumor-stromal junctional, and negative. PD-L1 expression patterns may be related to mechanistic hypotheses. For example, in tumors with a scattered expression pattern, PD-L1 expression is driven not by mutation, but by 9p24 amplification in the oncogenic signaling pathway. In tumors with a tumor-stromal junctional pattern, adaptive resistance exists rather than epithelial-mesenchymal transition (EMT).

[0280] The PD-L1 expression patterns in commercially available NSCLC tumors correlate with the PD-L1 H-score, as shown in Figure 2. Substantial differences in PD-L1 expression levels exist among the different patterns. For example, extremely high H-scores were observed in scattered pattern samples, suggesting a possible dependence of these tumors on PD-L1 inhibition.

[0281] The PD-L1 expression pattern observed in commercially available NSCLC tumors was also seen in the biopsies. Figure 3 shows the PD-L1 expression pattern in test biopsies corresponding to patients treated with nivolumab monotherapy, and this pattern corresponds to the same pattern observed in commercially available NSCLC tumors.

[0282] The likelihood of a false negative PD-L1 is influenced by pattern category, pre-analysis variability, and biopsy size. For example, tumor-stromal junction patterns are heterogeneous and particularly prone to false negatives. Therefore, there is a need for biomarkers that can be used to facilitate the classification of NSCLC tumors and predict tumor response to specific therapeutic agents.

[0283] A correlation was observed between PD-L1 expression patterns and nivolumab efficacy (Figure 4). The majority of complete responders with grade 3 tumors exhibited scattered PD-L1 expression patterns and high PD-L1 H-scores. Therefore, identifying biomarkers specific to scattered PD-L1 expression patterns could be used to identify patients suitable for nivolumab treatment based on the presence or absence of these biomarkers.

[0284] In commercially available NSCLC tumors that produced the data shown in Figure 5, tumors with scattered or heterogeneous PD-L1 patterns were associated with more abundant immunoinfiltration (higher PD-L1 H-score), suggesting that immunoinfiltration may be useful as a specific biomarker for NSCLC tumors exhibiting scattered expression patterns (Figure 5).

[0285] Multiple IHC experiments revealed a specific association between tumor cells and immune cell subsets. PD-L1 labeling indicated scattered PD-L1 expression in tumors. CD68 detection showed that the macrophage layer at the tumor-stromal junction contributes to the formation of "borderline" activated T cells, while CD3 detection showed that T cells were moderately abundant but largely confined to the stroma (Figure 6).

[0286] PD-L1 expression patterns correlate with genomic data (Figure 7). Panel A in Figure 7 shows that PD-L1 expression levels correlate with RNA sequencing data, but RNA sequencing data alone does not provide the geographical context of PD-L1 expression patterns observed by IHC. Panel B in Figure 7, showing exome sequencing data, shows that scattered PD-L1 expression patterns correlate with higher mutation levels.

[0287] Higher mutational loads were also associated with inflammatory tumors (Figure 8). Panel A shows overall inflammation as measured using the "CI score," which is the intensity score of chronic inflammatory infiltrates. Panel B shows PD-L1+ inflammation as measured using the "PD-L1+ CI score," which is the intensity score of the relative proportion of PD-L1+ immune infiltrates. A correlation exists between the number of missense mutations and overall inflammation in NSCLC tumors.

[0288] We evaluated the mutation frequencies of different biomarkers (TP53, STK11, KEAP1, KRAS, EGFR, and MET) against the observed PD-L1 expression patterns (Figure 9). The results showed that expression in negative PD-L1 tumor cells and low PD-L1 mRNA expression were associated with the presence of the SKT11 mutation. The presence of mutant STK11 correlated with the presence of the "N" (PD-L1 negative) expression pattern. The presence of mutant STK11 did not correlate with the presence of the "D" (scattered) pattern (a pattern seen in the majority of responders to nivolumab therapy). Therefore, the presence of the STK11 mutation can be used as a negative biomarker for the treatment of NSCLC tumors with nivolumab (i.e., its presence would predict no or low responsiveness to nivolumab). Conversely, the presence of wild-type STK11 (or absence of the mutation) can be used as a positive selection biomarker for nivolumab treatment.

[0289] Mutation-induced loss of STK11 is predicted to enhance mTOR signaling. Lung adenocarcinomas with KRAS and STK11 mutations (both mouse models and human tumors) show decreased PD-L1 expression and reduced T cell infiltration. Proposed mechanisms of immunosuppression mediated by mutations in SKT11 may include a switch to glycolytic metabolism that increases lactate production, and high-frequency co-mutations in KEP1 that trigger an anti-inflammatory transcriptional program.

[0290] Immunoprint analysis of 24 NSCLC tumor samples, in which levels of FOLR2, VSIG4, CD163, CLEC4D, CSF1R, CD86, MS4A1, CD79B, CD19, KIR2DS4, CD3E, CCR4, CCR8, and CD8A were analyzed, and the samples were classified by inflammation pattern (sigClass). Samples were classified into low ("sigClass low"), intermediate ("sigClass med"), and high ("sigClass hi") inflammation. Samples were also classified by the presence or absence of the STK11 mutation ("STK11 mut") ("STK11 wt") (Figure 10). Furthermore, samples were classified based on their PD-L1 expression pattern as negative ("PDL1_Pattern 2 Negative"), scattered ("PDL1_Pattern 2 Scattered"), heterogeneous ("PDL1_Pattern 2 Heterogeneous"), and tumor-stromal junction ("PDL1_Pattern 2 TS"). Tumors with scattered PD-L1 expression patterns showed high inflammation and were STK11 wild-type. PD-L1 negative tumors formed two groups: intermediate inflammation and low inflammation. There was no clear distinction in the inflammation level of PD-L1 negative tumors based on STK11 mutation status. All tumors with mutant STK11 were also PD-L1 negative.

[0291] These data indicate that PD-L1 expression patterns are associated with unique phenotypic and genetic backgrounds. Scattered PD-L1 expression correlates with inflammatory TME and high mutational load. Furthermore, the presence of STK11 mutations identifies a subset of PD-L1-negative tumors. These findings confirm the suitability of STK11 as a biomarker for identifying subsets of PD-L1-positive tumors, as well as the potential to combine histopathological and genomic data to identify features that define NSCLC subsets, along with varying prospects for responsiveness to immunotherapy.

[0292] Example 2 An open-label, randomized phase 3 clinical trial was conducted to test the efficacy of first-line anti-PD-1 monoclonal antibody (nivolumab) treatment in patients with PD-L1-positive NSCLC. Patients with previously untreated stage IV or relapsed NSCLC and a PD-L1 tumor expression level of 1% or higher received up to 6 cycles of either nivolumab (3 mg / kg body weight intravenously every 2 weeks) or platinum-based chemotherapy (every 3 weeks).

[0293] In a post-hoc analysis, patients' tumor cells were analyzed for PD-L1 expression, as well as wild-type or mutant STK11, KRAS, CDKN2A, PTPND, CUBN, and / or HERC1. Patient survival was followed for 25 months.

[0294] After treatment, patients carrying STK11 mutations were found to have a lower progression-free survival (PFS) than patients with wild-type STK11. This was observed in patients with any non-synonymous STK11 mutation (Figure 12A) and patients with nonsense, frameshift, or splicing STK11 mutations (Figure 12B). STK11 mutation-carrying patients with additional KRAS mutations also showed reduced responsiveness to anti-PD-1 antibody treatment (Figure 13). When patients were stratified based on NSCLC type, patients with non-squamous NSCLC and any STK11 mutation showed lower PFS than patients with wild-type STK11, regardless of KRAS status (Figures 14A-14B).

[0295] STK11 mutation status and responsiveness were compared with tumor PD-L1 expression levels (Figures 15A-15F). Two patients who showed partial response after anti-PD-1 antibody treatment were found to have STK11 mutations and high PD-L1 levels (Figure 15B). However, in the anti-PD-1 antibody treatment group, PD-L1 expression levels were similar between the wild-type and STK11 mutant subgroups (Figure 15E).

[0296] In addition to STK11, patients' tumors were monitored for TP53, CDKN2A, PTPND, CUBN, and HERC1 status (Figures 16A-16D). Patients with mutations in both TP53 and KRAS showed a higher PFS after treatment with first-line anti-PD-1 monoclonal antibody therapy than patients with wild-type TP53 (Figure 16A). Patients with mutations in CDKN2A also showed a higher PFS than patients with wild-type CDKN2A (Figure 16B), and similar results were observed in patients with PTPND, CUBN, and HERC1 mutations (Figures 16C-16D).

[0297] In general, there were no significant differences in TMB when considering all patients' STK11 status or focusing only on patients with KRAS mutations (Figures 17A-17B). However, in wild-type STK11 patients, overall responsiveness may be slightly increased in patients with higher TMB compared to those with lower TMB (Figure 17C).

[0298] Example 3 An open-label, randomized phase 3 clinical trial was conducted to test the efficacy of second-line anti-PD-1 monoclonal antibody (nivolumab) treatment in patients with non-squamous NSCLC that had progressed during or after platinum-based dual chemotherapy. Patients were given either nivolumab (3 mg / kg body weight intravenously every two weeks) or docetaxel (75 mg / m²). 2 The drug was administered once every three weeks at the specified dose.

[0299] In post-hoc analysis, patients' tumor cells were analyzed for STK11 and KRAS mutation status. As seen in Example 2 above, patients with STK11 mutations generally showed lower overall PFS than patients with wild-type STK11 (Figures 18A-18D). This was the case when subjects had any non-synonymous STK11 mutation (Figures 18A-18B) or nonsense, frameshift, or splicing STK11 mutation (Figures 18D-18E), as well as when subjects had wild-type KRAS (Figures 18A and 18C) or a KRAS mutation (Figure 18B or 18D).

[0300] Example 4 An open-label, randomized phase 3 clinical trial was conducted to test the efficacy of a second-line anti-PD-1 monoclonal antibody (nivolumab) in patients with squamous cell carcinoma (NSCLC) that had progressed during or after platinum-based dual chemotherapy. Patients were given either nivolumab (3 mg / kg body weight intravenously every two weeks) or docetaxel (75 mg / m²). 2 The drug was administered once every three weeks at the specified dose.

[0301] In a post-hoc analysis, the STK11 mutation status of patients' tumor cells was examined. The incidence of STK11 mutations in the patient population was low (n=5), and patients with STK11 mutations showed a higher overall PFS after anti-PD-1 antibody treatment compared to patients with wild-type STK11 (Figure 19). Further analysis of more patients will confirm the usefulness of these initial observations.

[0302] This application claims the benefits of U.S. Provisional Application No. 62 / 513,831, filed June 1, 2017 (which is incorporated herein by reference in its entirety).

Claims

1. An antibody or its antigen-binding portion ("anti-PD-1 antibody") for treating a subject suffering from a tumor, wherein the subject has been identified as having a wild-type STK11 gene.

2. The anti-PD-1 antibody according to claim 1, wherein the subject has been identified as having a mutant marker gene selected from the group consisting of KRAS, TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

3. An antibody or its antigen-binding portion ("anti-PD-1 antibody") for treating a subject suffering from a tumor, wherein the subject is identified as having a mutant marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

4. The anti-PD-1 antibody according to claim 2 or 3, wherein the marker gene includes a nonsynonymous mutation, a nonsense mutation, a frameshift mutation, or a splicing mutation.

5. The anti-PD-1 antibody according to any one of claims 1 to 4, wherein the tumor is derived from lung cancer.

6. The anti-PD-1 antibody according to claim 5, wherein the tumor is derived from small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

7. The anti-PD-1 antibody according to any one of claims 1 to 6, wherein PD-L1 expression is detected in the tumor before administration of the anti-PD-1 antibody.

8. The anti-PD-1 antibody according to claim 7, wherein the tumor expresses PD-L1 in a scattered or heterogeneous pattern.

9. The anti-PD-1 antibody according to any one of claims 1 to 8, wherein the tumor has a tumor mutational load (TMB) state of high TMB.

10. The tumor TMB status is determined by sequencing the nucleic acids in the tumor and then identifying genomic changes in the sequenced nucleic acids, wherein the genomic changes include one or more changes selected from the group consisting of somatic mutations, nonsynonymous mutations, missense mutations, base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and any combination thereof. The anti-PD-1 antibody according to claim 9.

11. The aforementioned tumor exhibits high levels of inflammation. The anti-PD-1 antibody according to any one of claims 1 to 10.

12. The anti-PD-1 antibody according to any one of claims 1 to 11, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

13. The anti-PD-1 antibody according to any one of claims 1 to 12, wherein the anti-PD-1 antibody is administered once every 1, 2, or 3 weeks in a dose ranging from at least about 0.1 mg / kg to at least about 10.0 mg / kg body weight.

14. The anti-PD-1 antibody or its antigen-binding portion is administered once every 1, 2, 3, or 4 weeks in a flat dose of approximately 240 mg or approximately 480 mg. The anti-PD-1 antibody according to any one of claims 1 to 13.

15. A kit for treating subjects suffering from tumors, (a) Anti-PD-1 antibody in doses ranging from approximately 4 mg to approximately 500 mg; and (b) A kit comprising instructions for administering the anti-PD-1 antibody to subjects identified as having the wild-type STK11 gene.

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