Methods of treating tumor

Abstract

To provide a method for treating a subject afflicted with a tumor.SOLUTION: A method comprises administering to a subject a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding portion thereof or an anti-PD-L1 antibody or an antigen-binding portion thereof, wherein the subject is identified as having a tumor with a high inflammatory gene signature score and a high tumor mutation burden (TMB) status. In some embodiments, the high inflammatory gene signature score is determined by measuring the expression of a panel of inflammatory genes in a tumor sample obtained from the subject, wherein the inflammatory gene panel comprises CD274 (PD-L1), CD8A, LAG3, and STAT1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Application No. 62 / 825,549, filed March 28, 2019, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention provides methods of treating a subject with a tumor using immunotherapy. [Background technology]

[0003] Background of the Invention Human cancers harbor numerous genetic and epigenetic alterations that produce potential neoantigens that can be recognized by the immune system (Sjoblom et al., Science (2006) 314(5797):268-274). The adaptive immune system, consisting of T and B lymphocytes, has potent anticancer properties, with broad capacity and exquisite specificity to respond to diverse tumor antigens. Furthermore, the immune system exhibits considerable plasticity and memory components. The successful utilization of all these attributes of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

[0004] To date, cancer immunotherapy has largely focused on approaches to enhance antitumor immune responses through adoptive transfer of activated effector cells, immunization against relevant antigens, or the provision of nonspecific immune stimulators such as cytokines. However, over the past decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have begun to provide novel immunotherapeutic approaches for cancer treatment, including the development of antibodies such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement, 2013) that specifically bind to the programmed death-1 (PD-1) receptor and block the inhibitory PD-1 / PD-1 ligand pathway (Topalian et al., 2012a, b; Topalian et al., 2014; Hamid et al., 2013; Hamid and Carvajal, 2013; McDermott and Atkins, 2013).

[0005] PD-1 is an important immune checkpoint receptor expressed by activated 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, programmed death-ligand-1 (PD-L1) and programmed death-ligand-2 (PD-L2), have been identified that are expressed on antigen-presenting cells and many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Patents 8,008,449 and 7,943,743), and the use of antibody inhibitors of the PD-1 / PD-L1 interaction for cancer treatment has entered clinical trials (Brahmer et al., 2010; Topalian et al., 2012a; Topalian et al., 2014; Hamid et al., 2013; Brahmer et al., 2012; Flies et al., 2011; Pardoll, 2012; Hamid and Carvajal, 2013).

[0006] 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 prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of antitumor T cell function (US Patent 8,008,449; Wang et al., 2014). Nivolumab has demonstrated activity in a variety of advanced solid tumors, including renal cell carcinoma (renal adenocarcinoma or adrenal neoplasia), melanoma, and non-small cell lung cancer (NSCLC) (Topalian et al., 2012a; Topalian et al., 2014; Drake et al., 2013; WO 2013 / 173223). Summary of the Invention [Problem to be solved by the invention]

[0007] The immune system and its response to immunotherapy are complex. Furthermore, anti-cancer drugs can vary in effectiveness based on unique patient characteristics. Therefore, there is a need for targeted treatment strategies that identify patients who are more likely to respond to specific anti-cancer drugs, thus improving clinical outcomes for patients diagnosed with cancer. [Means for solving the problem]

[0008] Summary of the Invention Certain embodiments of the present invention relate to methods of treating a human subject diagnosed with a tumor, comprising: (i) identifying a subject exhibiting (a) a high inflammatory signature score and (b) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested; and (ii) administering to the subject an anti-PD-1 antibody; wherein the inflammatory signature score is determined by measuring expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject; and wherein the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0009] Certain embodiments of the present invention relate to methods of treating a human subject diagnosed with a tumor, comprising administering to the subject an anti-PD-1 antibody, wherein the subject is identified prior to administration as exhibiting (i) a high inflammatory signature score and (ii) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested; wherein the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject; and wherein the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0010] In certain embodiments, the method further comprises measuring the TMB status of a biological sample obtained from the subject prior to administration.

[0011] Certain embodiments of the present invention relate to methods for identifying a human subject having a tumor suitable for anti-PD-1 antibody treatment, comprising: (i) measuring (a) an inflammatory signature score of a tumor sample obtained from the subject and (b) a TMB status of a biological sample obtained from the subject; and (ii) administering an anti-PD-1 antibody to the subject if the subject exhibits a high inflammatory signature score and a TMB status comprising at least about 10 mutations per megabase of the genome examined; wherein the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in the tumor sample obtained from the subject; and wherein the inflammatory gene panel comprises CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0012] In certain embodiments, the inflammatory gene panel consists of less than about 20, less than about 18, less than about 15, less than about 13, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, or less than about 5 inflammatory genes. In certain embodiments, the inflammatory gene panel consists essentially of (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, or 15 additional inflammatory genes.

[0013] In some embodiments, the additional inflammatory gene is selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCR5, CD27, CD274, CD276, CMKLR1, CXCL10, CXCL11, CXCL9, CXCR6, GZMA, GZMK, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DRA, HLA-DRB1, HLA-E, ICOS, IDO1, IFNG, IRF1, NKG7, PDCD1LG2, PRF1, PSMB10, TIGIT, and any combination thereof.

[0014] In certain embodiments, the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1. In certain embodiments, the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0015] In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score that is higher than an average inflammatory signature score, where the average inflammatory signature score is determined by averaging the expression of the panel of inflammatory genes in tumor samples obtained from a population of tumor-bearing subjects.

[0016] In certain embodiments, the average inflammatory signature score is determined by averaging the expression of the panel of inflammatory genes in tumor samples from a population of subjects.

[0017] In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score that is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the average inflammatory signature score. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score that is at least about 50% higher than the average inflammatory signature score. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score that is at least about 75% higher than the mean inflammatory signature score.

[0018] In some embodiments, the tumor sample is a tumor tissue biopsy sample. In some embodiments, the tumor sample is formalin-fixed, paraffin-embedded tumor tissue or fresh-frozen tumor tissue. In some embodiments, the expression of inflammatory genes in the inflammatory gene panel is determined by the presence of inflammatory gene mRNA, the presence of proteins encoded by inflammatory genes, or both. In some embodiments, the presence of inflammatory gene mRNA is determined using reverse transcriptase PCR. In some embodiments, the presence of proteins encoded by inflammatory genes is determined using IHC assay. In some embodiments, the IHC assay is an automated IHC assay.

[0019] In some embodiments, TMB status is determined by sequencing nucleic acids in tumors and identifying genomic alterations in the sequenced nucleic acids. In some embodiments, the genomic alterations include one or more somatic mutations. In some embodiments, the genomic alterations include one or more non-synonymous mutations. In some embodiments, the genomic alterations include one or more missense mutations. In some embodiments, the genomic alterations include 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.

[0020] In one embodiment, the TMB status of the tumor is determined by FOUNDATIONONE® CDX TM The assay comprises at least 10 mutations, at least about 11 mutations, at least about 12 mutations, at least about 13 mutations, at least about 14 mutations, at least about 15 mutations, at least about 16 mutations, at least about 17 mutations, at least about 18 mutations, at least about 19 mutations, at least about 20 mutations, at least about 21 mutations, at least about 22 mutations, at least about 23 mutations, at least about 24 mutations, at least about 25 mutations, at least about 26 mutations, at least about 27 mutations, at least about 28 mutations, at least about 29 mutations, or at least about 30 mutations per megabase of the genome examined.

[0021] In some embodiments, biological sample is tumor tissue biopsy sample.In some embodiments, tumor tissue is formalin-fixed, paraffin-embedded tumor tissue or fresh-frozen tumor tissue.In some embodiments, biological sample is liquid biopsy sample.In some embodiments, biological sample comprises one or more of blood, serum, plasma, exoRNA, circulating tumor cell, ctDNA and cfDNA.

[0022] In some embodiments, the TMB status is determined by genome sequencing. In some embodiments, the TMB status is determined by exome sequencing. In some embodiments, the TMB status is determined by genome profiling.

[0023] In certain embodiments, the genomic profile comprises at least about 20 genes, at least about 30 genes, at least about 40 genes, at least about 50 genes, at least about 60 genes, at least about 70 genes, at least about 80 genes, at least about 90 genes, at least about 100 genes, at least about 110 genes, at least about 120 genes, at least about 130 genes, at least about 140 genes, at least about 150 genes, at least about 160 genes, at least about 170 genes, at least about 180 genes, at least about 190 genes, at least about 200 genes, at least about 210 genes, at least about 220 genes, at least about 230 genes, at least about 240 genes, at least about 250 genes, at least about 260 genes, at least about 270 genes, at least about 280 genes, at least about 290 genes, at least about 300 genes, at least about 310 genes, at least about 320 genes, at least about 330 genes, at least about 340 genes, at least about 350 genes, at least about 360 genes, at least about 370 genes, at least about 380 genes, at least about 390 genes, at least about 400 genes, at least about 410 genes, at least about 420 genes, at least about 430 genes, at least about 440 genes, at least about 450 genes, at least about 460 genes, at least about 470 genes, at least about 480 genes, at least about 490 genes, at least about 500 genes, at least about 510 genes, at least about 520 genes, at least about 530 genes, at least about 540 genes, at least about 550 genes, at least about 560 genes, at least about 570 genes, at least about 5 Each of the genomic profiles comprises at least about 260 genes, at least about 270 genes, at least about 280 genes, at least about 290 genes, at least about 300 genes, at least about 305 genes, at least about 310 genes, at least about 315 genes, at least about 320 genes, at least about 325 genes, at least about 330 genes, at least about 335 genes, at least about 340 genes, at least about 345 genes, at least about 350 genes, at least about 355 genes, at least about 360 genes, at least about 365 genes, at least about 370 genes, at least about 375 genes, at least about 380 genes, at least about 385 genes, at least about 390 genes, at least about 395 genes, or at least about 400 genes. In some embodiments, the genomic profile comprises at least about 265 genes. In some embodiments, the genomic profile comprises at least about 315 genes. In some embodiments, the genomic profile comprises at least about 354 genes.

[0024] ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MIT F、PDCD1LG2(PD-L2)、RBM10、STAT4、ABL2、BRCA1、CHEK2、FANCD2、GATA4 JAK3, MLH1, PDGFRA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JU N, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf 39) KAT6A(MYST 3) MR 11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLl1, KDM5A MSH2, PIK3C2B, ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11. KDM5C, MSH6, PIK3CA, RPTOR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, AMER1(FAM123B), C11orf 30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT C, CARD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PIK3R1, SDHA, TET2, AR, CBFB, CTNN B1, FGF10, GPR124, KEL, MYCL(MYC). L1) PIK3R2 SDHB TGFBR2 ARAF CBL CUL3 FGF14 GRIN2A KIT MYCN PLCG2 SDHC TNFAIP3 ARFRP1 CCND1C YLD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFRSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, PO LD1, SETD2, TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, TOP2A, ARID2, CCNE1, DIC ER1, FGF4, HGF, KMT2D(MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274(PD-L1), DNMT3A, FGF6, HNF1A, and 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, TSHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOT CH3, PRKDC, SMARCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1(MEK1), NRAS , PTCH1, SNCAIP, WISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2(MEK2), NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PT PN11, SOX10, XPO1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, S OX9, ZNF217, BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFl1, FRS2, INPP4B, MDM4, PAK3, RAD51, S In one embodiment, the TMB status is determined by one or more genes selected from the group consisting of POP, BCL6, CDKN2C, 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.TM It is measured by an assay.

[0025] In certain embodiments, the method further comprises identifying genomic alterations in one or more of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6, and MYB.

[0026] In certain embodiments, the tumor has a high neoantigen load. In certain embodiments, the subject has an expanded T cell repertoire.

[0027] In some embodiments, the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. In some embodiments, the anti-PD-1 antibody binds to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody or a portion thereof. In some embodiments, the anti-PD-1 antibody comprises a heavy chain constant region of human IgG1 or IgG4 isotype. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0028] 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 of body weight approximately once every one, two, or three weeks. In some embodiments, the anti-PD-1 antibody is administered at a dose of at least about 3 mg / kg of body weight approximately once every two weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, or at least about 550 mg. In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a flat dose of about 240 mg. In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a flat dose of about 480 mg.

[0029] In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose once every 1, 2, 3, or 4 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 240 mg once every 2 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 480 mg once every 4 weeks.

[0030] In certain embodiments, the anti-PD-1 antibody is administered as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. In certain embodiments, the anti-PD-1 antibody is formulated for intravenous administration. In certain embodiments, the anti-PD-1 antibody is administered at a subtherapeutic dose.

[0031] In some embodiments, the method further comprises administering an antibody or antigen-binding fragment thereof ("anti-CTLA-4 antibody") that specifically binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab. In some embodiments, the anti-CTLA-4 antibody is ipilimumab. In some embodiments, the anti-CTLA-4 antibody is tremelimumab.

[0032] In some embodiments, the anti-CTLA-4 antibody is administered at a dose ranging from 0.1 mg / kg to 20.0 mg / kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of 1 mg / kg body weight once every 6 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of 1 mg / kg body weight once every 4 weeks.

[0033] In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg. In some embodiments, the anti-CTLA-4 antibody is administered in a flat dose about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0034] In some embodiments, the tumor is derived from a cancer selected from the group consisting of hepatocellular carcinoma, gastroesophageal carcinoma, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof. In some embodiments, the tumor is derived from hepatocellular carcinoma. In some embodiments, the tumor is derived from gastroesophageal carcinoma. In some embodiments, the tumor is derived from melanoma.

[0035] In some embodiments, the tumor is recurrent. In some embodiments, the tumor is refractory. In some embodiments, the tumor becomes refractory after at least one prior treatment comprising the administration of at least one anticancer agent. In some embodiments, the at least one anticancer agent comprises a standard treatment agent. In some embodiments, the at least one anticancer agent comprises immunotherapy.

[0036] In some embodiments, the tumor is locally advanced. In some embodiments, the tumor is metastatic.

[0037] In some embodiments, the administration treats tumors. In some embodiments, the administration reduces tumor size. In some embodiments, the 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. In some 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.

[0038] In some embodiments, the subject experiences stable disease after administration. In some embodiments, the subject experiences a partial response after administration. In some embodiments, the subject experiences a complete response after administration.

[0039] One aspect of the present invention relates to a kit for treating a subject with a tumor, the kit comprising: (a) a single dose of an anti-PD-1 antibody ranging from about 4 mg to about 500 mg; and (b) instructions for using the anti-PD-1 antibody in any of the methods disclosed herein. In some embodiments, the kit further comprises an anti-CTLA-4 antibody. In some embodiments, the kit further comprises an anti-PD-L1 antibody. In some embodiments, the kit further comprises a comprehensive genomic profiling assay. In some embodiments, the comprehensive genomic profiling assay is FOUNDATIONONE® CDX TM It is a genomic profiling assay.

[0040] Other features and advantages of the present invention will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all cited information, including scientific articles, newspaper articles, GenBank entries, patents, and patent applications cited throughout this specification, are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 is a schematic diagram of the study design for exploratory endpoint biomarker evaluation of the efficacy of NIVO in sorafenib (SOR)-pretreated ("SOR-experienced") and -naive ("SOR-naive") patients with advanced hepatocellular carcinoma (HCC) in clinical trial NCT01658878.

[0042] [Figure 2-1]Figures 2A and 2B are waterfall plots illustrating the best reduction from baseline in target lesions (%) for all subjects in the overall population (Figure 2A) and the SOR-experienced population (Figure 2B), where subjects in each plot are labeled according to PD-L1 status. Figures 2C and 2D are graphical representations of overall survival (months) for patients in the overall population (SOR-naive and SOR-experienced; Figure 2C) and the SOR-experienced population alone (Figure 2D) in patients with tumor cell PD-L1 ≥ 1% or < 1%, as indicated. The number of patients at risk in each PD-L1 group is shown below the x-axis. [Figure 2-2] Same as above

[0043] [Figure 3-1] Figures 3A-3D are plots showing the correlation between best overall response and the percentage of cells expressing T cell markers selected from CD3 (Figure 3A), CD4 (Figure 3B), CD8 (Figure 3C), and FOXP3 (Figure 3D) for all populations (SOR-naive and SOR-experienced). [Figure 3-2] Same as above

[0044] [Figure 4-1] Figures 4A-4D are graphical representations showing overall survival for the total population (SOR-naive and SOR-experienced) stratified into tertiles based on lowest, intermediate, or highest levels of expression of T-cell markers selected from CD3 (Figure 4A), CD4 (Figure 4B), CD8 (Figure 4C), and FOXP3 (Figure 4D). The number of patients at risk for each stratification group is shown below the x-axis. [Figure 4-2] Same as above

[0045] [Figure 5] Figures 5A-5B are plots showing the correlation between best overall response and the percentage of cells expressing macrophage markers selected from CD68 (Figure 5A) and CD163 (Figure 5B) for all populations (SOR-naive and SOR-experienced).

[0046] [Figure 6]6A-6B are graphical representations showing overall survival for the total population (SOR-naive and SOR-experienced) stratified into tertiles based on lowest, intermediate, or highest levels of expression of T-cell markers selected from CD68 (FIG. 6A) and CD163 (FIG. 6B). The number of patients at risk for each stratification group is shown below the x-axis.

[0047] [Figure 7] Figure 7A is a plot showing the correlation between best overall response and the 4-gene signature score described herein. Figure 7B is a graphical representation showing overall survival for the entire population (SOR-naive and SOR-experienced) stratified based on the lowest, intermediate, or highest 4-gene inflammatory signature score. The number of patients at risk for each stratification group is shown below the x-axis.

[0048] [Figure 8] Figure 8 is a schematic diagram of the study design for the exploratory endpoint biomarker evaluation of efficacy of nivolumab treatment with and without ipilimumab in patients with chemotherapy-refractory gastroesophageal cancer in the Phase I / II clinical trial NCT01928394.

[0049] [Figure 9] Figures 9A-9B are plots showing the correlation between best overall response and tumor PD-L1 expression (Figure 9A) and PD-L1 combined positivity score (CPS; Figure 9B), as defined herein, for subjects treated with nivolumab 3 mg / kg monotherapy or nivolumab 1 mg / kg plus ipilimumab 3 mg / kg, nivolumab 3 mg / kg plus ipilimumab 1 mg / kg, or nivolumab 1 mg / kg plus ipilimumab 1 mg / kg.

[0050] [Figure 10-1]Figures 10A-10F are graphical representations of overall survival for patients in all treatment arms, stratified by tumor PD-L1 expression of ≥1% or <1% (Figure 10A), ≥5% or <5% (Figure 10B), ≥10% or <10% (Figure 10C) or by PD-L1 CPS of ≥1 or <1 (Figure 10D), ≥5 or <5 (Figure 10E), ≥10 or <10 (Figure 10F), as indicated. The number of patients at risk in each PD-L1 group is shown below the x-axis. [Figure 10-2] Same as above

[0051] [Figure 11-1] Figures 11A-11D are graphical representations of overall survival for patients in the nivolumab 1 mg / kg + ipilimumab 3 mg / kg treatment arm, stratified by tumor PD-L1 expression of ≥ 1% or < 1% (Figure 11A) or by PD-L1 CPS of ≥ 1 or < 1 (Figure 11B), ≥ 5 or < 5 (Figure 11C), or ≥ 10 or < 10 (Figure 11D), as indicated. The number of patients at risk in each PD-L1 group is shown below the x-axis. [Figure 11-2] Same as above

[0052] [Figure 12] Figures 12A-12D are plots showing the correlation between best overall response and the CD8 T cell signature (Figure 12A), PD-L1 transcript (Figure 12B), Ribas 10-gene signature (Figure 12C), and 4-gene inflammatory signature (Figure 12D) described herein.

[0053] [Figure 13] FIG. 13 shows the ROC analysis and benefit of the 4-gene immune signature.

[0054] [Figure 14] Figure 14 is a schematic diagram of the study design for exploratory endpoint biomarker evaluation of efficacy of nivolumab monotherapy, ipilimumab monotherapy, and nivolumab / ipilimumab combination treatment in patients with unresectable stage III or IV melanoma in the NCT01721772 and NCT01844505 trials.

[0055] [Figure 15-1] Figures 15A-15D are Kaplan-Meier plots of the key findings, progression-free survival (PFS; Figures 15A and 15C) and overall survival (OS; Figures 15B and 15D), of the intention-to-treat (ITT) population from NCT01721772 (Figures 15A-15B) and NCT01844505 (Figures 15C-15D). [Figure 15-2] Same as above

[0056] [Figure 16] Figures 16A-16C are bar graphs showing sample dispositions of subjects treated with nivolumab or dacarbazine in NCT01721772 and assessed for TMB (Figure 16A); or treated with nivolumab + ipilimumab combination therapy, nivolumab monotherapy, or ipilimumab monotherapy in NCT01844505 and assessed for TMB (Figure 16B) or 4-gene signature score (Figure 16C). The total number in each group is shown above each bar.

[0057] [Figure 17] Figure 17 is a plot showing the correlation between best overall response and the 4-gene inflammatory signature score described herein in subjects receiving nivolumab / ipilimumab combination treatment, nivolumab monotherapy, or ipilimumab monotherapy in the NCT01844505 trial.

[0058] [Figure 18-1] Figures 18A-18C are graphical representations showing progression-free survival of subjects receiving nivolumab / ipilimumab combination therapy (Figure 18A), nivolumab monotherapy (Figure 18B), or ipilimumab monotherapy (Figure 18C), where subjects are stratified by high 4-gene inflammatory signature score ("high ISS") or low 4-gene inflammatory signature score ("low ISS"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 18D shows the corresponding hazard ratios. [Figure 18-2] Same as above

[0059] [Figure 19-1] Figures 19A-19C are graphical representations showing overall survival (OS) of subjects receiving nivolumab / ipilimumab combination therapy (Figure 19A), nivolumab monotherapy (Figure 19B), or ipilimumab monotherapy (Figure 19C), where subjects have a high 4-gene inflammatory signature score ("high ISS") or a low 4-gene inflammatory signature score ("low ISS"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 19D shows the corresponding hazard ratios. [Figure 19-2] Same as above

[0060] [Figure 20] Figure 20 is a plot showing the relationship between best overall response and TMB, as described herein, in subjects receiving nivolumab monotherapy or dacarbazine in the NCT01844505 trial.

[0061] [Figure 21] Figures 21A-21B are graphical representations showing progression-free survival of subjects receiving nivolumab monotherapy (Figure 21A) or dacarbazine (Figure 21B), where subjects are stratified by high TMB ("TMB-high") or low TMB ("TMB-low"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 21C shows the corresponding hazard ratios.

[0062] [Figure 22] Figures 22A-22B are graphical representations showing overall survival of subjects receiving nivolumab monotherapy (Figure 22A) or dacarbazine (Figure 22B), where subjects are stratified by high TMB ("TMB-high") or low TMB ("TMB-low"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 22C shows the corresponding hazard ratios.

[0063] [Figure 23]Figure 23 is a plot showing the relationship between best overall response and TMB, as described herein, in subjects receiving nivolumab / ipilimumab combination treatment, nivolumab monotherapy, or ipilimumab monotherapy in the NCT01844505 trial.

[0064] [Figure 24-1] Figures 24A-24C are graphical representations showing progression-free survival of subjects receiving nivolumab / ipilimumab combination treatment (Figure 24A), nivolumab monotherapy (Figure 24B), or ipilimumab monotherapy (Figure 24C), where subjects are stratified by high TMB ("TMB-high") or low TMB ("TMB-low"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 24D shows the corresponding hazard ratios. [Figure 24-2] Same as above

[0065] [Figure 25-1] Figures 25A-25C are graphical representations showing overall survival of subjects receiving nivolumab / ipilimumab combination treatment (Figure 25A), nivolumab monotherapy (Figure 25B), or ipilimumab monotherapy (Figure 25C), where subjects are stratified by high TMB ("TMB-high") or low TMB ("TMB-low"). The number of patients at risk for each stratification group is shown below the x-axis. Figure 25D shows the corresponding hazard ratios. [Figure 25-2] Same as above

[0066] [Figure 26] Figures 26A-26C are scatter plots showing the relationship between 4-gene inflammatory signature score and TMB for subjects receiving nivolumab / ipilimumab combination therapy (Figure 26A), nivolumab monotherapy (Figure 26B), or ipilimumab monotherapy (Figure 26C). DETAILED DESCRIPTION OF THE INVENTION

[0067] Detailed Description of the Invention The present invention provides a method of treating a human subject with a tumor, comprising: (i) identifying a subject with (a) a high inflammatory signature score and (b) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested; and (ii) administering a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, to the subject. The present invention also provides a method of treating a human subject with a tumor, comprising administering a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the subject has been identified as having (i) a high inflammatory signature score and (ii) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested prior to administration. In certain embodiments, the method further comprises measuring the TMB status of a biological sample obtained from the subject prior to administration. The present invention also provides a method for identifying a subject having a tumor suitable for treatment with a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, comprising: (i) measuring (a) an inflammatory signature score of a tumor sample obtained from the subject and (b) a TMB status of a biological sample obtained from the subject; and (ii) if the subject has a high inflammatory signature score and a TMB status comprising at least about 10 mutations per megabase of the genome tested, administering a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, to the subject.

[0068] I. Terminology So that the present invention may be more readily understood, some terms are first defined. Unless otherwise expressly stated herein, each of the following terms has the meaning indicated: Additional definitions are set forth throughout this specification.

[0069] "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 of skill in the art. Preferred routes of administration for immunotherapy, e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies, include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, e.g., by injection or infusion. As used herein, the term "parenteral administration" refers to methods of administration other than enteral and topical administration, usually 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. Other non-parenteral routes include oral, topical, epithelial, or mucosal administration routes, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times and / or over one or more extended periods of time.

[0070] As used herein, an "adverse event" (AE) is any undesirable and generally unintended or undesirable sign (including abnormal laboratory findings), symptom, or disease associated with the use of a medical treatment. For example, an adverse event may be associated with the activation of the immune system or the expansion of immune system cells (e.g., T cells) in response to the treatment. A medical treatment may have one or more associated AEs, and each AE may be of the same or different severity levels. Reference to a method that can "alter an adverse event" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0071] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin or antigen-binding portion thereof that specifically binds to an antigen and comprises at least two heavy (H) chains and at least two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region comprises three constant domains, C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (herein VL The light chain constant region contains one constant domain, C L Includes V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L comprises three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Thus, the term "anti-PD-1 antibody" includes intact antibodies and antigen-binding portions of intact antibodies having two heavy chains and two light chains that specifically bind to PD-1. Non-limiting examples of antigen-binding portions are provided elsewhere in this specification.

[0072] 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 heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce immunogenicity in humans. Unless expressly stated and indicated otherwise by context, the term "antibody" includes antigen-binding fragments or portions of any of the above immunoglobulins, including monovalent and bivalent fragments or portions and single-chain antibodies.

[0073] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from different species. Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0074] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., essentially identical in primary sequence, which display a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are examples of isolated antibodies. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0075] 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 invention 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" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used interchangeably.

[0076] A "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDRs of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In some embodiments of a humanized form of an antibody, some, most, or all of the amino acids outside the CDRs are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within the CDRs 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 similar to that of the original antibody.

[0077] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0078] An "anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, an anti-PD-L1 antibody specifically binds to PD-L1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

[0079] An "antigen-binding portion" (also referred to as an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, such as an anti-PD-1 antibody or anti-PD-L1 antibody described herein, include: (i) V L , V H (ii) a Fab fragment (a fragment resulting from papain cleavage) or similar monovalent fragment, consisting of the LC and CH1 domains; (iii) a F(ab') fragment (a fragment resulting from pepsin cleavage) or similar bivalent fragment, comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody; L and V HFv fragment consisting of domains, (v) V H (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, optionally linked by synthetic linkers. In addition, the two domains V of the Fv fragment are L and V H are encoded by separate genes, but V L and V H The regions can be joined using recombinant methods by synthetic linkers that allow them to be formed as a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0080] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that can invade nearby tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0081] The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of developing or recurring a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "treatment" of a subject refers to any type of intervention or process or administration of an active agent performed on a subject with the goal of ameliorating, alleviating, ameliorating, arresting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions or biochemical manifestations associated with a disease.

[0082] "Programmed death-1" (PD-1) refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed on pre-activated T cells 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, as well as analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863.

[0083] "Programmed death-ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon 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, as well as analogs that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7. The human PD-L1 protein is encoded by the human CD274 gene (NCBI Gene ID: 2912).

[0084] As used herein, a PD-1 or PD-L1 "inhibitor" refers to any molecule that blocks, reduces, or otherwise limits the interaction of PD-1 and PD-L1 and / or the activity of PD-1 and / or PD-L1. In some embodiments, the inhibitor is an antibody or an antigen-binding fragment of an antibody. In other embodiments, the inhibitor comprises a small molecule.

[0085] As used herein, "T cell surface glycoprotein CD8 alpha chain" or "CD8A" refers to an integral membrane glycoprotein involved in immune responses and fulfilling multiple functions in responding to both external and internal attacks. In T cells, CD8a primarily functions as a coreceptor for MHC class I molecule / peptide complexes. CD8A simultaneously interacts with the T cell receptor (TCR) and MHC class I proteins presented by antigen-presenting cells (APCs). CD8a then recruits the Src kinase LCK to the vicinity of the TCR-CD3 complex. LCK then phosphorylates various substrates, triggering various intracellular signaling pathways that ultimately lead to lymphokine production, adhesion, and activation of cytotoxic T lymphocytes (CTLs). This mechanism enables CTLs to recognize and eliminate infected and tumor cells. In NK cells, the presence of CD8A homodimers on the cell surface provides a survival mechanism that allows for the conjugation and lysis of multiple target cells. The CD8A homodimeric molecule also promotes the survival and differentiation of activated lymphocytes into memory CD8 T cells. The complete CD8a amino acid sequence is available under UniProtKB identification number P01732. The human CD8a protein is encoded by the human CD8a gene (NCBI Gene ID: 925).

[0086] As used herein, "lymphocyte activation gene-3," "LAG3," "LAG-3," or "CD223" refers to a type I transmembrane protein expressed on the cell surface of activated CD4+ and CD8+ T cells and subsets of NK and dendritic cells. The LAG-3 protein is closely related to CD4, a coreceptor for T helper cell activation. Both molecules contain four extracellular Ig-like domains and require a ligand, major histocompatibility complex (MHC) class II, for functional activity. The LAG-3 protein is expressed only on the cell surface of activated T cells, and its cleavage from the cell surface terminates LAG-3 signaling. LAG-3 can also be found as a soluble protein that does not bind to MHC class II. LAG-3 also plays an important role in promoting regulatory T cell (Treg) activity and negatively regulating T cell activation and proliferation. Both natural and induced Tregs increase LAG-3 expression, which is necessary for maximal suppressive function. The complete human LAG-3 amino acid sequence can be found under UniProtKB identification number P18627. The human LAG-3 protein is encoded by the human LAG3 gene (NCBI Gene ID: 3902).

[0087] As used herein, "signal transducer and activator of transcription 1-alpha / beta" or "STAT1" refers to a signal transducer and transcription activator that mediates cellular responses to interferons (IFNs), the cytokine KITLG / SCF, and other cytokines and growth factors. Following binding of type I IFNs (IFN-alpha and IFN-beta) to their cell surface receptors, signal transduction via protein kinases leads to activation of Jak kinases (TYK2 and JAK1) and tyrosine phosphorylation of STAT1 and STAT2. Phosphorylated STATs dimerize and associate with ISGF3G / IRF-9 to form a complex called the ISGF3 transcription factor, which enters the nucleus. ISGF3 binds to IFN-stimulated response elements (ISREs) and activates transcription of IFN-stimulated genes (ISGs), driving cells into an antiviral state. In response to type II IFNs (IFN-gamma), STAT1 is tyrosine and serine phosphorylated. It then forms a homodimer called IFN-gamma-activating factor (GAF), translocates to the nucleus, and binds to IFN-gamma-activating sequence (GAS) to drive target gene expression and induce an antiviral state in the cell. STAT1 becomes activated in response to KITLG / SCF and KIT signaling. STAT1 can also mediate cellular responses to activated FGFR1, FGFR2, FGFR3, and FGFR4. The complete human STAT1 amino acid sequence is available under UniProtKB identification number P42224. Human STAT1 protein is encoded by the human STAT1 gene (NCBI Gene ID: 6772).

[0088] "Cytotoxic T-lymphocyte antigen-4" (CTLA-4) refers to an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385.

[0089] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In a preferred embodiment, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0090] The use of the term "flat dose" in connection with the methods and dosages of the present invention refers to a dose that is administered regardless of the patient's weight or body surface area (BSA). A flat dose is therefore provided as an absolute amount, rather than a mg / kg dose of an agent (e.g., an anti-PD-1 antibody). For example, a 60 kg person and a 100 kg person would receive the same dose of antibody (e.g., 240 mg of anti-PD-1 antibody).

[0091] The use of the term "fixed dose" in relation to the methods of the invention means that two or more different antibodies (e.g., an anti-PD-1 antibody and an anti-CTLA-4 antibody, or an anti-PD-L1 antibody and an anti-CTLA-4 antibody) in a single composition are present in a specific (fixed) ratio relative to each other in the composition. 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 1:20 ... The mg ratio of the first antibody (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) to the second antibody (e.g., anti-CTLA-4 antibody) is 0:1, 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. For example, a 3:1 ratio of anti-PD-1 antibody and anti-CTLA-4 antibody could mean that a vial could contain about 240 mg of anti-PD-1 antibody and 80 mg of anti-CTLA-4 antibody, or about 3 mg / ml of anti-PD-1 antibody and 1 mg / ml of anti-CTLA-4 antibody.

[0092] As used herein, the term "weight-based dose" means that the dose to be 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, the appropriate amount of anti-PD-1 antibody for administration (i.e., 180 mg) can be calculated and used.

[0093] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with other therapeutic agents, protects a subject from developing disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom-free periods, or prevention of functional or disability impairment due to disease morbidity. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in assays of the agent's activity in in vitro assays.

[0094] By way of example, an "anti-cancer agent" promotes cancer regression in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, or prevention of functional impairment or disability due to disease. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0095] For example, in tumor treatment, a therapeutically effective amount of an anticancer agent preferably inhibits cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared with untreated subjects. In other preferred embodiments of the present invention, tumor regression can be observed and continued for at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Regardless of these final measurements of therapeutic effectiveness, the evaluation of immunotherapy drugs must also take into account immune-related response patterns.

[0096] "Immune response," as understood in the art, generally refers to a biological response in a vertebrate to foreign agents or abnormalities, e.g., cancer cells, which protects the organism from these agents and the diseases they cause. Immune responses are mediated by one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses are mediated by T cells, e.g., effector T cells, Th cells, CD4 + cells, CD8 + This includes, for example, the activation or inhibition of T cells or Treg cells or the activation or inhibition of any other cell of the immune system, for example, NK cells.

[0097] "Immune-related response pattern" refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or modifying innate immune processes. This response pattern is characterized by a beneficial therapeutic effect after an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of classical chemotherapy agents would be classified as disease progression and is synonymous with drug failure. Therefore, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0098] As used herein, the terms "treat" and "treatment" refer to any type of intervention or process or administration of an active agent to a subject for the purpose of ameliorating, alleviating, ameliorating, arresting, slowing, or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease, or improving overall survival. Treatment can be of subjects with a disease or subjects not having the disease (e.g., for prophylaxis).

[0099] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom periods, an increase in overall survival (the length of time a patient diagnosed with a disease, such as cancer, is still alive from the date of diagnosis or initiation of treatment), or prevention of functional impairment or disability due to disease morbidity. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is any amount that prevents the onset or recurrence of disease when administered alone or in combination with other therapeutic agents to a subject at risk of developing or experiencing disease recurrence. The ability of a therapeutic agent to promote disease regression or prevent the onset or recurrence of disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in assays of the agent's activity in in vitro assays.

[0100] For example, an anti-cancer drug is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" refers to the administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, that results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, an increase in overall survival, prevention of functional or disability impairment due to disease, or other improvement of disease symptoms in a patient. Furthermore, 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 cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal levels resulting from the administration of a drug.

[0101] As an example of tumor treatment, a therapeutically effective amount or dosage of a drug inhibits 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 controls. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell proliferation or tumor growth, i.e., inhibits cell proliferation or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described herein. Alternatively, this property of a composition can be assessed by testing the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro using assays known to those skilled in the art. In some embodiments described herein, tumor regression can be observed and sustained for at least about 20 days, at least about 40 days, or at least about 60 days.

[0102] As used herein, the term "tumor mutation burden" (TMB) refers to the number of somatic mutations in a tumor's genome and / or the number of somatic mutations per area of ​​the tumor's genome. Germline (inherited) variants are excluded when determining TMB because the immune system is likely to recognize them as self. Tumor mutation burden (TMB) may also be used interchangeably with "tumor mutation load," "tumor mutational burden," or "tumor mutational load."

[0103] TMB is a genetic analysis of the tumor genome and can therefore be measured by applying sequencing methods well known to those skilled in the art. Tumor DNA can be compared with DNA from patient-matched normal tissue to rule out germline mutations or polymorphisms.

[0104] In some embodiments, TMB is determined by tumor DNA sequencing using high-throughput sequencing technology, such as next-generation sequencing (NGS) or NGS-based methods. In some embodiments, the NGS-based method is whole genome sequencing (WGS), whole exome sequencing (WES), or FOUNDATIONONE CDX. TMand comprehensive genomic profiling (CGP) of cancer gene panels, such as the MSK-IMPACT clinical trial. In some embodiments, TMB as used herein refers to the number of somatic mutations per megabase (Mb) of sequenced DNA. In some embodiments, TMB is measured using the number of non-synonymous mutations, such as missense mutations (i.e., specific amino acid changes in proteins) and / or nonsense mutations (which cause premature termination and therefore shortening of protein sequences), identified by normalizing matched tumors to germline samples to exclude any inherited germline genetic alterations. In other embodiments, TMB is measured using the total number of missense mutations in tumors. To measure TMB, a sufficient amount of sample is required. In some embodiments, tissue samples (e.g., at least 10 slides) are used for evaluation. In some embodiments, TMB is expressed as NsM per megabase (NsM / Mb). One megabase represents 1 million base pairs.

[0105] TMB status can be a numerical or relative value, eg, high, medium, or low; within the highest quantile or upper tertile of a control set.

[0106] As used herein, the term "high TMB" refers to a number of somatic mutations in a tumor genome 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 360, at least 370, at least 380, 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 least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least 680, at least 690, at least 700, at least 710, at least 720, at least 7 In some embodiments, the marker has 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 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. And in certain embodiments, high TMB has a score of at least 243.

[0107] In other embodiments, "high TMB" refers to TMB within the highest quantile of control TMB values. For example, all subjects with evaluable TMB data are grouped according to the quantile distribution of TMB, i.e., subjects are ranked from the highest to the lowest number of genetic alterations and divided into a specified number of groups. In some embodiments, all subjects with evaluable TMB data are ranked and divided into three, with "high TMB" being within the upper tertile of control TMB values. In certain embodiments, the tertile boundaries are 0<100 genetic alterations; 100-243 genetic alterations; and >243 genetic alterations. It is understood that once ranked, subjects with evaluable TMB data can be divided into any number of groups, for example, quartiles, quintiles, etc.

[0108] In certain embodiments, "high TMB" refers to a TMB of at least about 20 mutations / tumor, at least about 25 mutations / tumor, at least about 30 mutations / tumor, at least about 35 mutations / tumor, at least about 40 mutations / tumor, at least about 45 mutations / tumor, 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, or at least about 100 mutations / tumor. In certain embodiments, "high TMB" refers to a TMB of at least about 105 mutations / tumor, at least about 110 mutations / tumor, at least about 115 mutations / tumor, at least about 120 mutations / tumor, at least about 125 mutations / tumor, at least about 130 mutations / tumor, at least about 135 mutations / tumor, at least about 140 mutations / tumor, at least about 145 mutations / tumor, at least about 150 mutations / tumor, at least about 175 mutations / tumor, or at least about 200 mutations / tumor. In certain embodiments, a tumor with high TMB has at least about 100 mutations / tumor.

[0109] "High TMB" can also be used, for example, in mutation assays such as FOUNDATIONONE® CDX TM It can also be referred to as the number of mutations per megabase of the sequenced tumor genome, as measured by the assay. In one embodiment, high TMB is measured using FOUNDATIONONE® CDX TM "High TMB" refers to at least about 9, at least about 10, at least about 11, at least 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 mutations per megabase of the genome as measured by the assay. In certain embodiments, "high TMB" refers to 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 16, at least about 17, at least about 18, at least about 19, or at least about 20 mutations per megabase of the genome as measured by the assay. TM At least 10 mutations per megabase of the genome sequenced by the assay.

[0110] As used herein, the term "intermediate TMB" refers to a tumor genome with or near the normal or average number of somatic mutations, and the term "low TMB" refers to a tumor genome with a number of somatic mutations that is less than the normal or average number of somatic mutations. In certain embodiments, a "high TMB" has a score of at least 243, a "intermediate TMB" has a score of 100 to 242, and a "low TMB" has a score of less than 100 (or 0 to 100). "Intermediate or low TMB" refers to, for example, FOUNDATIONONE® CDX TM Refers to fewer than 9 mutations per megabase of the sequenced genome as measured by the assay.

[0111] As used herein, the term "control TMB value" may be the TMB value shown in Table 9.

[0112] In certain embodiments, TMB status may be correlated with smoking status. In particular, subjects who are current or former heavy smokers have more genetic alterations, e.g., missense mutations, than subjects who have never smoked.

[0113] Tumors with high TMB may also have a high neoantigen load. 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 as foreign (or non-self) by the immune system. Transcription of a gene in a tumor genome with a somatic mutation results in mutant mRNA that, when translated, produces a mutant protein, which is then processed, transported to the ER lumen, binds to the MHC class I complex, and promotes T cell recognition of the neoantigen. Neoantigen recognition can promote T cell activation, clonal expansion, and differentiation into effector and memory T cells. Neoantigen load can be correlated with TMB. In some embodiments, TMB is assessed as a surrogate for measuring tumor neoantigen load. The TMB status of a tumor can be used, alone or in combination with other factors, to determine the likelihood that a patient will benefit from a particular anticancer agent or a particular type of treatment or therapy, for example, a combination therapy comprising (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. In certain embodiments, high TMB status (or high TMB) indicates an enhanced likelihood of benefiting from cancer immunity and can therefore be used to identify patients who are likely to benefit from a combination therapy comprising (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. Similarly, tumors with high tumor neoantigen load and high TMB are more likely to be immunogenic than tumors with low neoantigen load and low TMB. Furthermore, high neoantigen / high TMB tumors are more likely to be recognized as non-self by the immune system and therefore induce immune-mediated anti-tumor responses. In certain embodiments, high TMB status and high neoantigen load indicate an enhanced likelihood of benefiting from cancer immunity, for example, a combination therapy comprising (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. As used herein, the term "benefit from treatment" refers to an improvement in one or more of overall survival, progression-free survival, partial response, complete response, and response rate, and may also include a reduction in tumor growth or size, a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom-free intervals, or prevention of functional impairment or disability due to disease.

[0114] Other factors, such as environmental factors, may be correlated with TMB status. For example, smoking status of patients with NSCLC correlates with TMB distribution, with current or former smokers having a higher median TMB compared with never-smokers. See Peters et al., AACR, April 1-5, 2017, Washington, DC. The presence of driver mutations in NSCLC tumors correlated with younger age, female gender, and non-smoker status. See Singal et al., ASCO, June 1-5, 2017; Chicago, IL. A trend toward lower TMB was observed with the presence of driver mutations such as EGFR, ALK, or KRAS (P=0.06). Davis et al., AACR, April 1-5, 2017, Washington, DC.

[0115] As used herein, the term "somatic mutation" refers to an acquired change in DNA that occurs after conception. Somatic mutations can occur in any cell of the body other than germ cells (sperm and eggs) and therefore are not passed on to offspring. These changes can, but do not always, cause cancer or other diseases. The term "germline mutation" refers to a genetic change in the body's germ cells (egg or sperm) that is incorporated into the DNA of all cells in the offspring's body. Germline mutations are passed from parents to offspring. They are also called "inherited mutations." In analyzing TMB, germline mutations are considered a "baseline" and are subtracted from the number of mutations found in tumor biopsy samples used to determine TMB in tumors. Because germline mutations are found in all cells of the body, their presence can be determined through less invasive sampling than tumor biopsies, such as blood or saliva. Germline mutations can increase the risk of developing certain cancers and may play a role in response to chemotherapy.

[0116] The terms "measuring" or "measured" or "measurement," when referring to TMB status, refer to the determination of a measurable amount of somatic mutations in a subject's biological sample. It is recognized that measurement can be performed by sequencing nucleic acids in the sample, such as cDNA, mRNA, exoRNA, ctDNA, and cfDNA. Measurement can be performed on the subject's sample and / or one or more control samples, and can be detected de novo or correspond to a previous determination. Measurement can be performed using, for example, PCR, qPCR, Sanger sequencing, genomic profiling (including comprehensive gene panels), exome sequencing, genome sequencing, and / or any other method disclosed herein, as known to those skilled in the art. In some embodiments, measurement identifies genomic alterations in the sequenced nucleic acid. Genomic (or gene) profiling methods can include a panel of a predetermined set of genes, e.g., 150-500 genes, and in some cases, genomic alterations assessed in a panel of genes correlate with the assessed total somatic mutations. As used herein, when referring to sequencing, the term "gene" refers to DNA coding regions (e.g., exons), DNA non-coding regions associated with the coding regions (e.g., introns and promoters), and mRNA transcripts.

[0117] As used herein, the term "genomic alteration" refers to a change (or mutation) in the nucleotide sequence of a tumor genome, which 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 certain embodiments, the genomic alteration measured in biological samples is a missense mutation.

[0118] As used herein, the term "whole genome sequencing" or "WGS" refers to a method of sequencing the entire genome. As used herein, the term "whole exome sequencing" or "WES" refers to a method of sequencing all of the protein-coding regions (exons) of the genome.

[0119] As used herein, a "cancer gene panel," "hereditary cancer panel," "comprehensive cancer panel," or "multigene cancer panel" refers to a method of sequencing a subset of target cancer genes, including coding regions, introns, promoters, and / or mRNA transcripts. In some embodiments, a CGP includes sequencing 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, or at least about 50 target cancer genes.

[0120] The terms "genomic profiling assay," "global genomic profiling," or "CGP" refer to an assay that analyzes a panel of genes and selects introns for in vitro diagnosis. CGP is a combination of NGS and targeted bioinformatics analysis to screen for mutations in known clinically relevant cancer genes. This method can be used to capture mutations that are missed by "hotspot" (e.g., BRCA1 / BRCA2 mutations or microsatellite marker) testing. In some embodiments, the CGP further includes one or more mRNA transcripts, non-coding RNAs, and / or promoter regions. In some embodiments, the genes in the panel are cancer-associated genes. In other embodiments, the genomic profiling assay is performed using FOUNDATION ONE (登録商標) It is an assay.

[0121] The term "harmonization" refers to a study conducted to determine the comparability between two or more scales and / or diagnostic tests. Harmonization studies provide a systematic approach to address the issue of how diagnostic tests compare with each other and their compatibility when used to determine the biomarker status of a patient's tumor. Generally, at least one well-characterized scale and / or diagnostic test is used as the standard for comparison with the others. Concordance assessments are often used in harmonization studies.

[0122] As used herein, the term "concordance" refers to the degree of agreement between two measurements and / or diagnostic tests. Agreement can be established using both qualitative and quantitative methods. Quantitative methods for assessing agreement vary based on the type of measurement. A particular measurement can be expressed as 1) an absolute / dichotomous variable or 2) a continuous variable. "Absolute / dichotomous variables" (e.g., above or below the TMB cutoff) can be used to assess agreement using percentage agreement, such as overall percent agreement (OPA), positive percent agreement (PPA), or negative percent agreement (NPA). "Continuous variables" (e.g., TMB by WES) use Spearman's rank correlation or Pearson's correlation coefficient (r), which takes the value -1≦r≦+1 to assess agreement across a range of values ​​(note: r=+1 or -1 means that the variables are perfectly correlated). The term "analytical concordance" refers to the degree of agreement between the performance of two assays or diagnostic tests to guide clinical use (e.g., the identification of biomarkers, genomic alteration types and genomic signatures, and test reproducibility assessment). The term "clinical concordance" refers to the degree of agreement between how two assays or diagnostic tests correlate with clinical results.

[0123] The term "microsatellite instability" or "MSI" refers to a change in the DNA of a cell (e.g., a tumor cell) in which the number of repeats of a microsatellite (a short, repetitive sequence of DNA) differs from the number of repeats present in the inherited DNA. MSI can be microsatellite-high (MSI-H) or microsatellite-low (MSI-L). Microsatellites are short, tandem DNA repeats of 1 to 6 base pairs. They are prone to DNA replication errors and are repaired by mismatch repair (MMR). Therefore, microsatellites are good indicators of genomic instability, particularly defective mismatch repair (dMMR). MSI is usually diagnosed by screening five microsatellite markers (BAT-25, BAT-26, NR21, NR24, and NR27). MSI-H refers to the presence of at least two unstable markers (or ≥30% of the markers if a large panel is used) among the five microsatellite markers analyzed. MSI-L means instability of one MSI marker (or 10%–30% of markers in a large panel). MSS means no unstable microsatellite markers.

[0124] The term "biological sample" as used herein refers to biological material isolated from a subject. Biological sample can include any biological material suitable for, for example, determining TMB by sequencing nucleic acid in tumor (or circulating tumor cells) and identifying genomic alterations in the sequenced nucleic acid. Biological sample can be any suitable biological tissue or fluid, such as tumor tissue, blood, plasma and serum. In some embodiments, the sample is a tumor tissue biopsy sample, such as formalin-fixed, paraffin-embedded (FFPE) tumor tissue or fresh-frozen tumor tissue. In other embodiments, the biological sample is a liquid biopsy sample, which in some embodiments includes one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA and cfDNA.

[0125] As used herein, the terms "about once every week," "about once every two weeks," or any other similar dosing interval term are intended to be approximations. "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 also apply to, for example, 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 certain embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose may be administered on any day of the first week, and the subsequent dose may be administered on any day of 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 of the week (e.g., Monday) in the first week, and the next dose is administered on the same day of the week (i.e., Monday) in the sixth or twelfth week, respectively.

[0126] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof. As used herein, the singular forms "a," "an," and "the" should be understood to refer to "one or more" of any described or listed components.

[0127] 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, which depends in part on how 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, as practiced in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10%. 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 value. When a particular value or composition is provided in the specification and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be presumed to be within an acceptable error range for that particular value or composition.

[0128] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include every integer value within the described range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0129] Abbreviations used herein are defined throughout the specification. Table 1 provides a list of additional abbreviations. [Table 1]

[0130] Various aspects of the invention are described in further detail in the following subsections.

[0131] II. Methods of the Invention The present invention relates to a method of treating a tumor in a human subject, comprising administering to the subject a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the tumor exhibits (i) a high inflammatory signature score prior to administration and (ii) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested prior to administration. In certain embodiments, the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject, wherein the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0132] II.A. Inflammatory Gene Panel In certain embodiments, the inflammatory gene panel consists of fewer than about 20, fewer than about 19, fewer than about 18, fewer than about 17, fewer than about 16, fewer than about 15, fewer than about 14, fewer than about 13, fewer than about 12, fewer than about 11, fewer than about 10, fewer than about 9, fewer than about 8, fewer than about 7, fewer than about 6, or fewer than about 5 inflammatory genes. In certain embodiments, the inflammatory gene panel consists of fewer than 20 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 19 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 18 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 17 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 16 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 15 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 14 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 13 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 12 genes. In certain embodiments, the inflammatory gene panel consists of fewer than 11 genes. In some embodiments, the inflammatory gene panel consists of fewer than 10 genes. In some embodiments, the inflammatory gene panel consists of fewer than 9 genes. In some embodiments, the inflammatory gene panel consists of fewer than 8 genes. In some embodiments, the inflammatory gene panel consists of fewer than 7 genes. In some embodiments, the inflammatory gene panel consists of fewer than 6 genes. In some embodiments, the inflammatory gene panel consists of fewer than 5 genes. In some embodiments, the inflammatory gene panel consists of 4 genes. In some embodiments, the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1. In some embodiments, the inflammatory gene panel consists of CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0133] In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1) and CD8A and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1) and LAG3 and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1) and STAT1 and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

[0134] In certain embodiments, the inflammatory gene panel consists essentially of (or consists of) (i) CD8A and LAG3 and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes. In certain embodiments, the inflammatory gene panel consists essentially of (or consists of) (i) CD8A and STAT1 and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

[0135] In certain embodiments, the inflammatory gene panel consists essentially of (or consists of) (i) LAG3 and STAT1 and (ii) 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, 16 additional inflammatory genes, or 17 additional inflammatory genes.

[0136] In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, and LAG3 and (ii) 1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

[0137] In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, and STAT1 and (ii) 1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

[0138] In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), LAG3, and STAT1 and (ii) 1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

[0139] In one embodiment, the inflammatory gene panel consists essentially of (or consists of) (i) CD274, CD8A, LAG3, and STAT1 and (ii) 1 additional inflammatory gene, 2 additional inflammatory genes, 3 additional inflammatory genes, 4 additional inflammatory genes, 5 additional inflammatory genes, 6 additional inflammatory genes, 7 additional inflammatory genes, 8 additional inflammatory genes, 9 additional inflammatory genes, 10 additional inflammatory genes, 11 additional inflammatory genes, 12 additional inflammatory genes, 13 additional inflammatory genes, 14 additional inflammatory genes, 15 additional inflammatory genes, or 16 additional inflammatory genes.

[0140] In some embodiments, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) one additional inflammatory gene. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) two additional inflammatory genes. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) three additional inflammatory genes. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) four additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) five additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) six additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) seven additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) eight additional inflammatory genes. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 9 additional inflammatory genes. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 10 additional inflammatory genes. In part, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 11 additional inflammatory genes.In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 12 additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 13 additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 14 additional inflammatory genes. In some cases, the inflammatory gene panel consists essentially of (or consists of) (i) CD274 (PD-L1), CD8A, LAG3, and STAT1, and (ii) 15 additional inflammatory genes.

[0141] Various genes associated with inflammation are known in the art and can be included in the inflammatory gene panels disclosed herein. For example, additional inflammatory genes can be selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CCR5, CD27, CD274, CD276, CMKLR1, CXCL10, CXCL11, CXCL9, CXCR6, GZMA, GZMK, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DRA, HLA-DRB1, HLA-E, ICOS, IDO1, IFNG, IRF1, NKG7, PDCD1LG2, PRF1, PSMB10, TIGIT, and any combination thereof.

[0142] In certain embodiments, the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1. In certain embodiments, the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1.

[0143] II.B.1. Inflammatory Signature Score As used herein, the inflammatory signature score is measured as the combined expression level of genes present in an inflammatory gene panel, e.g., comprising, consisting essentially of, or consisting of CD274 (PD-L1), CD8A, LAG3, and STAT1, in a sample obtained from a subject. Any biological sample containing one or more tumor cells can be used in the methods disclosed herein. In some embodiments, the sample is selected from a tumor biopsy sample, a blood sample, a serum sample, or any combination thereof. In some embodiments, the sample is a tumor biopsy sample taken from a subject prior to administration of an anti-PD-1 antibody. In a specific embodiment, the sample obtained from a subject is a formalin-fixed tumor biopsy sample. In some embodiments, the sample obtained from a subject is a paraffin-embedded tumor biopsy sample. In some embodiments, the sample obtained from a subject is a fresh-frozen tumor biopsy sample.

[0144] Any method known in the art for measuring a particular gene or group of genes can be used in the methods of the invention. In one embodiment, expression of one or more of the inflammatory genes in the inflammatory gene panel is determined by detecting the presence of mRNA transcribed from the inflammatory gene, the presence of a protein encoded by the inflammatory gene, or the presence of both.

[0145] In some embodiments, the expression of one or more inflammatory genes is determined by measuring the level of inflammatory gene mRNA in a sample obtained from a subject, for example, by measuring the level of one or more of LAG3 mRNA, PD-L1 mRNA, CD8A mRNA, and STAT1 mRNA. In some embodiments, the inflammatory gene score is determined by measuring the levels of LAG3 mRNA, PD-L1 mRNA, CD8A mRNA, and STAT1 mRNA in a sample obtained from a subject. Any method known in the art can be used to measure the level of inflammatory gene mRNA. In some embodiments, the inflammatory gene mRNA is measured using reverse transcriptase PCR. In some embodiments, the inflammatory gene mRNA is measured using RNA in situ hybridization.

[0146] In some embodiments, the expression of one or more inflammatory genes is determined by measuring the level of inflammatory gene proteins in a sample obtained from a subject, for example, measuring the level of one or more of PD-L1, CD8A, LAG-3, and STAT1. In some embodiments, the inflammatory gene score is determined by measuring the levels of PD-L1, CD8A, LAG-3, and STAT1 in a sample obtained from a subject. Any method known in the art can be used to measure the level of inflammatory gene proteins. In some embodiments, the inflammatory gene proteins are measured using immunohistochemistry (IHC) assay. In some embodiments, the IHC is automated IHC.

[0147] In some embodiments, the expression of one or more inflammatory genes in the inflammatory gene panel is normalized to the expression of one or more housekeeping genes. In some embodiments, the one or more housekeeping genes are comprised of genes whose expression is relatively constant across different tumor types in different subjects.

[0148] In some embodiments, raw gene expression values ​​are normalized according to standard gene expression profiling (GEP) protocol.In these embodiments, gene expression signature score can be calculated as the median or average of the log2-transformed normalized and adjusted expression values ​​of all target genes in signature, and can be displayed on a linear scale.In some embodiments, score has positive or negative value according to whether gene expression is up-regulated or down-regulated under the condition of measurement.

[0149] In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score that is greater than the control inflammatory signature score. In certain embodiments, the control inflammatory signature score is a mean inflammatory signature score. In certain embodiments, the mean inflammatory signature score is determined by measuring the expression of genes present in an inflammatory gene panel in tumor samples obtained from a subject population and calculating the mean for the subject population. In certain embodiments, each member of the subject population has the same tumor as the subject to which an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or any combination thereof is administered. In specific embodiments, the mean inflammatory signature score is about -0.07, about -0.06, -0.05, about -0.04, about -0.03, or about -0.02. In a specific embodiment, the mean inflammatory signature score is about -0.04. In certain embodiments, the mean inflammatory signature score is about -0.0434.

[0150] In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the average inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 25% higher than the average inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 30% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 35% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 40% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 45% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 50% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 55% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 60% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 65% higher than the mean inflammatory signature score.In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 70% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 75% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 80% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 85% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 90% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 95% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 100% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 125% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 150% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 175% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 200% higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 225% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 250% higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 275% higher than the mean inflammatory signature score.In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 300% higher than the mean inflammatory signature score.

[0151] In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.25x, at least about 1.30x, at least about 1.35x, at least about 1.40x, at least about 1.45x, at least about 1.50x, at least about 1.55x, at least about 1.60x, at least about 1.65x, at least about 1.70x, at least about 1.75x, at least about 1.80x, at least about 1.85x, at least about 1.90x, at least about 1.95x, at least about 2x, at least about 2.25x, at least about 2.50x, at least about 2.75x, at least about 3x, at least about 3.25x, at least about 3.50x, at least about 3.75x, or at least about 400x higher than the average inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.25 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.30 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.35 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.40 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.45 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.50 times higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.55 times higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.60 times higher than the mean inflammatory signature score. In some embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.65 times higher than the mean inflammatory signature score.In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.70 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.75 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.80 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.85 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.90 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 1.95 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 2-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 2.25-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 2.50-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 2.75-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 3-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 3.25-fold higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 3.50 times higher than the mean inflammatory signature score.In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 3.75 times higher than the mean inflammatory signature score. In certain embodiments, a high inflammatory score is characterized by an inflammatory signature score that is at least about 4 times higher than the mean inflammatory signature score.

[0152] In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 0.5, wherein the inflammatory signature score is determined by the methods disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 0.75, wherein the inflammatory signature score is determined by the methods disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 1.0, wherein the inflammatory signature score is determined by the methods disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 1.25, wherein the inflammatory signature score is determined by the methods disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 1.50, wherein the inflammatory signature score is determined by the methods disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 1.75, wherein the inflammatory signature score is determined by a method disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 2.0, wherein the inflammatory signature score is determined by a method disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 2.25, wherein the inflammatory signature score is determined by a method disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 2.5, wherein the inflammatory signature score is determined by a method disclosed herein. In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 2.75, wherein the inflammatory signature score is determined by a method disclosed herein.In certain embodiments, a high inflammatory signature score is characterized by an inflammatory signature score of at least about 3.0, where the inflammatory signature score is determined by the methods disclosed herein.

[0153] II.B. Tumor Mutation Burden (TMB) One embodiment of the present invention relates to a method of treating a human subject diagnosed with a tumor, comprising administering to the subject a PD-1 inhibitory antibody, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the subject has been identified prior to administration as exhibiting (i) a high inflammatory signature score and (ii) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested. The present invention is based on the direct relationship between tumor immunogenicity and TMB and / or neoantigen load.

[0154] As tumors grow, somatic mutations not present in germline DNA accumulate. TMB refers to the number of somatic mutations in the tumor genome and / or the number of somatic mutations per area of ​​the tumor genome (after accounting for germline variant DNA). The acquisition of somatic mutations, and therefore high TMB, can be influenced by mechanisms such as exposure to exogenous mutagens (e.g., smoking) 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). "Non-synonymous mutations" herein refer to nucleotide mutations that change the amino acid sequence of a protein. Both missense and nonsense mutations can be non-synonymous mutations. "Missense mutations" herein refer to non-synonymous point mutations in which a single nucleotide change results in a codon encoding a different amino acid. A "nonsense mutation" herein refers to a nonsynonymous point mutation in which a codon is changed to a premature stop codon leading to truncation of the resulting protein.

[0155] In some embodiments, somatic mutations are expressed at the RNA and / or protein level, resulting in neoantigens (also referred to as neoepitopes).Neoantigens can be responsible for immune-mediated anti-tumor responses.For example, neoantigen recognition can promote T cell activation, clonal proliferation and differentiation into effector and memory T cells.

[0156] As tumors progress, most or all tumor cells may harbor early clonal mutations (or "headquarters mutations"), while late mutations (or "branch mutations") may arise only in 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 "headquarters" mutations may be more widely distributed in the tumor genome than "branch" mutations, resulting in higher numbers of T cells reactive against the clonal neoantigens (McGranahan et al., (2016)351:1463-1469). In general, tumors with high TMB may also have a high neoantigen load, leading to increased tumor immunogenicity and increased T cell reactivity and antitumor responses. Thus, cancers with high TMB may respond well to immunotherapy, for example, treatment with anti-PD-1 or anti-PD-L1 antibodies.

[0157] Advances in sequencing technology have made it possible to assess the genomic mutation landscape of tumors. Any sequencing method known in the art can be used to sequence nucleic acids from tumor genomes (e.g., obtained from biological samples from tumor-bearing subjects). In some embodiments, PCR or qPCR methods, Sanger sequencing methods, or next-generation sequencing ("NGS") methods (e.g., genomic profiling, exome sequencing, or genome sequencing) can be used to measure TMB. In some embodiments, TMB status is measured using genomic profiling. Genomic profiling involves the analysis of nucleic acids from tumor samples, including coding and non-coding regions, and can be performed using methods with integrated optimized nucleic acid selection, read alignment, and mutation calling. In some embodiments, gene profiling provides next-generation sequencing (NGS)-based analysis of tumors, which can be optimized on a cancer-by-cancer, gene-by-gene, and / or location-by-location basis. Genomic profiling can integrate the use of multiple alignment methods or algorithms, fine-tuned here, to optimize the performance of the sequencing method, particularly in methods based on massively parallel sequencing of multiple diverse genetic events in multiple diverse genes. Genomic profiling can provide a comprehensive analysis of a subject's cancer genome with clinical-grade quality, and the output of the genetic analysis can be contextualized with relevant scientific and medical knowledge to improve the quality and efficiency of cancer treatment.

[0158] II.B.1. Genomic profiling Genomic profiling involves panels of predefined sets of genes, including as few as 5 genes or 1000 genes, from about 25 genes to about 750 genes, from about 100 genes to about 800 genes, from about 150 genes to about 500 genes, from about 200 genes to about 400 genes, or from about 250 genes to about 350 genes or so. In some embodiments, 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 other embodiments, the genomic profile comprises at least 325 genes. In certain embodiments, the genomic profile includes at least 315 cancer-associated genes and introns in 28 genes (FOUNDATIONONE®) or the complete DNA coding sequence of 406 genes, introns in 31 rearranged genes, and RNA sequences (cDNA) of 265 genes (FOUNDATIONONE® Heme). In other embodiments, the genomic profile includes 26 genes and 1000 associated mutations (EXODX® Solid Tumor). In yet other embodiments, the genomic profile includes 76 genes (Guardant360). In yet other embodiments, the genomic profile includes 73 genes (Guardant360). In other embodiments, the genomic profile includes 354 genes and introns in 28 genes for rearrangement (FOUNDATIONONE® CDX TMIn one embodiment, the genomic profile is FOUNDATIONONE® F1CDx. In another embodiment, the genomic profile comprises 468 genes (MSK-IMPACT TM ). One or more genes can be added to the genomic profile as more genes are identified as being associated with oncology.

[0159] II.B.1.a. 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 testing to identify genomic alterations (base substitutions, insertions and deletions, copy number alterations, and rearrangements) and select genomic signatures (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 2 and 3. See FOUNDATIONONE: Technical Specifications, Foundation Medicine, Inc., available at FoundationMedicine.com (last accessed March 16, 2018), incorporated herein by reference in its entirety. [Table 2-1] [Table 2-2] [Table 3]

[0160] II.B.1.b. 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 4. See Plasma-Based Solid Tumor Mutation Panel Liquid Biopsy, Exosome Diagnostics, Inc., available at exosomedx.com (last accessed March 25, 2019). [Table 4]

[0161] II.B.1.c. Guardant360 Assay In one embodiment, TMB status is determined using the Guardant360 assay. The Guardant360 assay measures mutations in at least 73 genes (Table 5), 23 indels (Table 6), 18 CNVs (Table 7), and 6 fusion genes (Table 8). See GuardantHealth.com (last accessed March 25, 2019). [Table 5] [Table 6] [Table 7] [Table 8]

[0162] II.B.1.d. 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. A list of TruSight Tumor 170 Assay genes is shown in Tables 12-14. [Table 9] [Table 10] [Table 11]

[0163] II.B.1.e. FOUNDATIONONE® F1CDx Assay FOUNDATIONONE® CDX TM("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 select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutation burden (TMB), using DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens. F1CDx has been approved by the U.S. Food and Drug Administration (FDA) for several oncology indications, including NSCLC, melanoma, breast cancer, colorectal cancer, and ovarian cancer.

[0164] The F1CDx assay uses a single DNA extraction method from routine FFPE biopsy samples or surgical resection specimens, with 50–1000 ng of DNA subjected to whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, one promoter region, one non-coding (ncRNA), and selected intronic regions from 34 commonly rearranged genes (21 of which also contain coding exons). Tables 12 and 13 provide a complete list of genes included in the F1CDx assay. In total, the assay detects alterations in 324 genes. The ILLUMINA® HiSeq 4000 platform is used to sequence the hybrid capture-selected libraries at high and uniform depth (targeting >500x median coverage, with >99% exons at >100x coverage). 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 alterations (amplifications and homozygous gene deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures, including microsatellite instability (MSI) and tumor mutation burden (TMB), are reported. [Table 12-1] [Table 12-2] [Table 13]

[0165] The F1CDx assay identifies a variety of alterations, including substitutions, insertions / deletions, and CNAs in gene and / or intron sequences. The F1CDx assay has been previously identified as being consistent with externally validated NGS assays and the FOUNDATIONONE® (F1 LDT) assay. FOUNDATIONONE® CDX, available at FoundationMedicine.com, is incorporated herein by reference in its entirety. TM : Technical Information, FoundationMedicine, Inc. (Last accessed March 25, 2019).

[0166] II.B.1.f. MSK-IMPACT TM In one embodiment, the TMB status is determined by MSK-IMPACT TM Assay: MSK-IMPACT TM The assay uses next-generation sequencing to analyze the mutation status of 468 genes. Target genes are analyzed using the ILLUMINA HISEQ TM The device captures and sequences the data. TM The assay is USFDA approved for the detection of somatic mutations and microsatellite instability in solid malignant neoplasms. TMA complete list of the 468 genes analyzed by the assay is shown in Table 14. See Evaluation of Automatic Class III Designation for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets): Decision Summary, United States Food and Drug Administration, November 15, 2017, available at accessdata.fda.gov. [Table 14-1] [Table 14-2] [Table 14-3]

[0167] II.B.1.g. NEOGENOMICS® NEOTYPE TM Assay In one embodiment, the TMB is NEOGENOMICS® NEOTYOPE TM In one embodiment, TMB is determined using a NEOTYPE assay. TM In one embodiment, TMB is determined using the NEOTYPE Solid Tumor Profile. The NEOGENOMICS assay measures the number of nonsynonymous DNA coding sequence changes per megabase of sequenced DNA.

[0168] II.B.1.h. ONCOMINE TM Tumor mutation burden assay In some embodiments, TMB is administered by THERMOFISHER SCIENTIFIC® ONCOMINE TM In some embodiments, TMB is determined using a tumor mutation assay. TM ONCOMINE TM Determined using tumor mutation assays. TM ONCOMINE TM The 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. TM ONCOMINE TM A complete list of 408 genes analyzed by the tumor mutation assay is shown in Table 15 (see Iontorrent, Oncomine Tumor Mutation Load Assay Flyer, available at assets.thermofisher.com / TFS-Assets / CSD / Flyers / oncomine-tumor-mutation-load-assay-flyer.pdf (last accessed March 25, 2019)). [Table 15-1] [Table 15-2]

[0169] II.B.1.i. NOVOGENE TM NOVOPM TM Assay In one embodiment, TMB is administered by NOVOGENE TM NOVOPM TM In one embodiment, TMB is determined using an assay. TMNOVOPM TM Determined using a cancer panel assay. TM NOVOPM TM The cancer panel assay is a comprehensive NGS cancer panel that analyzes the complete coding regions of 548 genes and the introns of 21 genes, representing approximately 1.5 Mb of DNA and relevant to the diagnosis and / or treatment of solid tumors according to National Comprehensive Cancer Network (NCCN) guidelines and medical literature. The assay detects SNVs, InDels, fusions, and copy number variations (CNVs) genomic aberrations.

[0170] II.B.1.j. Other TMB assays In some embodiments, TMB is determined using a TMB assay provided by CARIS® Life Sciences. In some embodiments, TMB is determined using the PESONALIS® ACE ImmunoID assay. In some embodiments, TMB is determined using PGDX® CANCERXOME TM - Determined using the R assay.

[0171] In yet other particular embodiments, genomic profiling detects all mutation types, i.e., single nucleotide variants, insertions / deletions (indels), copy number variations and rearrangements, e.g., translocations, expression and epigenetic markers.

[0172] A comprehensive gene panel often comprises predetermined genes that are selected based on the type of tumor being analyzed.Therefore, the genomic profile used to measure TMB status can be selected based on the type of tumor that the subject has.In some embodiments, the genomic profile can comprise a set of genes that are specifically related to solid tumors.In other embodiments, the genomic profile can comprise a set of genes that are specifically related to hematological tumors and sarcomas.

[0173] ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, PD CD1LG2, RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDGF RA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTO R, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf39), KAT6A(MYST3), MRE11 A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLI1, KDM5A, MSH2, PIK3C2B ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PIK3CA, RPTOR. TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, AMER 1(FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1 T1, TERT(Type), APC, CARD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PIK3R 1, SDHA, TET2, AR, CBFB, CTNNB1, FGF10, GPR124, KEL, MYCL(MYCL1), PIK3R2. SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC, and TNF AIP3, ARFRP1, CCND1, CYLD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFRSF1 4. ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1, SETD2, TOP 1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, and TOP2A ARID2, CCNE1, DICER1, FGF4, HGF, KMT2D(MLL2), NFE2L2, PPP2R1A, SLIT2, T P53, 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, TSHR , ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMARC B1, 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, BA RD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BC L2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, The TMB analysis may further include one or more genes selected from the group consisting of CDKN2C, 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. In other embodiments, the TMB analysis further includes identification of genomic alterations in one or more of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6, and MYB.

[0174] ABL1, 12B, ABL2, ACTB, ACVR1, ACVR1B, and AG O2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, AMER1(FAM123B). WTX, AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1, AR HGAP26(GRAF) ARID1A ARID1B ARID2 ARID5B ARv7 ASMTL ASXL1 ASXL2, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXIN2, AXL, B2M, BABAM 1, BAP1, BARD1, BBC3, BCL10, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2 BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA 2, BRD4, BRIP1, BRIP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf 30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD11, CARM1, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCN E1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC42, CDC73, CDH1, CDK12, CD K4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBPA, CENPA, CHD 2 CHD4, CHEK1, CHEK2, CIC, CIITA, CKS1B, CPS1, CREBBP, CRKL, CRLF2, CSDE1, CSF1R, CSF3R, CTCF, CLTA-4, CTNN B1, CTNNA1, CTNNB1, CUL3, CUL4A, CUX1, CXCR4, CYLD, CYP17A1, CYSLTR2, DAXX, DCUN1D1, DDR1, DDR2, DDX3X, DH 2, 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-ALK, EP300, EPAS 1. EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC1, ERCC2, ERCC3, E RCC4, ERCC5, ERF, ERG, ERRFI1, ERRFl1, ESR1, 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, FGF1 4. FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGF R4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH1, FOXA1, FOXL2, FOXO1, FOXO3, FOXP1, F RS2、FUBP1、FYN、GABRA6、GADD45B、GATA1、GATA2、GATA3、GATA4、GATA6、GEN1、GID4(C17orf 39) GID4(C17orf39), GLI1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRIN2A, GRM3, GSK3B, GTSE1 H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, HER-2 / NEU;ERBB2、HGF、HIST1H1C、HIST1H1D、HIST1H1E、HIST1H2A C、HIST1H2AG、HIST1H2AL、HIST1H2AM、HIST1H2BC、HIST1H2BD、HIST1H2BJ、HIST1H2BK、HIST1H2BO、HIST1H3A、HIST1H3B HIST1H3C、HIST1H3D、HIST1H3E、HIST1H3F、HIST1H3G、HIST1H3H、HIST1H3I、HIST1H3J、HIST2H3C、HIST2H3D、HIST3H3<h2 style=";text-align:left;direction:ltr">HLA-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(MYST) 3) KAT6A(MYST3) KDM2B KDM4C KDM5A KDM5C KDM6A KDR KEAP1 KEL KIF5B KIT KLF4 KLHL6 KMT2A KMT2A(MLL) KMT2B KMT2C KM T2C(MLL3)、KMT2D、KMT2D(MLL2)、KNSTRN、KRAS、LAMP1、LATS1、LATS 2、LEF1、LMO1、LRP1B、LRRK2、LTK、LYN、LZTR1、MAF、MAFB、MAGED1、MA GI2、MALT1、MAP2K1、MAP2K1(MEK1)、MAP2K2、MAP2K2(MEK2)、MAP2K4、MAP3、MAP3K1、MAP3K13、MAP3K14、MAP3K6、MAP3K7、MAPK1、MAPK3、M APKAP1、MAX、MCL1、MDC1、MDM2、MDM4、MED12、MEF2B、MEF2C、MEK1、ME N1、MERTK、MET、MGA、MIB1、MITF、MKI67、MKNK1、MLH1、MLLT3、MPL、MRE 11A、MRE11A、MSH2、MSH3、MSH6、MSI1、MSI2、MST1、MST1R、MTAP、MTOR、MUTYH、MYC、MYCL、MYCL(MYC) L1)、MYCL(MYCL1)、MYCL1、MYCN、MYD88、MYO18A、MYOD1、NBN、NCOA 3、NCOR1、NCOR2、NCSTN、NEGR1、NF1、NF2、NFE2L2、NFKBIA、NKX2-1 、NKX3-1、NOD1、NOTCH1、NOTCH2、NOTCH3、NOTCH4、NPM1、NRAS、NRG 1、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、<h2 style=";text-align:left;direction:ltr">SPTA1、SRC、SRSF2、STAG2、STAT3、STAT4、STAT5A、STAT5B、STAT6、STK11、ST K19、STK40、SUFU、SUZ12、SYK、TAF1、TAP1、TAP2、TBL1XR1、TBX3、TCEB1、TCF 3, TCF3(E2A), TCF7L2, TCL1A(TCL1), TEK, TERC, TERT, TERT, TET1, TET2, TFRC, TGFBR1, TGFBR2, TIPARP, TLL2, TMEM127, TMEM30A, TMPRSS2, TMS B4XP8(TMSL3)、TNFAIP3、TNFRSF11A、TNFRSF14、TNFRSF17、TOP1、TOP2A、TP 53、TP53BP1、TP63、TRAF2、TRAF3、TRAF5、TRAF7、TSC1、TSC2、TSHR、TUSC3、T YK2、TYRO3、U2AF1、U2AF2、UPF1、VEGFA、VHL、VTCN1、WDR90、WHSC1、WHSC1(M) MSETまたはNSD2)、WHSC1L1、WISP3、WT1、WWTR1、XBP1、XIAP、XPO1、XRCC2、YAP1、 YES1、YY1AP1、ZBTB2、ZFHX3、ZMYM3、ZNF217、ZNF24(ZSCAN3)、ZNF703、ZRSR 2、0082、SEPT9、81RC2、81RC3、81RC5、8AI3、8CL10、8CL118、8CL11A、8CL2、8 CL2L1、8CL2L2、8CL3、8CL6、8CL9、8CR、8LM、8LNK、8MPR1A、8RD3、8TK、8U818 、A8L2、ACVR2A、ADAMTS2、AFF1、AFF3、AKAP9、ARNT、ATF1、AURK8、AURKC、CASC S、CDH11、CDH2、CDH20、CDH5、CMPK1、COL1A1、CRBN、CREB1、CRTC1、CSMD3、CY P2C19、CYP2D6、DCC、DDIT3、DEK、DPYD、DST、EP400、EXT1、EXT2、FAM123B、FA NCJ、FLl1、FN1、FOX01、FOX03、FOXP4、FZR1、G6PD、GDNF、GRM8、HCAR1、HFN1A 、HIF1A、HLF、HOOK3、HSP90A81、ICK、IGF2R、IKBKB、IL2、IL21R、IL6ST、ING4、ITGA10, ITGA9, ITGB2, ITGB3, KAT6A, KAT6B, KLF6, KOR, LCK, LIFR, LPHN3, LPP, LRP18, LTF, M8D1, MAF8, MAGEA1, MAGl1, MAML2, MAPK8, MARK1, MARK4, MLL, MLL2, MLL3, MLLT10, MMP2, MN1, MTC, MTOT, , MTR, MTRR, MUC1, MY8, MYH11, MYH9, NCOA1, NCOA2, NCOA4, NFK81, NFK82, NIN, NLRP1, NUMA1, NUP214, P8RM1, P8X1, PAX?, PAX3, PAX8, PAXS, PDE4DIP, P DGF8, PER1, PGAP3, PHOX28, PIK3C28, PKHD1, PLAG1, PLCG1, PLEKHGS, PML, POU5F1, PSIP1, PTGS2, RADSO, RALGDS, RHOH, RNASEL, RNF2, RNF213, RPS6KA2 , RRM1, SAMD9, SBDS, SMUG1, SOHO, SOX11, SSX1, STK36, SYNE1, T8X22, TAF1L, TAL1, TCF12, TCF7L1, TFE3, TGF8R2, TGM7, TH8S1, TIMP3, TLR4, TLX1, TNK2, TPR, TRIM24, TRIM33, TRIP11, TRRAP, U8R5, UGT1A1, USP9X, WAS, WRN, XP01, XPA, XPC, ZNF384, ZNF521, and any combination thereof.

[0175] These are ABL1, 12B, ABL2, ACTB, ACVR1, and ACVR 1B, AGO2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, AMER1(FAM12). 3B, WTX, AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1 ARHGAP26(GRAF) ARID1A ARID1B ARID2 ARID5B ARv7 ASMTL ASX L1, ASXL2, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXIN2, AXL, B2M, BAB AM1, BAP1, BARD1, BBC3, BCL10, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2 L2, BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRC A2, BRD4, BRIP1, BRIP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf 30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD11, CARM1, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCN E1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC42, CDC73, CDH1, CDK12, CD K4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBPA, CENPA, CHD 2 CHD4, CHEK1, CHEK2, CIC, CIITA, CKS1B, CPS1, CREBBP, CRKL, CRLF2, CSDE1, CSF1R, CSF3R, CTCF, CLTA-4, CTNN B1, CTNNA1, CTNNB1, CUL3, CUL4A, CUX1, CXCR4, CYLD, CYP17A1, CYSLTR2, DAXX, DCUN1D1, DDR1, DDR2, DDX 3X, 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-ALK, E P300, EPAS1, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC1, ERCC2 ERCC3, ERCC4, ERCC5, ERF, ERG, ERRFI1, ESR1, ETS1, ETV1, ETV4, ETV5, ETV6, EWSR1 EXOSC6, EZH1, EZH2, FAF1, FAM175A, FAM46C, FAM58A, FANCA, FANCC, FANCD2, FANCE, FANCF, F ANCG, FANCI, FANCL, FAS, FAS(TNFRSF6), FAT1, FBXO11, FBXO31, FBXW7, FGF1, FGF10, FGF12. FGF14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGFR1, FGFR2, FGFR3 FGFR4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH1, FOXA1, FOXL2, FOXO1, FOXO3, FOXP1 、FRS2、FUBP1、FYN、GABRA6、GADD45B、GATA1、GATA2、GATA3、GATA4、GATA6、GEN1、GID4(C17orf 39) GID4(C17orf39), GLI1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRIN2A, GRM3, GSK3B, GTS E1, H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, HER-2 / NEU;ERBB2, HGF, HIST1H1C, HIST1H1D, HIST1H1E, and HIST 1H2AC、HIST1H2AG、HIST1H2AL、HIST1H2AM、HIST1H2BC、HIST1H2BD、HIST1H2BJ、HIST1H2BK、HIST1H2BO、HIST1H3A、HI ST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HIST2H3C, HIST2H3D<h2 style=";text-align:left;direction:ltr">HIST3H3, HLA-A, HLA-B, HNF1A, HOXB13, HRAS, HSD3B1, HSP90AA1, ICK, ICOSLG, ID3, IDH1, IDH2, IFNGR1, IGF1, IGF1R, IGF2, IKBKE, IKZF1, IKZF2, IKZF3, IL 10、IL7R、INHA、INHBA、INPP4A、INPP4B、INPP5D(SHIP)、INPPL1、INSR、IRF1、I RF2、IRF4、IRF8、IRS1、IRS2、JAK1、JAK2、JAK3、JARID2、JUN、K14、KAT6A(MYST) 3) KAT6A(MYST3) KDM2B KDM4C KDM5A KDM5C KDM6A KDR KEAP1 KEL KIF5B KIT KLF4 KLHL6 KMT2A KMT2A(MLL) KMT2B KMT2C KM T2C(MLL3)、KMT2D、KMT2D(MLL2)、KNSTRN、KRAS、LAMP1、LATS1、LATS 2、LEF1、LMO1、LRP1B、LRRK2、LTK、LYN、LZTR1、MAF、MAFB、MAGED1、MA GI2、MALT1、MAP2K1、MAP2K1(MEK1)、MAP2K2、MAP2K2(MEK2)、MAP2K4、MAP3、MAP3K1、MAP3K13、MAP3K14、MAP3K6、MAP3K7、MAPK1、MAPK3、M APKAP1、MAX、MCL1、MDC1、MDM2、MDM4、MED12、MEF2B、MEF2C、MEK1、ME N1、MERTK、MET、MGA、MIB1、MITF、MKI67、MKNK1、MLH1、MLLT3、MPL、MRE 11A、MRE11A、MSH2、MSH3、MSH6、MSI1、MSI2、MST1、MST1R、MTAP、MTOR、MUTYH、MYC、MYCL、MYCL(MYC) L1)、MYCL(MYCL1)、MYCL1、MYCN、MYD88、MYO18A、MYOD1、NBN、NCOA3、NCOR1、NCOR2、NCSTN、NEGR1、NF1、NF2、NFE2L2、NFKBIA、NKX 2-1、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、<h2 style=";text-align:left;direction:ltr">SPRED1、SPTA1、SRC、SRSF2、STAG2、STAT3、STAT4、STAT5A、STAT5B、STAT6、S TK11、STK19、STK40、SUFU、SUZ12、SYK、TAF1、TAP1、TAP2、TBL1XR1、TBX3、TC EB1, TCF3, TCF3(E2A), TCF7L2, TCL1A(TCL1), TEK, TERC, TERT, TERT, TET1, TET2, TFRC, TGFBR1, TGFBR2, TIPARP, TLL2, TMEM127, TMEM30A, TMPRS S2, TMSB4XP8(TMSL3), TNFAIP3, TNFRSF11A, TNFRSF14, TNFRSF17, TOP1, TOP2A, TP53, TP53BP1, TP63, TRAF2, TRAF3, TRAF5, TRAF7, TSC1, TSC2, TSHR, T USC3、TYK2、TYRO3、U2AF1、U2AF2、UPF1、VEGFA、VHL、VTCN1、WDR90、WHSC1、W HSC1(MMSETまたはNSD2)、WHSC1L1、WISP3、WT1、WWTR1、XBP1、XIAP、XPO1、XRCC2 、YAP1、YES1、YY1AP1、ZBTB2、ZFHX3、ZMYM3、ZNF217、ZNF24(ZSCAN3)、ZNF70 3、ZRSR2、0082、SEPT9、81RC2、81RC3、81RC5、8AI3、8CL10、8CL118、8CL11A、 8CL2、8CL2L1、8CL2L2、8CL3、8CL6、8CL9、8CR、8LM、8LNK、8MPR1A、8RD3、8TK 、8U818、A8L2、ACVR2A、ADAMTS2、AFF1、AFF3、AKAP9、ARNT、ATF1、AURK8、AURK C、CASCS、CDH11、CDH2、CDH20、CDH5、CMPK1、COL1A1、CRBN、CREB1、CRTC1、CS MD3、CYP2C19、CYP2D6、DCC、DDIT3、DEK、DPYD、DST、EP400、EXT1、EXT2、FAM12 3B、FANCJ、FLl1、FN1、FOX01、FOX03、FOXP4、FZR1、G6PD、GDNF、GRM8、HCAR1、 HFN1A、HIF1A、HLF、HOOK3、HSP90A81、ICK、IGF2R、IKBKB、IL2、IL21R、IL6ST、ING4, ITGA10, ITGA9, ITGB2, ITGB3, KAT6A, KAT6B, KLF6, KOR, LCK, LIFR, LPHN3, LPP, LRP18, LTF, M8D1, MAF8, MAGEA1, MAGl1, MAML2, MAPK8, MARK1, MARK4, MLL, MLL2, MLL3, MLLT10, MMP2, MN1, MT, C, MTOT, MTR, MTRR, MUC1, MY8, MYH11, MYH9, NCOA1, NCOA2, NCOA4, NFK81, NFK82, NIN, NLRP1, NUMA1, NUP214, P8RM1, P8 X1, PAX?, PAX3, PAX8, PAXS, PDE4DIP, PDGF8, PER1, PGAP3, PHOX28, PIK3C28, PKHD1, PLAG1, PLCG1, PLEKHGS, PML, POU5F 1, PSIP1, PTGS2, RADSO, RALGDS, RHOH, RNASEL, RNF2, RNF213, RPS6KA2, RRM1, SAMD9, SBDS, SMUG1, SOHO, SOX11, SSX1, S TK36, SYNE1, T8X22, TAF1L, TAL1, TCF12, TCF7L1, TFE3, TGF8R2, TGM7, TH8S1, TIMP3, TLR4, TLX1, TNK2, TPR, TRIM24, TR 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, 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 selected from the group consisting of IM33, TRIP11, TRRAP, U8R5, UGT1A1, USP9X, WAS, WRN, XP01, XPA, XPC, ZNF384, ZNF521, and any combination thereof.

[0176] In other embodiments, the genomic profile comprises one or more genes selected from the genes listed in Tables 2-15.

[0177] II.B.2. TMB status In some embodiments, the TMB status based on genomic profiling is highly correlated with the TMB status based on whole exome or whole genome sequencing.The evidence provided herein shows 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 efficient means of measuring TMB status without compromising the prognostic quality of TMB status.

[0178] TMB can be measured using tissue biopsy samples, or alternatively, circulating tumor DNA (ctDNA), cfDNA (cell-free DNA), and / or liquid biopsy samples. ctDNA can be used to measure TMB status by whole-exome or whole-genome sequencing or genomic profiling using available methods, for example, GRAIL, Inc.

[0179] In certain embodiments, a subject may be identified as suitable for the anti-PD-1 therapy disclosed herein 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 certain embodiments, a 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 370, at least 380, 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 least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least In some embodiments, the marker has 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 any integer score between 200 and 300 or higher. In other embodiments, high TMB has any integer score between 210 and 290 or higher. In other embodiments, high TMB has any integer score between 220 and 280 or higher. In other embodiments, high TMB has any integer score between 230 and 270 or higher. In other embodiments, high TMB has any integer score between 235 and 265 or higher.

[0180] 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 control TMB value. In some embodiments, the subject's TMB status is within the highest quantile of the control TMB value. In other embodiments, the subject's TMB status is within the upper tertile of the control TMB value.

[0181] 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 has a 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 has a 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 certain embodiments, a tumor has a high TMB status if it has at least about 100 mutations / tumor.

[0182] In certain embodiments, the tumor has been genetically, e.g., genomically sequenced by a TMB assay, e.g., FOUNDATIONONE® CDX TMA tumor has high TMB status if it has at least about 5 mutations per megabase (mutations / Mb), at least about 6 mutations / Mb, at least about 7 mutations / Mb, at least about 8 mutations / Mb, at least about 9 mutations / Mb, at least about 10 mutations / Mb, at least about 11 mutations / Mb, at least about 12 mutations / Mb, at least about 13 mutations / Mb, at least about 14 mutations / Mb, at least about 15 mutations / Mb, at least about 20 mutations / Mb, at least about 25 mutations / Mb, at least about 30 mutations / Mb, at least about 35 mutations / Mb, at least about 40 mutations / Mb, at least about 45 mutations / Mb, at least about 50 mutations / Mb, at least about 75 mutations / Mb, or at least about 100 mutations / Mb of the genome sequenced by the assay. In certain embodiments, a tumor has high TMB status if it has at least about 5 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 10 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 11 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 12 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 13 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 14 mutations / Mb. In certain embodiments, a tumor has high TMB status if it has at least about 15 mutations / Mb.

[0183] Because mutation numbers vary by tumor type and other methods (see Q4 and Q5), the values ​​associated with "TMB high" and "TMB low" may differ across tumor types.

[0184] II.C. Antibodies The present invention relates to a method of treating a human subject with cancer, comprising administering to the subject a PD-1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the subject is administered anti-PD-1 monotherapy, e.g., wherein the subject is not administered one or more additional anti-cancer agents. In some embodiments, the subject is administered combination therapy, e.g., wherein the subject is administered an anti-PD-1 antibody and one or more additional anti-cancer agents. In some embodiments, the subject is administered combination therapy comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody.

[0185] In other embodiments of the invention, the anti-PD-L1 antibody is replaced with an anti-PD-1 antibody. In certain embodiments, the method comprises administering to the subject an anti-PD-L1 antibody. In certain embodiments, the subject is administered an anti-PD-L1 monotherapy. In certain embodiments, the subject is administered a combination therapy comprising an anti-PD-L1 antibody and a second anti-cancer agent, such as an anti-CTLA-4 antibody.

[0186] II.C.1. Anti-PD-1 Antibodies Useful in the Invention 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 disclosed in U.S. Patent 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Patent 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) a specific binding affinity of 1×10 to human PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(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) stimulates an antigen-specific memory response; (i) stimulates an antibody response; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in certain embodiments, at least five, of the above characteristics.

[0187] Other anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patents 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 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, and WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825 and WO2017 / 133540, each of which is incorporated herein by reference in its entirety.

[0188] 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; also known as toripalimab; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; also known as tislelizumab; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847; 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), BCD-100 (Biocad; Kaplon et al., mAbs 10(2):183-203 (2018) and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825 and WO2017 / 133540).

[0189] In one embodiment, the anti-PD-1 antibody is nivolumab, 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 blocking downregulation of anti-tumor T cell function (U.S. Patent 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0190] In other embodiments, 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. Patents 8,354,509 and 8,900,587.

[0191] Anti-PD-1 antibodies useful in the compositions and methods of the present disclosure also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein, e.g., nivolumab, for binding to human PD-1 (see, e.g., U.S. Patents 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 the other cross-competing antibody to a particular epitope region. Because these cross-competing antibodies bind to the same epitope region of PD-1, they are expected to have functional properties very similar to those of the control antibody, e.g., nivolumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0192] In some embodiments, the antibody that cross-competes with the human PD-1 antibody, nivolumab, or binds to the same epitope region as human PD-1 is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0193] Anti-PD-1 antibodies useful in the compositions and methods of the invention also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0194] Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and prevent the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits similar functional properties of ligand binding inhibition and immune system upregulation as the whole antibody. In one embodiment, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0195] In one embodiment, the anti-PD-1 antibody is administered at a dose ranging from 0.1 mg / kg to 20.0 mg / kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks, e.g., 0.1 mg / kg to 10.0 mg / kg body weight once every 2, 3, or 4 weeks. In another embodiment, the anti-PD-1 antibody is administered at a dose of about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or 10 mg / kg body weight once every 2 weeks. In another embodiment, the anti-PD-1 antibody is administered at a dose of about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or 10 mg / kg body weight once every 3 weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 5 mg / kg body weight about once every three weeks. In another embodiment, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 3 mg / kg body weight about once every two weeks. In another embodiment, the anti-PD-1 antibody, e.g., pembrolizumab, is administered at a dose of about 2 mg / kg body weight about once every three weeks.

[0196] Anti-PD-1 antibodies useful in the present invention can be administered as a flat dose. In certain embodiments, the anti-PD-1 antibody is administered in a flat dose of about 100 to about 1000 mg, about 100 to about 900 mg, about 100 to about 800 mg, about 100 to about 700 mg, about 100 to about 600 mg, about 100 to about 500 mg, about 200 to about 1000 mg, about 200 to about 900 mg, about 200 to about 800 mg, about 200 to about 700 mg, about 200 to about 600 mg, about 200 to about 500 mg, about 200 to about 480 mg, or about 240 to about 480 mg. In certain embodiments, the anti-PD-1 antibody is administered at a flat dose of at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg, with a dosing interval of about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In other embodiments, the anti-PD-1 antibody is administered at a flat dose of about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, or about 200 mg to about 500 mg at dosing intervals of about 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0197] In one embodiment, the anti-PD-1 antibody is administered as a flat dose of about 200 mg about once every three weeks. In another embodiment, the anti-PD-1 antibody is administered as a flat dose of about 200 mg about once every two weeks. In another embodiment, the anti-PD-1 antibody is administered as a flat dose of about 240 mg about once every two weeks. In one embodiment, the anti-PD-1 antibody is administered as a flat dose of about 480 mg about once every four weeks.

[0198] In some embodiments, nivolumab is administered at a flat dose of about 240 mg once every two weeks. In some embodiments, nivolumab is administered at a flat dose of about 240 mg once every three weeks. In some embodiments, nivolumab is administered at a flat dose of about 360 mg once every three weeks. In some embodiments, nivolumab is administered at a flat dose of about 480 mg once every four weeks.

[0199] In some embodiments, pembrolizumab is administered in a flat dose of about 200 mg about once every two weeks. In some embodiments, pembrolizumab is administered in a flat dose of about 200 mg about once every three weeks. In some embodiments, pembrolizumab is administered in a flat dose of about 400 mg about once every four weeks.

[0200] In some embodiments, the PD-1 inhibitor is a small molecule. In some embodiments, the PD-1 inhibitor comprises Miramolecule. In some embodiments, the PD-1 inhibitor comprises a macrocyclic peptide. In some embodiments, the PD-1 inhibitor comprises BMS-986189. In some embodiments, the PD-1 inhibitor comprises an inhibitor disclosed in International Publication WO2014 / 151634, which is incorporated herein by reference in its entirety. In some embodiments, the PD-1 inhibitor comprises INCMGA00012 (Incyte Corporation). In some embodiments, the PD-1 inhibitor comprises a combination of an anti-PD-1 antibody and a PD-1 small molecule inhibitor disclosed herein.

[0201] II.C.2. Anti-PD-L1 Antibodies Useful in the Invention In certain embodiments, an anti-PD-L1 antibody is substituted for the anti-PD-1 antibody in any of the methods disclosed herein. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the invention. Examples of anti-PD-L1 antibodies useful in the compositions and methods of the invention include those disclosed in U.S. Patent 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent 9,580,507 has been shown to exhibit one or more of the following characteristics: (a) a 1x10 affinity to human PD-L1 as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D (b) bind to human PD-L1 in a mixed lymphocyte reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in certain embodiments, at least five, of the foregoing characteristics.

[0202] 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®; also known as MPDL3280A, RG7446; see U.S. Patent No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; Imfinzi) TM, also known as MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; also known as BAVENCIO®, 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), BGB-A333 (Beigene; Desai et al., JCO 36 (15suppl):TPS3113 (2018)) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

[0203] In one embodiment, the PD-L1 antibody is atezolizumab (Tecentriq®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0204] In one embodiment, the PD-L1 antibody is durvalumab (Imfinzi TM Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0205] In one embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0206] Anti-PD-L1 antibodies useful in the compositions and methods disclosed herein also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein, e.g., atezolizumab, durvalumab, and / or avelumab, for binding to human PD-L1. In some 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 they bind to the same epitope region of the antigen and sterically hinder the binding of the other cross-competing antibody to a particular epitope region. Because these cross-competing antibodies bind to the same epitope region of PD-L1, they are expected to have functional properties that are very similar to those of the control antibody, e.g., atezolizumab and / or avelumab. 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, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0207] In some embodiments, the human PD-L1 antibody, as atezolizumab, durvalumab, and / or avelumab, cross-competes with or binds to the same epitope region as human PD-L1 antibodies, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0208] Anti-PD-L1 antibodies useful in the compositions and methods of the invention also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0209] Anti-PD-L1 antibodies suitable for use in the disclosed compositions and methods are those that bind to PD-L1 with high specificity and affinity, block PD-1 binding, and prevent the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits similar functional properties of receptor binding inhibition and immune system upregulation as the whole antibody. 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.

[0210] The anti-PD-L1 antibody useful in the present invention can be any PD-L1 antibody that specifically binds to PD-L1, for example, an antibody that cross-competes with durvalumab, avelumab, or atezolizumab for binding to human PD-1, for example, an antibody that binds to the same epitope as durvalumab, avelumab, or atezolizumab. In certain embodiments, the anti-PD-L1 antibody is durvalumab. In other embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0211] In some embodiments, the anti-PD-L1 antibody is administered at a dose ranging from about 0.1 mg / kg to about 20.0 mg / kg of body weight once about every 2, 3, 4, 5, 6, 7, or 8 weeks, including about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg.

[0212] In one embodiment, the anti-PD-L1 antibody is administered at a dose of about 15 mg / kg body weight about once every three weeks. In another embodiment, the anti-PD-L1 antibody is administered at a dose of about 10 mg / kg body weight about once every two weeks.

[0213] In other embodiments, the anti-PD-L1 antibodies useful in the present invention are administered as a flat dose of about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 700 mg to about 1300 mg, about 800 mg to about 1200 mg, about 700 mg to about 900 mg, or about 1100 mg to about 1300 mg. In some embodiments, the anti-PD-L1 antibody is administered as a flat dose of at least about 240 mg, at least about 300 mg, at least about 320 mg, at least about 400 mg, at least about 480 mg, at least about 500 mg, at least about 560 mg, at least about 600 mg, at least about 640 mg, at least about 700 mg, at least about 720 mg, at least about 800 mg, at least about 840 mg, at least about 880 mg, at least about 900 mg, at least 960 mg, at least about 1000 mg, at least about 1040 mg, at least about 1100 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1300 mg, at least about 1360 mg, or at least about 1400 mg, administered at dosing intervals of about 1, 2, 3, or 4 weeks. In some embodiments, the anti-PD-L1 antibody is administered as a flat dose of about 1200 mg about once every 3 weeks. In another embodiment, the anti-PD-L1 antibody is administered as a flat dose of about 800 mg about once every two weeks. In another embodiment, the anti-PD-L1 antibody is administered as a flat dose of about 840 mg about once every two weeks.

[0214] In some embodiments, atezolizumab is administered in a flat dose of about 1200 mg about once every three weeks. In some embodiments, atezolizumab is administered in a flat dose of about 800 mg about once every two weeks. In some embodiments, atezolizumab is administered in a flat dose of about 840 mg about once every two weeks.

[0215] In one embodiment, avelumab is administered in a flat dose of about 800 mg once about every two weeks.

[0216] In some embodiments, durvalumab is administered at a dose of about 10 mg / kg about once every two weeks. In some embodiments, durvalumab is administered as a flat dose of about 800 mg / kg about once every two weeks. In some embodiments, durvalumab is administered as a flat dose of about 1200 mg / kg about once every three weeks.

[0217] In some embodiments, the PD-L1 inhibitor is a small molecule. In some embodiments, the PD-L1 inhibitor comprises a miramolecule. In some embodiments, the PD-L1 inhibitor comprises a macrocyclic peptide. In some embodiments, the PD-L1 inhibitor comprises BMS-986189.

[0218] In some embodiments, the PD-L1 inhibitor is a compound of formula (I): [ka] [In the formula, R 1 ~R 13 is the amino acid side chain, and R a ~R n is hydrogen, methyl, or forms a ring with the vicinal R group, and R 14 -C(O)NHR 15 where R 15 is a glycine residue substituted with hydrogen or optionally with additional glycine residues and / or tails that can improve pharmacokinetic properties. In some embodiments, the PD-L1 inhibitor includes a compound disclosed in International Publication WO2014 / 151634, which is incorporated herein by reference in its entirety. In some embodiments, the PD-L1 inhibitor comprises a compound disclosed in International Publication Nos. WO2016 / 039749, WO2016 / 149351, WO2016 / 077518, WO2016 / 100285, WO2016 / 100608, WO2016 / 126646, WO2016 / 057624, WO2017 / 151830, WO2017 / 176608, WO2018 / 085750, WO2018 / 237153, or WO2019 / 070643, each of which is incorporated herein by reference in its entirety.

[0219] In some embodiments, the PD-L1 inhibitor comprises a small molecule PD-L1 inhibitor disclosed in International Publication Nos. WO2015 / 034820, WO2015 / 160641, WO2018 / 044963, WO2017 / 066227, WO2018 / 009505, WO2018 / 183171, WO2018 / 118848, WO2019 / 147662, or WO2019 / 169123, each of which is incorporated herein by reference in its entirety.

[0220] In some embodiments, the PD-L1 inhibitor comprises a combination of an anti-PD-L1 antibody disclosed herein and a PD-L1 small molecule inhibitor disclosed herein.

[0221] II.C.3. Anti-CTLA-4 antibody Anti-CTLA-4 antibodies known in the art can be used in the compositions and methods of the present invention. The anti-CTLA-4 antibodies of the present invention bind to human CTLA-4 in a manner that disrupts the interaction between CTLA-4 and human B7 receptor. Because the interaction between CTLA-4 and B7 leads to the inactivation of CTLA-4 receptor-bearing T cells, disrupting the interaction effectively induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhances, or prolongs an immune response.

[0222] Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Patent 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patents 5,977,318, 6,051,227, 6,682,736, and 7,034,121 and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504, each of which is incorporated herein by reference in its entirety. The anti-CTLA-4 human monoclonal antibodies disclosed in U.S. Patent 6,984,720 have been shown to exhibit one or more of the following characteristics: a denaturing activity of at least about 10% as determined by Biacore analysis; 7 M -1 or about 10 9 M -1 or about 10 10 M -1 ~10 11 M -1 or higher equilibrium association constant (K a (b) specifically binds human CTLA-4 with a binding affinity reflected by at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 The dynamic association constant (k a );(c) at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 The dynamic dissociation constant (k d and (d) inhibit the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the above characteristics.

[0223] In certain embodiments, the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as Yervoy®, MDX-010, 10D1; see U.S. Patent 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO2016 / 196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO2000 / 037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In a specific embodiment, the anti-CTLA-4 antibody is ipilimumab.

[0224] In a specific embodiment, the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein. Ipilimumab is a fully human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0225] In a specific embodiment, the CTLA-4 antibody is tremelimumab.

[0226] In a specific embodiment, the CTLA-4 antibody is MK-1308.

[0227] In a specific embodiment, the CTLA-4 antibody is AGEN-1884.

[0228] Anti-CTLA-4 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies disclosed herein, e.g., ipilimumab and / or tremelimumab, for binding to human CTLA-4. In certain embodiments, the anti-CTLA-4 antibody binds to the same epitope as any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab and / or tremelimumab. The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically hinder the binding of the other cross-competing antibody to a specific epitope region. Because these cross-competing antibodies bind to the same epitope region of CTLA-4, they are expected to have functional properties very similar to those of the control antibody, e.g., ipilimumab and / or tremelimumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab and / or tremelimumab in standard CTLA-4 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO2013 / 173223).

[0229] In some embodiments, the human CTLA-4 antibody, as ipilimumab and / or tremelimumab, cross-competes with or binds to the same epitope region as human CTLA-4 antibody, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0230] Anti-CTLA-4 antibodies useful in the compositions and methods of the invention also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0231] Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are those that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and interfere with the interaction of CTLA-4 with the human B7 receptor. In any of the compositions or methods disclosed herein, an anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits similar functional properties of inhibiting the interaction of CTLA-4 with the human B7 receptor and upregulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and / or tremelimumab for binding to human CTLA-4.

[0232] In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from 0.1 mg / kg to 10.0 mg / kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1 mg / kg or 3 mg / kg body weight once every 3, 4, 5, or 6 weeks. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3 mg / kg body weight once every 2 weeks. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 1 mg / kg body weight once every 6 weeks.

[0233] In certain embodiments, the anti-CTLA-4 antibody, or antigen-binding portion thereof, is administered as a flat dose of about 10 to about 1,000 mg, about 10 to about 900 mg, about 10 to about 800 mg, about 10 to about 700 mg, about 10 to about 600 mg, about 10 to about 500 mg, about 100 mg to about 1,000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 480 mg, or about 240 mg to about 480 mg. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is at least about 60 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 160 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg In another embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose of at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg. In another embodiment, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose about once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0234] In some embodiments, ipilimumab is administered at a dose of about 3 mg / kg about once every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10 mg / kg about once every 3 weeks. In some embodiments, ipilimumab is administered at a dose of about 10 mg / kg about once every 12 weeks. In some embodiments, ipilimumab is administered four times.

[0235] II.C.1. Combination Therapy In certain embodiments, the anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody are administered in a therapeutically effective amount. In certain embodiments, the method comprises administering therapeutically effective amounts of an anti-PD-1 antibody and an anti-CTLA-4 antibody. In other embodiments, the method comprises administering therapeutically effective amounts of an anti-PD-L1 antibody and an anti-CTLA-4 antibody. Any of the anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies disclosed herein can be used in the method. In certain embodiments, the anti-PD-1 antibody comprises nivolumab. In certain embodiments, the anti-PD-1 antibody comprises pembrolizumab. In certain embodiments, the anti-PD-L1 antibody comprises atezolizumab. In certain embodiments, the anti-PD-L1 antibody comprises durvalumab. In certain embodiments, the anti-PD-L1 antibody comprises avelumab. In certain embodiments, the anti-CTLA-4 antibody comprises ipilimumab. In certain embodiments, the anti-CTLA-4 antibody comprises ipilimumab-tremelimumab.

[0236] In some embodiments, (a) the anti-PD-1 antibody or anti-PD-L1 antibody and (b) the anti-CTLA-4 antibody are each administered about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, or about once every 6 weeks. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered about once every 2 weeks, about once every 3 weeks, or about once every 4 weeks, and the anti-CTLA-4 antibody is administered about once every 6 weeks. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered on the same day as the anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered on a different day from the anti-CTLA-4 antibody.

[0237] In some embodiments, the anti-CTLA-4 antibody is administered at a dose ranging from about 0.1 mg / kg to about 20.0 mg / kg body weight approximately once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 0.1 mg / kg, about 0.3 mg / kg, about 0.6 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 3 mg / kg, about 6 mg / kg, about 9 mg / kg, about 10 mg / kg, about 12 mg / kg, about 15 mg / kg, about 18 mg / kg, or about 20 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg approximately once every 4 weeks. In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg approximately once every 6 weeks.

[0238] In certain embodiments, the anti-CTLA-4 antibody is administered in a flat dose. In certain embodiments, the anti-CTLA-4 antibody is administered in a flat dose ranging from at least about 40 mg to at least about 1600 mg. In certain embodiments, the anti-CTLA-4 antibody is administered in a flat dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg. In some embodiments, the CTLA-4 antibody is administered in a uniform dose of at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 320 mg, at least about 360 mg, at least about 400 mg, at least about 440 mg, at least about 480 mg, at least about 520 mg, at least about 560 mg, or at least about 600 mg. In some embodiments, the CTLA-4 antibody is administered in a uniform dose of at least about 640 mg, at least about 720 mg, at least about 800 mg, at least about 880 mg, at least about 960 mg, at least about 1040 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1360 mg, at least about 1440 mg, or at least about 1600 mg. In certain embodiments, the anti-CTLA-4 antibody is administered in a flat dose at least about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0239] In one embodiment, the anti-PD-1 antibody is administered at a dose of about 2 mg / kg about once every three weeks, and the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 3 mg / kg about once every two weeks, and the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 6 mg / kg about once every four weeks, and the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg about once every six weeks.

[0240] In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every three weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 240 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 480 mg about once every four weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks.

[0241] In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every three weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 240 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks. In one embodiment, the anti-PD-1 antibody is administered in a flat dose of about 480 mg about once every four weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks.

[0242] In one embodiment, the anti-PD-L1 antibody is administered at a dose of about 10 mg / kg about once every two weeks, and the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-L1 antibody is administered at a dose of about 15 mg / kg about once every three weeks, and the anti-CTLA-4 antibody is administered at a dose of about 1 mg / kg about once every six weeks.

[0243] In one embodiment, the anti-PD-L1 antibody is administered in a flat dose of about 800 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks. In one embodiment, the anti-PD-L1 antibody is administered in a flat dose of about 1200 mg about once every three weeks, and the anti-CTLA-4 antibody is administered in a dose of about 1 mg / kg about once every six weeks.

[0244] In one embodiment, the anti-PD-L1 antibody is administered in a flat dose of about 800 mg about once every two weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks. In one embodiment, the anti-PD-L1 antibody is administered in a flat dose of about 1200 mg about once every three weeks, and the anti-CTLA-4 antibody is administered in a flat dose of about 80 mg about once every six weeks.

[0245] In one embodiment, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 3 mg / kg and the anti-CTLA-4 antibody is administered on the same day at about 1 mg / kg for four doses, once every three weeks, followed by a flat dose of 240 mg about once every two weeks or 480 mg about once every four weeks. In one embodiment, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 1 mg / kg and the anti-CTLA-4 antibody is administered on the same day at about 3 mg / kg for four doses, followed by a flat dose of 240 mg about once every two weeks or 480 mg about once every four weeks.

[0246] II.C.1. Additional Anticancer Therapies In some embodiments of the present invention, the methods disclosed herein further comprise administering an anti-PD-1 antibody (or anti-PD-L1 antibody) and an additional anti-cancer therapy. In certain embodiments, the methods comprise administering an anti-PD-1 antibody (or anti-PD-L1 antibody), an anti-CTLA-4 antibody, and an additional anti-cancer therapy. The additional anti-cancer therapy can include any therapeutic target and / or any standard of care known in the art for treating a tumor in a subject, as disclosed herein. In certain embodiments, the additional anti-cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In certain embodiments, the additional anti-cancer therapy comprises chemotherapy, including any chemotherapy disclosed herein. In certain embodiments, the additional anti-cancer therapy comprises immunotherapy. In certain embodiments, the additional anticancer therapy comprises administration of an antibody or antigen-binding portion thereof that specifically binds to LAG-3, TIGIT, TIM3, NKG2a, OX40, ICOS, MICA, CD137, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof.

[0247] II.D. Tumor In some embodiments, the tumor is derived from a cancer selected from the group consisting of hepatocellular carcinoma, gastroesophageal carcinoma, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof.In some embodiments, the tumor is derived from hepatocellular carcinoma, wherein the tumor has a high inflammatory signature score.In some embodiments, the tumor is derived from hepatocellular carcinoma, wherein the tumor has a high inflammatory signature score, and the tumor has a TMB status of at least about 10 mutations per megabase of the tested gene.In some embodiments, the tumor is derived from gastroesophageal carcinoma, wherein the tumor has a high inflammatory signature score.In some embodiments, the tumor is derived from gastroesophageal carcinoma, wherein the tumor has a high inflammatory signature score, and the tumor has a TMB status of at least about 10 mutations per megabase of the tested gene.In some embodiments, the tumor is derived from melanoma, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is derived from melanoma, wherein the tumor has a high inflammatory signature score and the tumor has a TMB status of at least about 10 mutations per megabase of the genes tested.In some embodiments, the tumor is derived from bladder cancer, wherein the tumor has a high inflammatory signature score.In some embodiments, the tumor is derived from bladder cancer, wherein the tumor has a high inflammatory signature score and the tumor has a TMB status of at least about 10 mutations per megabase of the genes tested.In some embodiments, the tumor is derived from lung cancer, wherein the tumor has a high inflammatory signature score.In some embodiments, the tumor is derived from lung cancer, wherein the tumor has a high inflammatory signature score and the tumor has a TMB status of at least about 10 mutations per megabase of the genes tested.In some embodiments, the tumor is derived from kidney cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is derived from kidney cancer, wherein the tumor has a high inflammatory signature score, and the tumor has a TMB status of at least about 10 mutations per megabase of the tested gene.In some embodiments, the tumor is derived from head and neck cancer, wherein the tumor has a high inflammatory signature score.In some embodiments, tumor is derived from head and neck cancer, wherein tumor has high inflammatory signature score, and tumor has TMB status of at least about 10 mutations per megabase of tested genes.In some embodiments, tumor is derived from colon cancer, wherein tumor has high inflammatory signature score.In some embodiments, tumor is derived from colon cancer, wherein tumor has high inflammatory signature score, and tumor has TMB status of at least about 10 mutations per megabase of tested genes.

[0248] In some embodiments, the subject has undergone one, two, three, four, five, or more prior cancer treatments. In other embodiments, the subject is treatment naive. In some embodiments, the subject has progressed on another cancer treatment. In some embodiments, the prior cancer treatment comprises immunotherapy. In other embodiments, the prior cancer treatment comprises chemotherapy. In some embodiments, the tumor has recurred. In some embodiments, the tumor is metastatic. In other embodiments, the tumor is not metastatic. In some embodiments, the tumor has progressed locally.

[0249] In some embodiments, the subject has received prior therapy for treating the tumor, and the tumor is relapsed or refractory. In some embodiments, at least one prior therapy comprises standard therapy. In some embodiments, at least one prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, at least one prior therapy comprises chemotherapy. In some embodiments, the subject has received prior immuno-oncology (IO) therapy for treating the tumor, and the tumor is relapsed or refractory. In some embodiments, the subject has received more than one prior therapy for treating the tumor, and the subject is relapsed or refractory. In other embodiments, the subject is receiving anti-PD-1 or anti-PD-L1 antibody therapy.

[0250] In some embodiments, the preceding treatment comprises chemotherapy. In some embodiments, the chemotherapy comprises a platinum-based treatment. In some embodiments, the platinum-based treatment comprises a platinum-based anti-neoplastic agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In some embodiments, the platinum-based treatment comprises cisplatin. In certain embodiments, the platinum-based treatment comprises carboplatin.

[0251] In some embodiments, the at least one prior treatment is selected from the group consisting of a platinum-based doublet chemotherapy, a taxane (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab (Avastin®), erlotinib (Tarceva®), crizotinib (Xalkori®), cetuximab (Erbitux®), and any combination thereof. In some embodiments, the at least one prior treatment is selected from the group consisting of a platinum-based doublet chemotherapy.

[0252] In some embodiments, the subject has experienced disease progression after at least one prior treatment. In some embodiments, the subject has received at least two prior treatments, at least three prior treatments, at least four prior treatments, or at least five prior treatments. In some embodiments, the subject has received at least two prior treatments. In some embodiments, the subject has experienced disease progression after at least two prior treatments. In some embodiments, the at least two prior treatments include a first prior treatment and a second prior treatment, wherein the subject has experienced disease progression after the first prior treatment and / or the second prior treatment, wherein the first prior treatment includes surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof; and the second prior treatment includes surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, the first prior treatment includes platinum-based doublet chemotherapy, and the second prior treatment includes single-agent chemotherapy. In some embodiments, the single-agent chemotherapy includes docetaxel.

[0253] II.E. Pharmaceutical Compositions and Dosages Therapeutic agents of the present invention may comprise compositions, such as pharmaceutical compositions, containing antibodies and / or cytokines and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, carriers for antibody-containing compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion), while carriers for antibody- and / or cytokine-containing compositions are suitable for non-parenteral, e.g., oral, administration. In certain embodiments, subcutaneous injections are based on Halozyme Therapeutics' ENHANZE® drug delivery technology (see U.S. Patent No. 7,767,429, incorporated herein by reference in its entirety). ENHANZE® uses a co-formulation of an antibody and a recombinant human hyaluronidase enzyme (rHuPH20), which eliminates the traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously via an extracellular matrix (see U.S. Patent No. 7,767,429). Pharmaceutical compositions of the invention may contain one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Thus, in some embodiments, pharmaceutical compositions of the invention may further contain a recombinant human hyaluronidase enzyme, such as rHuPH20.

[0254] In some embodiments, the method comprises administering an anti-PD-1 antibody (or anti-PD-L1 antibody) and an anti-CTLA-4 antibody, wherein the anti-PD-1 antibody (or anti-PD-L1 antibody) is administered at a fixed dose with the anti-CTLA-4 antibody in a single composition. In some embodiments, the anti-PD-1 antibody is administered at a fixed dose with the anti-CTLA-4 antibody. In some embodiments, the anti-PD-L1 antibody is administered at a fixed dose with the anti-CTLA-4 antibody in a single composition. In some embodiments, the ratio of the anti-PD-1 antibody (or anti-PD-L1 antibody) to the anti-CTLA-4 antibody is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:130, about 1:140, about 1:150, about 1:160, about 1:170, about 1:180, about 1:190, about 1:200, about 1:210, about 1:220, about 1:230, about 1:240, about 1:250, about 1:260, about 1:270, about 1:280, about 1:290, about 1:300, about 1:310, about 1:320, about 1:330, about 1:340, about 1:350, about 1:360, about 1:370, about 1:380, about 1:390, about 1:410, about 1:420, about 1:430, about 1:440, about 1:450, about 1:460, about 1:470, about 1:480, about 1:500, about 1:510, about 1:520, about 1:530, about 1:540, about 1:550, about 1:560 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1 or about 2:1.

[0255] Although high-dose nivolumab monotherapy up to 10 mg / kg every 2 weeks has been achieved without reaching the maximum tolerated dose (MTD), the substantial toxicity reported in other trials of checkpoint inhibitors and antiangiogenic therapies (see, e.g., Johnson et al., 2013 ; Rini et al., 2011 ) supports the selection of nivolumab doses below 10 mg / kg.

[0256] Treatment is continued as long as clinical benefit is observed or until unacceptable toxicity or disease progression occurs. However, in certain embodiments, the administered dose of the anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody is significantly lower than the approved dose of the agent, i.e., a subtherapeutic dose. The anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody may be administered at a dose shown to produce maximal efficacy as monotherapy in clinical trials, for example, about 3 mg / kg of nivolumab administered once every 3 weeks (Topalian et al., 2012a; Topalian et al., 2012), or at a significantly lower dose, i.e., a subtherapeutic dose.

[0257] 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 non-human antibodies. Dosage and frequency can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are generally administered at relatively infrequent intervals over a long period of time. Some patients may receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until disease symptoms are reduced or terminated, and preferably until the patient shows partial or complete improvement of the disease. Thereafter, the patient can be administered a preventive regimen.

[0258] The actual dosage level of the active ingredient of the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is not excessively toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and administration method. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention 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, sex, weight, condition, general health, and medical history of the patient being treated, and equivalent factors well known in the medical field. The composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods well known in the art. As will be recognized by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0259] III. Kit Also within the scope of the present invention are kits containing (a) an anti-PD-1 antibody or an anti-PD-L1 antibody for therapeutic use. The kit will generally include a label indicating the intended use of the contents of the kit and instructions for use. The term label includes any writing or recorded material provided on or with the kit, or which otherwise accompanies the kit. Accordingly, the present invention provides kits for treating a subject with a tumor, comprising: (a) a single dose of an anti-PD-1 antibody in the range of 0.1-10 mg / kg body weight, or a single dose of an anti-PD-L1 antibody in the range of 0.1-20 mg / kg body weight; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. The present invention further provides a kit for treating a subject with a tumor, comprising: (a) a single dose of an anti-PD-1 antibody in the range of about 4 mg to about 500 mg, or a single dose of an anti-PD-L1 antibody in the range of about 4 mg to about 2000 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. In certain embodiments, the present invention provides a kit for treating a subject with a tumor, comprising: (a) a single dose of an anti-PD-1 antibody in the range of 200 mg to 800 mg, or a single dose of an anti-PD-L1 antibody in the range of 200 mg to 1800 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein.

[0260] In certain embodiments for treating a human patient, the kit comprises an anti-human PD-1 antibody disclosed herein, e.g., nivolumab or pembrolizumab. In certain embodiments for treating a human patient, the kit comprises an anti-human PD-L1 antibody disclosed herein, e.g., atezolizumab, durvalumab, or avelumab.

[0261] In certain embodiments, the kit further comprises an anti-CTLA-4 antibody. In certain embodiments for treating a human patient, the kit comprises an anti-human CTLA-4 antibody disclosed herein, e.g., ipilimumab, tremelimumab, MK-1308, or AGEN-1884.

[0262] In some embodiments, the kit further comprises an inflammatory gene panel assay disclosed herein. In some embodiments, the kit further comprises instructions for administering an anti-PD-1 antibody or an anti-PD-L1 antibody to a subject identified by the methods disclosed herein as having a high inflammatory signature score. In other embodiments, the kit further comprises an anti-CTLA-4 antibody and instructions for administering (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody to a subject identified by the methods disclosed herein as having a high inflammatory signature score.

[0263] In some embodiments, the kit further comprises a comprehensive genomic profiling assay as disclosed herein. In some embodiments, the kit comprises FOUNDATIONONE® CDX TM and a genomic profiling assay. In certain embodiments, the kit further comprises instructions for administering an anti-PD-1 antibody or an anti-PD-L1 antibody to a subject identified by the methods disclosed herein as having a high TMB status, e.g., a TMB status of at least about 10 mutations / Mb of the sequenced genome. In other embodiments, the kit further comprises an anti-CTLA-4 antibody and instructions for administering (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody to a subject identified by the methods disclosed herein as having a high TMB status, e.g., a TMB status of at least about 10 mutations / Mb of the sequenced genome.

[0264] In one embodiment, the kit comprises (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, (b) an inflammatory gene panel assay disclosed herein, (c) a comprehensive genomic profiling assay disclosed herein, and (d) instructions for administering the anti-PD-1 antibody or anti-PD-L1 antibody to a subject identified by a method disclosed herein as having (a) a high inflammatory signature score and (b) a high TMB status, e.g., a TMB status of at least about 10 mutations / Mb of the sequenced genome. In other embodiments, the kit comprises (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody, (c) an inflammatory gene panel assay disclosed herein, (d) a comprehensive genomic profiling assay disclosed herein, and (e) instructions for administering (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody to a subject identified by a method disclosed herein as having (a) a high inflammatory signature score and (b) a high TMB status, e.g., a TMB status of at least about 10 mutations / Mb of the sequenced genome.

[0265] All of the references cited above and all references cited herein are incorporated by reference in their entirety.

[0266] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0267] Example 1: Evaluation of inflammatory biomarkers correlated with clinical outcome in nivolumab-treated patients with advanced hepatocellular carcinoma Liver cancer is the fourth leading cause of cancer-related deaths worldwide, with the majority of liver cancers being hepatocellular carcinoma (HCC). There are few effective treatment options for patients with advanced HCC, and agents that can achieve robust and durable responses remain an unmet need in HCC. Clinical trials of approved first-line and second-line targeted therapies report median overall survival ranging from 10.7 to 13.6 months and 10.2 to 10.6 months, respectively (see Abou-Alfa et al., N Engl J Med. 379(1):54-63 (2018); Bruix et al., Lancet 389(10064):56-66 (2017); Llovet et al., N Engl J Med. 359(4):378-90 (2008); and Kudo et al., Lancet. 391(10126):1163-73 (2018)). Nivolumab ("NIVO") binds to the PD-1 receptor, which is primarily expressed on activated T cells, thereby preventing the binding of PD-L1 and PD-L2 ligands expressed on tumor cells. Nivolumab has demonstrated durable responses, manageable safety, and long-term survival in patients with advanced HCC, regardless of etiology and whether they had prior sorafenib (SOR) therapy, in clinical trial NCT01658878 (see El-Khoueiry et al., Lancet. 389:2492-2502 (2017)). NIVO is approved for SOR-experienced patients with HCC in many countries, including the United States, based on the results of clinical trial NCT01658878.

[0268] This example relates to findings from an exploratory biomarker analysis of nivolumab-treated patients with advanced HCC from clinical trial NCT01658878.

[0269] Test Design

[0270] These data pertain to cohorts 1 and 2 of clinical trial NCT01658878, which combined included a total of 262 subjects (Figure 1). Cohort 1 included 80 SOR-naive subjects, and cohort 2 included 182 SOR-experienced subjects. Eleven subjects in cohort 1 and 37 subjects in cohort 2 received 0.1–10 mg / kg nivolumab in the dose-escalation analysis portion. Sixty-nine subjects in cohort 1 and 145 subjects in cohort 2 received 3 mg / kg nivolumab in the dose-expansion analysis portion. After initial treatment, 154 subjects in cohort 2 (9 subjects from the dose-escalation study and 145 subjects from the dose-expansion study) received maintenance nivolumab at 3 mg / kg.

[0271] The primary endpoints of clinical trial NCT01658878 were safety and tolerability (dose escalation) and objective response rate (ORR; dose expansion). Secondary endpoints included ORR (dose escalation), disease control rate, time to response, duration of response, and overall survival. Exploratory endpoints included biomarker assessments as described herein.

[0272] Data generated from clinical trial NCT01658878, including an ORR of 14.3% and a duration of response (DOR) of at least 12 months in 50% of subjects, contributed to the USFDA approval of nivolumab for the treatment of SOR-experienced patients with HCC.

[0273] Eligible subjects were those with (i) histologically confirmed advanced HCC not amenable to curative resection; (ii) Child-Pugh score ≤7 (gradual increase) or ≤6 (enlarging); (iii) progression on or intolerance to or refusal of at least one prior line of systemic therapy; (iv) AST and ALT ≤5x upper limit and bilirubin ≤3mg / dL; (v) for HBV-infected patients, viral load <100 IU / mL and concurrent effective antiviral therapy; and (vi) for HCV-infected patients, active or resolved infection as evidenced by detectable HCV RNA or antibodies. Subjects with a history of hepatic encephalopathy, previous or current clinically significant ascites, or active HBV and HCV coinfection were excluded.

[0274] Pre-treatment tumor samples (fresh or archived) were obtained from patients in the titration and expansion phase receiving 3 mg / kg nivolumab (saved for IHC) or 0.1-10 mg / kg nivolumab (saved for RNA sequencing).

[0275] Biomarker evaluation

[0276] Samples were analyzed using (i) IHC to assess PD-L1, PD-1, T cell markers (CD3, CD4, CD8, FOXP3), and macrophage markers (CD68, CD163); and (ii) RNA sequencing to assess tumor inflammatory signatures. Biomarkers were assessed by an anonymous independent review committee (per RECIST v1.1) for association with clinical outcomes, including BOR, over overall survival. Analyses were performed using a standard Limma and Cox regression framework.

[0277] Biomarker analysis

[0278] PD-L1

[0279] In the overall population, 195 subjects had evaluable PD-L1 data (SOR-naive, n=58; SOR-experienced, n=137; Table 16). Clinically meaningful responses were observed in all subjects, including those with PD-L1 <1%, with six subjects having complete responses. In the overall population, numerically higher objective response rates were observed in subjects with PD-L1 ≥1% versus PD-L1 <1%, with overlapping 95% confidence intervals. The SOR-experienced population had a similar ORR to the overall population.

[0280] Deep responses were observed in the overall population, regardless of PD-L1 status (Figures 2A-2B). Tumor cell PD-L1 expression in at least 1% of tumor cells significantly correlated with overall survival (Figure 2C; P = 0.032). In general, positive PD-L1 expression in at least 1% of tumor cells correlated with longer overall survival in subjects who experienced SOR, although this difference was not statistically significant in any case (Figure 2D). [Table 16]

[0281] No significant differences in tumor PD-L1 expression were observed when stratified by region (Asian vs. non-Asian; data not shown).

[0282] T cell markers

[0283] The expression profiles of T cell markers CD3, CD8, CD4, and FOX-3 were analyzed in tumor samples obtained from subjects before nivolumab administration. CD3-positive cell frequency was observed to correlate with response (CR / PR compared with SD; P = 0.03; Figure 3A). No significant correlation was observed between CD4-, CD8-, or FOXP3-positive cell frequency and response (Figures 3B–3D). In the tumor microenvironment, CD3-positive cell frequency was higher than that of other T cell markers evaluated (data not shown). No significant differences in T cell marker distribution were observed when stratified by viral infection (HBV- or HCV-infected vs. uninfected; data not shown) or region (Asian vs. non-Asian; data not shown).

[0284] Tumor inflammation, as measured by CD3 or CD8 expression, had a non-significant trend toward improved overall survival (Figures 4A-4B; P=0.08), as did, to a lesser extent, CD4 or FOXP3 expression (Figures 4C-4D).

[0285] Macrophage markers

[0286] The expression profiles of macrophage markers CD68 and CD163 were analyzed in tumor samples obtained from subjects before nivolumab administration. No correlation was observed between CD68 and CD163 expression and clinical outcome (Figures 5A-5B and 6A-6B). Furthermore, no significant differences in macrophage marker distribution were observed when stratified by viral infection (HBV- or HCV-infected or uninfected; data not shown) or region (Asian vs. non-Asian; data not shown).

[0287] Tumor immune gene signature

[0288] For a subset of subjects with available data (n=37), gene expression profiling was performed using RNA sequencing to assess tumor immune infiltration and inflammatory signatures (Table 17). Several inflammatory signatures, such as the disclosed 4-gene inflammatory signature (including CD274 (PD-L1), CD8A, LAG3, and STAT1), the Gajewski 13-gene inflammatory signature, the Merck 6-gene interferon gamma signature, the NanoString interferon gamma biology signature, and the NanoString T cell exhaustion signature, were significantly correlated with response and improved overall survival (Table 17). Notably, the mean 4-gene inflammatory signature score described herein was observed to be significantly higher in patients experiencing a partial response compared with stable disease (p=0.05; Figure 7A). Furthermore, the mean median 4-gene inflammatory score significantly correlated with improved overall survival (p=0.01; Figure 7B). [Table 17] 1. Danilova L, et al. Proc Natl Acad Sci. 2016;113:E7769-E7777; 2. Spranger S, et al. Nature. 2015;523:231-235; 3. Ayers M, et al. J Clin Invest. 2017;127:2930-2940.

[0289] No significant differences in the 4-gene inflammatory signature scores were found when stratified by viral infection (HBV- or HCV-infected or uninfected; data not shown) or region (Asian vs. non-Asian; data not shown).

[0290] In clinical trial NCT01658878 cohorts 1 and 2, durable responses were observed in both SOR-naive and SOR-experienced patients, regardless of tumor cell PD-L1 status. In this retrospective analysis of pretreatment tumor samples from patients with advanced HCC, tumor cell PD-L1 expression correlated with OS; however, this correlation was not significant in SOR-experienced patients. CD3 + T cell frequency correlated with response to nivolumab, and there was a trend toward improved survival and CD3 and CD8 positivity. High scores on several inflammatory signatures, including the 4-gene inflammatory signature, correlated with response and improved overall survival.

[0291] Example 2: Correlation of PD-L1 combined positive score and immune gene signature with efficacy of nivolumab ± ipilimumab in patients with metastatic gastroesophageal cancer Combination therapy including nivolumab (NIVO) and ipilimumab (IPI) has demonstrated therapeutically meaningful antitumor activity and a manageable safety profile in patients with chemotherapy-refractory gastroesophageal cancer in a phase 1 / 2 trial (NCT01928394; Janjigian YY, et al. J Clin Oncol. 2018;36:2836-2844). In the current exploratory analysis from clinical trial NCT01928394, the expression of select immune gene signatures was evaluated to determine whether they correlated with the efficacy of nivolumab monotherapy in combination with ipilimumab.

[0292] Test Design

[0293] Subjects with locally advanced or metastatic gastric / esophageal / GEJ cancer who were refractory to ≥1 prior chemotherapy were randomly assigned to one of the following: nivolumab 3 mg / kg intravenously (NIVO3) every 2 weeks (n = 59); nivolumab 1 mg / kg + ipilimumab 3 mg / kg (NIVO1 + IPI3) every 3 weeks for 4 cycles (n = 49); or nivolumab 3 mg / kg + ipilimumab 1 mg / kg (NIVO3 + IPI1) every 3 weeks for 4 cycles (n = 52) (Figure 8). All combination regimens were followed by NIVO3 every 2 weeks until disease progression or unacceptable adverse events (AEs).

[0294] The primary endpoint was the objective response rate (ORR), defined as the best complete or partial response divided by the number of treated patients according to RECIST version 1.1. Secondary endpoints included overall survival (OS), progression-free survival (PFS), time to response, duration of response (DOR), and safety. Tumor response was assessed using imaging every 6 weeks until 24 weeks, then every 12 weeks until disease progression or treatment discontinuation. Survival was monitored continuously while patients were receiving treatment and every 3 months after treatment discontinuation. Exploratory endpoints included correlation of tumor PD-L1 expression with efficacy and safety.

[0295] Key eligibility criteria for the esophagogastric cancer cohort included a diagnosis of locally advanced or metastatic gastric, esophageal, or GEJ adenocarcinoma that had progressed on or was intolerant to at least one chemotherapy regimen; measurable disease as assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.118; Eastern Cooperative Oncology Group performance status of 0 or 1; and adequate organ function. Patients with human epidermal growth factor receptor 2-positive tumors were eligible if they had received prior treatment with trastuzumab. Key exclusion criteria included suspected autoimmune disease; hepatitis B virus or human immunodeficiency virus infection; conditions requiring corticosteroids or other immunosuppressive medications; and prior immune checkpoint inhibitor treatment.

[0296] Biomarker analysis

[0297] PD-L1 expression

[0298] Biological samples were collected from subjects prior to immunotherapy, and a subset of subject samples was available for PD-L1 expression analysis (Table 18). [Table 18] a Three patients in the dose-escalation phase of NIVO1+IPI1 were also included in this analysis. CR, complete response; ECOG, Eastern Cooperative Oncology Group; NE, not evaluable; PD, progressive disease; PR, partial response; SD, stable disease.

[0299] PD-L1 immunohistochemistry (IHC) was used to assess PD-L1 expression on tumors and tumor-associated immune cells. As used in this example, tumor PD-L1 expression represents the percentage of viable tumor cells that exhibit partial or complete membrane PD-L1 staining. Tumor PD-L1 expression is calculated according to Formula II:

number

[0300] The combined positive score (CPS) incorporates both tumor and tumor-associated immune cell PD-L1 expression. The CPS is calculated according to Formula III.

number

[0301] PD-L1 expression by CPS (Figure 9B) was observed to correlate better with response than PD-L1 expression on tumor cells (Figure 9A). PD-L1 expression by CPS had a higher prevalence than PD-L1 expression on tumor cells, regardless of the cutoff, and correlated better with response at higher cutoffs (Table 19). At higher cutoffs, PD-L1 expression by CPS showed a stronger correlation with overall survival than tumor PD-L1 expression (Figures 10A-10F). [Table 19] a PD-L1 expression on tumor cells; b PD-L1 expression by CPS; c For tumor PD-L1 expression, cutoffs are shown as percentages. For CPS, cutoffs are shown as scores. NA, not applicable; ORR, objective response rate.

[0302] In the nivolumab 1 mg / kg + ipilimumab 3 mg / kg treatment arm, PD-L1 expression by PS had a higher prevalence than PD-L1 expression on tumor cells, regardless of the cutoff, and correlated well with response at higher cutoffs. Furthermore, PD-L1 expression by CPS showed a strong correlation with overall survival at higher cutoffs (Figures 11A-11D). This correlation was more pronounced in patients treated with nivolumab 1 mg / kg + ipilimumab 3 mg / kg, consistent with patients receiving all regimens combined (see Figures 10D-10F). [Table 20] a PD-L1 expression on tumor cells; b PD-L1 expression by CPS; c For tumor PD-L1 expression, cutoffs are shown as percentages. For CPS, cutoffs are shown as scores; d Only one patient had tumor PD-L1 ≥ 5% and ≥ 10%.

[0303] Gene profiling analysis

[0304] Biological samples were collected from subjects prior to immunotherapy, and a subset of subject samples was available for gene expression profiling analysis (Table 21). [Table 21]

[0305] Various gene expression signatures were analyzed in the available samples (Table 22). All gene expression signatures showed a tendency to correlate with response (Table 22). Notably, significant correlations were observed with the disclosed 4-gene inflammatory signature (including CD274 (PD-L1), CD8A, LAG3, and STAT1; Figure 12D), CD8 T cell signature (Figure 12A), PD-L1 transcript (Figure 12B), and Ribas 10-gene interferon gamma signature (Figure 12C), with the 4-gene inflammatory signature showing the strongest correlation with response (patients with CR / PR, n=4; Table 22). Despite the small number of responding patients in this analysis (n=4), good discrimination was demonstrated with an AUC (90% [95% CI, 77-100]). [Table 22] a P-values ​​and false discovery rates are from studies using nine pre-specified signatures and genes. False discovery rate-adjusted P-values. bAn estimate of the false discovery rate for a given number of tests / hypotheses. c Given the small sample size, the exploratory P-values ​​are intended to indicate the relative performance of various signatures in terms of correlation with the response. 1. Siemers NO, et al. PLoS One. 2017;12:e0179726; 2. Spranger S, et al. Nature. 2015;523:231-235; 3. Ayers M, et al. J Clin Invest. 2017;127:2930-2940.

[0306] In this exploratory analysis, we observed that inflammatory gene signature expression correlated with response to nivolumab monotherapy and combination therapy with ipilimumab, indicating the existence of actionable biological factors that can be targeted by cancer immunotherapeutics. Example 3: Genomic analysis and immunotherapy in advanced melanoma

[0307] Nivolumab (NIVO) and ipilimumab (IPI) are immune checkpoint inhibitors with different but complementary activities. Combination therapy including nivolumab and ipilimumab, as well as monotherapy with nivolumab and ipilimumab, are approved for the treatment of unresectable or metastatic melanoma.

[0308] High tumor mutational burden (TMB) or high inflammatory gene expression is associated with improved clinical outcomes of immune checkpoint blockade in several tumor types. TMB is a clinically relevant biomarker that may correlate with response to nivolumab / ipilimumab combination therapy in lung cancer and castration-resistant prostate cancer, response to nivolumab monotherapy in urothelial carcinoma, lung cancer, and melanoma, and response to ipilimumab in melanoma.

[0309] Studies across multiple tumor types, including melanoma, have shown that anti-PD-1 treatment correlates with T cell inflammatory gene expression profiles.

[0310] This example reports the results of an exploratory analysis of the correlation between a novel inflammatory gene signature and clinical outcomes of nivolumab / ipilimumab combination treatment and nivolumab and ipilimumab monotherapy in melanoma, alone and in combination with TMB.

[0311] Test Design

[0312] This example reports data pooled from two clinical trials, NCT01721772 and NCT01844505. In NCT01721772, 418 subjects with confirmed, unresectable, previously untreated, stage III or IV melanoma without BRAF mutations were randomly assigned (1:1) to receive either nivolumab 3 mg / kg body weight every 2 weeks plus dacarbazine vs. placebo every 3 weeks (n=210, 206 treated) or dacarbazine 1000 mg per body surface area every 3 weeks plus nivolumab vs. placebo every 2 weeks (n=208, 205 treated) administered intravenously. Randomization was stratified by tumor PD-L1 status (positive vs. negative or undetermined) and metastatic stage (M0, M1a, or M1b vs. M1c, as defined by the American Joint Committee on Cancer and International Union against Cancer tumor-node-metastasis system).

[0313] The primary endpoint was overall survival. Secondary endpoints included investigator-assessed progression-free survival, objective response rate, tumor PD-L1 expression, and health-related quality of life. Exploratory endpoints included safety, pharmacokinetics, and biomarker analysis.

[0314] In NCT01844505, 945 patients with previously untreated unresectable stage III or IV melanoma were randomly assigned in a 1:1:1 ratio to one of the following regimens: (i) nivolumab at 3 mg / kg body weight every 2 weeks (plus ipilimumab-matching placebo) (n=316, 313 treated); (ii) nivolumab at 1 mg / kg every 3 weeks plus ipilimumab at 3 mg / kg every 3 weeks for 4 doses, then nivolumab at 3 mg / kg every 2 weeks from cycle 3 onwards (n=314, 313 treated); or nivolumab at 3 mg / kg every 3 weeks for 4 doses (plus nivolumab-matching placebo) (n=315, 311 treated) (Figure 14). Both nivolumab and ipilimumab were administered by intravenous infusion.

[0315] Randomization was stratified by tumor PD-L1 status (positive vs. negative or undetermined), BRAF mutation status (V600 mutation-positive vs. wild-type), and American Joint Committee on Cancer stage (M0, M1a, or M1b vs. M1c). Treatment continued until disease progression (as defined by RECIST, version 1.1), unacceptable toxicity, or consent withdrawal.

[0316] Progression-free survival and overall survival were coprimary endpoints. Secondary endpoints included objective response rate, tumor PD-L1 expression, and health-related quality of life. Exploratory endpoints included safety, pharmacokinetics, and biomarker analysis.

[0317] Three-year follow-up of NCT01721772 demonstrated a durable survival benefit with nivolumab monotherapy in patients with previously untreated BRAF wild-type advanced melanoma (ORR, % (95% CI): 43% (95% CI, 36.1–49.8) NIVO; 14% (9.9–19.9) dacarbazine; median PFS, months (95% CI): 5.1 (3.5–12.2) NIVO; 2.2 (2.1–2.5) dacarbazine; and median OS, months (95% CI): 37.5 (25.5–11.2) NIVO; 11.2 (9.6–13.0) dacarbazine) (Figures 15A–15B).

[0318] Four-year follow-up of NCT01844505 demonstrated a long-term, durable survival benefit with first-line nivolumab / ipilimumab combination treatment and nivolumab monotherapy in patients with advanced melanoma (ORRb, % (95% CI): 58% (52.6-63.8) NIVO+IPI; 45% (39.1-50.3) NIVO; 19% (14.9-23.8) ) IPI; median PFS, months (95% CI): 11.5 (8.7-19.3) NIVO + IPI; 6.9 (5.1-10.2) NIVO; 2.9 (2.8-3.2) IPI; and median OS, months (95% CI): NR (38.2-NR) NIVO + IPI; 36.9 (28.3-NR) NIVO; 19.9 (16.9-24.6) IPI) (Figures 15C-15D). NCT01844505 was not powered for formal statistical comparisons of nivolumab / ipilimumab combination treatment and nivolumab monotherapy.

[0319] the purpose

[0320] The objective of this analysis was to correlate the inflammatory signature with clinical response, PFS, and OS in patients with TMB- and nivolumab-based immuno-oncology (IO) therapy. For the inflammatory signature analysis, pretreatment tumor samples were analyzed using RNA sequencing to estimate relative tumor inflammation using the expression of four key genes—CD274 (PD-L1), CD8a, LAG3, and STAT1—that comprise the four-gene inflammatory signature described here. The correlation of the four-gene inflammatory signature score with PFS and OS was assessed in the NCT01844505 samples using the relative median score to define high versus low four-gene inflammatory signature scores (median = -0.0434).

[0321] For TMB analysis, pretreatment tumor samples were analyzed by whole-exome sequencing using the median of total missense mutations to define high vs. low TMB (median = 162 in NCT01721772 and 208 in NCT01844505). The relationship between TMB and clinical response, PFS, and OS was assessed using the 4-year follow-up data from NCT01721772 and NCT01844505. A summary of sample distribution for both trials is shown in Tables 23A and 23B and Figures 16A-16C. [Table 23] [Table 24]

[0322] result

[0323] The distribution of 4-gene inflammatory signature scores was higher in patients responding to treatment with nivolumab / ipilimumab combination therapy, nivolumab monotherapy, and ipilimumab monotherapy (Figure 17). Across all treatment arms, longer PFS was observed in patients with high inflammatory signature scores versus low inflammatory signature scores (Figures 18A-18D). Across all treatment arms, longer OS was also observed in patients with high inflammatory signature scores versus low inflammatory signature scores (Figures 19A-19D).

[0324] For the NCT01721772 samples, median TMB values ​​were numerically higher in patients who responded to treatment with nivolumab (Figure 20). In the nivolumab arm, prolonged PFS was observed in patients with high versus low TMB (Figures 21A-21C). In the nivolumab arm, prolonged OS was observed in patients with high versus low TMB (Figures 22A-22C).

[0325] For the NCT01844505 samples, median TMB was numerically higher in patients who responded to treatment with nivolumab / ipilimumab combination therapy, nivolumab monotherapy, and ipilimumab monotherapy (Figure 23). Across all treatment arms, longer PFS was observed in patients with high versus low TMB (Figures 24A-24D). Across all treatment arms, longer OS was observed in patients with high versus low TMB (Figures 25A-25D).

[0326] The 4-gene inflammatory signature and TMB were not correlated (r = 0.27 [95% CI, 0.15-0.38], all arms combined) and appear to be independent markers of response to cancer immunotherapy (Figures 26A-26C). High inflammation and high TMB were associated with increased CR / PR with nivolumab + ipilimumab and nivolumab monotherapy, respectively, compared with ipilimumab monotherapy.

[0327] In previously untreated metastatic melanoma, a high 4-gene inflammatory signature score was observed to be associated with clinical response and prolonged survival to cancer immunotherapy.

[0328] High TMB correlated with improved response and increased PFS and OS in patients treated with cancer immunotherapy (nivolumab monotherapy, nivolumab / ipilimumab combination therapy, or ipilimumab monotherapy). In NCT01844505, treatment with nivolumab monotherapy or nivolumab / ipilimumab combination therapy was associated with improved response and longer survival than ipilimumab monotherapy, regardless of TMB status. TMB status did not correlate with differences in outcomes with dacarbazine in NCT01721772.

[0329] The 4-gene inflammatory signature score and TMB were not correlated and were independently associated with increased response to cancer immunotherapy.

Claims

1. 1. A method of treating a human subject having a tumor, comprising: (i) identifying a subject exhibiting (a) a high inflammatory signature score and (b) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested; and (ii) administering to the subject an anti-PD-1 antibody; wherein the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject; and the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

2. A method of treating a human subject having a tumor, comprising administering to the subject an anti-PD-1 antibody, wherein the subject is identified prior to administration as exhibiting (i) a high inflammatory signature score and (ii) a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of genes tested; wherein the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject; and the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

3. 3. The method of claim 1 or 2, further comprising measuring the TMB status of a biological sample obtained from the subject prior to administration.

4. 1. A method for identifying a human subject having a tumor suitable for anti-PD-1 antibody treatment, comprising: (i) measuring (a) an inflammatory signature score of a tumor sample obtained from the subject and (b) a TMB status of a biological sample obtained from the subject; and (ii) administering an anti-PD-1 antibody to the subject if the subject exhibits a high inflammatory signature score and a TMB status comprising at least about 10 mutations per megabase of the genome examined; wherein the inflammatory signature score is determined by measuring the expression of a panel of inflammatory genes (an "inflammatory gene panel") in a tumor sample obtained from the subject; and the inflammatory gene panel includes CD274 (PD-L1), CD8A, LAG3, and STAT1.

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

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

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

8. 6. The method of any of claims 1 to 5, wherein the inflammatory gene panel consists essentially of CD274 (PD-L1), CD8A, LAG3, and STAT1.

9. The method of any one of claims 1 to 5, wherein the inflammatory gene panel consists of CD274 (PD-L1), CD8A, LAG3, and STAT1.

10. 10. The method of any of claims 1-9, wherein a high inflammatory signature score is characterized by an inflammatory signature score that is higher than an average inflammatory signature score, wherein the average inflammatory signature score is determined by averaging the expression of the panel of inflammatory genes in tumor samples obtained from a population of tumor-bearing subjects.

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

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

13. 13. The method of any of claims 10-12, wherein a high inflammatory signature score is characterized by an inflammatory signature score that is at least about 50% higher than the average inflammatory signature score.

14. 14. The method of any of claims 10-13, wherein a high inflammatory signature score is characterized by an inflammatory signature score that is at least about 75% higher than the average inflammatory signature score.

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

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

17. 17. The method of any of claims 1 to 16, wherein expression of the inflammatory genes in the inflammatory gene panel is determined by the presence of inflammatory gene mRNA, the presence of proteins encoded by the inflammatory genes, or both.

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

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

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

21. 21. The method of any of claims 1-20, wherein TMB status is determined by sequencing nucleic acid in the tumor and identifying genomic alterations in the sequenced nucleic acid.

22. 22. The method of claim 21, wherein the genomic alteration comprises one or more somatic mutations.

23. 23. The method of claim 21 or 22, wherein the genomic alteration comprises one or more non-synonymous mutations.

24. 24. The method of any of claims 21 to 23, wherein the genomic alteration comprises one or more missense mutations.

25. 25. The method of any of claims 21 to 24, 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.

26. Tumor TMB status is determined by FOUNDATIONONE® CDX TM 26. The method of any of claims 1-25, comprising at least 10 mutations, at least about 11 mutations, at least about 12 mutations, at least about 13 mutations, at least about 14 mutations, at least about 15 mutations, at least about 16 mutations, at least about 17 mutations, at least about 18 mutations, at least about 19 mutations, at least about 20 mutations, at least about 21 mutations, at least about 22 mutations, at least about 23 mutations, at least about 24 mutations, at least about 25 mutations, at least about 26 mutations, at least about 27 mutations, at least about 28 mutations, at least about 29 mutations, or at least about 30 mutations per megabase of the genome examined as measured by the assay.

27. 27. The method of any one of claims 3 to 26, wherein the biological sample is a tumor tissue biopsy sample.

28. 25. The method of claim 24, wherein the tumor tissue is formalin-fixed, paraffin-embedded tumor tissue or fresh-frozen tumor tissue.

29. 27. The method of any of claims 3 to 26, wherein the biological sample is a liquid biopsy sample.

30. 27. The method of any of claims 3 to 26, wherein the biological sample comprises one or more of blood, serum, plasma, exoRNA, circulating tumor cells, ctDNA, and cfDNA.

31. 31. The method of any of claims 1 to 30, wherein TMB status is determined by genome sequencing.

32. 31. The method of any of claims 1 to 30, wherein TMB status is determined by exome sequencing.

33. 31. The method of any of claims 1 to 30, wherein TMB status is determined by genomic profiling.

34. The genomic profile may comprise at least about 20 genes, at least about 30 genes, at least about 40 genes, at least about 50 genes, at least about 60 genes, at least about 70 genes, at least about 80 genes, at least about 90 genes, at least about 100 genes, at least about 110 genes, at least about 120 genes, at least about 130 genes, at least about 140 genes, at least about 150 genes, at least about 160 genes, at least about 170 genes, at least about 180 genes, at least about 190 genes, at least about 200 genes, at least about 210 genes, at least about 220 genes, at least about 230 genes, at least about 240 genes, at least about 250 genes, at least about 260 genes, 34. The method of claim 33, wherein the gene comprises at least about 270 genes, at least about 280 genes, at least about 290 genes, at least about 300 genes, at least about 305 genes, at least about 310 genes, at least about 315 genes, at least about 320 genes, at least about 325 genes, at least about 330 genes, at least about 335 genes, at least about 340 genes, at least about 345 genes, at least about 350 genes, at least about 355 genes, at least about 360 genes, at least about 365 genes, at least about 370 genes, at least about 375 genes, at least about 380 genes, at least about 385 genes, at least about 390 genes, at least about 395 genes, or at least about 400 genes.

35. 34. The method of claim 33, wherein the genomic profile comprises at least about 265 genes.

36. 34. The method of claim 33, wherein the genomic profile comprises at least about 315 genes.

37. 34. The method of claim 33, wherein the genomic profile comprises at least about 354 genes.

38. ゲノムプロファイルTHIS、HYD3、HY22、SHORE、SHR1 2(DAY2)、DY10、SYS4、DY20、DY H11、SHA2、HHH2、HARSH4、SH3 LIKE1、SHASH、SHA11、SHASH1 、HAR2、HHH、HHHH、HHHH、20、 THIS、DAYSYS、SAYS、SYS1、 CHA4、SHARSH、DASHY、CHA4(S17). 39)、SHASH(SHASH 3)、SHASH 11、DWH1、DY43、SYS、HY22、3 ROCK1、SHAKH、DWHHH、F11、SHR5 、SY2、SYS323、SYS1、SYS1、S 33、CH11、SHYLOVE2、HYDY11、 DA56、SHASH、SHASH、SHASH、 SHY3、SHY、SHYSY、SYS、DYS1 3, CHRIST、HYSYS、SYS33、SYS1 SHARE、SHARE1(SHA123)、S11 30(SYS)、SYS3、SYS1、SYSY、SHYS 、SHRISH、SYS1S1、SHAS(プロモーターのみ)、2 1000000000000000000000000000000000000000000 HYS、SYS1、SHYS、SHYS2、SHYS、SHYS 11、FIS10、FIS124、SHICH、FIM4(F10). 11) 、SHASHY2、SHASH、SHI22、 DASH、SHAR3、HAN14、DAN 20、SYS、KHKH2、SHYS、 ROCK3、ROCK1、SHO11 、CH19、CHH3、CHHICH、FY88、 DASH2、SHASH、SHASHS14、SHA1 2、DAM23、DYS33 、DY20(LOVE)、D11、DY01、SYS TH2、SH11、DY13、SHR3、 22、CH33、SHICH3H、CHY2H(CHAR3 ). ROSE2、ROSE1、ROSE1、ROSE4、 FIRE、CHY20(LOVE2)、DY202、S 2010、SYSY2、SYS53、SYSY1、6 DA274(DASH1)、DYS3、 NHYH、HYSY、HYHYH、HYHY1、SMAD2, TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD 3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKAR1A, SMAD 4, TSHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMAR CA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMA RCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VH L, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1 (MEK1), NRAS, PTCH1, SNCAIP , WISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2(MEK2), NSD1, PTEN, SOCS1 , WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO 1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL 2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2 L2, CDKN2B, ERRFl1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2 35. The method of claim 33 or 34, wherein the gene expression vector comprises 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, RBI, STAT3, and any combination thereof.

39. TMB status is FOUNDATIONONE® CDX TM 39. The method of any of claims 1 to 38, measured by an assay.

40. 40. The method of any of claims 1-39, further comprising identifying genomic alterations in one or more of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6 and MYB.

41. 41. The method of any of claims 1 to 40, wherein the tumor has a high neoantigen load.

42. 42. The method of any of claims 1-41, wherein the subject's T cell repertoire is expanded.

43. 43. The method of any of claims 1-42, wherein the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1.

44. The method of any of claims 1 to 43, wherein the anti-PD-1 antibody binds to the same epitope as nivolumab.

45. 45. The method of any of claims 1 to 44, wherein the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody or portion thereof.

46. 46. ​​The method of any of claims 1-45, wherein the anti-PD-1 antibody comprises a heavy chain constant region that is of the human IgG1 or IgG4 isotype.

47. 47. The method of any of claims 1 to 46, wherein the anti-PD-1 antibody is nivolumab.

48. 47. The method of any of claims 1 to 46, wherein the anti-PD-1 antibody is pembrolizumab.

49. 49. The method of any of claims 1-48, 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 of body weight about once every 1 week, 2 weeks, or 3 weeks.

50. 50. The method of claim 49, wherein the anti-PD-1 antibody is administered in a dose of at least about 3 mg / kg body weight about once every two weeks.

51. 49. The method of any of claims 1-48, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a uniform dose.

52. 52. The method of any of claims 1-48 and 51, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a uniform dose of at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, or at least about 550 mg.

53. 53. The method of any of claims 1-48, 51 and 52, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 240 mg.

54. 53. The method of any of claims 1-48, 51 and 52, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 480 mg.

55. 55. The method of any of claims 1-48 and 51-54, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a flat dose once every 1, 2, 3, or 4 weeks.

56. 56. The method of any of claims 1-48, 51, 52, and 55, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered in a flat dose of about 240 mg about once every two weeks.

57. 53. The method of any of claims 1-48, 51 and 52, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered in a flat dose of about 480 mg administered once about every four weeks.

58. 58. The method of any of claims 1-57, wherein the anti-PD-1 antibody is administered for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

59. 59. The method of any of claims 1-58, wherein the anti-PD-1 antibody is formulated for intravenous administration.

60. 60. The method of any of claims 1-59, wherein the anti-PD-1 antibody is administered in a sub-therapeutic dose.

61. 61. The method of any of claims 1-60, further comprising administering an antibody or antigen-binding fragment thereof that specifically binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ("anti-CTLA-4 antibody").

62. 62. The method of claim 61, wherein the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4.

63. 63. The method of claim 61 or 62, wherein the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab.

64. 64. The method of any of claims 61 to 63, wherein the anti-CTLA-4 antibody is ipilimumab.

65. 64. The method of any of claims 61 to 63, wherein the anti-CTLA-4 antibody is tremelimumab.

66. 66. The method of any of claims 61-65, wherein the anti-CTLA-4 antibody is administered at a dose ranging from 0.1 mg / kg to 20.0 mg / kg of body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks.

67. 67. The method of any of claims 61-66, wherein the anti-CTLA-4 antibody is administered in a dose of 1 mg / kg body weight once every six weeks.

68. 67. The method of any of claims 61-66, wherein the anti-CTLA-4 antibody is administered in a dose of 1 mg / kg body weight once every four weeks.

69. 66. The method of any of claims 61-65, wherein the anti-CTLA-4 antibody is administered in a uniform dose.

70. 70. The method of claim 69, wherein the anti-CTLA-4 antibody is administered in a uniform dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg.

71. 71. The method of claim 69 or 70, wherein the anti-CLTA-4 antibody is administered in a flat dose about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

72. 72. The method of any of claims 1-71, wherein the tumor is derived from a cancer selected from the group consisting of hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof.

73. 73. The method of any of claims 1 to 72, wherein the tumor is derived from hepatocellular carcinoma.

74. 73. The method of any of claims 1 to 72, wherein the tumor is derived from a gastroesophageal cancer.

75. 73. The method of any of claims 1 to 72, wherein the tumor is derived from melanoma.

76. 76. The method of any of claims 1 to 75, wherein the tumor is recurrent.

77. 77. The method of any of claims 1 to 76, wherein the tumor is refractory.

78. 78. The method of any of claims 1-77, wherein the tumor is refractory after at least one prior treatment comprising administration of at least one anti-cancer agent.

79. 79. The method of claim 78, wherein the at least one anti-cancer agent comprises a standard of care therapy.

80. 80. The method of claim 78 or 79, wherein the at least one anti-cancer agent comprises an immunotherapy.

81. 81. The method of any of claims 1-80, wherein the tumor is locally advanced.

82. 82. The method of any of claims 1-81, wherein the tumor is metastatic.

83. 83. The method of any of claims 1-82, wherein the administration treats a tumor.

84. 84. The method of any of claims 1-83, wherein the administration reduces tumor size.

85. 85. The method of claim 84, wherein the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size before administration.

86. 86. The method of any of claims 1-85, wherein 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.

87. 87. The method of any of claims 1-86, wherein the subject exhibits stable disease after administration.

88. 87. The method of any of claims 1-86, wherein the subject exhibits a partial response following administration.

89. 87. The method of any of claims 1-86, wherein the subject exhibits a complete response following administration.

90. 1. A kit for treating a subject having a tumor, comprising: (a) a single dose ranging from about 4 mg to about 500 mg of an anti-PD-1 antibody; and (b) instructions for using an anti-PD-1 antibody in the method of any of claims 1-89. Includes a kit.

91. 91. The kit of claim 90, further comprising an anti-CTLA-4 antibody.

92. 92. The kit of claim 90 or 91, further comprising an anti-PD-L1 antibody.

93. 93. The kit of any of claims 90 to 92, further comprising a comprehensive genomic profiling assay.

94. Comprehensive Genomic Profiling Assays FOUNDATIONONE® CDX TM 94. The kit of claim 93, which is a genomic profiling assay.

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