Tumor treatment method using anti-PD-1 antibody

Abstract

The present disclosure provides a method for treating a subject suffering from a tumor, comprising administering to the subject an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (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 a wild-type STK11 gene. [Selection diagram] None

Description

[Technical Field]

[0001] The present disclosure relates to a method for treating tumors, comprising administering an anti-programmed death-1 (PD-1) antibody to a subject, wherein the subject has wild-type STK11. [Background technology]

[0002] Human cancers have numerous genetic and epigenetic alterations that generate potential neoantigens that can 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 potent anticancer capabilities due to its broad ability to respond to various tumor antigens and exquisite specificity. Furthermore, the immune system exhibits high plasticity and memory organization. Successfully harnessing all of these attributes of the adaptive immune system would make immunotherapy unique among all cancer treatment modalities.

[0003] PD-1 is an important immune checkpoint receptor expressed by T and B cells and 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 have been identified: programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2), which are expressed on antigen-presenting cells and many human cancers. Binding to PD-1 has been shown to downregulate T cell activation and cytokine secretion.

[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 Carcinoma, recently accessed May 14, 2014, available at: www.nccn.org / professionals / physician_gls / pdf / nscl.pdf). NSCLC is relatively insensitive to chemotherapy, but patients with stage IV disease who demonstrate good performance status (PS) benefit from treatment with chemotherapy agents, including platinum agents (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. Summary of the Invention

[0006] The present disclosure provides a method for treating a subject suffering from a tumor, the method comprising: (i) determining the mutational status of the STK11 gene in the subject; and (ii) administering to the subject an antibody or antigen-binding portion thereof (an "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 aspect, the present disclosure relates to a method for treating a subject suffering from a tumor, the method comprising administering an anti-PD-1 antibody to the subject, wherein the subject is identified as having a wild-type STK11 gene. In another aspect, the present disclosure relates to a method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody therapy, the method comprising: (i) determining the mutational status of the STK11 gene in the subject; and (ii) administering to the subject an anti-PD-1 antibody if the STK11 gene is wild-type. In certain embodiments, the method further comprises 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 aspect, the present disclosure relates to a method of treating a subject suffering from a tumor, comprising: (i) determining a mutation in a marker gene in the subject; and then (ii) if the marker gene is mutated, administering an anti-PD-1 antibody to the subject; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In another aspect, the present disclosure relates to a method of 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 mutated marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In another aspect, the present disclosure relates to a method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody therapy, 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 is mutated; the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. In one embodiment, the TP53 gene is mutated. In one embodiment, the CDKN2A gene is mutated. In one embodiment, the PTPND, CUBN, and HERC1 gene are mutated.

[0008] In certain embodiments, the marker gene comprises a non-synonymous mutation. In certain embodiments, the marker gene comprises a nonsense, frameshift, or splicing mutation.

[0009] In certain embodiments, the tumor is derived from lung cancer. In certain embodiments, the tumor is derived from small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In certain embodiments, the tumor is derived from NSCLC. In certain embodiments, the tumor is derived from non-squamous NSCLC. In other embodiments, the tumor is derived from squamous NSCLC.

[0010] In certain embodiments, the method further comprises detecting PD-L1 expression in the tumor prior to administration. In certain embodiments, the tumor expresses PD-L1 in a diffuse pattern. In certain embodiments, the tumor expresses PD-L1 in a heterogeneous pattern.

[0011] In certain embodiments, the mutation status of the STK11 gene is determined by sequencing the STK11 gene.

[0012] In certain embodiments, the tumor exhibits a high tumor mutation burden (TMB) status. In certain embodiments, the TMB status of the tumor is determined by sequencing nucleic acid in the tumor and identifying genomic alterations in the sequenced nucleic acid.

[0013] In certain embodiments, the tumor exhibits high inflammation, hi certain embodiments, the inflammation is measured by expression of STK11.

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

[0015] In some embodiments, the anti-PD-1 antibody is administered at a dose ranging from at least about 0.1 mg / kg to at least about 10.0 mg / kg body weight approximately once every 1, 2, or 3 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered as a flat dose. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered as a flat dose or about 240 mg.

[0016] In some embodiments, the administration treats tumors. In some embodiments, the administration reduces tumor size. In some embodiments, 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 some embodiments, the subject exhibits partial remission after administration. In some embodiments, the subject exhibits complete remission after administration.

[0017] In another aspect, the present disclosure provides a kit for treating a subject suffering from a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from about 4 mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in the methods described herein. In certain embodiments, the kit further comprises an anti-PD-L1 antibody.

[0018] Implementation E1. A method of treating a subject suffering from a tumor, comprising: (i) determining the mutational status of the STK11 gene in said subject; and (ii) administering to said subject an antibody or 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").

[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 has been 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 therapy, comprising: (i) determining the mutation 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.

[0021] E4. The method of 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 (ii) administering to the subject an antibody or 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 is mutated; 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 to the subject an anti-PD-1 antibody, 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] E7. A method for identifying a subject suffering from a tumor 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.

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

[0026] E9. The method described in 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 comprises a non-synonymous mutation.

[0029] E12. The method according to any one of E4 to E11, wherein the marker gene comprises a nonsense, frameshift, or splicing mutation.

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

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

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

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

[0034] E17. The method according to E15, wherein said tumor is derived from squamous NSCLC.

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

[0036] E19. The method of E18, wherein said tumor expresses PD-L1 in a diffuse pattern.

[0037] E20. The method of E19, wherein the scattered pattern of PD-L1 expression is characterized by a PD-L1 H-score of about 60 to about 500, about 80 to about 480, about 100 to about 460, about 120 to about 440, about 140 to about 420, about 160 to about 400, about 180 to about 380, about 200 to about 360, about 200 to about 340, about 200 to about 320, or about 200 to about 300.

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

[0039] The method of E21, wherein the diffuse pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 200.

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

[0041] E24. The method of E23, wherein the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of about 1 to about 50, about 5 to about 45, about 10 to about 40, or about 15 to about 35, and wherein said PD-L1 expression is restricted to one or more distinct parts of the tumor.

[0042] E25. The method of 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 of E25, wherein the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 15.

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

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

[0046] E29. The method of E28, wherein said IHC assay is an automated IHC assay.

[0047] E30. The method of 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 of any one of E18 to 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 the tumor cells express PD-L1.

[0049] E32. The method of any one of E1-E31, wherein said tumor exhibits a tumor mutation burden (TMB) status that is high TMB.

[0050] E33. The method of E32, wherein said tumor TMB status is determined by sequencing nucleic acid in the tumor and identifying genomic alterations in said sequenced nucleic acid.

[0051] E34. The method of E33, wherein said genomic alterations comprise one or more somatic mutations.

[0052] E35. The method of E33 or E34, wherein the genomic alteration comprises one or more non-synonymous mutations.

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

[0054] E37. The method of 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 alterations (CNAs), gene rearrangements, and any combination thereof.

[0055] E38. The 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, the method of any one of E32 to E37, wherein the score is 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.

[0056] E39. The method of any one of E32-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 of any one of E32 to E39, wherein said high TMB is a score of at least 243.

[0058] E41. The method of any one of E32-E40, further comprising comparing the TMB status of said subject to a reference TMB value.

[0059] E42. The method of E41, wherein said subject's TMB status is within the highest quantile of reference TMB values.

[0060] E43. The method of E41, wherein said subject's TMB status is within the highest tertile of reference TMB values.

[0061] E44. The method of any one of E32-E43, wherein said TMB status is determined by genomic sequencing.

[0062] E45. The method of any one of E32-E43, wherein said TMB status is determined by exome analysis.

[0063] E46. The method of any one of E32-E45, wherein said TMB status is determined by genomic profiling.

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

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

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

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

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

[0069] E52. The method of 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 of any one of E1 to E52, wherein the anti-PD-1 antibody is nivolumab.

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

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

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

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

[0075] E58. The method of any one of E1-E54, and 57, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a flat dose of at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, or at least about 550 mg.

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

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

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

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

[0080] E63. The method of any one of E1-E62, wherein the anti-PD-1 antibody is administered at a sub-therapeutic dose.

[0081] E64. The method of any one of E1-E63, wherein said administering treats said tumor.

[0082] E65. The method of any one of E1-E64, wherein said administering reduces tumor size.

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

[0084] E67. The method of any one of E1-E66, wherein 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.

[0085] E68. The method of any one of E1-E67, wherein the subject exhibits stable disease after said administering.

[0086] E69. The method of any one of E1-E67, wherein said subject exhibits a partial response after said administering.

[0087] E70. The method of any one of E1-E67, wherein said subject exhibits a complete response after said administering.

[0088] E71. A kit for treating a subject suffering from a tumor, comprising: (a) an anti-PD-1 antibody at a dose ranging from about 4 mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in a method according to any one of E1 to E70; Includes a kit.

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

[0090] [Figure 1] Figures 1A-1D show immunohistochemistry (IHC) patterns of PD-L1 expression in NSCLC tumors. PD-L1 expression patterns can be divided into scattered (Figure 1A), heterogeneous (Figure 1B), tumor-stroma junction (Figure 1C), and negative (Figure 1D). [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-stroma junction (T)) as shown in Figures 1A-1D (Figure 2A), as well as the distribution of PD-L1 H-scores in two NSCLC subtypes (i.e., adenocarcinoma and squamous cell carcinoma) (Figure 2D). [Figure 3] Figures 3A-3C show IHC profiles corresponding to scattered (Figure 3A), tumor-stroma junction (Figure 3B), and negative (Figure 3C) PD-L1 expression patterns corresponding to biopsies from patients treated with nivolumab monotherapy. [Figure 4] Figure 4 shows the PD-L1 H-scores for patients receiving nivolumab monotherapy. The predominant PD-L1 pattern in most complete responders (CR) and partial responders (PR) is a sparse pattern. [Figure 5] Figures 5A-5B show the overall CI score (Figure 5A) and PD-L1 CI score (Figure 5B) by PD-L1 tumor predominant pattern. [Figure 6] Figure 6 shows multiple IHC images stained for PD-L1, CD68, and CD3. [Figure 7] Figures 7A-7B show PD-L1 expression in NSCLC tumors according to PD-L1 expression patterns measured using RNA sequencing (Figure 7A) and mutational burden in NSCLC tumors according to PD-L1 expression patterns measured using exome analysis (Figure 7B). [Figure 8] Figures 8A-8B show the correlation between the number of missense mutations in NSCLC tumors and overall inflammation as measured by the CI score (Figure 8A), and the correlation between the number of missense mutations in NSCLC tumors and PD-L1+ inflammation as measured by the PDLP1pos CI score (Figure 8B). [Figure 9]Figures 9A-9B show the mutation frequency in the PD-L1 expression patterns observed relative to different biomarkers (TP53, STK11, KEAP1, KRAS, EGFR, and MET) in Figure 9A. D = scattered, H = heterogeneous, I = tumor-stroma junction, N = negative. Figure 9B shows the presence ("y") or absence ("n") of STK11 mutations relative to PD-L1 expression measured by RNA sequencing (RNAseq). [Figure 10] Figures 10A-10C show the correlation between the presence ("y") or absence ("n") of STK11 mutations and PD-L1+CI score (Figure 10A). Numerical data corresponding to the information shown in Figure 10A is shown in Figure 10B. Figure 10C shows numerical data corresponding to the global inflammation score in NSCLC tumors according to the presence ("STK11-MUT") or absence ("STK11-WT") of STK11 mutations. [Figure 11] Figure 11 shows immunoprint analysis of 24 NSCLC tumor samples, analyzing levels of FOLR2, VSIG4, CD163, CLEC4D, CSF1R, CD86, MS4A1, CD79B, CD19, KIR2DS4, CD3E, CCR4, CCR8, and CD8A to classify samples according to inflammation patterns (sigClass). 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 STK11 mutation. In addition, samples were classified according to PD-L1 expression pattern as negative ("PDL1_pattern2 negative"), scattered ("PDL1_pattern2 scattered"), heterogeneous ("PDL1_pattern2 heterogeneous"), and tumor-stroma junction ("PDL1_pattern2TS"). [Figure 12]Figures 12A-12B are graphical representations of survival probability for advanced NSCLC subjects with wild-type STK11 (data labels 2 and 4) or mutant STK11 (data labels 1 and 3) treated with physician's choice of 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 to STK11, while Figure 12B includes only mutant STK11 subjects with nonsense, frameshift, or splicing mutations to STK11. The number of subjects at risk at each time point for each group is shown below the x-axis. [Figure 13] Figure 13 is a graphical representation of survival probability for advanced NSCLC subjects with KRAS mutations and wild-type STK11 (data labels 2 and 4) or mutant STK11 (data labels 1 and 3), who were treated with physician's choice of chemotherapy (data labels 1 and 2) or first-line 3 mg / kg anti-PD-1 antibody treatment (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. [Figure 14] Figures 14A-14B are graphical representations of survival probability for non-squamous NSCLC subjects with wild-type STK11 (data labels 2 and 4) or non-synonymous mutations in STK11 (data labels 1 and 3) treated with physician's choice of 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 subjects meeting this criteria, while Figure 14B includes only subjects who also have a KRAS mutation. The number of subjects at risk at each time point for each group is shown below the x-axis. [Figure 15]Figures 15A-15E are graphical representations showing the correlation between PDL1 expression levels and STK11 mutation status. Figures 15A and 15B show the distribution of WT or mutant STK11 subjects who experience a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) after treatment with a physician's choice of 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 experience a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) after treatment with a physician's choice of 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's choice chemotherapy (Figure 15D) or first-line 3 mg / kg anti-PD-1 antibody treatment (nivolumab; Figure 15E). The Y-axis shows the percentage of tumor cells expressing PD-L1 (Figures 15A-15E). Figure 15F is a table providing the status of specific mutations for the subset of subjects with STK11 mutations. [Figure 16]Figure 16A is a graphical representation of survival probability for subjects with advanced NSCLC harboring a KRAS mutation and wild-type TP53 (data labels 2 and 4) or mutant TP53 (data labels 1 and 3), who were treated with either a physician's choice of 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 probability for subjects with advanced NSCLC harboring wild-type (data labels 2 and 4) or mutant (data labels 1 and 3) CDKN2A (Figure 16B) or PTPND / CUBN / HERC1 (Figure 16C) who were treated with a physician's choice of 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). FIG. 16D is a screenshot showing the distribution of HERC1, CUBNM, and PTPRD mutations among the 1144 subjects analyzed. [Figure 17] Figures 17A-17B are distribution plots showing the association between tumor mutation burden and STK11 mutation status in all analyzed advanced NSCLC subjects (Figure 17A) or in the subpopulation that additionally harbored KRAS mutations (Figure 17B). Figure 17C is a graphical representation showing the distribution of WT or mutant STK11 subjects experiencing complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) following treatment with physician's choice chemotherapy or first-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab) in relation to tumor mutation burden (TMB; Figure 17C). [Figure 18]Figures 18A-18D are graphical representations of survival probability 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 second-line 3 mg / kg anti-PD-1 antibody therapy (nivolumab; data labels 3 and 4). Figure 18A includes all subjects with any nonsynonymous STK11 mutation; Figure 18B includes subjects with any nonsynonymous STK11 mutation and any KRAS mutation; Figure 18C includes all subjects with any nonsense, frameshift, or splicing mutation in STK11; and Figure 18D includes subjects with any nonsense, frameshift, or splicing mutation in STK11 and any KRAS mutation. The number of subjects at risk at each time point for each group is shown below the x-axis (Figures 18A-18D). [Figure 19] Figure 19 is a graphical representation of survival probability for 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 second-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 (Figure 19). DETAILED DESCRIPTION OF THE INVENTION

[0091] The present disclosure relates to a method for treating a subject suffering from a tumor, comprising: (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 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") or an antibody or antigen-binding portion thereof that specifically binds to programmed death-ligand 1 (PD-L1) and inhibits PD-1 activity (an "anti-PD-L1 antibody"). In one embodiment, the tumor is derived from NSCLC.

[0092] term In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms has the meaning set forth below. Additional definitions are set forth throughout this application.

[0093] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of 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, e.g., by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, the combination is administered by a non-parenteral route, in certain embodiments, orally. Other non-parenteral routes include, for example, intranasal, intravaginal, rectal, sublingual, or topical, epithelial, or mucosal routes of administration. Administration can also be, for example, one time, multiple times, and / or over one or more extended periods of time.

[0094] As used herein, an "adverse event" (AE) is an unfavorable, generally unintended, or undesired sign (including laboratory abnormalities), symptom, or disease associated with the administration of a medical treatment. For example, an adverse event may be associated with immune system activation or proliferation of immune system cells (e.g., T cells) in response to the treatment. A medical treatment may exhibit one or more associated AEs, each of which may exhibit the same or different levels of severity. A method that can "alter adverse events" refers to a treatment regimen that reduces the occurrence and / or severity of one or more AEs associated with the administration of a different treatment regimen.

[0095] "Antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, inter-connected by disulfide bonds. Each H chain contains a heavy chain variable region (herein referred to as V H and a heavy chain constant region. The heavy chain constant region comprises at least three constant domains, C H1 , C H2 , and C H3 Each light chain comprises a light chain variable region (referred to herein as V L and a light chain constant region. The light chain constant region comprises one constant domain, C L Includes V H and V L The regions are further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). H and V L contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0096] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, by way of example, natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully 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 dictates otherwise, the term "antibody" also includes any antigen-binding fragment or portion of the immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain antibodies.

[0097] An "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0098] The term "monoclonal antibody" ("mAb") refers to antibody molecules of single molecular composition, i.e., a non-naturally occurring preparation of antibody molecules that are essentially identical in primary sequence and that exhibit binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. MAbs can be prepared by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0099] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0100] "Humanized antibody" refers to an antibody in which any, most, or all of the non-human antibody regions other than the CDR domains have been replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, any, most, or all of the regions other than the CDR domains have been replaced with amino acids from a human immunoglobulin, while any, most, or all of the regions within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity and antigenic properties similar to those of the original antibody.

[0101] "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, e.g., whose variable region is derived from a murine antibody and whose constant region is derived from a human antibody.

[0102] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1.

[0103] An "antigen-binding portion" (also called an "antigen-binding fragment") of an antibody means one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody.

[0104] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can form malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0105] "Serine / threonine kinase 11" or "STK11" (also known as "polarity-associated protein LKB1," "renal cancer antigen NY-REN-19," "liver kinase B1," "EC 2.7.11.1," and "HLKB1") refers to a member of the serine / threonine kinase family that regulates cell polarity and functions as a tumor suppressor. STK11 controls the activity of AMP-activated protein kinase (AMPK) family members, thereby playing a role in various processes such as cell metabolism, cell polarity, apoptosis, and DNA damage response. STK11 is ubiquitously expressed, most highly in testis and fetal liver. STK11 is generally inactivated in NSCLC, particularly in tumors harboring KRAS mutations. As described herein, mutant STK11, e.g., loss of wild-type expression of STK11, correlates with reduced or aberrant PD-L1 expression in tumors derived from NSCLC. In some embodiments, the loss of mutant STK11, e.g., wild-type STK11 expression, occurs in tumors derived from NSCLC, and the tumors either express or do not express wild-type KRAS (e.g., the tumors either have or do not have KRAS mutations). In some embodiments, the STK11 mutant is an STK11 mutant 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 by reference in its entirety).

[0106] "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, with the majority of these mutations resulting in constitutive activation 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 the G1 and G2 phases. Loss-of-function mutations in CDKN2A are common in lung cancer. As used herein, "CUBN" refers to the gene encoding cubilin, a receptor for the intrinsic factor-vitamin B12 complex. As used herein, "HERC1" refers to the gene encoding the HECT and RLD domain-containing E3 ubiquitin protein ligase family member 1 (HERC1), a member of the HERC family. HERC1 activates guanine nucleotide exchange in ARF1 and Rab proteins and may be involved in membrane transport processes.

[0107] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from a disease or of recurrence of a disease by methods involving inducing, enhancing, suppressing, or modulating an immune response. "Treatment" or "therapy" of a subject refers to an intervention or method performed in a subject, or the administration of an active agent to said subject, to alleviate, ameliorate, suppress, delay, or prevent the onset, worsening, progression, severity, or recurrence of symptoms, complications, or conditions, or biochemical manifestations associated with a disease.

[0108] 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 measured by any method known in the art. In other embodiments, the PD-L1 expression is measured by automated IHC. Thus, a PD-L1-positive tumor can exhibit 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 that express PD-L1 on the cell surface.

[0109] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed primarily 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, isoforms, and species homologs of hPD-1, and analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863.

[0110] "Programmed death-ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion by binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0111] A "subject" includes either a human or a non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0112] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is the amount of drug, when used alone or in combination with another therapeutic agent, that protects a subject from developing the disease or promotes regression of the disease as indicated by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to the affliction of the disease. The efficacy of a therapeutic agent in promoting regression of the disease can be assessed using a variety of methods known to those skilled in the art, for example, by measuring the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0113] As used herein, a "sub-therapeutic dose" means a dose that is lower than the usual or typical dose of a therapeutic compound (e.g., an antibody) when administered alone to treat a hyperproliferative disease (e.g., cancer).

[0114] As an example, an "anti-cancer drug" promotes the regression of cancer in a subject or suppresses further tumor growth. In certain embodiments, a therapeutically effective amount of the drug promotes the regression of cancer until the cancer is eliminated. "Promoting the regression of cancer" means that administration of an effective amount of the drug, alone or in combination with an anti-tumor drug, results in a decrease 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 symptom-free periods, or prevention of disability or disability due to disease suffering. In addition, the terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (side effects) at the cellular, organ, and / or organismal levels resulting from the administration of a drug.

[0115] As an example for tumor treatment, a therapeutically effective amount of an anticancer agent 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 untreated subjects. In other embodiments of the present disclosure, tumor regression can be observed and continued for at least about 20 days, at least about 40 days, or at least about 60 days. Despite these ultimate measures of therapeutic efficacy, evaluation of immunotherapeutic drugs must also consider "immune-related" response patterns.

[0116] "Immune-related" response pattern refers to a clinical response pattern commonly observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or modulating endogenous immune processes. This response pattern is characterized by an initial increase in tumor burden or the appearance of new lesions, which would be classified as disease progression and synonymous with drug failure in the evaluation of conventional chemotherapy agents, followed by a beneficial therapeutic effect. Therefore, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0117] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is the amount of drug that, when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject exhibiting a pre-malignant state) or a subject at risk of cancer recurrence, inhibits the onset or recurrence of the cancer. In some embodiments, a prophylactically effective amount inhibits the onset or recurrence of cancer altogether. "Inhibiting" the onset or recurrence of cancer means reducing the likelihood of cancer progression or recurrence, or inhibiting the onset or recurrence of cancer altogether.

[0118] As used herein, the term "wild-type" refers to a gene having a nucleotide sequence encoding a protein exhibiting an amino acid sequence known in the art as a common sequence. For example, in one embodiment, a "wild-type" STK11 has a nucleotide sequence encoding an STK11 protein having an amino acid sequence identical to that of the protein having UniProt identifier Q15831-1. In one embodiment, a "wild-type" gene can have a mutation relative to the gene's conventional nucleotide sequence, so long as the mutation is synonymous (e.g., a nucleotide mutation that does not result in a change in the amino acid sequence of the resulting protein). Conversely, as used herein, a "mutated" or "mutant" gene refers to a gene having one or more nucleotide substitutions, insertions, or deletions that alter the resulting amino acid sequence (e.g., a non-synonymous mutation). A mutant gene may or may not be expressible. In one embodiment, a non-synonymous mutation is a "nonsense mutation" in which a nucleotide substitution or deletion results in a premature stop codon. In some embodiments, the non-synonymous mutation is a "frameshift mutation," in which the nucleotide substitution or deletion involves the insertion or deletion of a number of nucleotides that are not divisible by three, causing a shift in the translation of the sequence. In some embodiments, the non-synonymous mutation is a "splicing mutation," in which the nucleotide substitution or deletion prevents or creates a splice site.

[0119] As used herein, the term "tumor mutation burden" (TMB) refers to the number of somatic mutations in the genome of a tumor and / or the number of somatic mutations per region of the genome of a tumor. Germline (inherited) mutations are excluded when measuring TMB, because the immune system is likely to recognize them as its own. TMB is a genetic analysis of the genome of a tumor, and can be measured by 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 tumors, which are identified by standardizing the tumor to a germline sample to exclude inherited germline genetic changes. To measure TMB, a sufficient amount of sample is required. In one embodiment, a tissue sample (for example, a minimum of 10 sections) is used for evaluation.

[0120] The TMB status can be a numerical or relative value (eg, high, intermediate, or low) within the highest quantile or within a tertile of a reference set.

[0121] As used herein, the term "high TMB" refers to a number of somatic mutations in the genome of a tumor that exceeds the normal or average number of somatic mutations. In some embodiments, the 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, at least In some embodiments, high TMB exhibits a score 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 other embodiments, high TMB exhibits a score 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 some embodiments, high TMB exhibits a score of at least 243. In other embodiments, "high TMB" refers to TMB within the highest quantile of reference TMB values.For example, a "reference TMB value" can be determined by grouping the values ​​of all subjects with evaluable TMB data according to the quantile distribution of TMB, i.e., subjects are ranked from highest to lowest genetic alterations and classified into a certain number of groups. In one embodiment, all subjects with evaluable TMB data are ranked and classified into three groups, with "high TMB" being within the highest quantile of the reference TMB value. In one embodiment, the tertile boundaries are 0<100 genetic alterations; 100-243 genetic alterations; and >243 genetic alterations. It should be understood that once ranked, subjects with evaluable TMB data can be classified into any number of groups (e.g., quartiles, quintiles, etc.).

[0122] As used herein, the term "intermediate TMB" refers to a number of somatic mutations in a tumor genome that is at or above the normal or average number of somatic mutations, and the term "low TMB" refers to a number of somatic mutations in a tumor genome that is less than the normal or average number of somatic mutations. In certain embodiments, "high TMB" refers to a score of at least 243, "intermediate TMB" refers to a score of 100 to 242, and "low TMB" refers to a score of 100 or less (or 0 to 100).

[0123] In some embodiments, TMB status may be correlated with smoking status. In particular, current or former smokers (d) often exhibit more genetic alterations, e.g., missense mutations, than non-smoking subjects (d).

[0124] Tumors with high TMB may also exhibit high neoantigen loads. As used herein, the term "neoantigen" refers to a newly formed antigen not previously recognized by the immune system. A neoantigen can be a protein or peptide recognized by the immune system as foreign (or non-self). Transcription of a gene in the genome of a tumor carrying a somatic mutation generates a mutant mRNA, which, when translated, produces a mutant protein, which is subsequently processed and transported to the ER lumen, where it then binds to the MHC class I complex, promoting T cell recognition of the neoantigen. Recognition of the neoantigen can promote T cell activation, clonal expansion, and differentiation into effector and memory T cells.

[0125] The TMB status of a tumor can be used alone or in combination with other factors in determining whether a patient is likely to benefit from a particular anti-cancer agent or type of treatment or therapy, e.g., a cancer immunotherapy agent, e.g., an anti-PD-1 antibody or antigen-binding portion thereof, or an anti-PD-L1 antibody or antigen-binding portion thereof. In one embodiment, high TMB status (or high TMB) indicates a high likelihood of benefiting from cancer immunotherapy and can thus be used to identify patients who are more likely to benefit from treatment with an anti-PD-1 antibody or antigen-binding portion thereof. As used herein, the term "benefiting from treatment" refers to an improvement in one or more of overall survival, progression-free survival, partial response, complete response, and overall response rate, and can include a decrease in tumor growth or size, a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or incapacity due to disease affliction.

[0126] The terms "measuring," "measured," or "measurement" refer to examining the measurable amount of somatic mutations in a subject's biological sample, as they indicate the TMB status or mutation status of a gene. Measurements can be performed by sequencing nucleic acids in the sample, such as cDNA, mRNA, exoRNA, ctDNA, and cfDNA. Measurements can be performed on the subject's sample and / or a reference sample, and can be newly detected or correspond to previous measurements. Measurements can be performed using, for example, PCR, qPCR, Sanger sequencing, genomic profiling (including comprehensive gene panels), exome analysis, genome sequencing, and / or other methods described herein, as known to those skilled in the art. In some embodiments, the measurements identify genomic alterations in the sequenced nucleic acids. Genomic (or gene) profiling methods can involve a panel of a predetermined set of genes, e.g., 150 to 500 genes, and in some instances, the genomic alterations assessed in the gene panel correlate with the total somatic mutations assessed.

[0127] As used herein, the term "genomic alteration" refers to a change (or mutation) in the nucleotide sequence of a tumor genome (wherein the change is not present in the germline nucleotide sequence), and in some embodiments, is a non-synonymous mutation, including but not limited to, base pair substitution, base pair insertion, base pair deletion, copy number alteration (CNA), gene rearrangement, and any combination thereof. In some embodiments, the genomic alteration measured in a biological sample is a missense mutation.

[0128] As used herein, the term "biological sample" refers to a biological material isolated from a subject. A biological sample can include, for example, suitable biological material for investigating TMB by sequencing nucleic acid in tumors (or circulating tumor cells) and identifying genomic alterations in the sequenced nucleic acid. The biological sample can be 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, paraffin-embedded tumor tissue or fresh-frozen tumor tissue. In another embodiment, the biological sample is a liquid biopsy, including, in some embodiments, one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA, and cfDNA.

[0129] The use of the alternative (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of the described or listed components.

[0130] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, depending in part on the method by which the value or composition is measured or determined (i.e., the limitations of the measurement system). For example, "about" or "essentially comprising" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "essentially comprising" can mean within a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any 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 can mean up to an order of magnitude or up to five times the numerical value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be within an acceptable error range for the particular value or composition.

[0131] As used herein, the terms "about once every week," "about once every two weeks," or other similar administration interval terms refer to approximate numbers. "About once every week" can include every 7 days ± 1 day, i.e., every 6 to 8 days. "About once every two weeks" can include every 14 days ± 3 days, i.e., every 11 to 17 days. Similar approximations apply, for example, to about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, and about once every 12 weeks. In some embodiments, an administration interval of about once every 6 weeks or about once every 12 weeks means that the first dose can be administered on any day in the first week, followed by the next dose on any day in the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose is administered on a particular day (e.g., Monday) in week 1, followed by the next dose on the same day (i.e., Monday) in week 6 or week 12, respectively.

[0132] 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.

[0133] The use of the term "fixed dose" in reference to the methods of the present disclosure means that two or more different antibodies (e.g., an anti-PD-1 antibody and a second antibody) in a single composition are present in the composition, particularly in a (fixed) ratio relative to one another. In certain embodiments, the fixed dose is based on the weight (e.g., mg) of the antibody. In certain embodiments, the fixed dose is based on the concentration (e.g., mg / ml) of the antibody. In certain embodiments, the ratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about The ratio may be 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg of a first antibody (e.g., an anti-PD-1 antibody). For example, a 3:1 ratio of anti-PD-1 antibody and a second antibody may mean that a vial contains about 240 mg of anti-PD-1 antibody and 80 mg of the second antibody, or about 3 mg / ml of anti-PD-1 antibody and 1 mg / ml of the second antibody.

[0134] The use of the term "flat dose" with respect to the methods and doses of the present disclosure refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Thus, a flat dose is not provided as a mg / kg dose of an agent (e.g., an anti-PD-1 antibody), but rather as an absolute amount. For example, a 60 kg human and a 100 kg human would receive the same dose of antibody (e.g., 240 mg of an anti-PD-1 antibody).

[0135] As described herein, concentration ranges, percentage ranges, ratio ranges, or integer ranges should be understood to include the integers within the stated range, and fractions thereof, as appropriate (e.g., tenths and hundredths of integers), unless otherwise stated.

[0136] Various aspects of the disclosure are described in further detail in the following sections.

[0137] Methods of the present disclosure The present disclosure provides a method for treating a subject suffering from a tumor, comprising: (i) determining the mutation status of the STK11 gene in the subject; and (ii) administering to the subject an antibody or 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"). In one embodiment, the subject suffering from the tumor (e.g., NSCLC, e.g., non-squamous NSCLC) is administered the anti-PD-1 antibody if the STK11 gene is wild-type. 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), wherein the subject has been identified as having a wild-type STK11 gene, comprising administering an anti-PD-1 antibody to the subject. In certain aspects, the disclosure relates to a method for identifying a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC) suitable for anti-PD-1 antibody therapy, comprising: (i) determining the mutation status of the STK11 gene in said subject; and then (ii) administering an anti-PD-1 antibody to said subject if the STK11 gene is wild-type.

[0138] The present disclosure further provides a method for treating a subject suffering from a tumor, comprising: (i) determining the mutation status of the STK11 gene in the subject; and (ii) administering to the subject an antibody or antigen-binding portion thereof that specifically binds to programmed death-ligand 1 (PD-L1) and inhibits PD-1 activity (an "anti-PD-L1 antibody"). In one embodiment, the subject suffering from the tumor (e.g., NSCLC, e.g., non-squamous NSCLC) is administered an anti-PD-L1 antibody if the STK11 gene is wild-type. 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), wherein the subject has been identified as having a wild-type STK11 gene, comprising administering an anti-PD-L1 antibody to the subject. In certain aspects, the disclosure relates to a method for identifying a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC) that is suitable for anti-PD-L1 antibody treatment, comprising: (i) determining the mutation status of the STK11 gene in said subject; and then (ii) administering an anti-PD-L1 antibody to said subject if the STK11 gene is wild-type.

[0139] In another aspect, the disclosure relates to a method of treating a subject afflicted with a tumor (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 as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist, if the STK11 gene comprises a non-synonymous mutation. Another aspect of the present disclosure relates to a method for identifying a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC) that is unsuitable for anti-PD-1 antibody treatment, the method comprising: (i) determining the mutation status of the STK11 gene in the subject; and then (ii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist, if the STK11 gene comprises a non-synonymous mutation.

[0140] In another aspect, the disclosure relates to a method of treating a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC), comprising: (i) determining the mutation status of the STK11 gene in said subject; and (ii) identifying said subject as unsuitable for administration of an anti-PD-L1 antibody, e.g., not administering an anti-PD-L1 antibody to said subject, or terminating or increasing anti-PD-L1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-L1 antagonist, if the STK11 gene contains a non-synonymous mutation. Another aspect of the present disclosure relates to a method for identifying a subject suffering from a tumor (e.g., NSCLC, e.g., non-squamous NSCLC) that is unsuitable for anti-PD-L1 antibody treatment, the method comprising: (i) determining the mutation status of the STK11 gene in said subject; and (ii) if the STK11 gene comprises a non-synonymous mutation, identifying said subject as unsuitable for administration of an anti-PD-L1 antibody, e.g., not administering an anti-PD-L1 antibody to said subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-L1 antagonist.

[0141] In another aspect, the present disclosure relates to a method of treating a subject suffering from a tumor, comprising: (i) determining the mutation status of a marker gene in the subject; and then (ii) if the marker gene is mutated, administering an anti-PD-1 antibody to the subject; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Another aspect of the present disclosure relates to a method of 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 mutated marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof. Yet another aspect of the present disclosure relates to a method for identifying a subject suffering from a tumor suitable for anti-PD-1 antibody therapy, 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 is mutated; wherein the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof.

[0142] In another aspect, the disclosure relates to a method of treating a subject suffering from a tumor, comprising: (i) determining the mutation status of a marker gene in the subject; and then (ii) if the marker gene is mutated, administering an anti-PD-L1 antibody to the subject; 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 of treating a subject suffering from a tumor, wherein the subject is identified as having a mutated marker gene, and the marker gene is selected from the group consisting of TP53, CDKN2A, PTPND, CUBN, HERC1, and any combination thereof, comprising administering an anti-PD-L1 antibody to the subject. Yet another aspect of the present disclosure relates to a method for identifying a subject suffering from a tumor suitable for anti-PD-L1 antibody treatment, comprising: (i) determining a mutation in a marker gene in the subject; and then (ii) administering an anti-PD-L1 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.

[0143] In some embodiments, the subject has a mutation in TP53. In some embodiments, the subject has a mutation in CDKN2A. In some embodiments, the subject has a mutation in PTPND. In some embodiments, the subject has a mutation in CUBN. In some embodiments, the subject has a mutation in HERC1. In some embodiments, the subject has mutations in PTPND and CUBN. In some embodiments, the subject has mutations in PTPND and HERC1. In some embodiments, the subject has mutations in CUBN and HERC1. In some embodiments, the subject has mutations in PTPND, CUBN, and HERC1.

[0144] In some embodiments, the wild-type STK11 contains one or more synonymous mutations, wherein the mutations in the genomic sequence do not affect the sequence of the expressed protein. In some embodiments, the mutant STK11 contains a nonsynonymous mutation. In some embodiments, the mutant STK11 contains a nonsense mutation. In some embodiments, the mutant STK11 contains a frameshift mutation. In some embodiments, the mutant STK11 contains a splicing mutation. In some embodiments, the mutant STK11 is expressed as mRNA and as a protein. In some embodiments, the mutant STK11 protein is functional. In other embodiments, the mutant STK11 protein has reduced activity. In other embodiments, the mutant STK11 protein is non-functional.

[0145] In some embodiments, the mutant TP53 comprises a nonsynonymous mutation. In some embodiments, the mutant TP53 comprises a nonsense mutation. In some embodiments, the mutant TP53 comprises a frameshift mutation. In some embodiments, the mutant TP53 comprises a splicing mutation. In some embodiments, the mutant TP53 is expressed as mRNA and as a protein. In some embodiments, the mutant TP53 protein is functional. In other embodiments, the mutant TP53 protein has reduced activity. In other embodiments, the mutant TP53 protein is non-functional.

[0146] In some embodiments, the mutant CDKN2A comprises a nonsynonymous mutation. In some embodiments, the mutant CDKN2A comprises a nonsense mutation. In some embodiments, the mutant CDKN2A comprises a frameshift mutation. In some embodiments, the mutant CDKN2A comprises a splicing mutation. In some embodiments, the mutant CDKN2A is expressed as mRNA and as a protein. In some embodiments, the mutant CDKN2A protein is functional. In other embodiments, the mutant CDKN2A protein has reduced activity. In other embodiments, the mutant CDKN2A protein is non-functional.

[0147] In some embodiments, the mutant PTPND comprises a nonsynonymous mutation. In some embodiments, the mutant PTPND comprises a nonsense mutation. In some embodiments, the mutant PTPND comprises a frameshift mutation. In some embodiments, the mutant PTPND comprises a splicing mutation. In some embodiments, the mutant PTPND is expressed as mRNA and as a protein. In some embodiments, the mutant PTPND protein is functional. In other embodiments, the mutant PTPND protein has reduced activity. In other embodiments, the mutant PTPND protein is non-functional.

[0148] In some embodiments, the mutant CUBN comprises a nonsynonymous mutation. In some embodiments, the mutant CUBN comprises a nonsense mutation. In some embodiments, the mutant CUBN comprises a frameshift mutation. In some embodiments, the mutant CUBN comprises a splicing mutation. In some embodiments, the mutant CUBN is expressed as mRNA and as a protein. In some embodiments, the mutant CUBN protein is functional. In other embodiments, the mutant CUBN protein has reduced activity. In other embodiments, the mutant CUBN protein is non-functional.

[0149] In some embodiments, the mutant HERC1 comprises a nonsynonymous mutation. In some embodiments, the mutant HERC1 comprises a nonsense mutation. In some embodiments, the mutant HERC1 comprises a frameshift mutation. In some embodiments, the mutant HERC1 comprises a splicing mutation. In some embodiments, the mutant HERC1 is expressed as mRNA and as a protein. In some embodiments, the mutant HERC1 protein is functional. In other embodiments, the mutant HERC1 protein has reduced activity. In other embodiments, the mutant HERC1 protein is non-functional.

[0150] In some embodiments, the tumor originates from lung cancer. In some embodiments, the tumor originates from NSCLC. In some embodiments, the subject is a human patient. In some embodiments, the subject is a chemotherapy-naive patient (e.g., a patient who has not received any chemotherapy). In other embodiments, the subject for the combination therapy of the present invention has received another cancer treatment (e.g., chemotherapy), but is resistant or refractory to such another cancer treatment.

[0151] In certain embodiments, the present disclosure provides a method for treating a subject suffering from squamous NSCLC, comprising: (i) determining the mutation status of the STK11 gene in the subject; and then (ii) administering an anti-PD-1 antibody (or an anti-PD-L1 antibody) to the subject if the STK11 gene is mutated. In certain embodiments, the present disclosure relates to a method for treating a subject suffering from squamous NSCLC, comprising administering an anti-PD-1 antibody to the subject, wherein the subject has been identified as having a mutant STK11 gene. In certain embodiments, the present disclosure relates to a method for identifying a subject suffering from squamous NSCLC 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 mutated.

[0152] In another aspect, the disclosure relates to a method of treating a subject suffering from squamous NSCLC, comprising: (i) determining the mutation status of the STK11 gene in the subject; and (ii) administering an anti-PD-1 antibody (or anti-PD-L1 antibody) to the subject, or terminating or increasing anti-PD-1 antibody (or anti-PD-L1 antibody) treatment, if the STK11 gene contains a non-synonymous mutation.

[0153] In certain embodiments, a treatment of the present disclosure (e.g., administration of an anti-PD-1 antibody or anti-PD-L1 antibody) effectively increases a subject's survival. In certain embodiments, an anti-PD-1 antibody treatment of the present disclosure increases a subject's progression-free survival. In certain embodiments, an anti-PD-1 antibody treatment of the present disclosure increases a subject's progression-free survival compared to standard treatment. After administration of an anti-PD-1 antibody treatment, a subject afflicted with a tumor may exhibit an overall survival of at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0154] 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 of care (e.g., docetaxel) alone or 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 of care (e.g., docetaxel) alone or a different dosing schedule of combination therapy.

[0155] In some embodiments, the treatment of the present disclosure effectively increases the progression-free survival time of the subject.In some embodiments, the subject shows the progression-free survival time 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.

[0156] In one embodiment, administration of an anti-PD-1 antibody treats a tumor. In one embodiment, the administration reduces 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 other embodiments, the subject exhibits 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 after the first administration. In one embodiment, the subject exhibits stable disease after administration. In certain embodiments, the subject experiences a partial response following administration. In certain embodiments, the subject experiences a complete response following administration. In certain embodiments, the subject experiences an improved objective response rate (ORR) following administration compared to subjects treated with standard of care.

[0157] 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 is a never smoker. In one embodiment, the subject is a former smoker. In one embodiment, the subject is a current smoker. In one embodiment, the subject has squamous cancer cells. In one embodiment, the subject has non-squamous cancer cells.

[0158] In certain embodiments, the tumor displays a diffuse pattern of PD-L1 expression. In certain embodiments, the scattered pattern of PD-L1 expression is characterized by a PD-L1 H-score of about 60 to about 500, about 70 to about 490, about 80 to about 480, about 90 to about 470, about 100 to about 460, about 110 to about 450, about 120 to about 440, about 130 to about 430, about 140 to about 420, about 150 to about 410, about 160 to about 400, about 170 to about 390, about 180 to about 380, about 190 to about 370, about 200 to about 360, about 20 to about 350, about 200 to about 340, about 200 to about 330, about 200 to about 320, about 200 to about 310, or about 200 to about 300. In certain embodiments, the sparing pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 225, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 275, at least about 280, at least about 290, or at least about 300. In certain embodiments, the sparing pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 200. In other embodiments, the diffuse pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 240. In certain embodiments, the diffuse pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 260.

[0159] In certain embodiments, the tumor exhibits a heterogeneous pattern of PD-L1 expression. In certain embodiments, the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of about 1 to about 50, about 5 to about 45, about 10 to about 40, or about 15 to about 35, and the PD-L1 expression is restricted to one or more distinct portions of the tumor. In certain embodiments, 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. In certain embodiments, the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 15. In other embodiments, the heterogeneous pattern of PD-L1 expression is characterized by a PD-L1 H-score of at least about 20. In certain embodiments, the heterogeneous pattern of PD-L1 expression is characterized by a fraction of the tumor comprising 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 expressing PD-L1. In certain embodiments, 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 fraction of tumor cells express PD-L1.

[0160] In certain embodiments, the tumor exhibits PD-L1 expression at the tumor-stroma junction. In certain embodiments, PD-L1 expression at the tumor-stroma junction is characterized by PD-L1 expression by tumor cells adjacent to the junction (e.g., within about 1 cell diameter, about 2 cell diameters, about 3 cell diameters, about 4 cell diameters, about 5 cell diameters, about 6 cell diameters, about 7 cell diameters, about 8 cell diameters, about 9 cell diameters, or about 10 cell diameters). In certain embodiments, PD-L1 expression at the tumor-stroma junction is characterized by PD-L1 expression on the surface of the tumor.

[0161] The PD-L1 expression status of a tumor in a subject can be measured before administering a composition described herein or using a method described herein. In certain embodiments, 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 other embodiments, the PD-L1 expression status of the tumor is at least about 1%. In other embodiments, the PD-L1 expression status of the tumor is at least about 5%. In certain embodiments, the PD-L1 expression status of the tumor is at least about 10%. In certain embodiments, the PD-L1 expression status of the tumor is at least about 25%. In certain embodiments, the PD-L1 expression status of the tumor is at least about 50%.

[0162] In certain embodiments, the method of the disclosure comprises: (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 said tumor; and (iii) detecting a tumor in which 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%, and (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 certain embodiments, the method of the disclosure comprises: (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 certain embodiments, the method of the disclosure comprises: (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.

[0163] In certain embodiments, the method of the disclosure comprises: (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) if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 50% or less, identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist. In certain embodiments, the method of the present disclosure comprises: (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) if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 40% or less, identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the subject afflicted with a tumor (e.g., NSCLC, e.g., non-squamous NSCLC); and then (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 30% or less.In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 25% or less. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 20% or less. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 15% or less. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 10% or less.In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 5% or less. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 3% or less, identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer drug that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 2% or less. In certain embodiments, the method of the present disclosure comprises: (i) determining the mutation status of the STK11 gene in the subject; (ii) detecting PD-L1 expression in the tumor; and (iii) identifying the subject as unsuitable for administration of an anti-PD-1 antibody, e.g., not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, e.g., administering an anti-cancer agent that is not a PD-1 antagonist, if the STK11 gene contains a non-synonymous mutation and the tumor has PD-L1 expression of about 1% or less.

[0164] In certain embodiments, the tumor may exhibit high levels of inflammation. Increased inflammation may be indicative of a diffuse PD-L1 expression pattern. Thus, high tumor inflammation may be indicative of a response to anti-PD-1 antibody treatment.

[0165] Detection Method Certain embodiments of the present disclosure relate 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 a subject possesses a wild-type or mutant form of a marker gene. In certain embodiments, a target marker gene is sequenced using techniques 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 certain embodiments, the marker gene has a nonsynonymous mutation. In certain embodiments, the marker gene has a nonsense mutation. In certain embodiments, the marker gene has a frameshift mutation. In certain embodiments, the marker gene has a splicing mutation. In certain embodiments, the mutant marker gene is expressed. In other embodiments, the mutant marker gene is not expressed.

[0166] In some embodiments, the mutation status of marker gene is determined by detecting the expression of said marker gene.In some embodiments, the mutant form of target gene is not expressed, and the absence of generated mRNA and / or protein indicates the existence of the mutant form.In some embodiments, the mutation status of marker gene is determined by sequencing generated mRNA and / or generated protein.In some embodiments, the mutation status of marker gene is determined by immunohistochemistry for generated protein.

[0167] In some embodiments, the subject has one wild-type copy and one mutant copy of the marker gene.In some embodiments, the subject has two wild-type copies of the marker gene and no mutant copy of the marker gene.In some embodiments, the subject has two mutant copies of the marker gene and no wild-type marker gene.In some embodiments, the two mutant copies of the marker gene are identical.In some embodiments, the subject has two different mutant copies of the marker gene.

[0168] Certain embodiments of the present disclosure relate to determining and / or measuring the expression levels of one or more marker genes in a tumor of a subject. To assess PD-L1 expression, in one embodiment, a test tissue sample can be obtained from a patient in need of treatment. In another embodiment, assessing PD-L1 expression can be performed without obtaining a test tissue sample. In one embodiment, selecting an appropriate patient includes (i) providing a test tissue sample, optionally obtained from a patient suffering from cancer of said tissue, wherein said test tissue sample contains tumor cells and / or tumor-infiltrating inflammatory cells; and then (ii) assessing the proportion of cells in said test tissue sample that express PD-L1 on their cell surface based on assessing that the proportion of cells in said test tissue sample that express PD-L1 on their cell surface is higher than a predetermined threshold.

[0169] However, it should be understood that in methods involving measuring PD-L1 expression in a test tissue sample, the step involving providing a test tissue sample obtained from a patient is an optional step. Also, in certain embodiments, the "measuring" or "assessing" step to identify or determine 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 certain other embodiments, no modified method is involved, and PD-L1 expression is measured, for example, by reviewing test result reports from a laboratory. In certain embodiments, the steps of the method up to and including assessing PD-L1 expression provide intermediate results that can be provided to a physician or other healthcare provider for use in selecting appropriate candidates for anti-PD-1 antibodies or anti-PD-L1 antibody therapy. In certain embodiments, the step of providing the intermediate results is performed by a physician or other person performing under the guidance of a physician. In other embodiments, these steps are performed by an independent laboratory or by an independent person, for example, a laboratory technician.

[0170] In certain embodiments of any of the methods, the proportion of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In further embodiments, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection. In other embodiments, the proportion of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In further embodiments, the presence of PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In certain embodiments, PD-L1 expression is assayed by IHC. In all other embodiments of these methods, cell surface expression of PD-L1 is assayed using, for example, IHC or in vivo imaging.

[0171] Imaging technologies provide important 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), may foresee further applications of these technologies in cancer research. Some of these molecular imaging systems allow clinicians not only to see where tumors are located in the body, but also to visualize the expression and activity of specific molecules, cells, and biological processes that affect 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)). Combining the sensitivity and resolution of PET with antibody specificity, immunoPET imaging allows for the monitoring and assaying of 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 certain embodiments of any of the methods, PD-L1 expression is assayed by immunoPET imaging. In certain embodiments of any of the methods, the proportion of cells in the test tissue sample that express PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide on the surface of cells in the test tissue sample. In certain embodiments, the test tissue sample is an FFPE tissue sample.In other embodiments, the presence of a PD-L1 polypeptide is determined by an IHC assay. In further embodiments, the IHC assay is performed using an automated method. In certain embodiments, the IHC assay is performed using an anti-PD-L1 monoclonal antibody that binds to a PD-L1 polypeptide.

[0172] In one embodiment of the method, an automated IHC method is used to assay the expression of PD-L1 on the cell surface in an FFPE tissue sample. The present disclosure provides a method for detecting the presence of human PD-L1 antigen in a test tissue sample or quantifying the level of human PD-L1 antigen or the proportion of cells in a sample that express 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 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 a 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 can be used to quantify PD-L1 expression.

[0173] In certain embodiments, the automated IHC method includes (a) deparaffinizing and rehydrating embedded tissue sections in an automated stainer; (b) retrieving antigens using an antigen retrieval chamber and pH 6 buffer heated to 110°C for 10 minutes; (c) placing the reagents in the automated stainer; and then (d) running the automated stainer, which includes the steps of neutralizing endogenous peroxidase in the tissue sample; blocking nonspecific protein binding sites on the slide; incubating the slide with primary Ab; incubating with postprimary blocking agent; incubating with NovoLink polymer; adding chromogenic substrate and developing; and then counterstaining with hematoxylin.

[0174] To evaluate PD-L1 expression in tumor tissue samples, pathologists count the number of membrane PD-L1+ tumor cells in each field under a microscope, mentally estimate the percentage of cells that are positive, and then average these to arrive at a final percentage. Different staining intensities are defined as 0 / negative, 1+ / weak, 2+ / intermediate, and 3+ / strong. Typically, percentages are first assigned to the 0 and 3+ buckets, and then the intermediate 1+ and 2+ intensities are considered. For highly heterogeneous tissues, the sample is divided into zones, each zone is scored separately, and then combined into a single set of percentages. The percentages of negative and positive cells for different staining intensities are determined from each region, and the median value is assigned to each zone. A final percentage value is assigned to the tissue for each staining intensity category (negative, 1+, 2+, and 3+). The sum of all staining intensities must equal 100%. In one embodiment, the threshold number of cells required as 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 as PD-L1 positive is at least about 100 cells.

[0175] Staining is also assessed in tumor-infiltrating inflammatory cells (e.g., macrophages and lymphocytes). Often, macrophages serve as an internal positive control, as staining is seen in the majority of macrophages. While staining at 3+ intensity is not required, the absence of macrophage staining should not be considered to rule out technical failure. Macrophages and lymphocytes are assessed for cell membrane staining, and all samples are scored as positive or negative for each cell category. Staining is also characterized according to the nomenclature of immune cells inside / outside the tumor. "Inside" means that immune cells are present on the border of the tumor area without being physically intercalated within the tumor tissue and / or between tumor cells. "Outside" means that there is no physical association with the tumor; immune cells are found in the periphery or adjacent tissues associated with connective tissue.

[0176] In some embodiments of these scoring methods, the samples are scored by two pathologists working independently, and the scores are then combined. In other embodiments, the identification of positive and negative cells is scored using appropriate software.

[0177] The histoscore or "H-score" is used as a more quantitative measure of the IHC data. The histoscore is calculated as follows: Histology score = [(%tumor x1 (low intensity)) + (%tumor x2 (intermediate intensity)) + (%tumor x3 (high intensity)].

[0178] To determine the tissue score, the pathologist estimates the percentage of cells staining in each intensity category within the sample. Because expression of most biomarkers is heterogeneous, the tissue score is a more accurate representation of overall expression. The final tissue score ranges from 0 (no expression) to 300 (maximal expression).

[0179] Another method for quantifying PD-L1 expression in test tissue sample IHC is to determine the modified inflammatory score (AIS), which is defined as the inflammatory intensity by multiplying 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)).

[0180] Tumor mutation burden (TMB) Another aspect of the present disclosure relates to measuring the TMB of tumor tissue obtained from a subject. As tumors grow, somatic mutations not present in germline DNA accumulate. Tumor mutation burden (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 (after taking into account germline mutation DNA). The acquisition of somatic mutations, i.e., higher TMB, can be influenced by different mechanisms, such as exposure to exogenous mutagens (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-base substitutions (Vogelstein et al., Science (2013) 339:1546-1558). As used herein, "nonsynonymous mutation" refers to a nucleotide mutation that changes the amino acid sequence of a protein. Both missense and nonsense mutations can be nonsynonymous mutations. As used herein, a "missense mutation" refers to a nonsynonymous point mutation in which a single nucleotide change results in a codon that encodes a different amino acid. As used herein, a "nonsense mutation" refers to a nonsynonymous point mutation in which a codon is changed to a premature stop codon that results in truncation of the protein produced.

[0181] In one embodiment, somatic mutations can be expressed at the RNA and / or protein level, generating neoantigens (also called neoepitopes). Neoantigens can influence immune-mediated anti-tumor responses. For example, recognition of neoantigens can promote T cell activation, clonal expansion, and differentiation into effector and memory T cells.

[0182] As tumors grow, early clonal mutations (or "trunk mutations") may be carried to most or all tumor cells, whereas later mutations (or "branch mutations") may arise in only a subset of 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 are more widespread in the tumor genome than those derived from "branch" mutations and therefore may generate more T cells reactive to clonal neoantigens (McGranahan et al., (2016) 351:1463-1469). In general, tumors with high TMB also have high neoantigen burden, which may result in high tumor immunogenicity and high T cell reactivity and antitumor responses. Therefore, cancers with high TMB may respond well to immunotherapy, for example, treatment with anti-PD-1 or anti-PD-L1 antibodies.

[0183] Advances in sequencing technology allow for the assessment of tumor genomic mutational status. Sequencing methods known to those skilled in the art can be used to sequence nucleic acids from tumor genomes (e.g., obtained from biological samples from subjects suffering from tumors). In one embodiment, PCR or qPCR, Sanger sequencing, or next-generation sequencing (e.g., genomic profiling, exome analysis, or genome 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) from tumor samples and can be performed using methods including the selection of optimized nucleic acids to be incorporated, read alignment, and mutation calling. In one embodiment, genetic profiling provides next-generation sequencing (NGS)-based analysis of tumors, which can be optimized on a cancer-by-cancer, gene-by-gene, and / or site-by-site basis. Genomic profiling can incorporate the use of multiple, individually modified alignment methods or algorithms to optimize performance in sequencing methods, particularly methods that rely on massively parallel sequencing of many different genetic events in many different genes. Genomic profiling provides comprehensive analysis of a subject's cancer genome at clinical levels, and the results of genetic analysis can be relevant to relevant scientific and medical knowledge to improve the quality and efficiency of cancer treatment.

[0184] Genomic profiling relates to a predetermined set of gene panels comprising only 5 genes or 1000 genes, about 25 to about 750 genes, about 100 to about 800 genes, about 150 to about 500 genes, about 200 to about 400 genes, about 250 to about 350 genes. In one embodiment, the genomic profile comprises 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 genomic profile comprises at least 325 genes. In particular embodiments, the genomic profile comprises at least 315 cancer-associated genes and introns of 28 genes (FOUNDATIONONE®), or the entire DNA coding sequence of 406 genes, the introns of 31 rearranged genes, and the RNA sequence (cDNA) of 265 genes (FOUNDATIONONE® Heme). In another embodiment, the genomic profile comprises 26 genes and 1000 associated mutations (EXODX® Solid Tumor). In yet another embodiment, the genomic profile comprises 76 genes (Guardant360). In yet another embodiment, the genomic profile comprises 73 genes (Guardant360). In another embodiment, the genomic profile comprises 354 genes and introns of 28 genes for rearrangement (FOUNDATIONONE® CDX™). In one embodiment, the genomic profile is FOUNDATIONONE® F1CDx.In another embodiment, the genomic profile comprises 468 genes (MSK-IMPACT™). One or more genes can be added to the genomic profile as additional genes identified as being associated with oncology.

[0185] In yet another particular embodiment, said genomic profiling detects all variants, i.e., single nucleotide variants, insertions / deletions (indels), copy number variations, and rearrangements, e.g., translocations, expression, and epigenetic markers.

[0186] The comprehensive gene panel may include predetermined genes selected based on the type of tumor being analyzed. Thus, the genomic profile used to measure TMB status can be selected based on the type of tumor the subject is suffering from. In one embodiment, the genomic profile may specifically include a set of genes for solid tumors. In another embodiment, the genomic profile may specifically include a set of genes for hematological malignancies and sarcomas.

[0187] 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 In another embodiment, the TMB analysis includes one or more genes selected from the group consisting of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6, ETV7, ETV8, ETV9, ETV10, ETV11, ETV12, ETV13, ETV14, ETV15, ETV16, ETV17, ETV18, ETV19, ETV20, ETV21, ETV22, ETV23, ETV24, ETV25, ETV26, ETV27, ETV28, ETV29, ETV30, ETV31, ETV32, ETV33, ETV34, ETV35, ETV36, ETV37, ETV38, ETV39, ETV40, ETV41, ETV42, ETV43, ETV44, ETV45, ETV46, ETV47, ETV48, ETV49, ETV50, ETV51, ETV52, ETV53, ETV54, ETV55, ETV56, ETV57, ETV58, ETV59, ETV60, ETV61, ETV62, ETV63, ETV64, ETV65, ETV66, ETV67, ETV68, ETV69, ETV70, ETV71, ETV72, ETV73, ETV74, ETV75, ETV76, ETV77, ETV78, ETV79, ETV76, ETV79, ETV71, ETV72, ETV76, ETV79, ETV71, ETV72, ETV74, ETV75, ETV76, ETV78, ETV79, ETV79, ETV71, ETV71, ETV72, ETV75, ETV79, ETV79, ETV71,and MYB. In other embodiments, the mutation status of the SKT11 gene can be assessed as part of a TMB analysis or as described above.

[0188] In one embodiment, TMB status based on genomic profiling is highly correlated with TMB status based on whole exome sequencing or whole genome sequencing.

[0189] TMB can be measured using tissue biopsy samples or ctDNA and / or liquid biopsy samples. ctDNA can be used to measure TMB status by whole exome sequencing or whole genome sequencing or genomic profiling using available methods (e.g., GRAIL).

[0190] TMB status can be used, alone or in combination with other factors, as a means of predicting tumor responsiveness to treatment, particularly treatment with a cancer immunotherapeutic agent (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In one embodiment, the TMB status of a tumor alone is used to identify patients with tumors that are more likely to respond to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody. In other embodiments, PD-L1 status and TMB status are used to identify patients with tumors that are more likely to respond to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0191] In certain embodiments, the methods of the present disclosure further comprise measuring the TMB status of the subject prior to administering the anti-PD-1 antibody. In certain embodiments, the methods comprise administering an anti-PD-1 antibody to a subject harboring wild-type STK11 and exhibiting a high TMB status. In other embodiments, the methods comprise not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, if the subject harbors a mutant form of STK11 and exhibits a high TMB status. In certain embodiments, the methods of the present disclosure further comprise measuring the TMB status of the subject prior to administering the anti-PD-1 antibody. In certain embodiments, the methods comprise administering an anti-PD-1 antibody to a subject harboring wild-type STK11 and exhibiting an intermediate TMB status. In other embodiments, the methods comprise not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, if the subject harbors a mutant form of STK11 and exhibits an intermediate TMB status. In other embodiments, the method comprises not administering an anti-PD-1 antibody to the subject, or terminating or increasing anti-PD-1 antibody treatment, if the subject harbors a mutant form of STK11 and the subject exhibits a low TMB status.

[0192] FOUNDATIONONE® Assay The FOUNDATIONONE® assay is a comprehensive genomic profiling assay for solid tumors, including, but not limited to, lung, colon, and breast solid tumors, melanoma, and ovarian cancer. The FOUNDATIONONE® assay uses hybrid capture next-generation sequencing to identify genomic alterations (base substitutions, insertions and deletions, copy number changes, and rearrangements) and select genomic features (e.g., TMB and microsatellite instability). The assay covers 322 unique genes (including the entire coding regions of 315 cancer-related genes and selected introns from 28 genes). A complete list of FOUNDATIONONE® assay genes is provided in Tables 1 and 2. See FOUNDATIONONE: Technical Specifications (Foundation Medicine, Inc.), available at FoundationMedicine.com, last visited March 16, 2018, which is incorporated herein by reference in its entirety. Table 1: List of genes whose entire coding sequences are assayed in the FOUNDATIONONE® assay. [Table 1] TIFF0007780857000002.tif56125 Table 2: List of genes whose selected introns are assayed in the FOUNDATIONONE® assay. [Table 2]

[0193] 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 changes, and rearrangements) and select genomic features (e.g., TMB and microsatellite instability). The assay analyzes the coding regions of 406 genes, selected introns of 31 genes, and the 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 FOUNDATIONONE® HEME: Technical Specifications (Foundation Medicine, Inc.), available at FoundationMedicine.com, last visited March 16, 2018, and incorporated herein by reference in its entirety. Table 3: List of genes whose full coding sequences are assayed with the FOUNDATIONONE® Heme assay. [Table 3] TIFF0007780857000005.tif188133 TIFF0007780857000006.tif11133 Table 4: List of genes whose selected introns are assayed with the FOUNDATIONONE® Heme assay. [Table 4] Table 5: List of genes whose RNA sequences are assayed with the FOUNDATIONONE® Heme assay. [Table 5] TIFF0007780857000009.tif26128

[0194] 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 cancer pathways. The EXODX® Solid Tumor Assay is a plasma-based assay that does not require a tissue sample. The EXODX® Solid Tumor Assay covers 26 genes and 1,000 mutations. The specific genes covered by the EXODX® Solid Tumor Assay are listed in Table 6. See the Plasma-Based Solid Tumor Mutation Panel Liquid Biopsy (Exosome Diagnostics, Inc.), available at exosomedx.com, last visited March 16, 2018. Table 6: Genes covered in the EXODX® solid tumor assay. [Table 6]

[0195] 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 visited March 16, 2018. Table 7: Guardant360 assay genes. [Table 7] Table 8: Indels in the Guardant360 assay. [Table 8] Table 9: Guardant360 assay amplification (CNV). [Table 9] Table 10: Guardant360 assay fusions. [Table 10]

[0196] 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 simultaneously analyzes DNA and RNA and covers 170 genes associated with common solid tumors. The TruSight Tumor 170 Assay evaluates fusions, splice variants, insertions / deletions, single nucleotide variants (SNVs), and amplifications. The TruSight Tumor 170 Assay gene list is shown in Tables 11-13. Table 11: TruSight Tumor 170 Assay Genes (Amplified). [Table 11] Table 12: TruSight Tumor 170 Assay Genes (Fusions). [Table 12] Table 13: TruSight Tumor 170 Assay Genes (Small Variants). [Table 13]

[0197] FOUNDATIONONE® F1CDx Assay FOUNDATIONONE® CDX™ ("F1CDx") is a next-generation sequencing-based in vitro diagnostic device for the detection of substitution, insertion, and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and selected gene rearrangements, as well as genomic features (including microsatellite instability (MSI) and tumor mutation burden (TMB)) using DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples. F1CDx has been 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.

[0198] The F1CDx assay uses a single DNA extraction method from routine FFPE biopsy or surgical resection samples, of which 50–1000 ng is subjected to whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-associated genes, one promoter region, one non-coding (ncRNA), and selected intronic regions from 34 commonly rearranged genes (21 of which contain coding exons). Tables 14 and 15 provide a complete list of genes included in the F1CDx assay. In total, the assay detects alterations in a total of 324 genes. Using the ILLUMINA® HiSeq4000 platform, hybrid capture-selected libraries are sequenced to high uniform depth (targeting a median coverage of >500X with >100X coverage and >99% of exons). The sequence data are then processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number changes (amplifications and homozygous gene deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic features (including microsatellite instability (MSI) and tumor mutation burden (TMB)) are reported. Table 14: Genes with full coding exon regions included in FOUNDATIONONE® CDX® for the detection of substitutions, insertions and deletions (indels), and copy number alterations (CNAs). [Table 14] Table 15: Genes with selected intron regions, one gene with a 3'UTR, one gene with a promoter region, and one ncRNA gene for the detection of gene rearrangements. [Table 15]

[0199] The F1CDx assay identifies a variety of alterations in gene and / or intron sequences, including substitutions, insertions / deletions, and CNAs. The F1CDx assay was previously identified as concordant with an externally validated NGS assay and the FOUNDATIONONE® (F1 LDT) assay. See FOUNDATIONONE® CDX®: Technical Information (Foundation Medicine, Inc.), available at FoundationMedicine.com, last visited March 16, 2018 (incorporated herein by reference in its entirety).

[0200] 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 mutational status of 468 genes. Target genes are captured and sequenced on an ILLUMINA® HISEQ® instrument. The MSK-IMPACT® assay is cleared 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]

[0201] NEOGENICS® NEOTYPE® Assay In some embodiments, TMB is determined using the NEOGENICS® NEOTYOPE® Assay. In some embodiments, the TMB is determined using the NEOTYPE® Discovery Profile. In some embodiments, the TMB is determined using the NEOTYPE® Solid Tumor Profile. The NEOGENICS® Assay measures the number of nonsynonymous DNA coding sequence changes per megabase of sequenced DNA.

[0202] ONCOMINE® Tumor Mutation Burden Assay In some embodiments, TMB is determined using the THERMOFISHER SCIENTIFIC® ONCOMINE® Tumor Mutation Assay. In some embodiments, TMB is determined using the THERMOFISHER SCIENTIFIC® ION TORRENT® ONCOMINE® Tumor Mutation Assay. The ION TORRENT® ONCOMINE® Tumor Mutation Assay is a targeted NGS assay that quantifies somatic mutations to determine tumor mutation burden. The assay covers 1.7 Mb of DNA.

[0203] NOVOGENE® NOVOPM® Assay In some embodiments, TMB is measured using the NOVOGENE® NOVOPM® assay. In some embodiments, TMB is measured using the NOVOGENE® NOVOPM® cancer panel assay. The NOVOGENE® NOVOPM® cancer panel assay analyzes the entire coding region of 548 genes and the introns of 21 genes (representing approximately 1.5 Mb of DNA), and is a comprehensive NGS cancer panel suitable for the diagnosis and / or treatment of solid tumors according to National Comprehensive Cancer Network (NCCN) guidelines and medical literature. The assay detects genomic abnormalities such as SNVs, indels, fusions, and copy number variations (CNVs).

[0204] Other TMB assays In some embodiments, TMB is determined using a TMB assay from CARIS® Life Sciences. In some embodiments, TMB is determined using the PESONALIS® ACEIMMUNOID assay. In some embodiments, TMB is determined using the PGDX® CANCERXOME®-R assay.

[0205] In yet another specific embodiment, said genomic profiling detects all variants, i.e., single nucleotide variants, insertions / deletions (indels), copy number variations, and rearrangements, e.g., translocations, expression, and epigenetic markers.

[0206] A comprehensive gene panel often includes predetermined genes selected based on the tumor type being analyzed. Thus, the genomic profile used to measure TMB status can be selected based on the tumor type the subject has. In one embodiment, the genomic profile can specifically include a set of genes for solid tumors. In another embodiment, the genomic profile can specifically include a set of genes for hematological malignancies and sarcomas.

[0207] 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 In other embodiments, the TMB analysis includes one or more genes selected from the group consisting of: 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 any combination thereof.and identifying genomic alterations in one or more of MYB.

[0208] In another embodiment, the genomic profile is selected from the group consisting of 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 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.

[0209] In another embodiment, the genomic profiling assay is performed using a gene encoding a genomic protein selected from the group consisting of 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, 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 about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or at least about 150, or at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or ... At least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, or at least about 300 genes.

[0210] In another embodiment, the genomic profile comprises one or more genes selected from the genes set forth in Tables 1-16.

[0211] In one embodiment, TMB status based on genomic profiling is highly correlated with TMB status based on whole exome or whole genome sequencing.The evidence provided herein demonstrates that the use of genomic profiling assays such as F1CDx assays is consistent with whole exome and / or whole genome sequencing assays.These data support the use of genomic profiling assays as a more effective way to measure TMB status without compromising the quality of TMB status prediction.

[0212] TMB can be measured using tissue biopsy samples, or circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and / or liquid biopsy samples. ctDNA can be used to measure TMB status following whole-exome or whole-genome sequencing or genomic profiling using available methods (e.g., GRAIL).

[0213] A subject is identified as suitable for immunotherapy (e.g., with an anti-PD-1 antibody, or antigen-binding portion thereof, or an anti-PD-L1 antibody, or antigen-binding portion thereof) based on measuring TMB status and identifying high TMB. In certain embodiments, the TMB score is calculated as the total number of nonsynonymous missense mutations in the tumor, as measured by whole-exome sequencing or whole-genome sequencing. In one embodiment, 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 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 510, at least 520, at least 530, at least 540, at least 550, at least 560, at least 570, at least 580, at least 590, at least 600, at have 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 other embodiments, 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 certain embodiments, high TMB has a score of at least 243. In other embodiments, high TMB has a score of at least 244. In certain embodiments, high TMB has a score of at least 245. In other embodiments, high TMB has a score of at least 246. In other embodiments, high TMB has a score of at least 247. In other embodiments, high TMB has a score of at least 248. In other embodiments, high TMB has a score of at least 249. In other embodiments, high TMB has a score of at least 250. In other embodiments, high TMB has an integer score of 200-300 or higher. In other embodiments, high TMB has an integer score of 210-290 or higher. In other embodiments, high TMB has an integer score of 220-280 or higher. In other embodiments, high TMB has an integer score of 230-270 or higher. In other embodiments, high TMB has an integer score of 235-265 or higher.

[0214] Alternatively, high TMB can be a relative value rather than an absolute value. In some embodiments, the subject's TMB status is compared to a reference TMB value. In one embodiment, the subject's TMB status is within the highest quantile of the reference TMB value. In other embodiments, the subject's TMB status is within the highest tertile of the reference TMB value.

[0215] In some embodiments, TMB status is expressed as the number of mutations per sample, per cell, per exome, or per length of DNA (e.g., Mb). In some embodiments, a tumor exhibits high TMB status 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 high TMB status 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 high TMB status if it has at least about 100 mutations / tumor.

[0216] In certain embodiments, the tumor 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 16 mutations / Mb, at least about 17 mutations / Mb, at least about 18 mutations / Mb, at least about 19 mutations / Mb, at least about 20 mutations / Mb, at least about 21 mutations / Mb, at least about 22 mutations / Mb, at least about 23 mutations / Mb, at least about 24 mutations / Mb, at least about 25 mutations / Mb, at least about 26 mutations / Mb, at least about 27 mutations / Mb, at least about 28 mutations / Mb, at least about 29 mutations / Mb, at least about 30 mutations / Mb, at least about 31 mutations / Mb, at least about 32 mutations / Mb, at least about 33 mutations / Mb, at least about 34 mutations / Mb, at least about 35 mutations / Mb, at least about 36 mutations / Mb, at least about 37 mutations / Mb, at least about 38 mutations / Mb, at least about 39 mutations / Mb, at least about 40 mutations / Mb, at least about 41 mutations / Mb, at least about 42 mutations / Mb, at least about 43 mutations / Mb, at least about 44 mutations / Mb, at least about 45 mutations / Mb, at least about 46 mutations / Mb A tumor exhibits high TMB status when it has 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. In certain embodiments, a tumor exhibits high TMB status when it has at least about 5 mutations / Mb. In one embodiment, a tumor exhibits high TMB status when it has at least about 10 mutations / Mb. In certain embodiments, a tumor exhibits high TMB status when it has at least about 11 mutations / Mb. In certain embodiments, a tumor exhibits high TMB status when it has at least about 12 mutations / Mb. In certain embodiments, a tumor exhibits high TMB status if it has at least about 13 mutations / Mb. In certain embodiments, a tumor exhibits high TMB status if it has at least about 14 mutations / Mb. In one embodiment, a tumor exhibits high TMB status if it has at least about 15 mutations / Mb.

[0217] Because mutation numbers vary by tumor type and other methods (see Q4 and Q5), the numbers associated with "TMB high" and "TMB low" may differ by tumor type.

[0218] non-small cell lung cancer This method can treat tumors at any stage. In some embodiments, the tumor is derived from NSCLC at any stage. There are at least seven stages used for NSCLC: latent (hidden) stage, stage 0 (carcinoma in vivo), 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 lining of the airways.

[0219] In one embodiment, the method treats stage I non-squamous NSCLC. Stage I NSCLC is divided into stage IA and stage IB. In stage IA, the tumor is present only in the lungs and is 3 centimeters or less. In stage IB, the cancer has not spread to lymph nodes, and one or more of the following is true: 1) the tumor is 3 centimeters or more but not larger than 5 centimeters; 2) the cancer has spread to the main bronchi and is at least 2 centimeters below where the trachea connects to the bronchi; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) part of the lung has collapsed in the area where the trachea connects to the bronchi, or pneumonia (lung inflammation) has occurred.

[0220] In another embodiment, the method of the present disclosure treats stage II non-squamous NSCLC.Stage II NSCLC is classified into stage IIA and IIB.In stage IIA, the cancer may or may not have spread to lymph nodes.If the cancer has spread to lymph nodes, the cancer only spreads to the lymph nodes in the same side of the chest as the tumor (the lymph nodes in the lung or in the lung or near the trachea), and one or more of the following are true: 1) the tumor is 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 that covers the lung; or 4) part of the lung has collapsed in the area where the trachea connects to the bronchus, or has pneumonia (lung inflammation). The tumor is also considered stage IIA if the cancer has not spread to the lymph nodes and one or more of the following is true: 1) the tumor is larger than 5 centimeters but not larger than 7 centimeters; 2) the cancer has spread to the main bronchi and is at least 2 centimeters below where the trachea joins the bronchi; 3) the cancer has spread to the innermost layer of the membrane that covers the lung; or 4) part of the lung has collapsed in the area where the trachea joins the bronchi or has developed pneumonia (inflammation of the lung). In stage IIB, the cancer may or may not have spread to the lymph nodes. If the cancer has spread to lymph nodes, the cancer has spread only to lymph nodes on the same side of the chest as the tumor (lymph nodes in the lung or near the trachea) and one or more of the following is 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 joins the bronchi; 3) the cancer has spread to the innermost layer of the membrane that covers the lung; or 4) part of the lung has collapsed in the area where the trachea joins the bronchi or has pneumonia (inflammation of the lung).The tumor is also considered stage IIB if the cancer has not spread to the lymph nodes and one or more of the following is true: 1) the tumor is larger than 7 centimeters; 2) the cancer has spread to the main bronchi, chest wall, diaphragm, or nerves that control the diaphragm (and is at least 2 centimeters below where the trachea connects to the bronchi); 3) the cancer has spread to the membrane around the heart or the membrane that covers the chest wall; 4) the entire lung has collapsed or has developed pneumonia (inflammation of the lungs); or 5) there are one or more other tumors in the same lobe of the lung.

[0221] In other embodiments, any method of the present disclosure treats stage III non-squamous NSCLC. Stage IIIA is classified into three categories. These three categories are based on 1) tumor size; 2) the location of the tumor, and 3) whether lymph nodes (if any) are affected by cancer. In the first type of stage IIIA NSCLC, the cancer has only spread to lymph nodes on the same side of the chest as the tumor, and the affected lymph nodes are near the sternum or where the bronchi enter the lungs. Additionally: 1) the tumor can be of any size; 2) it can collapse part of the lung (where the trachea joins the bronchi) or the entire lung or cause pneumonia (inflammation of the lung); 3) there can be one or more other tumors in the same lobe of the lung; and 4) the cancer can spread to any of the following: a) the main bronchus (not the area where the trachea joins the bronchi), b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes around the lung or membranes that line the chest wall, e) the membranes around the heart. In the second type of Stage IIIA NSCLC, the cancer has spread to lymph nodes in the lung ipsilateral to the tumor, and the affected lymph nodes are within the lung or near the bronchi. Additionally: 1) the tumor may be of any size; 2) the entire lung may have collapsed or developed pneumonia (inflammation of the lungs); 3) there may be one or more additional tumors 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 bronchus (not the area where the trachea joins the bronchi), b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lungs or covering the chest wall, e) the heart or membranes around it, f) the major blood vessels leading to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (voice box), j) the sternum or skeleton, or k) the carina (where the trachea joins the bronchi). In a third type of Stage IIIA NSCLC, the cancer has not spread to 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) the nerves that control the larynx (voice box), f) the sternum or skeleton, or g) the carina (where the trachea joins the bronchi).Stage IIIB is divided into two categories based on 1) tumor size, 2) where the tumor is found, and 3) whether the lymph nodes are affected. In the first type of Stage IIIB NSCLC, the cancer has spread to lymph nodes on the opposite side of the chest from the tumor. Furthermore, 1) the tumor may be of any size; 2) a portion of the lung (where the trachea connects to the bronchi) or the entire lung may be collapsed or have pneumonia (lung inflammation); 3) one or more additional tumors may be present in any of the lobes of the lung affected by the 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 lungs or covering the chest wall, e) the heart or the membranes surrounding it, f) the major blood vessels leading to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (voice box), j) the sternum or bones, or k) the carina (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 ipsilateral to the tumor. The affected lymph nodes are near the sternum or where the bronchi enter the lungs. Additionally, 1) the tumor can be of any size; 2) there can be separate tumors in different lobes of the same lung; and 3) the cancer has spread to any of the following: a) the heart, b) the major blood vessels leading to or from the heart, c) the trachea, d) the esophagus, e) the nerves controlling the larynx (voice box), f) the sternum or skeleton, or g) the carina (where the trachea joins the bronchi).

[0222] In some embodiments, 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 lymph nodes. One or more of the following are true in stage IV NSCLC: 1) there is one or more tumors in both lungs; 2) cancer is found in the fluid around the lung or heart; and 3) cancer has spread to other parts of the body (for example, the brain, liver, adrenal gland, kidney or bone).

[0223] In another embodiment, the NSCLC treatable by the present method is squamous cell (epidermoid) carcinoma (squamous cell NSCLC). Approximately 25% to 30% of all lung cancers are known to be squamous cell carcinomas. These cancers begin in the squamous epithelium (flat cells that line the airways in the lungs). They are often associated with a smoking history and tend to be found in the central part of the lung (bronchi) near the major airways.

[0224] In one embodiment of the 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 antigen-binding portion thereof, is administered at a subtherapeutic dose.

[0225] Anti-PD-1 or anti-PD-L1 antibodies useful in the present 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 described in U.S. Patent No. 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) a 1x10 mAb concentration of 1x10 mAb or more, as determined by surface plasmon resonance using a Biacore biosensor system; -7(b) binds to human PD-1 with a KD of M or less; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (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) binds to human PD-1 and cynomolgus 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 the present disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in certain embodiments, at least five, of the foregoing characteristics.

[0226] Other anti-PD-1 monoclonal antibodies are described in, e.g., 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 Nos. 017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540, each of which is incorporated by reference in its entirety.

[0227] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (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), cemiplimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al., J. Hematol. 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; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (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).

[0228] In one embodiment, the anti-PD-1 antibody is nivolumab, a fully 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 anti-tumor T cell function (see U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0229] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0230] Anti-PD-1 antibodies that can be used in the compositions and methods of the present disclosure also include isolated antibodies that specifically bind to human PD-1 and cross-compete for binding to human PD-1 with the anti-PD-1 antibodies described herein, such as nivolumab (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; WO 2013 / 173223). In certain embodiments, 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 hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody (e.g., nivolumab) due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays (e.g., Biacore analysis, ELISA assays, or flow cytometry) (see, e.g., WO2013 / 173223).

[0231] In some embodiments, the antibody (nivolumab) that cross-competes with human PD-1 for binding to a human PD-1 antibody or 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, modified, 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.

[0232] Anti-PD-1 antibodies that can be used in the compositions and methods of the disclosure also include antigen-binding portions of said antibodies. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0233] Anti-PD-1 antibodies suitable for use in the compositions and methods of the present disclosure are those that bind to PD-1 with high specificity and affinity, inhibit binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods described herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor, inhibits ligand binding, and exhibits functional properties similar to those of a whole antibody in upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0234] Anti-PD-1 or anti-PD-L1 antibodies useful in the present disclosure In certain embodiments, the anti-PD-1 antibody used in the methods can be replaced with another PD-1 or anti-PD-L1 antagonist. For example, an anti-PD-L1 antibody can be substituted for the use of an anti-PD-1 antibody in the methods described herein, because anti-PD-L1 antibodies inhibit the interaction of PD-1 and PD-L1, thereby exhibiting a similar effect on the PD-1 signaling pathway. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the disclosure. Examples of anti-PD-L1 antibodies useful in the compositions and methods of the disclosure include those described in U.S. Patent No. 9,580,507. Anti-PD-L1 human monoclonal antibodies are described in U.S. Patent No. 9,580,507, which have been shown to exhibit one or more of the following properties: (a) a 1x10 -density of PD-L1 or PD-L1 as determined by surface plasmon resonance using a Biacore biosensor system; -7 (b) binds to human PD-L1 with a KD of M or less; (b) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increases interferon-γ production in an MLR assay; (d) increases IL-2 secretion in an MLR assay; (e) activates antibody responses; and (f) reverses the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in certain embodiments, at least five, of the above characteristics.

[0235] In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (12A4, also known as MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; TECENTRIQ®; MPDL3280A, also known as RG7446; see U.S. Patent No. 8,217,149; Herbst et al. (2013) J Clin Oncol 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 Co.; see, e.g., WO2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

[0236] In one embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0237] In one embodiment, the PD-L1 antibody is durvalumab (IMFINZI®). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0238] In one embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0239] 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.

[0240] Anti-PD-L1 antibodies that can be used in the compositions and methods of the present disclosure also include isolated antibodies that specifically bind to human PD-L1 and cross-compete for binding to human PD-L1 with the anti-PD-L1 antibodies described herein, e.g., 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 (e.g., 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 that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody (e.g., 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 (e.g., Biacore analysis, ELISA assays, or flow cytometry) (see, e.g., WO2013 / 173223).

[0241] In some embodiments, antibodies (atezolizumab, durvalumab, and / or avelumab) that cross-compete for binding to human PD-L1 with a human PD-L1 antibody or that bind to the same epitope region of a human PD-L1 antibody are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric, modified, 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.

[0242] Anti-PD-L1 antibodies that can be used in the compositions and methods of the disclosure also include antigen-binding portions of said antibodies. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0243] Anti-PD-L1 antibodies suitable for use in the compositions and methods of the disclosure are those that bind to PD-1 with high specificity and affinity, inhibit PD-1 binding, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods described herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-L1 receptor, inhibits ligand binding, and exhibits functional properties similar to those of a whole antibody in upregulating the immune system. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0244] 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 Standard Treatment Guidelines in Oncology (NCCN GUIDELINES®), which provide detailed, up-to-date information on standard treatments for a wide variety of cancers (see NCCN GUIDELINES® (2014) (available at: www.nccn.org / professionals / physician_gls / pdf / nscl.pdf, recently accessed May 14, 2014).

[0245] NSCLC is the leading cause of cancer death in the United States and worldwide, surpassing breast, colon, and prostate cancers combined. In the United States, an estimated 228,190 new cases of lung and bronchial cancer will be diagnosed in the United States, and 159,480 will die from the disease (Siegel et al. (2014) CA Cancer J Clin 64(1):9-29). The majority of patients (approximately 78%) will be diagnosed with advanced / recurrent or metastatic disease. Metastasis from lung cancer to the adrenal gland is common, occurring in approximately 33% of patients with such metastases. NSCLC treatments have shown gradually improved OS, but benefit remains steady (median OS for late-stage patients is only 1 year). Progression after 1L therapy occurs in almost all of these subjects, and the 5-year survival rate is only 3.6%, representing a refractory state. 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, available at: www.nccn.org / professionals / physician_gls / pdf / nscl.pdf (recently accessed May 14, 2014).

[0246] Surgery, radiation therapy (RT), and chemotherapy are three modalities commonly used to treat patients with NSCLC. As a class, NSCLC is relatively insensitive to chemotherapy and RT compared to small cell carcinoma. Generally, for patients with stage I or II disease, surgical resection, with chemotherapy used both pre- and post-operatively, offers the best chance of cure. 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.

[0247] Patients with stage IV disease who demonstrate favorable performance status (PS) benefit from chemotherapy. Many drugs, including platinum-based agents (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine, are effective in treating stage IV NSCLC. Combinations using many of these drugs result in 1-year survival rates of 30% to 40%, superior to single agents. 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 targeting 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 harboring a mutant ALK fusion gene. Cetuximab (Erbitux®) is a monoclonal antibody that targets EGFR.

[0248] Due to the complete lack of treatment options after first-line (1L) therapy, a need exists among certain patients with squamous NSCLC (representing up to 25% of all NSCLCs). Single-agent chemotherapy is the standard of care after progression with platinum-based doublet chemotherapy (Pt-doublet), resulting in a median OS of approximately 7 months. While erlotinib can be used less frequently, docetaxel remains the standard of care in this line of treatment. Pemetrexed also produces clinically equivalent efficacy but has been shown to have significantly fewer side effects than docetaxel in the second-line (2L) treatment of patients with advanced NSCLC (Hanna et al. (2004) J Clin Oncol 22:1589-97). No treatments are currently approved for use in lung cancer beyond the third-line (3L) treatment. Pemetrexed and bevacizumab are not approved for squamous NSCLC, and molecular targeted therapies have limited application. These unmet needs in advanced lung cancer are exacerbated by the recent failure of STIMUVAX® to improve OS in Oncothyreon and Merck KgaA's phase 3 trial, the inability of ArQule and Daiichi Sankyo's c-Met kinase inhibitor (tivantinib) to meet survival endpoints, the failure of Eli Lilly's ALIMTA® in combination with Roche's AVASTIN® to improve OS in a late-stage trial, and the failure of the small molecule VEGF-R antagonist motesanib to meet clinical endpoints in Amgen and Takeda Pharmaceutical's late-stage trial.

[0249] Immunotherapy for lung cancer There is a clear need for effective agents for patients who have progressed on multiple targeted therapies and for treatments that extend long-term survival beyond the current standard of care. Recent approaches to immunotherapy, particularly immune checkpoint blockade (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, particularly those who may be more responsive to anti-PD-1 or anti-PD-L1 antibody treatment.

[0250] Pharmaceutical Compositions and Dosages Therapeutic agents of the present disclosure can be comprised in compositions, such as pharmaceutical compositions containing one or more antibodies and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In one embodiment, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Pharmaceutical compositions of the present disclosure can include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents).

[0251] The present disclosure provides dosing regimens capable of providing a desired response, e.g., maximal therapeutic response and / or minimal side effects. For administration of anti-PD-1 antibodies, the dose can range from about 0.01 to about 10 mg / kg, about 1 to about 9 mg / kg, about 2 to about 8 mg / kg, about 3 to about 7 mg / kg, about 3 to about 6 mg / kg, about 0.01 to about 5 mg / kg, or about 1 to about 3 mg / kg of the subject's body weight. For example, the dose can be about 0.1, about 0.3, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / kg of body weight. The dosing schedule is generally designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the antibody. Exemplary treatment regimens include administration about once every week, about once every two weeks, about once every three weeks, about once every four weeks, about once every month, or about once every three to six months or more. In some embodiments, the anti-PD-1 antibody (e.g., nivolumab) is administered to a subject about once every two weeks. The anti-PD-1 antibody may be administered in at least two doses, each of which is about 0.01 mg / kg to about 5 mg / kg, e.g., 3 mg / kg, with a two-week dosing interval between two doses. In some embodiments, the anti-PD-1 antibody may be administered in at least three, four, five, six, or seven doses (i.e., multiple administrations), each of which is about 0.01 mg / kg to about 10 mg / kg, e.g., 1 mg / kg, 3 mg / kg, or 6 mg / kg, with a two-week dosing interval between two adjacent doses. The dose and schedule can be modified during the course of treatment. In one embodiment, the dosing regimen for an anti-PD-1 antibody of the disclosure includes about 0.1 to about 5 mg / kg body weight, about 1 to about 5 mg / kg body weight, or about 1 to about 3 mg / kg body weight administered intravenously (the antibody is administered about every 14 to 21 days for up to about 6-week or about 12-week cycles until a complete response or until progressive disease is confirmed).In certain embodiments, the antibody therapy, or 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.

[0252] For administration of anti-PD-L1 antibodies, the dose can range from about 1 to about 20 mg / kg, about 1 to about 19 mg / kg, about 2 to about 18 mg / kg, about 3 to about 17 mg / kg, about 3 to about 16 mg / kg, about 4 to about 15 mg / kg, about 5 to about 14 mg / kg, about 6 to about 13 mg / kg, about 7 to about 12 mg / kg, or about 8 to about 12 mg / kg of the subject's body weight. For example, the dose can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 mg / kg of body weight. The dosing schedule is generally designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the antibody. Exemplary treatment regimens include administration about once every week, about once every two weeks, about once every three weeks, about once every four weeks, about once every month, or about once every three to six months or more. In one embodiment, the anti-PD-L1 antibody is administered to a subject about once every two weeks. The anti-PD-L1 antibody may be administered in at least two doses, each of which is about 6 mg / kg to about 18 mg / kg, e.g., 10 mg / kg, with a two-week interval between 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 administrations), each of which is about 6 mg / kg to about 18 mg / kg, e.g., 10 mg / kg or 15 mg / kg, with a two-week interval between two adjacent doses. The doses and schedules may be varied during the course of treatment. In one embodiment, a dosing regimen for an anti-PD-L1 antibody of the disclosure includes about 1 to about 18 mg / kg body weight, about 6 to about 15 mg / kg body weight, or about 10 to about 15 mg / kg body weight administered intravenously, where the antibody is administered about every 14 to 21 days for up to about 6-week or about 12-week cycles until complete response or until progressive disease is confirmed.In certain embodiments, the antibody therapy, or 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.

[0253] 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 in monotherapy. A dose of nivolumab of 0.001 mg / kg or more, typically 3 mg / kg or less, is a subtherapeutic dose. A subtherapeutic dose of an anti-PD-1 antibody used in the methods described herein is 0.001 mg / kg or more and 3 mg / kg or less. In certain embodiments, a subtherapeutic dose is about 0.001 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, or about 0.001 mg / kg to about 0.1 mg / kg of body weight. In some embodiments, the subtherapeutic dose is 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 receiving nivolumab at doses of 0.3 mg / kg to 10 mg / kg indicate that PD-1 occupancy may be dose-dependent within this dose range. Considering all doses, the mean occupancy was 85% (range 70% to 97%), with a mean steady-state occupancy of 72% (range 59% to 81%) (Brahmer et al. (2010) J Clin Oncol 28:3167-75). Thus, a dose of 0.3 mg / kg may allow sufficient exposure to produce maximal biological activity.

[0254] 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.

[0255] In certain embodiments, the dose of the anti-PD-1 antibody (or anti-PD-L1 antibody) is a flat dose in the pharmaceutical composition. In other embodiments, the methods of the disclosure can be used with a flat dose (a dose given to a patient regardless of the patient's weight). In certain embodiments, the flat dose of the anti-PD-1 antibody or antigen-binding portion thereof 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, a flat dose of pembrolizumab can be about 200 mg. In one embodiment, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 240 mg. In one embodiment, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 360 mg. In one embodiment, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of about 480 mg. In one embodiment, a flat dose of the anti-PD-1 antibody or antigen-binding portion thereof is administered about once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In one embodiment, 240 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every 2 weeks. In another embodiment, 360 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every 3 weeks. In another embodiment, 480 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every 4 weeks.

[0256] In certain embodiments, the flat dose of the anti-PD-L1 antibody or antigen-binding portion thereof 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, a flat dose of atezolizumab (TECENTRIQ®) may be about 1200 mg. For example, a flat dose of durvalumab (IMFINZI®) may be about 800 mg. For example, a flat dose of avelumab (BAVENCIO®) may be about 800 mg. In certain embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, is administered at a dose of about 800 mg. In certain embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1200 mg. In certain embodiments, the anti-PD-L1 antibody, or antigen-binding portion thereof, is administered at a dose of about 1600 mg. In certain embodiments, a flat dose of the anti-PD-L1 antibody, or antigen-binding portion thereof, is administered about once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In one embodiment, about 800 mg of the anti-PD-L1 antibody or antigen-binding fragment is administered once every two weeks. In another embodiment, about 1200 mg of the anti-PD-L1 antibody or antigen-binding fragment is administered once every four weeks.

[0257] The dosage and frequency vary depending on the half-life of the antibody in the subject. Generally, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and then non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, typically, relatively low doses are administered at relatively long intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the disease slows or stops, or until the patient shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient can be administered a preventive regimen.

[0258] The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure can be varied to obtain an amount of the active ingredient that is not too toxic to the patient and is effective for achieving the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration, the time of administration, the excretion 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, biology, weight, condition, overall health, and past medical history of the patient being treated, and other factors well known in the pharmaceutical field. The composition of the present disclosure can be administered by one or more routes of administration using one or more of a variety of methods well known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0259] kit Kits containing an anti-PD-1 antibody or an anti-PD-L1 antibody are also within the scope of the present invention. Kits generally include labeling and instructions indicating the intended use of the contents of the kit. The term "labeling" includes any description or record of material provided in, with, or accompanying the kit. Accordingly, the present disclosure provides kits for treating a subject suffering from a tumor, the kit comprising: (a) an anti-PD-1 antibody at a dose ranging from about 4 mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in the methods described herein. In certain embodiments for treating a human patient, the kit comprises an anti-human PD-1 antibody described herein, e.g., nivolumab or pembrolizumab. In certain embodiments, the kit further comprises an anti-PD-L1 antibody. In certain embodiments, the kit further comprises instructions for detecting the mutation status of STK11 in a tumor sample. In other embodiments, the kit further comprises instructions for detecting PD-L1 expression in a tumor sample.

[0260] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are hereby incorporated by reference. [Example]

[0261] STK11 mutations as biomarkers of nivolumab response PD-L1 is expressed in NSCLC tumors (e.g., commercially available NSCLC tumors) with different expression patterns (Figure 1). These patterns are designated as scattered, heterogeneous, tumor-stroma interface, and negative. PD-L1 expression patterns can be related to mechanistic hypotheses. For example, in tumors with a scattered pattern, PD-L1 expression is driven not by mutation but by 9p24 amplification in oncogenic signaling pathways. In tumors with a tumor-stroma interface pattern, adaptive resistance is present rather than epithelial-mesenchymal transition (EMT).

[0262] PD-L1 expression patterns in commercially available NSCLC tumors correlate with PD-L1 H-scores, as shown in Figure 2. Substantial differences exist in PD-L1 expression levels among different patterns. For example, extremely high H-scores were observed in scattered pattern samples, indicating the potential dependence of these tumors on PD-L1 inhibition.

[0263] The PD-L1 expression pattern seen in commercially available NSCLC tumors was also seen in the biopsies. Figure 3 shows the PD-L1 expression pattern in study biopsies corresponding to patients treated with nivolumab monotherapy, which corresponds to the same pattern seen in commercially available NSCLC tumors.

[0264] The likelihood of false-negative PD-L1 results is influenced by pattern category, preanalytical variability, and biopsy size. For example, tumor-stroma interface patterns are heterogeneous and particularly prone to false-negative results. Thus, there is a need for biomarkers that can be used to facilitate the classification of NSCLC tumors and predict tumor responsiveness to specific therapeutic agents.

[0265] A correlation was observed between PD-L1 expression patterns and nivolumab efficacy (Figure 4). Most complete responders with grade 3 tumors showed a scattered pattern of PD-L1 expression and a high PD-L1 H-score. Therefore, identification of a biomarker specific for the scattered PD-L1 expression pattern can be used to identify patients suitable for nivolumab treatment based on the presence / absence of this biomarker.

[0266] Immune infiltrates may be used as a specific biomarker for NSCLC tumors with a scattered expression pattern, as tumors with a scattered or heterogeneous PD-L1 pattern had more abundant immune infiltrates (higher PD-L1 H-scores) in commercially available NSCLC tumors that generated the data shown in Figure 5 (Figure 5).

[0267] Multiplex IHC experiments demonstrated specific associations between tumor cells and immune cell subsets. PD-L1 labeling revealed scattered PD-L1 expression in the tumor. CD68 detection indicated that the macrophage layer at the tumor-stroma interface contributed to the formation of "border" activated T cells, while CD3 detection indicated that T cells were moderately abundant but largely restricted to the stroma (Figure 6).

[0268] PD-L1 expression patterns correlate with genomic data (Figure 7). Panel A of Figure 7 shows that although the level of PD-L1 expression correlates with RNA-seq data, RNA-seq data alone does not provide a geographic context for the PD-L1 expression patterns observed by IHC. Panel B of Figure 7, showing exome analysis data, shows that a diffuse PD-L1 expression pattern correlates with a higher mutational burden.

[0269] Higher mutation burden was also associated with inflamed tumors (Figure 8). Panel A shows overall inflammation as measured using the "CI score," an intensity score of chronic inflammatory infiltrates. Panel B shows PD-L1+ inflammation as measured using the "PD-L1+CI score," an intensity score of the relative proportion of PD-L1+ immune infiltrates. There is an association between the number of missense mutations and overall inflammation in NSCLC tumors.

[0270] We assessed the mutation frequencies of different biomarkers (TP53, STK11, KEAP1, KRAS, EGFR, and MET) relative to the observed PD-L1 expression patterns (Figure 9). The results showed that negative PD-L1 expression in tumor cells and low PD-L1 mRNA expression were associated with the presence of SKT11 mutations. 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 (seen in most responders to nivolumab therapy). Thus, the presence of STK11 mutants may be used as a negative biomarker for the treatment of NSCLC tumors with nivolumab (i.e., its presence would predict non- or poor response to nivolumab). Conversely, the presence of wild-type STK11 (or the absence of mutant forms) may be used as a positive selection biomarker for treatment with nivolumab.

[0271] Mutational loss of STK11 is predicted to enhance mTOR signaling. Lung adenocarcinomas (both mouse models and human tumors) with KRAS and STK11 mutations exhibit reduced PD-L1 expression and reduced T cell infiltrates. Proposed mechanisms of immunosuppression mediated by mutations in SKT11 may include a switch to glycolytic metabolism that increases lactate production and frequent co-mutations in KEP1, which triggers an anti-inflammatory transcriptional program.

[0272] Immunoprint analysis of 24 NSCLC tumor samples analyzed for levels of FOLR2, VSIG4, CD163, CLEC4D, CSF1R, CD86, MS4A1, CD79B, CD19, KIR2DS4, CD3E, CCR4, CCR8, and CD8A was used to classify the samples by inflammatory pattern (sigClass). 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 STK11 mutation (Figure 10). Furthermore, samples were classified according to PD-L1 expression pattern as negative ("PDL1_pattern2 negative"), scattered ("PDL1_pattern2 scattered"), heterogeneous ("PDL1_pattern2 heterogeneous"), and tumor-stroma interface ("PDL1_pattern2TS"). Tumors with a scattered PD-L1 expression pattern exhibited high inflammation and were STK11 wild-type. PD-L1-negative tumors were divided into two groups: intermediate and low inflammation. There was no clear distinction in the inflammation level of PD-L1-negative tumors according to STK11 mutation status. All tumors with mutant STK11 were also PD-L1 negative.

[0273] These data indicate that PD-L1 expression patterns are associated with unique phenotypic and genetic backgrounds. Scattered PD-L1 expression correlates with an inflammatory TME and high mutational burden. 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 and the potential to combine histopathological and genomic data to identify characteristics that define NSCLC subsets with varying likelihood of response to immunotherapy.

[0274] Example 2 This 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 recurrent NSCLC and PD-L1 tumor expression levels of 1% or higher received up to six cycles of nivolumab (intravenously administered once every two weeks at a dose of 3 mg / kg of body weight) or platinum-based chemotherapy (intravenously administered once every three weeks).

[0275] In a post-hoc analysis, patients' tumor cells were analyzed for PD-L1 expression and wild-type or mutant STK11, KRAS, CDKN2A, PTPND, CUBN, and / or HERC1. Patient survival was followed for 25 months.

[0276] After treatment, patients harboring STK11 mutations were found to have a reduced progression-free survival (PFS) compared with patients with wild-type STK11. This was observed in patients with any of the nonsynonymous STK11 mutations (Figure 12A) and patients with nonsense, frameshift, or splicing STK11 mutations (Figure 12B). STK11 mutation-carrying patients who also had a KRAS mutation also showed a reduced response to anti-PD-1 antibody treatment (Figure 13). When patients were stratified based on NSCLC type, patients with non-squamous NSCLC and any of the STK11 mutations showed a lower PFS compared with patients with wild-type STK11, regardless of KRAS status (Figures 14A-14B).

[0277] STK11 mutation status and response were compared with tumor PD-L1 expression levels (Figures 15A-15F). Two patients who showed partial responses 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 in the wild-type and STK11 mutant subgroups (Figure 15E).

[0278] In addition to STK11, patient tumors were monitored for TP53, CDKN2A, PTPND, CUBN, and HERC1 status (Figures 16A-16D). Patients with both TP53 and KRAS mutations exhibited higher PFS than patients with wild-type TP53 after treatment with first-line anti-PD-1 monoclonal antibody therapy (Figure 16A). Patients with CDKN2A mutations also exhibited higher PFS than patients with wild-type CDKN2A (Figure 16B), as did patients with mutant forms of PTPND, CUBN, and HERC1 (Figures 16C-16D).

[0279] Overall, there were no clear differences in TMB based on STK11 status for all patients or when looking only at patients who also had KRAS mutations (Figures 17A-17B). However, among wild-type STK11 patients, there was a slight increase in overall response in patients with high TMB compared to patients with lower TMB (Figure 17C).

[0280] Example 3 This 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 doublet chemotherapy. Patients were randomized to receive either nivolumab (intravenously administered once every 2 weeks at a dose of 3 mg / kg of body weight) or docetaxel (75 mg / m 2 The patients were administered a single dose of 100mg of riboflavin every three weeks.

[0281] In a post-hoc analysis, patient tumor cells were analyzed for STK11 and KRAS mutation status. As seen in Example 2 above, patients with STK11 mutants generally showed lower overall PFS than patients with wild-type STK11 (Figures 18A-18D). This was the case whether subjects had any nonsynonymous STK11 mutations (Figures 18A-18B) or nonsense, frameshift, or splicing STK11 mutations (Figures 18D-18E), and whether subjects were KRAS wild-type (Figures 18A and 18C) or KRAS mutant (Figures 18B or 18D).

[0282] Example 4 This 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 squamous NSCLC that had progressed during or after platinum-based doublet chemotherapy. Patients were randomized to receive either nivolumab (intravenously administered once every 2 weeks at a dose of 3 mg / kg of body weight) or docetaxel (75 mg / m 2 The patients were administered a single dose of 100mg of riboflavin every three weeks.

[0283] In a post-hoc analysis, patient tumor cells were analyzed for STK11 mutation status. Although the incidence of STK11 mutations in the patient population was low (n=5), patients with STK11 mutant forms exhibited higher overall PFS than patients with wild-type STK11 after anti-PD-1 antibody treatment (Figure 19). Further analysis of a larger number of patients will confirm the usefulness of these initial observations.

[0284] This application claims the benefit of U.S. Provisional Application No. 62 / 513,831, filed June 1, 2017, which is incorporated herein by reference in its entirety.

Claims

1. 1. A composition comprising an antibody or antigen-binding portion thereof ("anti-PD-1 antibody") that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity for treating a subject afflicted with a tumor derived from non-small cell lung cancer (NSCLC), wherein the subject has been identified as having a wild-type STK11 gene and a mutant marker gene comprising PTPRD, CUBN, and HERC1, wherein the mutant marker gene comprises a nonsynonymous mutation, a nonsense mutation, a frameshift mutation, or a splicing mutation.

2. 10. The composition of claim 1, wherein the subject is further identified as having additional mutant marker genes including KRAS, TP53, CDKN2A, or any combination thereof.

3. The composition of claim 1 or 2, wherein PD-L1 expression is detected in the tumor prior to administration of the anti-PD-1 antibody.

4. The composition of claim 3, wherein the tumor expresses PD-L1 in a diffuse or heterogeneous pattern.

5. 5. The composition of any of claims 1 to 4, wherein the tumor has a tumor mutation burden (TMB) score of at least 243, wherein the tumor TMB score is determined by sequencing nucleic acids in the tumor and then identifying genomic alterations in the sequenced nucleic acids, wherein the genomic alterations comprise one or more alterations including somatic mutations, non-synonymous mutations, missense mutations, base pair substitutions, base pair insertions, base pair deletions, copy number alterations (CNAs), gene rearrangements, or any combination thereof.

6. The composition described in any of claims 1 to 5, wherein the anti-PD-1 antibody comprises nivolumab or an antigen-binding portion thereof.

7. The composition of any one of claims 1 to 5, wherein the anti-PD-1 antibody comprises nivolumab.

8. The composition of any one of claims 1 to 5, wherein the anti-PD-1 antibody comprises pembrolizumab or an antigen-binding portion thereof.

9. 9. The composition of any of claims 1-8, wherein the anti-PD-1 antibody is administered once every 1, 2, or 3 weeks at a dose ranging from 0.1 mg / kg to 10.0 mg / kg of body weight.

10. 10. The composition of any of claims 1-9, wherein the anti-PD-1 antibody is administered at a flat dose of 240 mg or 480 mg once every 1, 2, 3, or 4 weeks.

11. A kit for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), wherein the subject has been identified as having a wild-type STK11 gene and mutant marker genes including PTPRD, CUBN, and HERC1, wherein the mutant marker genes include a nonsynonymous mutation, a nonsense mutation, a frameshift mutation, or a splicing mutation; (a) an anti-PD-1 antibody at a dose ranging from 4 mg to 500 mg; and (b) a kit comprising instructions for administering the anti-PD-1 antibody according to any one of claims 1 to 10.

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