Methods for treating tumors

Abstract

The present invention provides a method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), comprising administering to the subject therapeutically effective amounts of (a) an anti-PD-1 antibody, or an antigen-binding portion thereof, or an anti-PD-L1 antibody, or an antigen-binding portion thereof, and (b) an anti-CTLA-4 antibody, or an antigen-binding portion thereof, wherein the tumor has a high tumor mutational burden (TMB) status. TMB status can be determined by sequencing nucleic acid in the tumor and identifying genomic alterations, e.g., somatic nonsynonymous mutations, in the sequenced nucleic acid.

Description

Technical Field

[0001] Technical Field The present invention provides a method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC) using immunotherapy.

Background Art

[0002] Background of the Invention Human cancers possess numerous genetic and epigenetic changes and produce neoantigens that may be recognizable by the immune system (Sjoblom et al., Science (2006) 314(5797):268-274). The adaptive immune system, consisting of T and B lymphocytes, has a powerful anti-cancer ability with broad capabilities and exquisite specificity to respond to diverse tumor antigens. Furthermore, the immune system exhibits considerable flexibility and a memory component. The successful utilization of all the traits of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

[0003] Until recently, cancer immunotherapy has been labor-intensive attempts to enhance anti-tumor immune responses by adoptive transfer of activated effector cells, immunization against appropriate antigens, or provision of non-specific immune stimulatory factors such as cytokines. However, intensive efforts over the past decade to develop specific immune checkpoint pathway inhibitors have begun to provide new immunotherapy approaches for treating cancer, including the development of antibodies such as nivolumab and pembrolizumab (previously lambrolizumab; USAN Council Statement, (2013)) that specifically bind to the programmed cell death-1 (PD-1) receptor and inhibit the 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).

[0004] PD-1 is an important immune checkpoint receptor expressed by activated T and B cells that mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2), have been identified. They are expressed on antigen-presenting cells and in many human cancers, and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Patent Nos. 8,008,449 and 7,943,743), and the use of PD-1 / PD-L1 interaction antibody inhibitors for treating cancer is in 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).

[0005] Nivolumab (formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby preventing downregulation of antitumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014). Nivolumab has been shown to be active in various advanced solid tumors, including renal cell carcinoma (renal adenocarcinoma or adrenal carcinoma), melanoma, and non-small cell lung cancer (NSCLC) (Topalian et al., 2012a; Topalian et al., 2014; Drake et al., 2013; WO 2013 / 173223).

[0006] The immune system and its response to immunotherapy are complex. Furthermore, the effectiveness of anticancer drugs can vary depending on the patient's characteristics. Therefore, there is a need for targeted therapy strategies that identify patients who are more likely to respond to specific anticancer drugs, thereby improving the clinical outcomes of patients diagnosed with cancer. [Overview of the project]

[0007] Summary of the Invention A particular aspect of the present invention provides a method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), comprising administering to the subject a therapeutically effective amount of (a) an antibody or its antigen-binding moiety that specifically binds to the programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity ("anti-PD-1 antibody") or an antibody or its antigen-binding moiety that specifically binds to programmed cell death-ligand 1 (PD-L1) and inhibits PD-1 activity ("anti-PD-L1 antibody") and (b) an antibody or its antigen-binding moiety that specifically binds to cytotoxic T lymphocyte antigen-4 (CTLA-4) ("anti-CTLA-4 antibody"), wherein the tumor has a tumor mutational burden (TMB) status of at least about 10 mutations per megabase of the gene being examined. In one embodiment, the method further comprises measuring the TMB status of a biological sample obtained from the subject before administration.

[0008] One aspect of the present invention provides a method for identifying subjects suffering from tumors derived from non-small cell lung cancer (NSCLC) who are suitable for combination therapy with (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, comprising measuring the TMB status of a biological sample of the subject, wherein the TMB status includes at least about 10 mutations per megabase of the genome examined, and the subject is identified as suitable for combination therapy. In one embodiment, the method further comprises administering therapeutically effective doses of the anti-PD-1 antibody and the anti-CLTA-4 antibody to the subject.

[0009] In one embodiment, the TMB status is determined by sequencing nucleic acids within the tumor and identifying genomic modifications in the sequenced nucleic acids. In one embodiment, the genomic modification includes one or more somatic mutations. In one embodiment, the genomic modification includes one or more non-synonymous mutations. In one embodiment, the genomic modification includes one or more missense mutations. In one embodiment, the genomic modification includes one or more modifications selected from the group consisting of base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and combinations thereof.

[0010] In one aspect, the TMB status of a tumor is FOUNDATIONONE® CDX (商標) The assay includes at least approximately 10 mutations, at least approximately 11 mutations, at least approximately 12 mutations, at least approximately 13 mutations, at least approximately 14 mutations, at least approximately 15 mutations, at least approximately 16 mutations, at least approximately 17 mutations, at least approximately 18 mutations, at least approximately 19 mutations, at least approximately 20 mutations, at least approximately 21 mutations, at least approximately 22 mutations, at least approximately 23 mutations, at least approximately 24 mutations, at least approximately 25 mutations, at least approximately 26 mutations, at least approximately 27 mutations, at least approximately 28 mutations, at least approximately 29 mutations, or at least approximately 30 mutations per megabase of the genome tested.

[0011] In one embodiment, the biological sample is a tumor tissue biopsy. In one embodiment, the tumor tissue is formalin-fixed, paraffin-embedded tumor tissue, or freshly frozen tumor tissue. In one embodiment, the biological sample is a liquid biopsy. In one embodiment, the biological sample comprises one or more of blood, serum, plasma, exoRNA, circulating tumor cells, ctDNA, and cfDNA.

[0012] In one embodiment, the TMB status is determined by genome sequencing. In another embodiment, the TMB status is determined by exome sequencing.

[0013] In some aspects, TMB status is determined by genome profiling. In some aspects, the genome profile includes at least approximately 20 genes, at least approximately 30 genes, at least approximately 40 genes, at least approximately 50 genes, at least approximately 60 genes, at least approximately 70 genes, at least approximately 80 genes, at least approximately 90 genes, at least approximately 100 genes, at least approximately 110 genes, at least approximately 120 genes, at least approximately 130 genes, at least approximately 140 genes, at least approximately 150 genes, at least approximately 160 genes, at least approximately 170 genes, at least approximately 180 genes, at least approximately 190 genes, at least approximately 200 genes, at least approximately 210 genes, at least approximately 220 genes, at least approximately 230 genes, at least approximately 240 genes, at least approximately 250 genes, and fewer Each includes approximately 260 genes, at least approximately 270 genes, at least approximately 280 genes, at least approximately 290 genes, at least approximately 300 genes, at least approximately 305 genes, at least approximately 310 genes, at least approximately 315 genes, at least approximately 320 genes, at least approximately 325 genes, at least approximately 330 genes, at least approximately 335 genes, at least approximately 340 genes, at least approximately 345 genes, at least approximately 350 genes, at least approximately 355 genes, at least approximately 360 genes, at least approximately 365 genes, at least approximately 370 genes, at least approximately 375 genes, at least approximately 380 genes, at least approximately 385 genes, at least approximately 390 genes, at least approximately 395 genes, or at least approximately 400 genes. In one embodiment, the genome profile includes at least approximately 265 genes. In one embodiment, the genome profile includes at least approximately 315 genes. In one embodiment, the genome profile includes at least approximately 354 genes.

[0014] ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, PDCD1LG2(PD-L2) RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDGFRA, RET, STK11, ACVR 1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF GID4(C17orf39), KAT6A(MYST3), MRE11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, G Ll1, KDM5A, MSH2, PIK3C2B, ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PI K3CA, RPTOR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, AMER1 (FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT(プロモーAPC, 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 CCND1 CYL D. FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFRSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1 SETD2, TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, TOP2A, ARID2, CCNE1, DICER1, F GF4, HGF, KMT2D(MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274(PD-L1), DNMT3A, FGF6, HNF1A, KRAS, and NFKBIA.PRDM1, SMAD2, TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKA R1A, SMAD4, TSHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMARCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1(MEK1), NRAS, P TCH1, SNCAIP, WISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2(MEK2), NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTP N11, SOX10, XPO1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFl1, FRS2, INPP4B, MDM4, PAK3, RAD51 It includes one or more genes selected from the group consisting of SPOP, 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 combinations thereof.

[0015] In one aspect, the TMB state is FOUNDATIONONE® CDX (商標) It is measured by assay.

[0016] In one embodiment, the method further includes identifying genomic modifications in one or more of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6, and MYB.

[0017] In one aspect, the tumor has a high neoantigen load. In another aspect, the subject has an increased T cell repertoire.

[0018] A particular aspect of the present invention is a method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), wherein (i) FOUNDATIONONE® CDX (商標) The present invention relates to a method for measuring the tumor's TMB status by assay, and (ii) administering therapeutically effective doses of anti-PD-1 antibody and anti-CTLA-4 antibody to the target, wherein the TMB status has at least approximately 10 mutations per megabase of the genome examined.

[0019] In one embodiment, NSCLC has a squamous epithelial tissue appearance. In another embodiment, NSCLC has a non-squamous epithelial tissue appearance.

[0020] In one embodiment, the anti-PD-1 antibody cross-competes with nivolumab or pembrolizumab for binding to human PD-1. In one embodiment, the anti-PD-1 antibody binds to the same epitope as nivolumab or pembrolizumab. In one embodiment, the anti-PD-1 antibody is a chimeric antibody, a humanized antibody, or a human monoclonal antibody. In one embodiment, the anti-PD-1 antibody contains a heavy chain constant region of a human IgG1 isotype or a human IgG4 isotype. In one embodiment, the anti-PD-1 antibody is nivolumab. In one embodiment, the anti-PD-1 antibody is pembrolizumab.

[0021] In one embodiment, the anti-PD-1 antibody is administered once every 2, 3, or 4 weeks at a dose ranging from 0.1 mg to 20.0 mg per kg of body weight. In another embodiment, the anti-PD-1 antibody is administered once every 3 weeks at a dose of 2 mg per kg of body weight. In another embodiment, the anti-PD-1 antibody is administered once every 2 weeks at a dose of 3 mg per kg of body weight.

[0022] In one embodiment, the therapeutically effective dose of anti-PD-1 antibody is a constant dose. In one embodiment, the therapeutically effective dose of anti-PD-1 antibody is a constant 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, anti-PD-1 antibody is administered as a constant dose approximately once every 1, 2, 3, or 4 weeks. In one embodiment, anti-PD-1 antibody is administered as a constant dose of about 200 mg once every 3 weeks. In one embodiment, anti-PD-1 antibody is administered as a constant dose of about 240 mg once every 2 weeks. In one embodiment, the anti-PD-1 antibody is administered once every four weeks at a constant dose of approximately 480 mg.

[0023] In some embodiments, the anti-PD-L1 antibody cross-competes with durvalumab, avelumab, or atezolizumab for binding to human PD-1. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as durvalumab, avelumab, or atezolizumab. In some embodiments, the anti-PD-L1 antibody is durvalumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0024] In one embodiment, the anti-PD-L1 antibody is administered once every 2, 3, or 4 weeks at a dose ranging from 0.1 mg to 20.0 mg per kg of body weight. In another embodiment, the anti-PD-L1 antibody is administered once every 3 weeks at a dose of 15 mg per kg of body weight. In another embodiment, the anti-PD-L1 antibody is administered once every 2 weeks at a dose of 10 mg per kg of body weight.

[0025] In one embodiment, the therapeutically effective dose of anti-PD-L1 antibody is a constant dose. In one embodiment, the therapeutically effective dose of anti-PD-L1 antibody is a constant 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 720 mg, at least about 800 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. In one embodiment, anti-PD-L1 antibody is administered as a constant dose approximately once every 1, 2, 3, or 4 weeks. In one embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 1200 mg once every three weeks. In another embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 800 mg once every two weeks.

[0026] 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.

[0027] In one embodiment, the anti-CTLA-4 antibody is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from 0.1 mg to 20.0 mg per kg of body weight. In another embodiment, the anti-CTLA-4 antibody is administered once every 6 weeks in a dose of 1 mg per kg of body weight. In another embodiment, the anti-CTLA-4 antibody is administered once every 4 weeks in a dose of 1 mg per kg of body weight.

[0028] In one embodiment, the therapeutically effective dose of anti-CTLA-4 antibody is a constant dose. In one embodiment, the therapeutically effective dose of anti-CTLA-4 antibody is a constant 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 one embodiment, anti-CTLA-4 antibody is administered as a constant dose approximately once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0029] In one embodiment, subjects demonstrate progression-free survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration.

[0030] In one embodiment, subjects demonstrate overall survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration.

[0031] In one embodiment, the subjects exhibit response rates of at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 100%.

[0032] In one embodiment, less than 1% of tumor cells express PD-L1.

[0033] Other features and advantages of the present invention will become apparent from the following detailed description and examples, but these examples should not be construed as limiting. All documents cited herein, including scientific literature, newspaper reports, GenBank registrations, patents and patent applications, are expressly incorporated herein by reference.

[0034] manner E1. A method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), comprising administering to the subject a therapeutically effective dose of (a) an antibody or its antigen-binding portion that specifically binds to the programmed cell death-1 (PD-1) receptor and inhibits PD-1 activity ("anti-PD-1 antibody") or an antibody or its antigen-binding portion that specifically binds to programmed cell death-ligand 1 (PD-L1) and inhibits PD-1 activity ("anti-PD-L1 antibody"), and (b) an antibody or its antigen-binding portion that specifically binds to cytotoxic T lymphocyte antigen-4 (CTLA-4) ("anti-CTLA-4 antibody"), wherein the tumor has a tumor mutational load (TMB) status of at least about 10 mutations per megabase of the gene examined.

[0035] E2. The method according to E1, further comprising measuring the TMB status of a biological sample obtained from a subject before administration.

[0036] E3. A method for identifying subjects with tumors derived from non-small cell lung cancer (NSCLC) who are suitable for combination therapy of (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, comprising measuring the TMB status of a biological sample of the subject, wherein the TMB status includes at least about 10 mutations per megabase of the genome examined, and the subject is identified as suitable for combination therapy.

[0037] E4. The method according to E3, further comprising administering therapeutically effective doses of anti-PD-1 antibody and anti-CTLA-4 antibody to the target.

[0038] E5. A method described in any of E1 to E4, wherein the TMB status is determined by sequencing nucleic acids in the tumor and identifying genomic modifications in the sequenced nucleic acids.

[0039] E6. The method described in E5, wherein the genome modification includes one or more somatic mutations.

[0040] E7. The method described in E5 or E6, wherein the genome modification includes one or more nonsynonymous mutations.

[0041] E8. A method described in any of E5 to E7, wherein the genome modification includes one or more missense mutations.

[0042] E9. A method according to any one of E5 to E8, wherein the genome modification comprises one or more modifications selected from the group consisting of base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and combinations thereof.

[0043] E10. The tumor's TMB status is FOUNDATIONONE(registered trademark) CDX (商標) The method described in any of E1 to E9, comprising at least approximately 10 mutations, at least approximately 11 mutations, at least approximately 12 mutations, at least approximately 13 mutations, at least approximately 14 mutations, at least approximately 15 mutations, at least approximately 16 mutations, at least approximately 17 mutations, at least approximately 18 mutations, at least approximately 19 mutations, at least approximately 20 mutations, at least approximately 21 mutations, at least approximately 22 mutations, at least approximately 23 mutations, at least approximately 24 mutations, at least approximately 25 mutations, at least approximately 26 mutations, at least approximately 27 mutations, at least approximately 28 mutations, at least approximately 29 mutations, or at least approximately 30 mutations per megabase of the genome tested, as measured by the assay.

[0044] E11. A method described in any of E2 to E10, wherein the biological sample is a tumor tissue biopsy.

[0045] E12. The method according to E11, wherein the tumor tissue is formalin-fixed, paraffin-embedded tumor tissue, or fresh-frozen tumor tissue.

[0046] E13. A method described in any of E2 to E11, wherein the biological sample is a liquid biopsy.

[0047] E14. A method according to any one of E2 to E11, wherein the biological sample comprises one or more of blood, serum, plasma, exoRNA, circulating tumor cells, ctDNA, and cfDNA.

[0048] E15. The TMB status is determined by genome sequencing, using one of the methods described in E1 to E14.

[0049] E16. A method described in any of E1 to E14, wherein the TMB state is determined by exome sequencing.

[0050] E17. The TMB status is determined by genomic profiling, as described in any of the methods from E1 to E14.

[0051] E18. The genome profile contains 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, and at least about 260 genes. The method described in E17, comprising 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.

[0052] E19. The method described in E17, which includes a genome profile of at least approximately 265 genes.

[0053] E20. The method described in E17, which includes a genome profile of at least approximately 315 genes.

[0054] E21. The method described in E17, wherein the genome profile includes at least approximately 354 genes.

[0055] E22.ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, PDCD1LG2(PD-L2), RBM1 0, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDGFRA, RET, STK11, ACVR1B, B RCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GI D4(C17orf39), KAT6A(MYST3), MRE11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLl1 KDM5A, MSH2, PIK3C2B, ROS1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PIK3 CA, RPTOR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, AMER1( FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT(プロモーAPC, 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 CCND1 CYLD FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFRSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1, SE TD2, TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, TOP2A, ARID2, CCNE1, DICER1, FGF4, H GF, KMT2D(MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274(PD-L1), 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, SMA D4, TSHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SM ARCB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1(MEK1), NRAS, PTCH1, SNCA IP, WISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2(MEK2), NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217 , BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFl1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, C The method described in E17 or E18, comprising one or more genes selected from the group consisting of DKN2C, 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 combinations thereof.

[0056] E23.TMB status is FOUNDATIONONE(registered trademark) CDX (商標) A method described in any of E1 to E22, measured by assay.

[0057] The method described in any of E1 to E24, further comprising identifying genomic modifications in one or more of ETV4, TMPRSS2, ETV5, BCR, ETV1, ETV6, and MYB.

[0058] E25. The tumor has a high neoantigen load, as described in any of the methods described in E1 to E24.

[0059] E26. A method described in any of E1 to E25, wherein the subject has an increased T cell repertoire.

[0060] E27. A method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), wherein (i) FOUNDATIONONE® CDX (商標) (ii) Measuring the tumor's TMB status by assay, and (ii) administering therapeutically effective doses of anti-PD-1 antibody and anti-CTLA-4 antibody to the target, wherein the TMB status has at least approximately 10 mutations per megabase of the genome examined.

[0061] E28. NSCLC having a squamous epithelial tissue appearance, as described in any of the methods from E1 to E27.

[0062] E29. A method described in any of E1 to E27, wherein NSCLC has a non-squamous epithelial tissue appearance.

[0063] E30. The method described in any of E1 to E29, wherein the anti-PD-1 antibody cross-competes with nivolumab or pembrolizumab for binding to human PD-1.

[0064] E31. The method described in any of E1 to E29, wherein the anti-PD-1 antibody binds to the same epitope as nivolumab or pembrolizumab.

[0065] E32. The method described in any of E1 to E30, wherein the anti-PD-1 antibody is a chimeric antibody, a humanized antibody, or a human monoclonal antibody.

[0066] E33. The method according to any one of E1 to E32, wherein the anti-PD-1 antibody comprises a heavy chain constant region of a human IgG1 isotype or a human IgG4 isotype.

[0067] E34. The method described in any of E1 to E33, wherein the anti-PD-1 antibody is nivolumab.

[0068] E35. The method described in any of E1 to E33, wherein the anti-PD-1 antibody is pembrolizumab.

[0069] E36. The method described in any of E1 to E35, wherein an anti-PD-1 antibody is administered once every 2, 3, or 4 weeks in a dose ranging from 0.1 mg to 20.0 mg per kg of body weight.

[0070] E37. The method described in any of E1 to E36, in which an anti-PD-1 antibody is administered once every three weeks at a dose of 2 mg per kg of body weight.

[0071] E38. The anti-PD-1 antibody is administered once every two weeks at a dose of 3 mg per kg of body weight, as described in any of the methods described in E1 to E36.

[0072] E39. A method described in any of E1 to E35, wherein the therapeutically effective dose of the anti-PD-1 antibody is a constant dose.

[0073] E40. The method according to E39, wherein the therapeutically effective dose of the anti-PD-1 antibody is a constant 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.

[0074] E41. The method described in E39 or E40, wherein an anti-PD-1 antibody is administered approximately once every 1, 2, 3, or 4 weeks in a fixed dose.

[0075] E42. The method described in any of E1 to E35, in which an anti-PD-1 antibody is administered once every three weeks at a constant dose of approximately 200 mg.

[0076] E43. The method described in any of E1 to E35, wherein an anti-PD-1 antibody is administered once every two weeks at a constant dose of approximately 240 mg.

[0077] E44. The method described in any of E1 to E35, wherein an anti-PD-1 antibody is administered once every four weeks at a constant dose of approximately 480 mg.

[0078] E45. A method described in any of E1 to E29, wherein an anti-PD-L1 antibody cross-competes with durvalumab, avelumab, or atezolizumab for binding to human PD-1.

[0079] E46. The method described in any of E1 to E29, wherein the anti-PD-L1 antibody binds to the same epitope as durvalumab, avelumab, or atezolizumab.

[0080] E47. The anti-PD-L1 antibody is durvalumab, as described in any of the methods from E1 to E29.

[0081] E48. The anti-PD-L1 antibody is avelumab, as described in any of the methods described in E1 to E29.

[0082] E49. The method described in any of E1 to E29, wherein the anti-PD-L1 antibody is atezolizumab.

[0083] E50. The method described in any of E45 to E49, wherein an anti-PD-L1 antibody is administered once every 2, 3, or 4 weeks in a dose ranging from 0.1 mg to 20.0 mg per kg of body weight.

[0084] E51. The method described in any of E45 to E49, in which an anti-PD-L1 antibody is administered once every three weeks at a dose of 15 mg per kg of body weight.

[0085] E52. The method described in any of E45 to E49, in which an anti-PD-L1 antibody is administered once every two weeks at a dose of 10 mg per kg of body weight.

[0086] E53. A method described in any of E1 to E29 and E45 to E49, wherein the therapeutically effective dose of the anti-PD-L1 antibody is a constant dose.

[0087] E54. The method according to E53, wherein the therapeutically effective dose of anti-PD-L1 antibody is a constant 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 720 mg, at least about 800 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.

[0088] E55. The method described in E53 or E54, wherein an anti-PD-L1 antibody is administered approximately once every 1, 2, 3, or 4 weeks in a fixed dose.

[0089] E56. The method described in any of E53 to E55, wherein an anti-PD-L1 antibody is administered once every three weeks at a constant dose of approximately 1200 mg.

[0090] E57. The method described in any of E53 to E55, wherein an anti-PD-L1 antibody is administered once every two weeks at a constant dose of approximately 800 mg.

[0091] E58. The method described in any of E1 to E57, wherein the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4.

[0092] E59. The method described in any of E1 to E57, wherein the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab.

[0093] E60. The anti-CTLA-4 antibody is ipilimumab, as described in any of the methods described in E1 to E59.

[0094] E61. The method described in any of E1 to E59, wherein the anti-CTLA-4 antibody is tremelimumab.

[0095] E62. The method described in any of E1 to E59, wherein an anti-CTLA-4 antibody is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from 0.1 mg to 20.0 mg per kg of body weight.

[0096] E63. The anti-CTLA-4 antibody is administered once every six weeks at a dose of 1 mg per kg of body weight, as described in any of the methods described in E1 to E59.

[0097] E64. The anti-CTLA-4 antibody is administered once every four weeks at a dose of 1 mg per kg of body weight, as described in any of the methods described in E1 to E59.

[0098] E65. A method described in any of E1 to E61, wherein the therapeutically effective dose of the anti-CTLA-4 antibody is a constant dose.

[0099] E66. The method according to E65, wherein the therapeutically effective dose of anti-CTLA-4 antibody is a constant 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.

[0100] E67. The method according to E65 or E66, wherein an anti-CLTA-4 antibody is administered approximately once every 2, 3, 4, 5, 6, 7, or 8 weeks in a fixed dose.

[0101] E68. A method described in any of E1 to E67, wherein the subject exhibits a progression-free survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration.

[0102] E69. A method described in any of E1 to E67, wherein the subject exhibits an overall survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration.

[0103] E70. A method according to any one of E1 to E69, wherein the subjects exhibit a response rate of at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 100%.

[0104] E71. The tumor is PD-L1 negative, according to any of the methods described in E1 to E70.

[0105] E72. A method described in any of E1 to E71, wherein the tumor has less than 1% PD-L1. [Brief explanation of the drawing]

[0106] [Figure 1]Figure 1 shows the clinical trial design for treating NSCLC. Participants were divided according to their PD-L1 expression status, i.e., PD-L1 expression of ≥1% and PD-L1 expression of <1%. Then, each group was divided into three subgroups (1:1:1): (i) a group receiving an anti-PD-1 antibody (e.g., nivolumab) at a dose of 3 mg / kg q2Q and an anti-CTLA-4 antibody (e.g., ipilimumab) at a dose of 1 mg / kg q6W (n=396 or n=187), (ii) a histological chemotherapy group (n=397 or n=186), and (iii) a group receiving only an anti-PD-1 antibody, e.g., nivolumab, at a constant dose of 240 mg q2W (n=396 or n=177). Subjects who had received histological chemotherapy were further stratified according to whether they had squamous (SQ) NSCLC or non-squamous (NSQ) NSCLC. Subjects with NSQ NSCLC who had received chemotherapy were administered pemetrexed (500 mg / m2) + cisplatin (75 mg / m2) or carboplatin (AUC 5 or 6) for 4 cycles or less at Q3W, with optional maintenance therapy of pemetrexed (500 mg / m2) after chemotherapy, or nivolumab (360 mg Q3W) + chemotherapy followed by maintenance therapy of nivolumab (360 mg Q3W) + pemetrexed (500 mg / m2). Subjects with SQ NSCLC who received chemotherapy were administered gemcitabine (1000 or 1250 mg / m2) + cisplatin (75 mg / m2) or gemcitabine (1000 mg / m2) + carboplatin (AUC 5) for 4 cycles or less at Q3W. TMB co-primary analysis was performed on a subset of patients randomized to nivolumab + ipilimumab or chemotherapy with evaluable TMB ≥ 10 mutations / Mb. [Figure 2] Figure 2 shows scatter plots of TMB and PD-L1 expression in all TMB-evaluable patients. The y-axis represents the number of mutations per megabase, and the x-axis represents PD-L1 expression. Symbols (dots) in the scatter plot may represent multiple data points, especially in patients with PD-L1 expression less than 1%.

[0107] [Figure 3]Figure 3A shows progression-free survival (PSD) with anti-PD-1 antibodies (e.g., nivolumab) and anti-CLTA-4 antibodies (e.g., ipilimumab) versus progression-free survival with chemotherapy in all randomized patients. Cl represents the confidence interval. HR represents the hazard ratio. Figure 3B shows progression-free survival (PSD) with anti-PD-1 antibodies (e.g., nivolumab) and anti-CLTA-4 antibodies (e.g., ipilimumab) versus progression-free survival with chemotherapy in TMB-evaluable patients. [Figure 4] Figure 4A shows the progression-free survival (PFS) with anti-PD-1 antibody (e.g., nivolumab) + anti-CLTA-4 antibody (e.g., ipilimumab) (Nivo + Ipi) versus progression-free survival (PFS) with chemotherapy (Chemo) in patients with TMB ≥ 10 mutations / Mb. 1-y PFS = 1-year progression-free survival; *95% CI, 0.43~0.77. Figure 4B shows the duration of response (DOR) with anti-PD-1 antibody (e.g., nivolumab) + anti-CLTA-4 antibody (e.g., ipilimumab) (Nivo + Ipi) versus duration of response with chemotherapy (Chemo) in patients with TMB ≥ 10 mutations / Mb. DOR: median duration of response, DOR;mo: median months of response; 1-y DOR: 1-year duration of response.

[0108] [Figure 5] Figure 5 shows the progression-free survival time with anti-PD-1 antibody (e.g., nivolumab) + anti-CLTA-4 antibody (e.g., ipilimumab) versus progression-free survival time with chemotherapy in patients with TMB < 10 mutations / Mb. [Figure 6] Figure 6A shows a subgroup analysis of progression-free survival in patients with TMB ≥ 10 mutations / Mb due to PD-L1 expression of ≥ 1%. PFS (%): Progression-free survival rate. Figure 6B shows a subgroup analysis of progression-free survival in patients with TMB ≥ 10 mutations / Mb due to PD-L1 expression greater than 1%. Figure 6C shows a subgroup analysis of progression-free survival in patients with TMB ≥ 10 mutations / Mb in squamous cell tumor histological type. Figure 6D shows a subgroup analysis of progression-free survival in patients with TMB ≥ 10 mutations / Mb in non-squamous cell tumor histological type. Figure 6E shows the characteristics of the selected subgroups.

[0109] [Figure 7] Figure 7 shows progression-free survival between anti-PD-1 antibody (e.g., nivolumab) monotherapy and chemotherapy in patients with TMB ≥ 13 mutations / Mb and tumor PD-L1 expression ≥ 1%. 95% Cl is 0.95 (0.64, 1.4). [Figure 8] Figure 8 shows progression-free survival in patients with TMB ≥ 10 mutations / Mb and tumor PD-L1 expression ≥ 1% against anti-PD-1 antibody (e.g., nivolumab) + anti-CLTA-4 antibody (e.g., ipilimumab) versus anti-PD-1 antibody (e.g., nivolumab) monotherapy and chemotherapy. The 95% confidence interval is 0.62 (0.44, 0.88) for nivolumab + ipilimumab versus chemotherapy.

[0110] [Figure 9] Figures 9A to 9C show progression-free survival (PFS; Figure 9A), objective response rate (ORR; Figure 9B), and duration of response (DOR; Figure 9C) after treatment with nivolumab plus chemotherapy or chemotherapy alone for patients with tumor PD-L1 expression of less than 1%. Figure 9D shows patient stratification based on baseline characteristics and associated unstratified hazard ratios (HRs) after treatment with either nivolumab plus chemotherapy ("Nivo+Chemo") or chemotherapy alone ("Chemo"). [Figure 10] Figures 10A and 10B show progression-free survival (PFS) in patients with tumor PD-L1 expression less than 1% and high TMB (≥10 mutations / Mb; Figure 10A) and low TMB (<10 mutations / Mb; Figure 10B) after treatment with nivolumab + ipilimumab (vertical dash), nivolumab + chemotherapy (circle), or chemotherapy alone (triangle) (Figures 10A-10B). Figure 10C shows duration of response (DOR) in patients with tumor PD-L1 expression less than 1% and high TMB (≥10 mutations / Mb) after treatment with nivolumab + ipilimumab (vertical dash), nivolumab + chemotherapy (circle), or chemotherapy alone (triangle).

[0111] [Figure 11] Figure 11 shows the distribution of treatment-related adverse events (TRAEs) in patients treated with either nivolumab plus chemotherapy (y-axis left) or nivolumab plus ipilimumab (y-axis right). Dark gray and black bars represent grade 1–2 TRAEs, while light gray bars represent grade 3–4 TRAEs. a. Selected AEs have immunological etiologies that may require monitoring / intervention. [Modes for carrying out the invention]

[0112] Detailed description of the invention The present invention provides a method for treating a subject suffering from a tumor derived from non-small cell lung cancer (NSCLC), comprising administering to the subject a combination therapy agent comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, wherein the tumor has a high tumor mutational load (TMB) status. In a particular embodiment, the tumor has a TMB of at least about 10 mutations per megabase of the gene examined.

[0113] The present invention also provides a method for identifying subjects with NSCLC-derived tumors who are suitable for combination therapy with (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, comprising measuring the TMB status of a biological sample of the tumor, wherein the tumor has a high TMB status and the subject is identified as suitable for combination therapy. In one embodiment, a subject identified as suitable for combination therapy has a tumor with a TMB of at least about 10 mutations per megabase of the genes tested.

[0114] term To make the descriptions herein easier to understand, some terms are defined first. Unless otherwise specified herein, the following terms have the meanings set forth below. Further definitions are provided throughout this specification.

[0115] "Administer" means the physical delivery of a composition containing a therapeutic agent to a subject using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for immunotherapeutic agents, such as anti-PD-1 antibodies or anti-PD-L1 antibodies, include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other non-enteral administration routes, such as by injection or infusion. As used herein, "non-enteral administration" means a method of administration other than enteral and local administration, which is usually by injection and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intrathoracic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions, as well as in vivo electroporation. Other non-enteral routes include oral, topical, epithelial, or mucosal administration routes, such as intranasal, intravaginal, intrarectal, sublingual, or topical administration. Administration may also be, for example, a single dose, multiple doses, and / or one or more doses over a long period of time.

[0116] As used herein, “adverse event” (AE) means an undesirable and generally unintended or undesirable sign (including abnormal laboratory findings), symptom, or disease associated with the use of a medical procedure. For example, an adverse event may be associated with the activation of the immune system or the proliferation of immune system cells (e.g., T cells) in response to a procedure. A medical procedure may have one or more associated AEs, each AE may have the same or different levels of severity. Reference to methods that can “modify an adverse event” means a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0117] An “antibody” (Ab) includes a glycoprotein immunoglobulin or its antigen-binding moiety, which specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain has a heavy chain variable region (V in this specification). H It includes the heavy chain constant region (abbreviated as C). The heavy chain constant region consists of three constant domains, C H1 , CH2 and C H3 Each light chain contains a variable light region (abbreviated herein as V L ), and a constant light region. The constant light region contains one constant domain, C L . V H and V L regions are further divided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V L contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins 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.

[0118] 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" means an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, by way of example, both natural antibodies 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, but is not limited thereto. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated otherwise and unless the context indicates another meaning, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of the above immunoglobulins, and also includes monovalent and bivalent fragments or portions and single-chain antibodies.

[0119] "Isolated antibody" means an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PD-1 substantially does not contain antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may cross-react to other antigens, such as PD-1 molecules from different species. Furthermore, an isolated antibody substantially does not contain other cellular material and / or chemical substances.

[0120] The term “monoclonal antibody” (mAb) refers to an antibody molecule with a single molecular composition, i.e., a naturally occurring preparation of antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma techniques, recombinant techniques, transgenic techniques, or other techniques known to those skilled in the art.

[0121] A “human” antibody (HuMAb) means an antibody having a variable region in which both the framework region and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, when the antibody includes a constant region, that constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may contain amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0122] A “humanized antibody” refers to an antibody in which some, almost all, or all of the amino acids outside the CDR of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one form of antibody humanization, some, almost all, or all of the amino acids outside the CDR are replaced with amino acids derived from human immunoglobulins, while some, almost all, or all of the amino acids within one or more CDRs remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not cause the antibody to lose its ability to bind to a particular antigen. “Humanized” antibodies retain similar antigen-binding specificity to the original antibody.

[0123] A "chimeric antibody" is an antibody in which the variable region originates from one species and the constant region originates from another species. For example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0124] An "anti-antigen" antibody is an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, an anti-PD-L1 antibody specifically binds to PD-L1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

[0125] The "antigen-binding portion" (also called the "antigen-binding fragment") of an antibody refers to one or more fragments of the antibody that possess the ability to specifically bind to the antigen to which it is bound by the full-length antibody.

[0126] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells within the body. Uncontrolled cell division and proliferation result in the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0127] The term “immunotherapy” means the treatment of a subject who is suffering from a disease or at risk of developing or relapsing the disease, by means of methods including inducing, enhancing, suppressing, or otherwise modifying the immune response. The “treatment” or “therapy” of a subject means any therapeutic intervention or method performed on the subject, or the administration of an active ingredient to the subject, for the purpose of restoring, alleviating, improving, inhibiting, delaying, or preventing the onset, progression, development, worsening, or relapse of symptoms, complications, or conditions, or biochemical signs associated with the disease.

[0128] "Programmed cell death-1" (PD-1) refers to an immunosuppressive 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 of hPD-1, isotypes and species homologs, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank accession number U64863.

[0129] Programmed cell death ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands of PD-1 that downregulate T cell activation and cytokine secretion upon binding to PD-1 (the other being PD-L2). The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants of hPD-L1, isotypes and species homologs, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank accession number Q9NZQ7.

[0130] "Cytotoxic T lymphocyte antigen-4" (CTLA-4) refers to an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is exclusively expressed in 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), its variants, isotypes and species homologs, and analogs that share at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found in GenBank accession number AAB59385.

[0131] The term "subject" includes humans or non-human animals. The term "non-human animals" includes, but is not limited to, non-human primates, vertebrates such as sheep and dogs, and rodents such as mice, rats, and guinea pigs. In a preferred embodiment, the subject is human. The terms "subject" and "patient" are used interchangeably herein.

[0132] With respect to the methods and dosages of the present invention, the use of the term “constant dose” means the dose administered to a patient regardless of the patient’s body weight or body surface area (BSA). Therefore, the constant dose is provided as an absolute amount of the drug (e.g., anti-PD-1 antibody), not as a mg / kg dose. For example, a 60kg person and a 100kg person may receive the same dose of antibody (e.g., 240mg of anti-PD-1 antibody).

[0133] The use of the term “fixed dose” in the method of the present invention means that two or more different antibodies (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody, or anti-PD-L1 antibody and anti-CTLA-4 antibody) in a single composition are present in the composition, particularly in a (fixed) ratio to each other. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In one embodiment, the ratio of mg of the first antibody (e.g., anti-PD-1 antibody or PD-L1 antibody) to mg of the second antibody (e.g., anti-CTLA-4 antibody) is at least approximately 1:1, approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:30, approximately 1:40, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, approximately 1:1 The ratios are approximately 00, 1:120, 1:140, 1:160, 1:180, 1:200, 200:1, 180:1, 160:1, 140:1, 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. For example, a 3:1 ratio of anti-PD-1 antibody to anti-CTLA-4 antibody could mean that the vial contains approximately 240 mg of anti-PD-1 antibody and approximately 80 mg of anti-CTLA-4 antibody, or approximately 3 mg / ml of anti-PD-1 antibody and approximately 1 mg / ml of anti-CTLA-4 antibody.

[0134] As used herein, the term “body weight-based dose” means that the dose administered to a patient is calculated based on the patient’s body weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-PD-1 antibody, an appropriate amount of anti-PD-1 antibody can be calculated for administration (i.e., 180 mg) and used.

[0135] The “therapeutic effective dose” or “therapeutic effective dose” of a drug or therapeutic agent is the amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or the prevention of impairment or disability due to the distress of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as human subjects in clinical trials, animal model systems to predict efficacy in humans, or assays of drug activity in in vitro assays.

[0136] For example, an “anticancer agent” promotes remission of cancer in a subject. In a preferred embodiment, a therapeutically effective dose of the drug promotes remission of cancer to the point of elimination of the cancer. “Promotion of cancer remission” means that when an effective dose of the drug is administered alone or in combination with an antitumor agent, the result is a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of functional impairment or disability caused by the suffering of the disease. In addition, the terms “effective” and “efficacy” in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy means the ability of a drug to promote remission of cancer in a patient. Physiological safety means the level of toxicity or other adverse physiological effects (side effects) at the cellular, organ, and / or biological level that result from the administration of the drug.

[0137] As an example of treating tumors, such as tumors derived from NSCLC, a therapeutically effective amount of anticancer agent can preferably inhibit 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 to an untreated subject. In another preferred embodiment of the present invention, tumor remission may be observed, which lasts for at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Apart from these final measurements of therapeutic effect, the evaluation of immunotherapeutic agents must also take into account immune-related response patterns.

[0138] "Immune response" refers to the biological response within a vertebrate to foreign factors or abnormal cells, such as cancerous cells, which protect the organism from these factors and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells, Th cells, CD4 + or CD8 + T cells or T reg This includes activation or inhibition of cells, or activation or inhibition of any other cells of the immune system, such as NK cells.

[0139] The “immune-associated response pattern” refers to the clinical response patterns frequently 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 beneficial therapeutic effects observed after an initial increase in tumor volume or the appearance of new lesions, which may be classified as disease progression and synonymous with drug failure in the evaluation of conventional chemotherapy agents. Therefore, proper evaluation of immunotherapeutic agents may require long-term monitoring of their effects on the target disease.

[0140] An “immunomodulator” or “immunoregulator” refers to a factor, such as a component of a signaling pathway, that may be involved in the modulation, control, or modification of the immune response. “Modification,” “control,” or “modification” of the immune response means any alteration of the activity of cells of the immune system or such cells (e.g., effector T cells, such as Th1 cells). Such modulation may manifest as an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system, including stimulation or suppression of the immune system. Both inhibitory and stimulative immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In some embodiments, immunomodulators target molecules on the surface of T cells. An “immunomodulatory target” or “immunomodulatory target” is a molecule, such as a cell surface molecule, that is targeted for binding by a substance, drug, component, compound, or molecule, and whose activity is modified by such binding. Examples of immunomodulatory targets include cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0141] "Immunotherapy" means the treatment of a subject who has, is at risk of contracting, or is at risk of relapsing with a disease, by means of inducing, enhancing, suppressing, or otherwise modifying the immune system or immune response. In one embodiment, immunotherapy includes the administration of antibodies to a subject. In another embodiment, immunotherapy includes the administration of small molecules to a subject. In yet another embodiment, immunotherapy includes the administration of cytokines or their analogues, variants, or fragments.

[0142] "Immune stimulating therapy" or "immunostimulatory therapy" refers to a treatment (procedure) that causes an increase (induction or enhancement) of the immune response in a target, such as to treat cancer.

[0143] "Enhancement of the endogenous immune response" means an increase in the effectiveness or potency of the existing immune response in the subject. This increase in effectiveness and potency can be achieved, for example, by suppressing mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0144] The therapeutically effective dose of a drug includes the “prophylactic effective dose,” which is the amount of the drug administered alone or in combination with an antitumor agent to a subject at risk of developing cancer or having cancer recurrence (e.g., a subject with a pre-malignant condition) that inhibits the development or recurrence of cancer. In a preferred embodiment, the prophylactic effective dose completely prevents the development or recurrence of cancer. To “inhibit” the development or recurrence of cancer means either to reduce the likelihood of cancer development or recurrence, or to completely prevent the development or recurrence of cancer.

[0145] As used herein, the term “tumor mutational load” (TMB) refers to the number of somatic mutations in the tumor genome and / or the number of somatic mutations per region of the tumor genome. Germline (genetic) variants are excluded when measuring TMB because their immune system is likely to recognize them as its own. Tumor mutational load (TMB) may also be used interchangeably with “tumor mutational load,” “tumor mutational burden,” or “tumor mutational load.”

[0146] Tumor-mediated biomass (TMB) is a genetic analysis of the tumor genome and can therefore be measured using sequencing methods known to those skilled in the art. Tumor DNA can be filtered to remove germline mutations or polymorphisms compared to DNA derived from normal tissue in the patient.

[0147] In one embodiment, the tumor biomass (TMB) is determined by sequencing the tumor DNA using high-throughput sequencing techniques, such as next-generation sequencing (NGS) or NGS-based methods. In one embodiment, the NGS-based method is whole-genome sequencing (WGS), whole-exome sequencing (WES), or FOUNDATIONONE® CDX (商標)The tumors are selected from comprehensive genomic profiling (CGP) of cancer gene panels, such as the MSK-IMPACT clinical trial. In one embodiment, TMB as used herein refers to the number of somatic mutations per megabase (Mb) of sequenced DNA. In one embodiment, TMB is measured using the total number of non-synonymous mutations, such as missense mutations (i.e., mutations in specific amino acids in a protein) and / or nonsense mutations (premature termination of a protein sequence, resulting in cleavage of the protein sequence), and identified by normalizing the contrasted tumors with germ cell samples to exclude heritable germline variations. In another embodiment, TMB is measured using the total number of missense mutations in the tumor. A sufficient amount of sample is required to measure TMB. In one embodiment, tissue samples (e.g., at least 10 slides) are used for evaluation. In one embodiment, TMB is expressed as NsMs per megabase (NsMs / Mb). One megabase represents one million bases.

[0148] The TMB status can be a numerical or relative value (e.g., high, medium, or low) within the highest quantile or tertile of the control set.

[0149] As used herein, the term “high TMB” means the number of somatic mutations in the tumor genome that exceeds a number of somatic mutations that are normal or average. In one embodiment, TMB is at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, and less At least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445 , having a score of 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 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 a specific embodiment, high TMB has a score of at least 243.

[0150] In another embodiment, “high TMB” refers to TMBs within the highest quantile of the control TMB value. For example, all subjects with evaluable TMB data are grouped according to the fractal distribution of TMB, i.e., subjects are ranked from the highest to the lowest number of gene variations and classified into a defined number of groups. In one embodiment, all subjects with evaluable TMB data are ranked and divided into three groups, and “high TMB” is within the highest quantile of the control TMB value. In a particular embodiment, the quartile boundaries are 0 < 100 gene variations; 100 to 243 gene variations; and > 243 gene variations. Once ranked, subjects with evaluable TMB data should be understood to be classifiable into one of the group numbers (e.g., quartile, quintile, etc.).

[0151] In one embodiment, “high TMB” means TMB with at least approximately 20 mutations / tumor, at least approximately 25 mutations / tumor, at least approximately 30 mutations / tumor, at least approximately 35 mutations / tumor, at least approximately 40 mutations / tumor, at least approximately 45 mutations / tumor, at least approximately 50 mutations / tumor, at least approximately 55 mutations / tumor, at least approximately 60 mutations / tumor, at least approximately 65 mutations / tumor, at least approximately 70 mutations / tumor, at least approximately 75 mutations / tumor, at least approximately 80 mutations / tumor, at least approximately 85 mutations / tumor, at least approximately 90 mutations / tumor, at least approximately 95 mutations / tumor, or at least approximately 100 mutations / tumor. In one embodiment, “high TMB” means TMB with at least approximately 105 mutations / tumor, at least approximately 110 mutations / tumor, at least approximately 115 mutations / tumor, at least approximately 120 mutations / tumor, at least approximately 125 mutations / tumor, at least approximately 130 mutations / tumor, at least approximately 135 mutations / tumor, at least approximately 140 mutations / tumor, at least approximately 145 mutations / tumor, at least approximately 150 mutations / tumor, at least approximately 175 mutations / tumor, or at least approximately 200 mutations / tumor. In a particular embodiment, a tumor with high TMB has at least approximately 100 mutations / tumor.

[0152] “High TMB” is also used in mutation assays, such as FOUNDATIONONE® CDX. (商標) This can mean the number of mutations per megabase of the sequenced tumor genome, as measured by the assay. In one embodiment, high TMB may refer to FOUNDATIONONE® CDX (商標) As measured by the assay, this means 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 genome. In certain embodiments, “High TMB” refers to FOUNDATIONONE® CDX (商標) The assay implies at least 10 mutations per megabase of the sequenced genome.

[0153] As used herein, the term “intermediate TMB” means a number of somatic mutations in the tumor genome that is normal or average, or a number of somatic mutations of that magnitude, and the term “low TMB” means a number of somatic mutations in the tumor genome that is less than normal or average. In certain embodiments, “high TMB” has a score of at least 243, “intermediate TMB” has a score of 100 to 242, and “low TMB” has a score of 100 or less (or 0 to 100). “Intermediate or low TMB” is, for example, FOUNDATIONONE® CDX (商標) This means fewer than 9 mutations per megabase of the sequenced genome, as measured by assays.

[0154] The term "control TMB value" as used in this specification may refer to the TMB values ​​shown in Table 9.

[0155] In one embodiment, TMB status may correlate with smoking status. In particular, subjects who currently smoke or have smoked in the past often have more gene mutations, such as missense mutations, than non-smokers.

[0156] Tumors exhibiting high TMB, such as those derived from NSCLC, may also have high neoantigen levels. As used herein, the term “neoantigen” refers to a newly formed antigen that was not previously recognized by the immune system. A neoantigen may be a protein or peptide recognized by the immune system as foreign (or non-self). Transcription of a gene in a tumor genome carrying a somatic mutation results in mutant mRNA, which, upon translation, produces a mutant protein, which is then processed, transported into the ER lumen, and binds to the MHC class I complex, facilitating T cell recognition of the neoantigen. Recognition of the neoantigen can promote T cell activation, clonal proliferation, and differentiation into effector and memory T cells. Neoantigen levels may correlate with TMB. In some embodiments, TMB is evaluated as a surrogate value for measuring tumor neoantigen levels. The tumor-myogenic body (TMB) status of a tumor, such as a tumor derived from NSCLC, can be used as a factor, alone or in combination with other factors, in determining whether a patient is likely to benefit from a particular anticancer agent or type of treatment, such as a combination therapy involving (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. In one embodiment, a high TMB status (or high TMB) indicates a high likelihood of benefiting from immuno-oncology and can therefore be used to identify patients who are likely to benefit from a combination therapy involving (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. Similarly, tumors with high neoantigen levels and high TMB are more likely to be immunogenic than tumors with low neoantigen levels and low TMB. In addition, tumors with high neoantigen / high TMB are more likely to be recognized as non-self by the immune system and therefore to induce an immune-mediated antitumor response. In one embodiment, high TMB status and high neoantigen levels suggest a high likelihood of benefiting from immuno-oncology, for example, combination therapies including (a) anti-PD-1 or anti-PD-L1 antibodies and (b) anti-CTLA-4 antibodies.As used herein, “to obtain a benefit from treatment” means an improvement in one or more of the following: overall survival, progression-free survival, partial response, complete response, and overall response rate, which may include a reduction in tumor growth or size, a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or impairment due to the suffering of the disease.

[0157] Other factors, such as environmental factors, may be related to TMB status. For example, smoking status in NSCLC patients correlated with TMB distribution, with current and former smokers having higher median TMB compared to non-smokers. See Peters et al., AACR, April 1-5, 2017, Washington, DC. The presence of driver mutations in NSCLC tumors was associated with juvenile, female, and non-smoking status. See Singal et al., ASCO, June 1-5, 2017; Chicago, IL. A tendency was observed to associate the presence of driver mutations such as EGFR, ALK, or KRAS with low TMB (P=0.06). See Davis et al., AACR, April 1-5, 2017, Washington, DC.

[0158] As used herein, the term “somatic mutation” refers to an acquired mutation in DNA that occurs after conception. Somatic mutations can occur in any cell of the body except germ cells (sperm and egg cells) and are therefore not inherited by children. These mutations may, but not necessarily, cause cancer or other diseases. The term “germ cell mutation” refers to a genetic change in germ cells (egg or sperm) that is incorporated into the DNA of any cell in the offspring's body. Germ cell mutations are inherited from parents to offspring and are also called hereditary mutations. In TMB analysis, germ cell mutations are considered the “baseline” and are subtracted from the number of mutations found in tumor biopsies to determine the TMB within the tumor. Because germ cell mutations are present in all cells of the body, their presence can be determined by less invasive sample collection methods than tumor biopsies, such as blood or saliva. Germ cell mutations may increase the risk of developing certain cancers and may play a role in the response to chemotherapy.

[0159] The terms “measure,” “measured,” or “measured” mean, when referring to the TMB (Therapeutic Microbiome) status, to examine the measurable amount of somatic variation in the biological sample of interest. Measurement may be recognized as being performed by sequencing nucleic acids in the sample, e.g., cDNA, mRNA, exoRNA, ctDNA, and cfDNA. Measurement may be performed on the sample of interest and / or control samples, and may, for example, be newly detected or correspond to previous measurements. Measurement may be performed using, for example, PCR, qPCR, Sanger sequencing, genome profiling (including comprehensive gene panels), exome sequencing, genome sequencing, and / or other methods known to those skilled in the art. In some embodiments, the measurement identifies genomic alterations in sequenced nucleic acids. Genomic (or gene) profiling may involve a panel of a given set of genes, e.g., 150–500 genes, and in some examples, the genomic alterations evaluated by the gene panel correlate with the total somatic variation being evaluated. When referring to sequencing, the term “gene” as used herein includes DNA coding regions (e.g., exons), non-coding regions of DNA associated with coding regions (e.g., introns and promoters), and mRNA transcripts.

[0160] As used herein, the term “genome modification” means a change (or mutation) in the nucleotide sequence of a tumor genome (a change not present in germline nucleotide sequences), and in some embodiments, includes, but is not limited to, base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and combinations thereof, which are non-synonymous mutations. In certain embodiments, the genome modification measured in a biological sample is a missense mutation.

[0161] 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 protein-coding regions (exons) of the genome.

[0162] As used herein, “cancer genome panel,” “hereditary cancer panel,” “comprehensive cancer panel,” or “multigene cancer panel” means a method for sequencing a subset of target oncogenes, including coding regions, introns, promoters, and / or mRNA transcripts. In one embodiment, a CGP involves 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 oncogenes.

[0163] The term “genome profiling assay,” “comprehensive genome profiling,” or “CGP” refers to an assay that analyzes a panel of genes and selects introns for in vitro diagnosis. CGP combines NGS and targeted bioinformatics analysis to screen for known oncogene mutations of clinical relevance. This method can be used to capture missed mutations by testing for “hotspots” (e.g., BRCA1 / BRCA2 mutations or microsatellite markers). In one embodiment, CGP further includes one or more mRNA transcripts, non-coding RNAs, and / or promoter regions. In one embodiment, the genes in the panel are cancer-related genes. In another embodiment, the genome profiling assay is performed using FOUNDATIONONE (登録商標) It is an assay.

[0164] The term “harmonization” refers to a test conducted to determine the comparability between two or more measurement methods and / or diagnostic tests. Harmonization tests provide a systematic approach to addressing how diagnostic tests compare to each other and the issue of compatibility when used to determine a patient’s tumor biomarker status. Generally, at least one well-characterized measurement and / or diagnostic test is used as a standard for comparison with other tests. Consensus assessment is often used in harmonization tests.

[0165] As used herein, “concordance” refers to the degree of agreement between two measurements and / or diagnostic tests. Concordance can be established using both qualitative and quantitative methods. The quantitative method for assessing concordance varies depending on the type of measurement. A particular measurement can be expressed as either 1) a categorical / binary variable or 2) a continuous variable. For “categorical / binary variables” (e.g., above or below the TMB cutoff), concordance can be assessed using percentage agreement such as overall percentage agreement (OPA), positive percentage agreement (PPA), or negative percentage agreement (NPA). For “continuous variables” (e.g., TMB by WES), Spearman’s rank correlation or Pearson’s correlation coefficient (r), taking values ​​of -1 ≤ r ≤ +1, is used to assess the agreement across the entire spectrum of values ​​(note: r = +1 or -1 means that each variable is perfectly correlated). The term “analytical agreement” refers to the degree of agreement between the performance of two assays or diagnostic tests to support clinical use (e.g., identification of biomarkers, genome modification types and genomic signatures, and assessment of test reproducibility). The term “clinical agreement” refers to the degree of agreement between two assays or diagnostic tests in how they correlate with clinical outcomes.

[0166] The term "microsatellite instability" or "MSI" refers to a change in the DNA of certain cells (such as tumor cells) where the number of microsatellites (short repeat sequences of DNA) differs from the number of repeats present in the DNA at the time of inheritance. MSI can be classified as high microsatellite instability (MSI-H) or low microsatellite instability (MSI-L). Microsatellites are short tandem DNA repeat sequences of 1–6 base pairs. They are prone to DNA replication errors and are repaired by mismatch repair (MMR). Therefore, microsatellites are a good indicator of genomic instability, particularly deficiencies in mismatch repair mechanisms (dMMR). MSI is typically diagnosed by screening for five microsatellite markers (BAT-25, BAT-26, NR21, NR24, and NR27). MSI-H indicates the presence of at least two instability markers among the five microsatellite markers analyzed (or more than 30% of markers when using a larger panel). MSI-L means instability of one MSI marker (or 10% to 30% of markers if a larger panel is used). MSS means that there are no unstable microsatellite markers.

[0167] As used herein, the term “biological sample” means biological material isolated from a subject. Examples of biological samples include any biological material suitable for determining TMB by sequencing nucleic acids in a tumor (or circulating tumor cells) and identifying genomic modifications in the sequenced nucleic acids. A biological sample may be any suitable biological tissue or liquid, such as tumor tissue, blood, plasma, and serum. In one embodiment, the sample is a tumor tissue biopsy, e.g., formalin-fixed paraffin-embedded (FFPE) tumor tissue or fresh-frozen tumor tissue. In another embodiment, the biological sample is, in some aspects, a liquid biopsy containing one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA, and cfDNA.

[0168] The terms “approximately once every week,” “approximately once every two weeks,” or other similar terms for dosing intervals as used herein mean approximate numbers. “Approximately once every week” may include every 7 days ± 1 day, i.e., every 6 days to every 8 days. “Approximately once every two weeks” may include every 14 days ± 3 days, i.e., every 11 days to every 17 days. Similar approximations apply, for example, to approximately once every 3 weeks, approximately once every 4 weeks, approximately once every 5 weeks, approximately once every 6 weeks, and approximately once every 12 weeks. In one embodiment, a dosing interval of approximately once every 6 weeks or approximately once every 12 weeks means that the first dose may be administered on any day in the first week, followed by the next dose on any day in the sixth or twelfth week, respectively. In other embodiments, an interval of approximately every 6 weeks or approximately every 12 weeks means that the first dose is administered on a specific day in the first week (e.g., Monday), followed by subsequent doses on the same day of the week in the sixth or twelfth week, respectively (i.e., Monday).

[0169] The use of alternative words (e.g., “or”) should be understood to mean one, both, or any combination thereof of the alternatives. The indefinite articles “a” or “an” used herein should be understood to mean any one or more listed or enumerated components.

[0170] The terms “about” or “essentially including” mean a value or composition that falls within the acceptable margin of error of a particular value or composition as determined by those skilled in the art, which may depend in part on how the value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “about” or “essentially including” may mean a standard deviation of 1 or less or greater than 1, according to the practice in the art. Alternatively, “about” or “essentially including” may mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or five times a certain value. When a particular value or composition is described in this application and claims, unless otherwise specifically stated, the meaning of “about” or “essentially including” should be considered to be within the acceptable margin of error of that particular value or composition.

[0171] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include, unless otherwise stated, all integer values ​​within the listed range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer).

[0172] A list of abbreviations is shown in Table 1. [Table 1] [Table 2]

[0173] Various aspects of the present invention are described in more detail in the following subsections.

[0174] Method of the present invention A particular aspect of the present invention relates to a method for treating a subject suffering from an NSCLC-derived tumor with a high TMB status, comprising administering to the subject a therapeutically effective amount of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. Another aspect of the present invention relates to a method for identifying a subject suffering from an NSCLC-derived tumor and suitable for combination therapy of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody, comprising measuring the TMB status of a biological sample of the subject, wherein the TMB status includes at least about 10 mutations per megabase of the genome examined, and the subject has been identified as suitable for combination therapy. The present invention is based on the fact that tumor immunogenicity is directly related to TMB and / or neoantigen levels.

[0175] As a tumor grows, somatic mutations not present in germ cell DNA accumulate. TMB refers to the number of somatic mutations in the tumor genome (after considering germ cell mutation DNA) and / or the number of somatic mutations per unit area of ​​the tumor genome. The acquisition of somatic mutations and the resulting higher TMB can be influenced by different mechanisms, such as exposure to exogenous mutation inducers (e.g., smoking or UV light exposure) and DNA mismatch repair mutations (e.g., MSI in colorectal and esophageal cancer). In solid tumors, approximately 95% of mutations are single nucleotide substitutions (Vogelstein et al., Science (2013) 339:1546-1558). As used herein, “non-synonymous mutation” refers to a nucleotide mutation that alters the amino acid sequence of a protein. Both missense and nonsense mutations can be non-synonymous mutations. As used herein, “missense mutation” refers to a non-synonymous point mutation in which a single nucleotide change results in a codon encoding a different amino acid. In this specification, “nonsense mutation” refers to a non-synonymous point mutation in which a codon is altered to an immature stop codon that results in the cleavage of the resulting protein.

[0176] In one embodiment, somatic mutations may be expressed at the RNA and / or protein level, resulting in the generation of neoantigens (also called neoepitopes). Neoantigens can influence immune-mediated antitumor responses. For example, recognition of neoantigens may promote T cell activation, clonal proliferation, and differentiation into effector and memory T cells.

[0177] When a tumor develops, early clonal mutations (or "trunk mutations") can be carried by almost all or all tumor cells, whereas later mutations (or "branched mutations") can occur only in tumor cells or parts of the tumor region (Yap et al., Sci Tranl Med (2012) 4:1-5; Jamai-Hanjani et al., (2015) Clin Cancer Res 21:1258-1266). As a result, neoantigens derived from clonal "trunk" mutations are more widely distributed in the tumor genome than "branched" mutations, which can lead to a large number of T cells that respond to clonal neoantigens (McGranahan et al., (2016) 351:1463-1469). Generally, tumors with high TMB also have a high neoantigen load, which can lead to high oncoimmunogenicity and increased T cell responsiveness and antitumor response. Thus, cancers with high TMB may respond well to immunotherapy, such as treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0178] Advances in sequencing technology enable the assessment of the genomic mutation status of tumors. Sequencing methods known to those skilled in the art can be used to sequence nucleic acids derived from tumor genomes (e.g., obtained from biological samples from subjects affected by tumors). In one embodiment, PCR or qPCR, Sanger sequencing, or next-generation sequencing (e.g., genomic profiling, exome sequencing, or genome sequencing) can be used to measure TMB. In one embodiment, TMB status is measured using genomic profiling. Genomic profiling involves analyzing nucleic acids (including coding and non-coding regions) derived from tumor samples and can be performed using methods including selection of optimized nucleic acids to be incorporated, read alignment, and mutation calling. In one embodiment, gene profiling provides next-generation sequencing (NGS)-based analysis of tumors, which can be optimized on a per-cancer, per-gene, and / or per-site basis. Genome profiling can incorporate the use of multiple individually modified alignment methods or algorithms to optimize performance in sequencing methods, particularly those involving massively parallel sequencing of numerous diverse genetic events across many different genes. Genome profiling provides comprehensive analysis of target cancer genomes at clinical-grade quality, and the results of the genetic analysis may be relevant to the scientific and medical knowledge needed to improve the quality and effectiveness of cancer treatment.

[0179] Genome profiling relates to a predetermined set of gene panels containing only 5 genes, 1000 genes, approximately 25 to 750 genes, approximately 100 to 800 genes, approximately 150 to 500 genes, approximately 200 to 400 genes, or approximately 250 to 350 genes. In one embodiment, the genome profile includes at least 300 genes, at least 305 genes, at least 310 genes, at least 315 genes, at least 320 genes, at least 325 genes, at least 330 genes, at least 335 genes, at least 340 genes, at least 345 genes, at least 350 genes, at least 355 genes, at least 360 genes, at least 365 genes, at least 370 genes, at least 375 genes, at least 380 genes, at least 385 genes, at least 390 genes, at least 395 genes, or at least 400 genes. In another embodiment, the genome profile includes at least 325 genes. In certain embodiments, the genome profile includes at least 315 cancer-related genes and 28 gene introns (FOUNDATIONONE®), or the whole DNA coding sequences of 406 genes, rearranged gene introns of 31 genes, and 265 gene RNA sequences (cDNA) (FOUNDATIONONE® Heme). In another embodiment, the genome profile includes 26 genes and 1000 associated mutations (EXODX® Solid Tumor). In yet another embodiment, the genome profile includes 76 genes (Guardant360). In yet another embodiment, the genome profile includes 73 genes (Guardant360). In yet another embodiment, the genome profile includes 354 genes and 28 gene introns for rearrangement (FOUNDATIONONE® CDX (商標)) includes. In a particular embodiment, the genome profile is FOUNDATIONONE® F1CDx. In another embodiment, the genome profile includes 468 genes (MSK-IMPACT (商標) ) and one or more genes may be added to the genomic profile as further genes identified as being related to oncology.

[0180] FOUNDATIONONE® Assay The FOUNDATIONONE® assay is a comprehensive genomic profiling assay for solid tumors, including, but not limited to, lung cancer, colon cancer, and breast cancer, as well as melanoma and ovarian cancer. The FOUNDATIONONE® assay uses hybrid capture next-generation sequencing to identify genomic modifications (base substitutions, insertions and deletions, copy number changes and rearrangements) and select genomic characteristics (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 derived from 28 genes. A complete list of FOUNDATIONONE® assay genes is shown in Tables 2 and 3. See FOUNDATIONONE:Technical Specifications, Foundation Medicine, Inc. (entirely incorporated herein by reference), available on FoundationMedicine.com, last accessed March 16, 2018.

[0181] [Table 3] [Table 4]

[0182] [Table 5]

[0183] EXODX® Solid Tumor Assay In one embodiment, TMB is measured using the EXODX® Solid Tumor Assay. The EXODX® Solid Tumor Assay is an exoRNA and cfDNA-based assay that detects actionable mutations in the cancer pathway. The EXODX® Solid Tumor Assay is a plasma-based assay that does not require tissue samples. The EXODX® Solid Tumor Assay covers 26 genes and 1000 mutations. Table 4 shows the specific genes covered by the EXODX® Solid Tumor Assay. See the Plasma-Based Solid Tumor Mutation Panel Liquid Biopsy (Exosome Diagnostics, Inc.) available at exosomedx.com, last accessed March 25, 2019.

[0184] [Table 6]

[0185] 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.

[0186] [Table 7]

[0187] [Table 8]

[0188] [Table 9]

[0189] [Table 10]

[0190] ILLUMINA® TruSight Assay In one embodiment, TMB is determined using the TruSight Tumor 170 assay (ILLUMINA). The TruSight Tumor 170 assay is a next-generation sequencing assay that covers 170 genes associated with common solid tumors, simultaneously analyzing DNA and RNA. The TruSight Tumor 170 assay evaluates fusions, splice variants, insertions / deletions, single nucleotide variants (SNVs), and amplifications. The list of genes for the TruSight Tumor 170 assay is shown in Tables 12 to 14.

[0191] [Table 11]

[0192] [Table 12]

[0193] [Table 13]

[0194] FOUNDATIONONE® F1CDx Assay FOUNDATIONONE (registered trademark) CDX (商標)(“F1CDx”) is a next-generation sequencing-based in vitro diagnostic device for detecting 324 genes and selected gene rearrangements, as well as substitution, insertion, and deletion changes (indels) and copy number changes (CNAs) in genomic characteristics (including microsatellite instability (MSI) and tumor mutational load (TMB)), using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. F1CDx is approved by the U.S. Food and Drug Administration (FDA) for use in several tumors, including NSCLC, melanoma, breast cancer, colorectal cancer, and ovarian cancer.

[0195] The F1CDx assay uses a single-pass DNA extraction method from standard FFPE biopsy or surgical excision specimens. 50–1000 ng of these samples are captured based on whole-genome shotgun library construction and hybridization, utilizing all coding exons, one promoter region, one non-coding (ncRNA), and selected intron regions from 309 cancer-related genes (21 of which contain coding exons). Tables 12 and 13 provide a complete list of genes included in F1CDx. In total, the assay detects alterations to a total of 324 genes. Using the ILLUMINA® HiSeq4000 platform, the libraries selected by hybrid capture are sequenced with high uniform depth (targeting a central coverage of >500X with >99% exon coverage at >100X). The sequence data is then processed using a customized analysis pipeline designed to detect all classes of genomic modifications, including base substitutions, indels, copy number changes (amplification and homozygous gene deletions), and selected genomic rearrangements (e.g., gene fusions). Furthermore, genomic characteristics including microsatellite instability (MSI) and tumor mutational load (TMB) have been reported.

[0196] [Table 14] [Table 15]

[0197] [Table 16]

[0198] The F1CDx assay identifies various modifications (including substitutions, insertions / deletions, and CNAs) in gene and / or intron sequences. The F1CDx assay has been identified as consistent with previously externally validated NGS assays and FOUNDATIONONE®(F1 LDT) assays. See FOUNDATIONONE®CDX-:Technical Information, Foundation Medicine, Inc. (entirely incorporated herein by reference), available on FoundationMedicine.com, last accessed March 25, 2019.

[0199] MSK-IMPACT (商標) In one aspect, the TMB state is MSK-IMPACT (商標) Evaluation is performed using an assay. MSK-IMPACT (商標) The assay uses next-generation sequencing to analyze the mutation status of 468 genes. The target gene is ILLUMINA HISEQ. (商標) It is captured by the device and sequenced. MSK-IMPACT (商標) The assay is approved by the U.S. FDA for the detection of somatic mutations and microsatellite instability in solid malignancies. MSK-IMPACT (商標)Table 14 shows the complete list of the 468 genes analyzed by the assay. See the Evaluation of Automatic Class III Designation for MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets): Decision Summary (U.S. Food and Drug Administration), available on accessdata.fda.gov as of November 15, 2017.

[0200] [Table 17] [Table 18] [Table 19]

[0201] NEOGENOMICS (registered trademark) NEOTYPE (商標) Assay In one aspect, TMB is NEOGENICS® NEOTYOPE (商標) Determined using an assay. In one embodiment, TMB is NEOTYPE (商標) It is determined using a discovery profile. In one embodiment, TMB is determined using a NEOTYPE solid tumor profile. The NEOGENICS assay measures the number of non-synonymous DNA coding sequence changes per megabase of sequenced DNA.

[0202] ONCOMINE (商標) Tumor mutational burden assay In one aspect, TMB is THERMOPISHER SCIENTIFIC® ONCOMINE (商標)It is determined using a tumor mutation assay. In one embodiment, TMB is THERMFOFISHER SCIENTIFIC® ION TORRENT (商標) ONCOMINE (商標) Determined using a tumor mutation assay. ION TORRENT (商標) ONCOMINE (商標) The tumor mutation assay is a targeted NGS assay that quantifies somatic mutations to determine tumor mutational load. This assay covers 1.7 Mb of DNA. THERMOFISHER SCIENTIFIC® ION TORRENT (商標) ONCOMINE (商標) Table 15 shows a complete list of the 408 genes analyzed by the tumor mutation assay (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).

[0203] [Table 20] [Table 21]

[0204] NOVOGENE (商標) NOVOPM (商標) Assay In one aspect, TMB is NOVOGENE (商標) NOVOPM (商標) Determined using an assay. In one embodiment, TMB is NOVOGENE (商標) NOVOPM (商標) Determined using a cancer panel assay. NOVOGENE (商標)The NOVOPM cancer panel assay analyzes the entire coding regions of 548 genes and the introns (representing approximately 1.5 Mb of DNA) of 21 genes, making it a comprehensive NGS cancer panel suitable for the diagnosis and / or treatment of solid tumors according to National Comprehensive Cancer Information Network (NCCN) guidelines and medical literature. The assay detects genomic abnormalities such as SNVs, indels (InDel), fusions, and copy number variations (CNVs).

[0205] Other TMB assays In one embodiment, TMB is determined using the TMB assay provided by CARIS® Life Sciences. In another embodiment, TMB is determined using the PESONALIS® ACE ImmunoID assay. In another embodiment, TMB is determined using the PGDX® CANCERXOME assay. (商標) - Determined using the R assay.

[0206] In yet another specific embodiment, the genome profiling detects all variants, i.e., single-nucleotide variants, insertions / deletions (indels), copy number variations, and rearrangements, such as translocations, expression, and epigenetic markers.

[0207] A comprehensive gene panel often includes predetermined genes selected based on the tumor type being analyzed. Therefore, the genomic profile used to measure TMB status can be selected based on the tumor type the subject is affected by. In one embodiment, the genomic profile may include a set of genes specific to solid tumors. In another embodiment, the genomic profile may include a set of genes specific to hematological malignancies and sarcomas.

[0208] Thanks for watching ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, PDCD1LG2, RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDG FROM RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf3). 9) KAT6A(MYST3), MRE11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRK L, 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, AMER1 (FAM123B), C11orf30(EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT(Environment), APC, CA RD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PIK3R1, SDHA, TET2, AR, CBFB, CTNNB1, FGF10, GPR124, KEL, MYCL (MYCL1), PIK3R2, SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC, TNFAIP3, ARFRP1, CCND1, CY LD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFRSF14, ARID1A, CCND2, DAXX, FGF23, GSK3B, KMT2A(MLL), NF1, POLD1, SE TD2, TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C(MLL3), NF2, POLE, SF3B1, TOP2A, ARID2, CCNE1, DICER1, FGF4, HGF KMT2D(MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274, DNMT3A, FGF6, HNF1A, KRAS, NFKBIA, PRDM1, SMAD2, TSC1.ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKAR1A, SMAD4, TSHR , ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMAR CB1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1, NRAS, PTCH1, SNCAIP, W ISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2, NSD1, PTEN, SOCS1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BC The analysis includes one or more genes selected from the group consisting of L6, 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 combinations thereof. In other embodiments, the TMB analysis includes ETV4, TMPRSS2, ETV5, BCR, ETV1,This further includes identifying genomic modifications in one or more of ETV6 and MYB.

[0209] These are ABL1, 12B, ABL2, ACTB, ACVR1, and ACVR1 B, AGO2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, AMER1( FAM123B, AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1, ARHGAP26(GRAF), ARID1A, ARID1B, ARID2, ARID 5B, ARv7, ASMTL, ASXL1, ASXL2, ATM, ATR, ATRX, AURKA, AURKB. AXIN1, AXIN2, AXL, B2M, BABAM1, BAP1, BARD1, BBC3, BCL10, BC L11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BR IP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD11, CARM1, CASP 8. CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC4 2. CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBPA CENPA, CHD2, CHD4, CHEK1, CHEK2, CIC, CIITA, CKS1B, CPS1, CREBBP, CRKL, CRLF2, CSDE1, CSF1R, CSF3R, CTCF, 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-A LK, EP300, EPAS1, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2, ERBB3 ERBB4, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERF, ERG, ERRFI1, ERRFl1, ESR 1, ETS1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXOSC6, EZH1, EZH2, FAF1, FAM175A FAM46C, FAM58A, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL FAS, FAS(TNFRSF6), FAT1, FBXO11, FBXO31, FBXW7, FGF1, FGF10, FGF12, FG F14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGFR1 FGFR2, FGFR3, FGFR4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH1, FOXA 1, FOXL2, Fキソ1, Fキソ3, FOXP1, FRS2, FUBP1, FYN, GABRA6, GADD45B, GATA1. GATA2、GATA3、GATA4、GATA6、GEN1、GID4(C17orf39)、GID4(C17orf39)、GLI 1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRIN2A, GR M3, GSK3B, GTSE1, H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, Hedgehog, HER -2 / NEU;ERBB2、HGF、HIST1H1C、HIST1H1D、HIST1H1E、HIST1H2AC、HIST1H2A G, HIST1H2AL, HIST1H2AM, HIST1H2BC, HIST1H2BD, HIST1H2BJ, HIST1H2BK, H IST1H2BO、HIST1H3A、HIST1H3B、HIST1H3C、HIST1H3D、HIST1H3E、HIST1H3F HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HIST2H3C, HIST2H3D, HIST3H3HLA-A, HLA-B, HNF1A, HOXB13, HRAS, HSD3B1, HSP90AA1, IK, ICOSLG, ID3, IDH1, IDH2, IFNGR1, IGF1, IGF1R, IGF2, IKBKE, IKZF1, IKZF2, IKZF3, IL10, IL7R, INHA, INHBA, INPP4A, INPP4B, INPP5D(SHIP), INPPL1, INSR, IRF1, IRF2, IRF4, I RF8, IRS1, IRS2, JAK1, JAK2, JAK3, JARID2, JUN, K14, KAT6A(MYST3), KAT6A(MYST3), KDM2B, KDM4C, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIF5B, KIT, KLF4, KLHL6, KMT2A, KMT2A(MLL), KMT2B, KMT2C, KMT2C(MLL3), KMT2D, KMT2D(MLL2) KNSTRN、KRAS、LAMP1、LATS1、LATS2、LEF1、LMO1、LRP1B、LRRK2、LTK、LYN、LZTR1、MAF、MAFB、MAGED1、MAGI2、MALT1、MAP 2K1、MAP2K1(MEK1)、MAP2K2、MAP2K2(MEK2)、MAP2K4、MAP3、MAP3K1、MAP3K13、MAP3K14、MAP3K6、MAP3K7、MAPK1、MAPK3 、MAPKAP1、MAX、MCL1、MDC1、MDM2、MDM4、MED12、MEF2B、MEF2C、MEK1、MEN1、MERTK、MET、MGA、MIB1、MITF、MKI67、MKNK1、 MLH1、MLLT3、MPL、MRE11A、MRE11A、MSH2、MSH3、MSH6、MSI1、MSI2、MST1、MST1R、MTAP、MTOR、MUTYH、MYC、MYCL” L1)、MYCL(MYCL1)、MYCL1、MYCN、MYD88、MYO18A、MYOD1、NBN、NCOA3 、NCOR1、NCOR2、NCSTN、NEGR1、NF1、NF2、NFE2L2、NFKBIA、NKX2-1、NK NS D1、NT5C2、NTHL1、NTRK1、NTRK2、NTRK3、NUF2、NUP93、NUP98、P2RY8、PAG1、PAK1、PAK3、PAK7、PALB2、PARK2、PARP1、PARP2、PARP3、PASK、PAX3、PAX5、PAX7、PBRM1、PC、PCBP1、PCLO、PDCD1、PDCD1(PD-1)、PDCD11、PDCD1LG2、PDCD1LG2(PD-L2)、PDGFRA、PDGFRB、PDK1、PDPK1、PGR、PHF6、PHOX2B、PIK3C2B、PIK3C2G、PIK3C3、PIK3CA、PIK3CB、PIK3CD、PIK3CG、PIK3R1、PIK3R2、PIK3R3、PIM1、PLCG2、PLK2、PMAIP1、PMS1、PMS2、PNRC1、POLD1、POLE、POT1、PPARG、PPM1D、PPP2、PPP2R1A、PPP2R2A、PPP4R2、PPP6C、PRDM1、PRDM14、PREX2、PRKAR1A、PRKCI、PRKD1、PRKDC、PRSS8、PTCH1、PTEN、PTP4A1、PTPN11、PTPN2、PTPN6(SHP-1)、PTPRD、PTPRO、PTPRS、PTPRT、QKI、R1A、RAB35、RAC1、RAC2、RAD21、RAD50、RAD51、RAD51B、RAD51C、RAD51D、RAD52、RAD54L、RAF1、RANBP2、RARA、RASA1、RASGEF1A、RB1、RBM10、RECQL、RECQL4、REL、RELN、RET、RFWD2、RHEB、RHOA、RICTOR、RIT1、RNF43、ROS1、RPS6KA4、RPS6KB1、RPS6KB2、RPTOR、RRAGC、RRAS、RRAS2、RTEL1、RUNX1、RUNX1T1、RXRA、RYBP、S1PR2、SDHA、SDHAF2、SDHB、SDHC、SDHD、SERP2、SESN1、SESN2、SESN3、SETBP1、SETD2、SETD8、SF3B1、SGK1、SH2B3、SH2D1A、SHOC2、SHQ1、SLIT2、SLX4、SMAD2、SMAD3、SMAD4、SMARCA1、SMARCA4、SMARCB1、SMARCD1、SMC1A、SMC3、SMO、SMYD3、SNCAIP、SOCS1、SOCS2、SOCS3、SOS1、SOX10、SOX17、SOX2、SOX9、SPEN、SPOP、SPRED1、SPTA1、SRC、SRSF2、STAG2、STAT3、STAT4、STAT5A、STAT5B、STAT6、STK11、STK19、ST K40、SUFU、SUZ12、SYK、TAF1、TAP1、TAP2、TBL1XR1、TBX3、TCEB1、TCF3、TCF3 (E2A)、TCF7L2、TCL1A(TCL1)、TEK、TERC、TERT、TERTプロモーター、TET1、TET2、TFRC、TGFBR1、TGFBR2、TIPARP、TLL2、TMEM127、TMEM30A、TMPRSS2、TMSB4XP8(T MSL3)、TNFAIP3、TNFRSF11A、TNFRSF14、TNFRSF17、TOP1、TOP2A、TP53、TP53 BP1、TP63、TRAF2、TRAF3、TRAF5、TRAF7、TSC1、TSC2、TSHR、TUSC3、TYK2、TYR O3、U2AF1、U2AF2、UPF1、VEGFA、VHL、VTCN1、WDR90、WHSC1、WHSC1(MMSETまたはNSD2)、WHSC1L1、WISP3、WT1、WWTR1、XBP1、XIAP、XPO1、XRCC2、YAP1、YES1、YY 1AP1、ZBTB2、ZFHX3、ZMYM3、ZNF217、ZNF24(ZSCAN3)、ZNF703、ZRSR2、0082、 SEPT9、81RC2、81RC3、81RC5、8AI3、8CL10、8CL118、8CL11A、8CL2、8CL2L1、8 CL2L2、8CL3、8CL6、8CL9、8CR、8LM、8LNK、8MPR1A、8RD3、8TK、8U818、A8L2、A CVR2A、ADAMTS2、AFF1、AFF3、AKAP9、ARNT、ATF1、AURK8、AURKC、CASCS、CDH11 、CDH2、CDH20、CDH5、CMPK1、COL1A1、CRBN、CREB1、CRTC1、CSMD3、CYP2C19、C YP2D6、DCC、DDIT3、DEK、DPYD、DST、EP400、EXT1、EXT2、FAM123B、FANCJ、FLl 1、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, PDGF8, PER1, PGAP3, PHOX28, PIK3C28, PKHD1, PLAG1, PLCG1, PLEKHGS, PML, POU5F1, PSIP1, PTGS2, RADSO, RALGDS, RHOH, RNASEL, RNF2, It includes one or more genes selected from the group consisting of 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 combinations thereof.

[0210] These are ABL1, 12B, ABL2, ACTB, and ACVR1. ACVR1B, AGO2, AKT1, AKT2, AKT3, ALK, ALOX, ALOX12B, AMER1, A MER1(FAM123BまたはWTX), AMER1(FAM123B), ANKRD11, APC, APH1A, AR, ARAF, ARFRP1, ARHGAP26(GRAF), ARID1A, ARID1B, ARID2. ARID5B, ARv7, ASMTL, ASXL1, ASXL2, ATM, ATR, ATRX, AURKA, AU RKB, AXIN1, AXIN2, AXL, B2M, BABAM1, BAP1, BARD1, BBC3, BCL1 0, BCL11B, BCL2, BCL2L1, BCL2L11, BCL2L2, BCL6, BCL7A, BCOR, BCORL1, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1 BRIP1(BACH1), BRSK1, BTG1, BTG2, BTK, BTLA, C11orf30(EMSY), C11orf30, C11orf30(EMSY), CAD, CALR, CARD11, CARM1, CA SP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CCT6B, CD22, CD274, CD274(PD-L1), CD276, CD36, CD58, CD70, CD79A, CD79B, CDC 42 CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2Ap14ARF, CDKN2Ap16INK4A, CDKN2B, CDKN2C, CEBP A, CENPA, CHD2, 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. DH2, DICER1, DIS3, DNAJB1, DNM2, DNMT1, DNMT3A, DNMT3B, DOT1L, DROSHA, DTX1, DUSP2, DUSP4, DUSP9, E2F3.EBF1, ECT2L, EED, EGFL7, EGFR, EIF1AX, EIF4A2, EIF4E, ELF3, ELP2, EML4, E ML4-ALK, EP300, EPAS1, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, EPHB4, ERBB2 ERBB3, ERBB4, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERF, ERG, ERRFI1, ERRFl 1, ESR1, ETS1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXOSC6, EZH1, EZH2, FAF1, FA M175A, FAM46C, FAM58A, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI FANCL, FAS, FAS(TNFRSF6), FAT1, FBXO11, FBXO31, FBXW7, FGF1, FGF10, FG F12, FGF14, FGF19, FGF2, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9 FGFR1, FGFR2, FGFR3, FGFR4, FH, FHIT, FLCN, FLI1, FLT1, FLT3, FLT4, FLYWCH 1, FOXA1, FOXL2, Fキソ1, Fキ3, FOXP1, FRS2, FUBP1, FYN, GABRA6, GADD45B. STEP1、STEP2、STEP3、STEP4、STEP6、GEN1、GID4(C17orf39)、GID4(C17orf3 9) GLI1, GLl1, GNA11, GNA12, GNA13, GNAQ, GNAS, GPR124, GPS2, GREM1, GRI N2A, GRM3, GSK3B, GTSE1, H3F3A, H3F3B, H3F3C, HDAC1, HDAC4, HDAC7, Hedge hog、HER-2 / NEU;ERBB2、HGF、HIST1H1C、HIST1H1D、HIST1H1E、HIST1H2AC、H IST1H2AG, HIST1H2AL, HIST1H2AM, HIST1H2BC, HIST1H2BD, HIST1H2BJ, HIS T1H2BK, HIST1H2BO, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HIST2H3C, HIST2H3DHIST3H3, HLA-A, HLA-B, HNF1A, HOXB13, HRAS, HSD3B1, HSP90AA1, ICK, ICOSLG, ID3, IDH1, IDH2, IFNGR1, IGF1, IGF1R, IGF2, IKBKE, IKZF1, IKZF2, IKZF3, IL10, IL7R, INHA, INHBA, INPP4A, INPP4B, INPP5D(SHIP), INPPL1, INSR, IRF1, IRF2 IRF4, IRF8, IRS1, IRS2, JAK1, JAK2, JAK3, JARID2, JUN, K14, KAT6A(MYST3), KAT6A(MYST3), KDM2B, KDM4C, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIF5B, KIT, KLF4, KLHL6, KMT2A, KMT2A(MLL), KMT2B, KMT2C, KMT2C(MLL3), KMT2D, KMT2D(ML) L2, KNSTRN, KRAS, LAMP1, LATS1, LATS2, LEF1, LMO1, LRP1B, LRRK2, LTK, LYN, LZTR1, MAF, MAFB, MAGED1, MAGI2, MALT1, MAP2K1, MAP2K1(MEK1), MAP2K2, MAP2K2(MEK2), MAP2K4, MAP3, MAP3K1, MAP3K13, MAP3K14, MAP3K6, MAP3K7, MAPK1, MAP K3、MAPKAP1、MAX、MCL1、MDC1、MDM2、MDM4、MED12、MEF2B、MEF2C、MEK1、MEN1、MERTK、MET、MGA、MIB1、MITF、MKI67、MKNK1 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、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 Promoter, 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, TSH, 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, MTC, MTOT, MTR, MTRR, MUC1, MY8, MYH11, MYH9, NCOA1, NCOA2, NCOA4, NFK81, NFK82, NIN, NLRP1, NUMA1, NUP214, P8RM1, P8X1, PAX-, PAX3, PAX8, PAXS, PDE4DIP, PDGF8, PER1, PGAP3, PHOX28, PIK3C28, PKHD1, PLAG1, PLCG 1, 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 The set includes 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 TPR, TRIM24, TRIM33, TRIP11, TRRAP, U8R5, UGT1A1, USP9X, WAS, WRN, XP01, XPA, XPC, ZNF384, ZNF521 and combinations thereof.

[0211] In another embodiment, the genome profile includes one or more genes selected from the genes listed in Tables 2-15.

[0212] In one embodiment, TMB status based on genomic profiling correlates strongly with TMB status based on whole exome sequencing or whole genome sequencing. The evidence provided herein shows that the use of genomic profiling assays, such as the F1CDx assay, is consistent with whole exome and / or whole genome sequencing assays. These data support the use of genomic profiling assays as a more effective method for measuring TMB status without compromising the quality of TMB status prediction.

[0213] TMB can be measured using tissue biopsy samples or circulating tumor DNA (ctDNA), cfDNA (cell-free DNA), and / or liquid biopsy samples. ctDNA can be used to measure TMB status according to available methods, such as GRAIL's method, by whole exome sequencing, whole genome sequencing, or genomic profiling.

[0214] Subjects are identified as suitable for combination therapy including (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody, based on the measurement of TMB status and the identification of high TMB. In one embodiment, the TMB score is calculated as the total number of non-synonymous missense mutations in the tumor, as measured by whole exome sequencing or whole genome sequencing. In one embodiment, high TMB is defined as at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, and less They all have a score of 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, or at least 500.In another embodiment, high TMB has a score of at least 215, at least 220, at least 221, at least 222, at least 223, at least 224, at least 225, at least 226, at least 227, at least 228, at least 229, at least 230, at least 231, at least 232, at least 233, at least 234, at least 235, at least 236, at least 237, at least 238, at least 239, at least 240, at least 241, at least 242, at least 243, at least 244, at least 245, at least 246, at least 247, at least 248, at least 249, or at least 250. In a particular embodiment, high TMB has a score of at least 243. In another embodiment, high TMB has a score of at least 244. In one embodiment, high TMB has a score of at least 245. In another embodiment, high TMB has a score of at least 246. In other embodiments, high TMB has a score of at least 247. In other embodiments, high TMB has a score of at least 248. In other embodiments, high TMB has a score of at least 249. In other embodiments, high TMB has a score of at least 250. In other embodiments, high TMB has an integer score of 200 to 300 or higher. In other embodiments, high TMB has an integer score of 210 to 290 or higher. In other embodiments, high TMB has an integer score of 220 to 280 or higher. In other embodiments, high TMB has an integer score of 230 to 270 or higher. In other embodiments, high TMB has an integer score of 235 to 265 or higher.

[0215] Alternatively, high TMB can be a relative value rather than an absolute value. In one embodiment, the subject's TMB state is compared to a control TMB value. In one embodiment, the subject's TMB state is within the highest quantile of the control TMB value. In another embodiment, the subject's TMB state is within the highest tertile of the control TMB value.

[0216] In one embodiment, the TMB status is expressed as the number of mutations per sample, per cell, per exome, or per unit length of DNA (e.g., Mb). In one embodiment, a tumor is defined as having at least approximately 50 mutations / tumor, at least approximately 55 mutations / tumor, at least approximately 60 mutations / tumor, at least approximately 65 mutations / tumor, and at A tumor has a high TMB state if it has approximately 70 mutations / tumor, at least approximately 75 mutations / tumor, at least approximately 80 mutations / tumor, at least approximately 85 mutations / tumor, at least approximately 90 mutations / tumor, at least approximately 95 mutations / tumor, at least approximately 100 mutations / tumor, at least approximately 105 mutations / tumor, at least approximately 110 mutations / tumor, at least approximately 115 mutations / tumor, or at least approximately 120 mutations / tumor. In one embodiment, a tumor has a high TMB state if it has at least approximately 125 mutations / tumor, at least approximately 150 mutations / tumor, at least approximately 175 mutations / tumor, at least approximately 200 mutations / tumor, at least approximately 225 mutations / tumor, at least approximately 250 mutations / tumor, at least approximately 275 mutations / tumor, at least approximately 300 mutations / tumor, at least approximately 350 mutations / tumor, at least approximately 400 mutations / tumor, or at least approximately 500 mutations / tumor. In a particular embodiment, a tumor has a high TMB state if it has at least approximately 100 mutations / tumors.

[0217] In one embodiment, the tumor is, for example, FOUNDATIONONE® CDX (商標)Genome sequencing is performed according to an assay, for example, at least about 5 mutations per megabase of a gene sequenced by a TMB assay, 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. When present, it has a high TMB state. In certain embodiments, a tumor has a high TMB state when it has at least about 5 mutations / Mb. In one embodiment, a tumor has a high TMB state when it has at least about 10 mutations / Mb. In certain embodiments, a tumor has a high TMB state when it has at least about 11 mutations / Mb. In certain embodiments, a tumor has a high TMB state when it has at least about 12 mutations / Mb. In certain embodiments, a tumor has a high TMB state when it has at least about 13 mutations / Mb. In certain embodiments, a tumor has a high TMB state when it has at least about 14 mutations / Mb. In certain embodiments, a tumor has a high TMB state when it has at least about 15 mutations / Mb.

[0218] Since the number of mutations can vary depending on the tumor type and other methods (see Q4 and Q5), the numerical values associated with "high TMB" and "low TMB" can differ depending on the tumor type.

[0219] PD-L1 status The TMB status can be used alone or in combination with other factors as a means to predict the response of tumors to combination therapies comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, and (b) an anti-CTLA-4 antibody. In certain embodiments, only the TMB status of the tumor is used to identify patients having tumors that are likely to respond to combination therapies comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. In other embodiments, the PD-L1 status and the TMB status are used to identify patients having tumors that are likely to respond to combination therapies comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. In certain embodiments, the tumor has less than 1% PD-L1 expression; for example, less than 1% of the tumor cells express PD-L1. In certain embodiments, the subject has a high TMB status (≧10 mutations (mut) / Mb) and a tumor PD-L1 expression level of less than 1%.

[0220] The PD-L1 expression status of tumors in a subject can be measured before administering the compositions described herein or using the methods described herein. PD-L1 expression can be determined by methods known in the art.

[0221] To evaluate PD-L1 expression, in one embodiment, a test tissue sample can be obtained from a patient who requires treatment. In another embodiment, the evaluation of PD-L1 expression can be performed without obtaining a test tissue sample. In certain embodiments, the selection of suitable patients includes (i) optionally, providing a test tissue sample obtained from a patient having a tumor derived from NSCLC, the test tissue sample including tumor cells and / or tumor infiltrating inflammatory cells; and (ii) evaluating the proportion of cells in the test tissue sample that express PD-L1 on the cell surface based on the evaluation that the proportion of cells in the test tissue sample that express PD-L1 on the cell surface is higher than a predetermined threshold.

[0222] However, in a method involving the measurement of PD-L1 expression in a test tissue sample, the step of providing a test tissue sample obtained from a patient should be understood to be an optional step. Also, in certain embodiments, the “measurement” or “evaluation” step for identifying or determining the number or percentage of cells in a test tissue sample expressing PD-L1 on the cell surface should be understood to be performed by a modified method for measuring PD-L1 expression, for example, by performing a reverse transcriptase polymerase chain reaction (RT-PCR) assay or an IHC assay. In other embodiments, without a modification step, PD-L1 expression is evaluated, for example, by reviewing a report of test results from a laboratory. In some embodiments, the steps of the method up to and including the evaluation of PD-L1 expression provide intermediate results that can be made available to a physician or other healthcare provider for use in selecting a suitable candidate combination therapy comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody. In some embodiments, the step of providing intermediate results is performed by a physician or another person performing under the direction of a physician. In other embodiments, these processes are carried out in an independent laboratory or by an independent person, such as a laboratory technician.

[0223] In certain embodiments of the methods of the present invention, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In further embodiments, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection. In other embodiments, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In further embodiments, the presence of PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, PD-L1 expression is assayed by IHC. In all other embodiments of these methods, cell surface expression of PD-L1 is assayed, for example, using IHC or in vivo imaging.

[0224] Imaging technologies provide essential tools in cancer research and treatment. Recent developments in molecular imaging systems, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence-mediated tomography (FMT), bioluminescence imaging (BLI), laser scanning confocal microscopy (LSCM), and multiphoton microscopy (MPM), foreshadow further applications of these technologies in cancer research. Some of these molecular imaging systems allow clinicians to visualize not only the location of tumors within the body, but also the expression and activity of specific molecules, cellular, and biological processes that influence tumor behavior and / or responsiveness to therapeutic agents (Condeelis and Weissleder, “In vivo imaging in cancer”, Cold Spring Harb. Perspect. Biol. 2(12):a003848 (2010)). Regarding antibody specificity, the combination of PET sensitivity and resolution allows immunoPET imaging to monitor and assay antigen expression, particularly in tissue samples (McCabe and Wu, “Positive progress in immunoPET—not just a coincidence,” Cancer Biother. Radiopharm. 25(3):253-61 (2010); Olafsen et al., “ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies),” Protein Eng. Des. Sel. 23(4):243-9 (2010)). In any particular embodiment of the method of the present invention, PD-L1 expression is assayed by immunoPET imaging. In any particular embodiment of the method of the present invention, the percentage of cells in a test tissue sample expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptides on the cell surface in the test tissue sample. In some embodiment, the test tissue sample is an FFPE tissue sample.In other embodiments, the presence of PD-L1 polypeptide is determined by an IHC assay. In further embodiments, the IHC assay is performed using an automated method. In some embodiments, the IHC assay is performed using an anti-PD-L1 monoclonal antibody that binds to PD-L1 polypeptide. In certain embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1 and combinations thereof. See WO / 2013 / 173223, which is incorporated herein by reference in its entirety.

[0225] In one embodiment of the method of the present invention, an automated IHC method is used to assay the expression of PD-L1 on the cell surface of FFPE tissue samples, for example, tissue samples taken from tumors derived from NSCLC. The presence of human PD-L1 antigen can be measured in the test tissue sample by contacting the test sample and a negative control sample (e.g., normal tissue) with a monoclonal antibody or a portion thereof that specifically binds to human PD-L1, under conditions that allow for the formation of a complex between the monoclonal antibody and human PD-L1. In a particular embodiment, the test and control tissue samples are FFPE samples. Next, the formation of the complex is detected, where the difference in complex formation between the test sample and the negative control sample indicates the presence of human PD-L1 antigen in the sample. Various methods are used to quantify PD-L1 expression.

[0226] In a particular embodiment, an automated IHC method includes activating an automated staining apparatus which includes the steps of (a) deparaffinizing and rehydrating embedded tissue sections in an automated staining apparatus; (b) activating the antigen using a decloaking chamber and pH 6 buffer heated to 110°C for 10 minutes; (c) setting the reagents in the automated staining apparatus; then (d) neutralizing endogenous peroxidase in the tissue sample; blocking nonspecific protein binding sites on the slide; incubating the slide with a primary antibody; incubating with a post-primary blocking agent; incubating with NovoLink polymer; adding a chromogen substrate and developing color; and then counterstaining with hematoxylin.

[0227] To evaluate PD-L1 expression in tumor tissue samples, pathologists examine membrane PD-L1 in each field of view using a microscope. + The number of tumor cells is counted, the percentage of positive cells is estimated mentally, and then these are averaged to obtain the final percentage value. Different staining intensities are defined as 0 / negative, 1+ / weak, 2+ / intermediate, and 3+ / strong. Generally, the percentage values ​​are assigned first to the 0 and 3+ buckets, then to the intermediate 1+ and 2+ intensities. For highly heterogeneous tissues, the sample is divided into sections, each section is scored separately, and then combined into a single set of percentage values. The percentages of negative and positive cells for each staining intensity are determined from each section, and the median is assigned to each section. The final percentage value is assigned to the tissue for each staining intensity category: negative, 1+, 2+, and 3+. The sum of all staining intensities must be 100%. In one embodiment, the threshold number of cells required to be PD-L1 positive is at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200 cells. In one embodiment, the threshold number of cells required to be PD-L1 positive is at least about 100 cells.

[0228] Staining is also evaluated in tumor-infiltrating inflammatory cells such as macrophages and lymphocytes. Macrophages often serve as internal positive controls, as staining is frequently observed in the majority of macrophages. While staining with 3+ intensity is not required, the absence of macrophage staining should not be considered a rule-of-failure. Macrophages and lymphocytes are evaluated for cell membrane staining and recorded only as positive or negative for each cell category for all samples. Staining is also characterized according to the naming of intratumoral / extratumoral immune cells. “Intratumoral” means that immune cells are present on the boundary of the tumor region without being physically inserted within and / or between tumor cells. “Extratumoral” means that there is no physical association with the tumor, and immune cells are found in peripheral or adjacent tissues associated with connective tissue.

[0229] In one embodiment of the method for assigning these scores, the samples are scored by two independently performing pathologists, and these scores are later combined. In another embodiment, the identification of positive and negative cells is scored using appropriate software.

[0230] The organization score (histoscore) is used as a more quantitative measure of IHC data. The organization score is calculated as follows: Tissue score = [(% tumor x 1 (low intensity)) + (% tumor x 2 (intermediate intensity)) + (% tumor x 3 (high intensity)].

[0231] To determine the tissue score, pathologists estimate the percentage of stained cells in each intensity category within the sample. Because the expression of many biomarkers is heterogeneous, the tissue score is a more accurate representation of overall expression. The final tissue score ranges from 0 (no expression) to 300 (maximum expression).

[0232] Another method for quantifying PD-L1 expression in test tissue samples (IHC) is to determine the modified inflammation score (AIS), which is defined as the intensity of inflammation by multiplying it by the percentage of PD-L1 expression from tumor-infiltrating inflammatory cells (Taube et al., “Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape,” Sci. Transl. Med. 4(127):127ra37 (2012)).

[0233] In one embodiment, the PD-L1 expression level of the tumor is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In another embodiment, the PD-L1 expression status of the tumor is at least about 1%. In yet another embodiment, the PD-L1 expression status of the tumor is at least about 5%. In one embodiment, the PD-L1 expression status of the tumor is at least about 10%. In one embodiment, the PD-L1 expression status of the tumor is at least about 25%. In a specific embodiment, the PD-L1 expression status of the tumor is at least about 50%.

[0234] As used herein, “PD-L1 positive” can be used interchangeably with “at least about 1% PD-L1 expression.” Accordingly, in one embodiment, a PD-L1 positive tumor may exhibit at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of tumor cells expressing PD-L1 as measured by automated IHC. In a particular embodiment, “PD-L1 positive” means the presence of at least 100 cells expressing PD-L1 on their cell surface.

[0235] In one embodiment, tumors derived from NSCLC that are PD-L1 positive and have high TMB may have a greater response to combination therapy of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody than tumors with high TMB only, PD-L1 positive expression only, or neither. In one embodiment, tumors derived from NSCLC have PD-L1 expression of at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In a particular embodiment, tumors derived from NSCLC with PD-L1 expression of 50% or more and a high TMB condition are more likely to respond to combination therapy of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody than tumors with high TMB only, PD-L1 expression of 50% or more only, or neither.

[0236] In certain embodiments, tumors in subjects suitable for immunotherapy, for example, combination therapy of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody as described herein, do not express PD-L1 (less than 1%, less than 2%, less than 3%, less than 4%, or less than 5% of membrane-bound PD-L1). In some embodiments, the methods of the present invention are independent of PD-L1 expression.

[0237] MSI status The TMB status can be used alone or in combination with other factors, such as the MSI status, as a means of predicting the response of NSCLC-derived tumors to combination therapy of (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody. In one embodiment, the MSI status is part of the TMB status. In another embodiment, the MSI status is measured separately from the TMB status.

[0238] Microsatellite instability (MSI) is a state of genetic hypermutability resulting from impaired DNA mismatch repair (MMR). The presence of MSI is evidence of a phenotype in which MMR is not functioning properly. In most cases, the genetic basis for the instability in MSI tumors is a hereditary germline mutation in one of the five human MMR genes: MSH2, MLH1, MSH6, PMS2, and PMS1. In certain embodiments, tumors derived from NSCLC (e.g., colon tumors) have high microsatellite instability (MSI-H) and have at least one mutation in the MSH2, MLH1, MSH6, PMS2, or PMS1 gene. In other embodiments, subjects receiving tumor treatment within the control group do not have microsatellite instability (MSS or MSI stability) and do not have mutations in the MSH2, MLH1, MSH6, PMS2, and PMS1 gene.

[0239] In one aspect, the subjects suitable for the combination therapy of (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody have high TMB status and MSI-H tumors derived from NSCLC. The MSI-H tumors described herein mean tumors exceeding at least about 30% of unstable MSI biomarkers. In one aspect, when germline changes are detected in at least two, at least three, at least four, or at least five MMR genes, the tumors derived from NSCLC are MSI-H. In other aspects, when germline changes are detected in at least 30% of more than five MMR genes, the tumors derived from NSCLC are MSI-H. In one aspect, the germline changes in the MMR genes are measured by polymerase chain reaction. In other aspects, tumors derived from NCSLC are MSI-H when at least one protein encoded by the DNA MMR gene is not detected in the tumor. In one aspect, at least one protein encoded by the DNA MMR gene is detected by immunohistochemistry.

[0240] The treatment method of the present invention The present invention relates to a method of treating a subject suffering from a tumor derived from NSCLC, comprising administering to the subject an effective amount of (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, wherein the tumor has a high TMB status. In certain aspects, the tumor has a TMB status of at least about 10 mutations per megabase. In one aspect, the method further comprises measuring the TMB status of a biological sample obtained from the subject prior to administration.

[0241] Certain cancer types, including lung cancer, have high TMB because of the high frequency of mutations (Alexandrov et al., Nature (2013) 500: 415-4). In one aspect, NSCLC has squamous epithelial tissue. In another aspect, NSCLC has non-squamous epithelial tissue.

[0242] The treatment methods described herein can provide improved clinical responses and / or clinical benefits for subjects affected by tumors originating from NSCLC, particularly those with tumors exhibiting high TMB. High TMB relates to novel antigen loading, i.e., the number of novel antigens and T cell reactivity, and is therefore associated with immune-mediated antitumor responses. Accordingly, high TMB is a factor that can be used, for example, to identify tumors (and patients with such tumors) that are likely to benefit from treatment with (a) anti-PD-1 antibody or anti-PD-L1 antibody and (b) anti-CLTA-4 antibody, either alone or in combination with other factors, compared to current standard treatment.

[0243] In one embodiment, subjects demonstrate progression-free survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration. In another embodiment, subjects demonstrate overall survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 1 year, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration. In yet another embodiment, the subjects exhibit a response rate of at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0244] Anti-PD-1 / anti-PD-L1 / anti-CTLA-4 antibody treatment A particular aspect of the present invention relates to a method for treating a subject suffering from a tumor derived from NSCLC, comprising administering to the subject (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody, wherein the tumor has a high TMB status, e.g., a TMB of at least about 10 mutations per megabase of the tested gene. The method further comprises measuring the TMB status of a biological sample obtained from the subject. Furthermore, the present invention intends to administer (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CLTA-4 antibody to a subject identified as suitable for such treatment, for example, based on the measurement of a high TMB of at least about 10 mutations per megabase of the tested gene.

[0245] In one embodiment, the anti-PD-1 antibody or its antigen-binding site cross-competes with nivolumab for binding to human PD-1. In another embodiment, the anti-PD-1 antibody or its antigen-binding site binds to the same epitope as nivolumab. In a particular embodiment, the anti-PD-1 antibody is nivolumab. In another particular embodiment, the anti-PD-1 antibody is pembrolizumab. Additional anti-PD-1 antibodies are described in other parts of this specification. In other embodiments, anti-PD-L1 antibodies or their antigen-binding sites useful for the methods described herein are described in other parts of this specification.

[0246] In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody or its antigen-binding site is a chimeric antibody, a humanized antibody, a human antibody, or its antigen-binding site. In another embodiment, the anti-PD-1 antibody or its antigen-binding site or the anti-PD-L1 antibody or its antigen-binding site includes the heavy chain constant region of a human IgG1 isotype or a human IgG4 isotype.

[0247] Anti-PD-1 antibodies useful in the present 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 described in U.S. Patent No. 8,008,449. The anti-PD-1 human antibodies described in U.S. Patent No. 8,008,449 have been proven to exhibit one or more of the following characteristics: (a) 1 × 10⁻¹⁶ as determined by surface plasmon resonance using a Biacore biosensor system. -7 M or less K D (b) binds to human PD-1; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an antibody response; and (j) inhibits tumor cell proliferation in vivo. Anti-PD-1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments, at least five of the above features.

[0248] Other anti-PD-1 monoclonal antibodies include, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Patent Publication 2016 / 0272708, and PCT Publications WO2012 / 145493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, and WO2016 / 19 As described in 7367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 194302, WO2017 / 040790, 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 in its entirety herein by reference).

[0249] In one embodiment, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), semiprimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as toripalimab; Si-Yang See 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; WO2015 / 085847; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu See 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; see Kaplon et al.The selection is made from the group consisting of , mAbs 10(2):183-203 (2018)), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540).

[0250] In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4(S228P)PD-1 immune checkpoint inhibitor antibody that selectively inhibits interaction with PD-1 ligands (PD-L1 and PD-L2) and therefore prevents downregulation of antitumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0251] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0252] The anti-PD-1 antibodies that can be used in the compositions and methods described herein also include isolated antibodies that specifically bind to human PD-1 and cross-compete with the anti-PD-1 antibodies described herein, e.g., nivolumab (e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; see WO2013 / 173223) for binding to human PD-1. In some 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 the antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to a particular epitope region. These cross-competing antibodies are expected to have functional properties remarkably similar to the control antibody (e.g., nivolumab) due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays (e.g., Biacore analysis, ELISA assay, or flow cytometry) (see, for example, WO2013 / 173223).

[0253] In certain embodiments, an antibody (nivolumab) that cross-competes for binding to human PD-1 or binds to the same epitope region of a human PD-1 antibody is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, modified antibodies, or humanized antibodies or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0254] The anti-PD-1 antibodies that can be used in the compositions and methods described herein also include the antigen-binding moiety of the antibody. It has been shown that the antigen-binding function of the antibody can be performed by a fragment of the full-length antibody.

[0255] An anti-PD-1 antibody suitable for use in the compositions and methods of the present invention is an antibody that binds to PD-1 with high specificity and affinity, inhibits the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods described herein, the anti-PD-1 "antibody" includes an antigen-binding moiety or fragment that binds to the PD-1 receptor, inhibits ligand binding, and exhibits functional properties similar to a complete antibody in upregulating the immune system. In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety cross-competes with nivolumab for binding to human PD-1.

[0256] In one embodiment, the anti-PD-1 antibody is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from 0.1 mg to 20.0 mg per kg of body weight, for example, once every 2, 3, or 4 weeks in a dose ranging from 0.1 mg to 10.0 mg per kg of body weight. In another embodiment, the anti-PD-1 antibody is administered once every 2 weeks in a dose of approximately 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg per kg of body weight. In yet another embodiment, the anti-PD-1 antibody is administered once every 3 weeks in a dose of approximately 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg per kg of body weight. In yet another embodiment, the anti-PD-1 antibody is administered once every approximately 3 weeks in a dose of approximately 5 mg per kg of body weight. In another embodiment, an anti-PD-1 antibody, such as nivolumab, is administered at a dose of approximately 3 mg per kg of body weight once every two weeks. In yet another embodiment, an anti-PD-1 antibody, such as pembrolizumab, is administered at a dose of approximately 2 mg per kg of body weight once every three weeks.

[0257] Anti-PD-1 antibodies useful for the present invention can be administered in a fixed dose. In one embodiment, the anti-PD-1 antibody is administered in a fixed dose of approximately 100 to 1000 mg, approximately 100 to 900 mg, approximately 100 to 800 mg, approximately 100 to 700 mg, approximately 100 to 600 mg, approximately 100 to 500 mg, approximately 200 to 1000 mg, approximately 200 to 900 mg, approximately 200 to 800 mg, approximately 200 to 700 mg, approximately 200 to 600 mg, approximately 200 to 500 mg, approximately 200 to 480 mg, or approximately 240 to 480 mg. In one embodiment, the anti-PD-1 antibody is administered at dosing intervals of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks in a constant dose of at least approximately 200 mg, at least approximately 220 mg, at least approximately 240 mg, at least approximately 260 mg, at least approximately 280 mg, at least approximately 300 mg, at least approximately 320 mg, at least approximately 340 mg, at least approximately 360 mg, at least approximately 380 mg, at least approximately 400 mg, at least approximately 420 mg, at least approximately 440 mg, at least approximately 460 mg, at least approximately 480 mg, at least approximately 500 mg, at least approximately 520 mg, at least approximately 540 mg, at least approximately 550 mg, at least approximately 560 mg, at least approximately 580 mg, at least approximately 600 mg, at least approximately 620 mg, at least approximately 640 mg, at least approximately 660 mg, at least approximately 680 mg, at least approximately 700 mg, or at least approximately 720 mg. In another embodiment, the anti-PD-1 antibody is administered at fixed doses of approximately 200 mg to 800 mg, 200 mg to 700 mg, 200 mg to 600 mg, and 200 mg to 500 mg at dosing intervals of approximately 1, 2, 3, or 4 weeks.

[0258] In one embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every three weeks. In another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every two weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 240 mg once every two weeks. In a specific embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 480 mg once every four weeks.

[0259] In one embodiment, nivolumab is administered at a constant dose of approximately 240 mg once every two weeks. In another embodiment, nivolumab is administered at a constant dose of approximately 240 mg once every three weeks. In another embodiment, nivolumab is administered at a constant dose of approximately 360 mg once every three weeks. In another embodiment, nivolumab is administered at a constant dose of approximately 480 mg once every four weeks.

[0260] In one embodiment, pembrolizumab is administered at a constant dose of approximately 200 mg once every two weeks. In another embodiment, pembrolizumab is administered at a constant dose of approximately 200 mg once every three weeks. In another embodiment, pembrolizumab is administered at a constant dose of approximately 400 mg once every four weeks.

[0261] Anti-PD-L1 antibodies useful in the present invention In certain embodiments, an anti-PD-L1 antibody may be used in place of an anti-PD-1 antibody in any of the methods described herein. Anti-PD-L1 antibodies known in the art may be used in the compositions and methods of the present invention. An example of an anti-PD-L1 antibody useful in the compositions and methods of the present invention is the antibody described in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody described in U.S. Patent No. 9,580,507 has been shown to exhibit one or more of the following characteristics: (a) 1 x 10⁻¹⁶ as measured by surface plasmon resonance using a Biacore biosensor system. -7 M or less K D (b) binds to human PD-L1; (c) increases T cell proliferation in mixed lymphocyte reaction (MLR) assays; (d) increases interferon-γ production in MLR assays; (e) stimulates an antibody response; and (f) reverses the effect of T regulatory cells in T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments, at least five of the above features.

[0262] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO2013 / 173223), atezolizumab (Roche; TECENTRIQ®; also known as MPDL3280A, RG7446; see U.S. 8,217,149; also see Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; IMFINZI (商標) Also known as MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; BAVENCIO®, also known as MSB-0010718C; see WO2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO2017 / 034916), BGB-A333 (BeiGene; Desai et al., JCO 36 (15suppl):TPS3113) The patient is selected from the group consisting of (see 2018) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0263] In a particular embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0264] In a particular embodiment, the PD-L1 antibody is durvalumab (IMFINZI (商標) Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0265] In a particular embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0266] Other anti-PD-L1 antibodies that can be used in the compositions and methods of the present invention include isolated antibodies that specifically bind to human PD-L1 and cross-compete with the anti-PD-L1 antibodies described 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 the antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to a particular epitope region. These cross-competing antibodies are expected to have functional properties remarkably similar to control antibodies (e.g., atezolizumab and / or avelumab) due to their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays such as Biacore analysis, ELISA assay, or flow cytometry (see, e.g., WO2013 / 173223).

[0267] In certain embodiments, antibodies that cross-compete with human PD-L1 antibodies such as atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1, or that bind to the same epitope region of human PD-L1 antibodies, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0268] Examples of anti-PD-L1 antibodies that can be used in the compositions and methods of the present invention include the antigen-binding portion of the above antibody. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.

[0269] Anti-PD-L1 antibodies suitable for use in the compositions and methods of the present invention are antibodies that bind to PD-1 with high specificity and affinity, inhibit PD-1 binding, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods described herein, the anti-PD-L1 "antibody" may be an antigen-binding moiety or fragment that binds to PD-L1 and exhibits functional properties similar to the whole antibody when inhibiting receptor binding and upregulating the immune system. In some embodiments, the anti-PD-L1 antibody or its antigen-binding moiety cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0270] An anti-PD-L1 antibody useful in the present invention may 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.

[0271] In one embodiment, anti-PD-L1 antibodies are administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in doses ranging from approximately 0.1 mg to approximately 20.0 mg per kg of body weight, specifically in doses of approximately 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0272] In one embodiment, the anti-PD-L1 antibody is administered at a dose of approximately 15 mg per kg of body weight once every three weeks. In another embodiment, the anti-PD-L1 antibody is administered at a dose of approximately 10 mg per kg of body weight once every two weeks.

[0273] In another embodiment, the anti-PD-L1 antibody useful for the present invention is a fixed dose. In one embodiment, the anti-PD-L1 antibody is administered in a fixed dose of approximately 200-1600 mg, approximately 200-1500 mg, approximately 200-1400 mg, approximately 200-1300 mg, approximately 200-1200 mg, approximately 200-1100 mg, approximately 200-1000 mg, approximately 200-900 mg, approximately 200-800 mg, approximately 200-700 mg, approximately 200-600 mg, approximately 700-1300 mg, approximately 800-1200 mg, approximately 700-900 mg, or approximately 1100-1300 mg. In one embodiment, the anti-PD-L1 antibody is administered at intervals of approximately 1, 2, 3, or 4 weeks in a constant dose of at least approximately 240 mg, at least approximately 300 mg, at least approximately 320 mg, at least approximately 400 mg, at least approximately 480 mg, at least approximately 500 mg, at least approximately 560 mg, at least approximately 600 mg, at least approximately 640 mg, at least approximately 700 mg, at least approximately 720 mg, at least approximately 800 mg, at least approximately 840 mg, at least approximately 880 mg, at least approximately 900 mg, at least approximately 960 mg, at least approximately 1000 mg, at least approximately 1040 mg, at least approximately 1100 mg, at least approximately 1120 mg, at least approximately 1200 mg, at least approximately 1280 mg, at least approximately 1300 mg, at least approximately 1360 mg, or at least approximately 1400 mg. In one embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 1200 mg once every approximately 3 weeks. In another embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 800 mg once every two weeks. In yet another embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 840 mg once every two weeks.

[0274] In one embodiment, atezolizumab is administered at a constant dose of approximately 1200 mg once every three weeks. In another embodiment, atezolizumab is administered at a constant dose of approximately 800 mg once every two weeks. In yet another embodiment, atezolizumab is administered at a constant dose of approximately 840 mg once every two weeks.

[0275] In one embodiment, avelumab is administered at a constant dose of approximately 800 mg once every two weeks.

[0276] In one embodiment, durvalumab is administered at a dose of approximately 10 mg per kg of body weight once every two weeks. In another embodiment, durvalumab is administered at a constant dose of approximately 800 mg once every two weeks. In another embodiment, durvalumab is administered at a constant dose of approximately 1200 mg once every three weeks.

[0277] Anti-CTLA-4 antibody Anti-CTLA-4 antibodies known in the art may be used in the compositions and methods of the present invention. The anti-CTLA-4 antibodies described herein bind to human CTLA-4 in such a way as to disrupt the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transmits signals that lead to the inactivation of T cells carrying the CTLA-4 receptor, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging an immune response.

[0278] A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is described in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patents No. 5,977,318, No. 6,051,227, No. 6,682,736 and No. 7,034,121, and in International Publications WO2012 / 122444, WO2007 / 113648, WO2016 / 196237 and WO2000 / 037504 (each of which is incorporated herein by reference in its entirety). The anti-CTLA-4 human monoclonal antibody described in U.S. Patent No. 6,984,720 has been shown to exhibit one or more of the following characteristics: (a) at least about 10 as measured by Biacore analysis. 7 M -1 or about 10 9 M -1 or about 10 10 M -1 from 10 11 M -1 or a higher equilibrium coupling constant (K a (b) Binds to human CTLA-4 with binding affinity as shown in (b) (b) at least about 10 3 , about 10 4 or about 10 5 M -1 S -1 The dynamic coupling 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 (d) increase IL-2 secretion in MLR assays; (e) stimulate an antibody response; and (f) 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 antibodies and exhibit at least one, at least two, or at least three of the above features.

[0279] In certain embodiments, the CTLA-4 antibody is selected from the group consisting of ipilimumab (YERVOY®, also known as MDX-010, 10D1; see U.S. Patent No. 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 certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0280] In certain embodiments, the CTLA-4 antibody is ipilimumab for use in the compositions and methods described herein. Ipilimumab is a fully human, IgG1 monoclonal antibody that inhibits the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with progressive melanoma.

[0281] In certain embodiments, the CTLA-4 antibody is tremelimumab.

[0282] In certain embodiments, the CTLA-4 antibody is MK-1308.

[0283] In certain embodiments, the CTLA-4 antibody is AGEN-1884.

[0284] Other anti-CTLA-4 antibodies usable in the compositions and methods of the present invention include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab and / or tremelimumab, for binding to human CTLA-4. In some 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 these antibodies bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to a particular epitope region. These cross-competing antibodies are expected to have functional properties remarkably similar to control antibodies (e.g., ipilimumab and / or tremelimumab) due to their binding to the same epitope region of CTLA-4. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab and / or tremelimumab in standard CTLA-4 binding assays such as Biacore analysis, ELISA assay, or flow cytometry (see, e.g., WO2013 / 173223).

[0285] In certain embodiments, antibodies that cross-compete with human CTLA-4 antibodies, such as ipilimumab and / or tremelimumab, for binding to human CTLA-4, or that bind to the same epitope region of human CTLA-4 antibodies, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, modified antibodies, or humanized or human antibodies. Such chimeric, modified, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0286] Examples of anti-CTLA-4 antibodies usable in the compositions and methods of the present invention include the antigen-binding moiety of the above-mentioned antibody. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.

[0287] An anti-CTLA-4 antibody suitable for use in the method or composition of the present invention is an antibody that binds to CTLA-4 with high specificity and affinity, inhibits the activity of CTLA-4, and disrupts the interaction between CTLA-4 and the human B7 receptor. In any of the compositions or methods described herein, the anti-CTLA-4 "antibody" may include an antigen-binding moiety or fragment that binds to CTLA-4, inhibits the interaction between CTLA-4 and the human B7 receptor, and exhibits functional properties similar to a whole antibody in upregulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or its antigen-binding moiety cross-competes with ipilimumab and / or tremelimumab for binding to human CTLA-4.

[0288] In one embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from approximately 0.1 mg to approximately 10.0 mg per kg of body weight. In another embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered once every 3, 4, 5, or 6 weeks in a dose of 1 mg or 3 mg per kg of body weight. In one embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered once every 2 weeks in a dose of 3 mg per kg of body weight. In yet another embodiment, the anti-PD-1 antibody or its antigen-binding site is administered once every 6 weeks in a dose of 1 mg per kg of body weight.

[0289] In one embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered in a fixed dose. In one embodiment, the anti-CTLA-4 antibody is administered in a fixed dose of approximately 10-1000 mg, approximately 10-900 mg, approximately 10-800 mg, approximately 10-700 mg, approximately 10-600 mg, approximately 10-500 mg, approximately 100-1000 mg, approximately 100-900 mg, approximately 100-800 mg, approximately 100-700 mg, approximately 100-600 mg, approximately 100-500 mg, approximately 100-480 mg, or approximately 240-480 mg. In one embodiment, the anti-CTLA-4 antibody or its antigen-binding site is present in amounts of at least approximately 60 mg, at least approximately 80 mg, at least approximately 100 mg, at least approximately 120 mg, at least approximately 140 mg, at least approximately 160 mg, at least approximately 180 mg, at least approximately 200 mg, at least approximately 220 mg, at least approximately 240 mg, at least approximately 260 mg, at least approximately 280 mg, at least approximately 300 mg, at least approximately 320 mg, at least approximately 340 mg, at least approximately 360 mg, and at least approximately 380 mg. g is administered in a constant dose of at least approximately 400 mg, at least approximately 420 mg, at least approximately 440 mg, at least approximately 460 mg, at least approximately 480 mg, at least approximately 500 mg, at least approximately 520 mg, at least approximately 540 mg, at least approximately 550 mg, at least approximately 560 mg, at least approximately 580 mg, at least approximately 600 mg, at least approximately 620 mg, at least approximately 640 mg, at least approximately 660 mg, at least approximately 680 mg, at least approximately 700 mg, or at least approximately 720 mg. In another embodiment, the anti-CTLA-4 antibody or its antigen-binding site is administered in a constant dose once every approximately 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0290] In one embodiment, ipilimumab is administered at a dose of approximately 3 mg per kg of body weight once every three weeks. In another embodiment, ipilimumab is administered at a dose of approximately 10 mg per kg of body weight once every three weeks. In another embodiment, ipilimumab is administered at a dose of approximately 10 mg once every 12 weeks. In another embodiment, ipilimumab is administered in four doses.

[0291] Cytokines In one embodiment, the method of the present invention comprises treating a subject suffering from a tumor derived from NSCLC, comprising administering (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody, and (c) a cytokine, wherein the tumor has a high TMB state, for example, the tumor has a TMB state of at least about 10 mutations per megabase of the gene examined. The cytokine may be cytokines known in the art or their variants. In one embodiment, the cytokine is selected from the group consisting of interleukin-2 (IL-2), IL-1β, IL-6, TNF-α, RANTES, monocyte chemoattractant protein (MCP-1), monocyte inflammatory proteins (MIP-1α and MIP-1β), IL-8, lymphotactin, fractalkine, IL-1, IL-4, IL-10, IL-11, IL-13, LIF, interferon-α, TGF-β, and combinations thereof. In one embodiment, the cytokine is a CD122 agonist. In a particular embodiment, the cytokine includes IL-2 or a variant thereof.

[0292] In one embodiment, the cytokine comprises one or more amino acid substitutions, deletions, or insertions compared to the wild-type cytokine amino acid sequence. In another embodiment, the cytokine comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions compared to the wild-type cytokine amino acid sequence.

[0293] In some embodiments, cytokines are modified, for example, to increase their activity and / or half-life. In certain embodiments, cytokines are modified by fusion of heterologous moieties into the cytokine. The heterologous moiety may be any structure, including polypeptides, polymers, small molecules, nucleotides or their fragments or analogs. In certain embodiments, the heterologous moiety includes polypeptides. In some embodiments, the heterologous moiety includes albumin or its fragments, albumin-conjugated polypeptide (ABP), XTEN, Fc, PAS, the C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin, or any combination thereof.

[0294] In certain embodiments, cytokines are modified by fusion of cytokines with polymers. In some embodiments, polymers include polyethylene glycol (PEG), polypropylene glycol (PPG), hydroxyethyl starch (HES), or any combination thereof. “PEG” or “polyethylene glycol” as used herein means encompassing water-soluble poly(ethylene oxide). Unless otherwise specified, “PEG polymer” or polyethylene glycol means encompassing any water-soluble poly(ethylene oxide). Unless otherwise specified, “PEG polymer” or polyethylene glycol is one in which substantially all (preferably all) monomer subunits are ethylene oxide subunits, however the polymer may include, for example, separate end-capping moieties or functional groups for bonding. PEG polymers for use in the present invention include one of the following two structures: for example, depending on whether terminal oxygens are substituted during synthetic transformation, “-(CH2CH2O) n-n "or "-(CH2CH2O) n-1 CH2CH2-”. As described above, in the case of PEG polymers, the variable (n) is in the range of approximately 3 to 4000, and the overall PEG terminal groups and structure can vary.

[0295] In one embodiment, the present invention relates to a method for treating a subject suffering from a tumor derived from NSCLC, comprising administering to the subject (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody, and (c) a CD122 agonist. In one embodiment, the method comprises administering to the subject (a) an anti-PD-1 antibody, (b) an anti-CTLA-4 antibody, and (c) a CD122 agonist. In another embodiment, the method comprises administering to the subject (a) an anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody, and (c) a CD122 agonist. In one embodiment, the CD122 agonist comprises IL-2 or a variant thereof. In one embodiment, the CD122 agonist comprises an IL-2 variant having at least one amino acid substitution compared to wild-type IL-2. In one embodiment, the CD122 agonist comprises IL-2 fused to PEG. In one embodiment, the CD122 agonist comprises an IL-2 variant having at least one amino acid substitution compared to wild-type IL-2, wherein the IL-2 variant is fused to PEG.

[0296] Combination therapy In certain embodiments, an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody are administered in a therapeutically effective dose. In one embodiment, the method comprises administering therapeutically effective doses of anti-PD-1 antibody and anti-CTLA-4 antibody. In other embodiments, the method comprises administering therapeutically effective doses of anti-PD-L1 antibody and anti-CTLA-4 antibody. Any of the anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody described herein can be used in the method of the present invention. In certain embodiments, the anti-PD-1 antibody comprises nivolumab. In one embodiment, the anti-PD-1 antibody comprises pembrolizumab. In one embodiment, the anti-PD-L1 antibody comprises atezolizumab. In one embodiment, the anti-PD-L1 antibody comprises durvalumab. In one embodiment, the anti-PD-L1 antibody comprises avelumab. In one embodiment, the anti-CTLA-4 antibody comprises ipilimumab. In one embodiment, the anti-CTLA-4 antibody comprises ipilimumab and tremelimumab.

[0297] In one embodiment, (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody are administered approximately every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks, respectively. In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody is administered approximately every two weeks, every three weeks, or every four weeks, and the anti-CTLA-4 antibody is administered approximately every six weeks. In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody is administered on the same day as the anti-CTLA-4 antibody. In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody is administered on a different day than the anti-CTLA-4 antibody.

[0298] In one embodiment, the anti-CTLA-4 antibody is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks in a dose ranging from approximately 0.1 mg to approximately 20 mg per kg of body weight. In another embodiment, the anti-CTLA-4 antibody is administered in doses of approximately 0.1 mg, 0.3 mg, 0.6 mg, 0.9 mg, 1 mg, 3 mg, 6 mg, 9 mg, 10 mg, 12 mg, 15 mg, 18 mg, or 20 mg per kg of body weight. In a specific embodiment, the anti-CTLA-4 antibody is administered once every 4 weeks in a dose of approximately 1 mg per kg of body weight. In another embodiment, the anti-CTLA-4 antibody is administered once every 6 weeks in a dose of approximately 1 mg per kg of body weight.

[0299] In one embodiment, the anti-CTLA-4 antibody is administered in a constant dose. In one embodiment, the anti-CTLA-4 antibody is administered in a constant dose ranging from at least about 40 mg to at least about 1600 mg. In one embodiment, the anti-CTLA-4 antibody is administered in a constant 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 one embodiment, the CTLA-4 antibody is administered in a constant dose of at least approximately 220 mg, at least approximately 230 mg, at least approximately 240 mg, at least approximately 250 mg, at least approximately 260 mg, at least approximately 270 mg, at least approximately 280 mg, at least approximately 290 mg, at least approximately 300 mg, at least approximately 320 mg, at least approximately 360 mg, at least approximately 400 mg, at least approximately 440 mg, at least approximately 480 mg, at least approximately 520 mg, at least approximately 560 mg, or at least approximately 600 mg. In one embodiment, the CTLA-4 antibody is administered in a constant dose of at least approximately 640 mg, at least approximately 720 mg, at least approximately 800 mg, at least approximately 880 mg, at least approximately 960 mg, at least approximately 1040 mg, at least approximately 1120 mg, at least approximately 1200 mg, at least approximately 1280 mg, at least approximately 1360 mg, at least approximately 1440 mg, or at least approximately 1600 mg. In one embodiment, the anti-CTLA-4 antibody is administered at a constant dose at least once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0300] In a particular embodiment, the anti-PD-1 antibody is administered at a dose of approximately 2 mg per kg of body weight once every three weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks. In another embodiment, the anti-PD-1 antibody is administered at a dose of approximately 3 mg per kg of body weight once every two weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a dose of approximately 6 mg per kg of body weight once every four weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks.

[0301] In a particular embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every approximately 3 weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every approximately 6 weeks. In another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every approximately 2 weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every approximately 6 weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 240 mg once every approximately 2 weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every approximately 6 weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a constant dose of 480 mg once every approximately 4 weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every approximately 6 weeks.

[0302] In one embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every three weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks. In another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 200 mg once every two weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a constant dose of 240 mg once every two weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks. In yet another embodiment, the anti-PD-1 antibody is administered at a constant dose of approximately 480 mg once every four weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks.

[0303] In a particular embodiment, the anti-PD-L1 antibody is administered at a dose of approximately 10 mg per kg of body weight once every two weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks. In another embodiment, the anti-PD-L1 antibody is administered at a dose of approximately 15 mg per kg of body weight once every three weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks.

[0304] In a particular embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 800 mg once every two weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks. In another embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 1200 mg once every three weeks, and the anti-CTLA-4 antibody is administered at a dose of approximately 1 mg per kg of body weight once every six weeks.

[0305] In a particular embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 800 mg once every two weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks. In another embodiment, the anti-PD-L1 antibody is administered at a constant dose of approximately 1200 mg once every three weeks, and the anti-CTLA-4 antibody is administered at a constant dose of approximately 80 mg once every six weeks.

[0306] In one embodiment, an anti-PD-1 antibody, such as nivolumab, is administered in four doses approximately every three weeks at a dose of approximately 3 mg per kg of body weight, and an anti-CTLA-4 antibody is administered on the same day at a dose of approximately 1 mg per kg of body weight. Subsequently, the anti-PD-1 antibody, such as nivolumab, is administered at a constant dose of 240 mg approximately every two weeks, or at a constant dose of 480 mg approximately every four weeks. In another embodiment, an anti-PD-1 antibody, such as nivolumab, is administered in four doses approximately every three weeks at a dose of approximately 1 mg per kg of body weight, and an anti-CTLA-4 antibody is administered on the same day at a dose of approximately 3 mg per kg of body weight. Subsequently, the anti-PD-1 antibody, such as nivolumab, is administered at a constant dose of 240 mg approximately every two weeks, or at a constant dose of 480 mg approximately every four weeks.

[0307] NSCLC NSCLC is a leading cause of cancer death in the United States and worldwide, exceeding the combined number of deaths from breast, colon, and prostate cancers. In the United States, 228,190 new cases of lung and bronchial NSCLC are diagnosed, and approximately 159,480 people die from the disease (Siegel et al. (2014) CA Cancer J Clin 64(1):9-29). The vast majority of patients (approximately 78%) are diagnosed with advanced / recurrent or metastatic NSCLC. Metastasis from lung cancer to the adrenal glands is common, with approximately 33% of metastatic patients having such metastases. NSCLC therapy has shown gradually improved overall survival (OS), but the benefit has plateaued (the mean OS for terminal patients is just one year). Progression occurs after first-line therapy (1L therapy) in almost all of these patients, and the 5-year survival rate is only 3.6% in the refractory group. Between 2005 and 2009, the relative five-year survival rate for lung cancer in the United States was 15.9% (NCCN Guidelines®, version 3.2014 - Non-Small Cell Lung Cancer, available at www.nccn.org / professionals / physician_gls / pdf / nscl.pdf, last accessed May 14, 2014).

[0308] The method of the present invention can treat NSCLC tumors at all stages. In a particular embodiment, the tumor originates from NSCLC at any stage. NSCLC has at least seven stages: latent stage, stage 0 (carcinoma confined to epithelium), stage I, stage II, stage IIIA, stage IIIB, and stage IV. In the latent stage, cancer cannot be confirmed by imaging or bronchoscopy. In stage 0, cancer cells are found on the inner wall of the airway.

[0309] In one embodiment, the method of the present invention treats stage I non-squamous NSCLC. Stage I NSCLC is divided into stages IA and IB. In stage IA, the tumor is confined to the lung and is 3 centimeters or less. In stage IB, the cancer has not metastasized to the lymph nodes and one or more of the following are met: 1) the tumor is larger than 3 centimeters but 5 centimeters or less; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchi; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) a portion of the lung has collapsed or developed non-infectious pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchi.

[0310] In another embodiment, the method of the present invention treats stage II non-squamous NSCLC. Stage II NSCLC is further divided into stages IIA and IIB. In stage IIA, the cancer is either metastatic to the lymph nodes or not. If the cancer has metastatic to the lymph nodes, the cancer has spread only to lymph nodes on the same side as the tumor in the chest, and the cancerous lymph nodes are located in the lung or near the bronchi and one or more of the following are true: 1) the tumor is 5 centimeters or less; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchi; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) a portion of the lung has collapsed or developed non-infectious pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchi. If the cancer has not spread to the lymph nodes, the tumor may also be considered Stage IIA, and one or more of the following conditions must be met: 1) the tumor is larger than 5 centimeters but no larger than 7 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchi; 3) the cancer has spread to the innermost layer of the membrane covering the lung; or 4) there is partial lung collapse or non-infectious pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchi. In Stage IIB, the cancer may or may not have metastasized to the lymph nodes. If cancer has spread to the lymph nodes, the cancer has spread only to lymph nodes on the same side as the tumor in the chest, and the lymph nodes containing the cancer are located in the lung or near the bronchi, and the following conditions are met: 1) the tumor is larger than 5 centimeters but no larger than 7 centimeters; 2) the cancer has spread to the main bronchus and is at least 2 centimeters below where the trachea connects to the bronchi; 3) the cancer has spread to the innermost layer of the membrane covering the lung, or 4) part of the lung has collapsed or developed non-infectious pneumonia (inflammation of the lung) in the area where the trachea connects to the bronchi.If the cancer has not spread to the lymph nodes, the tumor is considered stage IIB and one or more of the following are true: 1) the tumor is larger than 7 centimeters; 2) the cancer has spread to the main bronchus (at least 2 centimeters below where the trachea connects to the bronchi), the chest wall, the diaphragm, or the nerves that control the diaphragm; 3) the cancer has spread to the membrane around the heart or inside the chest wall; 4) the entire lung is collapsed or has developed non-infectious pneumonia (inflammation of the lung); or 5) there is one or more separate tumors in the same lung lobe.

[0311] In other embodiments, the methods of the present invention treat stage III non-squamous NSCLC. Stage IIIA is divided into three sections. These three sections are based on 1) tumor size; 2) tumor location; and 3) (if any) presence of cancer in lymph nodes. In the first type of stage IIIA NSCLC, the cancer has spread to lymph nodes on the same side as the tumor in the chest, with the cancerous lymph nodes located near the sternum or where the bronchi enter the lungs. Furthermore, 1) the tumor may be of any size; 2) part of the lung (where the trachea connects with the bronchi) or the entire lung is collapsed or has developed non-infectious pneumonia (inflammation of the lung); 3) there is one or more distinct tumors in the same lung lobe; and 4) the cancer has metastasized to any of the following: a) the main bronchi (excluding the parts where the bronchi connect); b) the chest well; c) the diaphragm and the nerves that control it; d) the membranes surrounding the lungs or the membranes covering the chest wall; e) the membranes surrounding the heart. In the second type of stage IIIA NSCLC, the cancer has metastasized to lymph nodes on the same side as the tumor in the chest, and the cancerous lymph nodes are located within the lung or near the bronchi. Furthermore, 1) the tumor may be of any size; 2) the entire lung is collapsed or non-infectious pneumonia (inflammation of the lung) has developed; 3) there is one or more distinct tumors in any of the lobules of the lung with cancer; and 4) the cancer may have metastasized to any of the following: a) the main bronchi (excluding the junctions between bronchi), b) the chest well, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lungs or the membranes covering the chest wall, e) the heart or membranes leading to the heart, f) the major blood vessels leading to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal organs), j) the sternum / chest bone or spine, or k) the carina (where the trachea merges with the bronchi). In the third type of stage IIIA NSCLC, the cancer has not metastasized to the lymph nodes, the tumor may be of any size, and the cancer has metastasized to one of the following: a) the heart, b) a major blood vessel connected to or from the heart, c) the trachea, d) the esophagus, e) the nerves that control the larynx (vocal organs), f) the sternum or spine, or g) the carina (where the trachea connects to the bronchi).Stage IIIB is divided into two types based on 1) the size of the tumor, 2) the location of the tumor, and 3) which lymph nodes the cancer is located in. In the first type of Stage IIIB NSCLC, the cancer has spread to the lymph nodes in the chest on the opposite side of the tumor. Furthermore, 1) the tumor may be of any size; 2) part of the lung (where the trachea joins the bronchi) or the entire lung is collapsed or non-infectious pneumonia (inflammation of the lung) has developed; 3) there is one or more distinct tumors in any of the lobules of the lung with cancer; and 4) the cancer has metastasized to any of the following: a) the main bronchi, b) the chest wall, c) the diaphragm and the nerves that control it, d) the membranes surrounding the lung or the membranes covering the chest wall, e) the heart or the membranes surrounding it, f) the major blood vessels that connect to or from the heart, g) the trachea, h) the esophagus, i) the nerves that control the larynx (vocal organs), j) the sternum or spine, or k) the carina (where the trachea joins the bronchi). In the second type of stage IIIB NSCLC, the cancer has metastasized to lymph nodes on the same side of the chest as the tumor. These cancerous lymph nodes are located near the sternum or where the bronchi enter the lungs. Furthermore, 1) the tumor can be of any size, 2) there are separate tumors in different lobes of the same lung, and 3) the cancer has metastasized to any of the following: a) the heart, b) major blood vessels connected to or from the heart, c) the trachea, d) the esophagus, e) the nerves that control the larynx (vocal organs), f) the sternum or spine, or g) the carina (where the trachea merges with the bronchi).

[0312] In one embodiment, the method of the present invention treats stage IV non-squamous NSCLC. In stage IV NSCLC, the tumors may be of any size, and the cancer has metastasized to the lymph nodes. In stage IV NSCLC, one or more of the following are met: 1) there is one or more tumors in both lungs; 2) the cancer is present in the fluids surrounding the lungs or heart; and 3) the cancer has metastasized to other parts of the body, such as the brain, liver, adrenal glands, kidneys, or bones.

[0313] In one embodiment, the subject has never smoked. In a specific embodiment, the subject has smoked in the past. In one embodiment, the subject is currently smoking. In a specific embodiment, the subject has squamous cell carcinoma cells. In a specific embodiment, the subject has non-squamous cell carcinoma cells.

[0314] Standard treatment for lung cancer In a particular aspect of the present invention, the subject has received at least one prior therapy for the treatment of a tumor derived from NSCLC. The at least one prior therapy may be any treatment known in the art for the treatment of NSCLC or tumors derived therefrom. In particular, the at least one prior therapy may be a standard treatment for the treatment of NSCLC.

[0315] Standard therapies for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Center Network (NCCN), a coalition of 21 leading cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines for Oncology (NCCN Guidelines®), which provide detailed and up-to-date information on standard therapies for various cancers (see NCCN Guidelines® (2014): available at www.nccn.org / professionals / physician_gls / f_guidelines.asp, last accessed May 14, 2014).

[0316] Surgery, radiotherapy (RT), and chemotherapy are the three most commonly used therapies to treat NSCLC patients. As a class, NSCLC is relatively less sensitive to chemotherapy and RT compared to small cell carcinoma. Generally, in patients with stage I or II disease, surgical resection offers the best chance of cure, and the use of chemotherapy both preoperatively and postoperatively is increasing. RT can also be used as adjuvant therapy, first-line local therapy, or palliative therapy for patients with resectable NSCLC.

[0317] Patients with stage IV disease who have a good performance status (PS) benefit from chemotherapy. Many drugs, including platinum-based drugs (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine, are useful for stage IV NSCLC. Combinations using many of these drugs result in a 30% to 40% one-year survival rate, which is superior to single drugs. Specific targeted therapies for the treatment of advanced lung cancer have also been developed. For example, bevacizumab (AVASTIN®) is an mAb that inhibits vascular endothelial growth factor A (VEGF-A). Erlotinib (TARCEVA®) is a small molecule TKI that targets the epidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is a small molecule TKI that targets ALK and MET and is used to treat NSCLC in patients carrying mutated ALK fusion genes. Cetuximab (ERBITUX®) is an mAb that targets EGFR.

[0318] Due to the limited treatment options after first-line (1L) therapy, there is a particularly unmet need in patients with squamous cell NSCLC (which accounts for up to 25% of all NSCLC cases). Monotherapy after progression with platinum-based combination therapy (Pt-doublet) is the standard treatment, resulting in a median overall survival (OS) of approximately 7 months. Docetaxel remains the benchmark drug for this treatment, but erlotinib is also available at a lower frequency. Pemetrexed has also shown comparable efficacy clinically and has been demonstrated to have significantly fewer side effects compared to docetaxel in second-line (2L) therapy for patients with advanced NSCLC (Hanna et al., J Clin Oncol 22:1589-97). Currently, there are no approved treatments for lung cancer beyond third-line (3L) therapy. Pemetrexed and bevacizumab are not approved for squamous cell NSCLC, and molecular targeted therapies have limited applicability. Recent development failures, including the failure of Oncothyreon and Merck KgaA's STIMUVAX® to improve overall survival (OS) in Phase 3 trials, the inability of ArQule and Daiichi Sankyo's c-Met kinase inhibitor tivantinib to meet survival endpoints, the failure of Eli Lilly's ALIMTA® and Roche's AVASTIN® combination to improve OS in late-stage trials, and the failure of Amgen and Takeda Pharmaceutical to meet clinical endpoints with the small molecule VEGF-R antagonist motesanib in late-stage trials, have created an unmet need in advanced lung cancer.

[0319] In certain embodiments, at least one prior therapy comprises standard treatment for the treatment of NSCLC or tumors derived therefrom. In some embodiments, at least one prior therapy comprises surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, at least one prior therapy comprises chemotherapy. In some embodiments, at least one prior therapy is selected from treatments comprising the administration of anticancer agents selected from the group consisting of platinum agents (e.g., cisplatin, carboplatin), taxane agents (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 certain embodiments, at least one prior therapy includes platinum-based doublet chemotherapy.

[0320] In one embodiment, the subject has experienced disease progression after at least one prior therapy. In a particular embodiment, the subject has received at least two prior therapies, at least three prior therapies, at least four prior therapies, or at least five prior therapies. In a particular embodiment, the subject has received at least two prior therapies. In one embodiment, the subject has experienced disease progression after receiving at least two prior therapies. In a particular embodiment, the at least two prior therapies include a first prior therapy and a second prior therapy, wherein the subject has experienced disease progression after the first prior therapy and / or the second prior therapy, wherein the first prior therapy includes surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof; and the second prior therapy includes surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof. In one embodiment, the first prior therapy includes platinum-based chemotherapy, and the second prior therapy includes monotherapy. In a particular embodiment, monotherapy includes docetaxel.

[0321] In some aspect of the present invention, the method of the present invention further comprises administering an additional anticancer therapy. The additional anticancer therapy may include any treatments known in the art for the treatment of NSCLC or tumors derived therefrom, and / or any standard treatments, as described herein. In some aspects, the additional anticancer therapy includes surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof. In some aspects, the additional anticancer therapy includes chemotherapy, including any chemotherapy described herein. In some aspects, the additional anticancer therapy includes immunotherapy. In some aspects, the additional anticancer therapy includes the administration of an antibody or its antigen-binding moiety that specifically binds to LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, MICA, CD137, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesoserine, CD27, GITR, or any combination thereof.

[0322] Anti-LAG-3 antibody A particular aspect of the present invention relates to a method for treating a subject suffering from a tumor with a high TMB status, comprising administering an immunotherapy agent to the subject, wherein the immunotherapy agent comprises an anti-LAG-3 antibody or its antigen-binding moiety. The method may further comprise measuring the TMB status of a biological sample obtained from the subject. Furthermore, the present invention intends to administer an anti-LAG-3 antibody or its antigen-binding moiety to a subject identified as suitable for such treatment, for example, based on the measurement of high TMB.

[0323] The anti-LAG-3 antibodies described herein bind to human LAG-3. Antibodies that bind to LAG-3 are described in International Publication WO / 2015 / 042246 and U.S. Patent Publications 2014 / 0093511 and 2011 / 0150892. An exemplary LAG-3 antibody useful in the present invention is 25F7 (described in U.S. Patent Publication 2011 / 0150892). A further exemplary LAG-3 antibody useful in the present invention is BMS-986016. In one embodiment, the anti-LAG-3 antibody useful in the composition cross-competes with 25F7 or BMS-986016. In another embodiment, the anti-LAG-3 antibody useful in the composition binds to the same epitope as 25F7 or BMS-986016. In yet another embodiment, the anti-LAG-3 antibody comprises six CDRs of 25F7 or BMS-986016.

[0324] Anti-CD137 antibody A particular aspect of the present invention relates to a method for treating a subject suffering from a tumor with a high TMB status, comprising administering an immunotherapy agent to the subject, wherein the immunotherapy agent comprises an anti-CD137 antibody or its antigen-binding moiety. The method may further comprise measuring the TMB status of a biological sample obtained from the subject. Furthermore, the present invention intends to administer an anti-CD137 antibody or its antigen-binding moiety to a subject identified as suitable for such treatment, for example, based on the measurement of high TMB.

[0325] Anti-CD137 antibodies specifically bind to and activate immune cells expressing CD137, stimulating the immune response against tumor cells, particularly the cytotoxic T cell response. Antibodies that bind to CD137 are described in U.S. Patent Publication 2005 / 0095244 and U.S. Patents 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863 and 6,210,669.

[0326] In one embodiment, the anti-CD137 antibody is urelumab (BMS-663513) as described in U.S. Patent No. 7,288,638 (20H4.9-IgG4 [10C7 or BMS-663513]). In one embodiment, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1) as described in U.S. Patent No. 7,288,638. In one embodiment, the anti-CD137 antibody is 4E9 or BMS-554271 as described in U.S. Patent No. 6,887,673. In one embodiment, the anti-CD137 antibody is the antibody described in U.S. Patent No. 7,214,493; No. 6,303,121; No. 6,569,997; No. 6,905,685; or No. 6,355,476. In one embodiment, the anti-CD137 antibody is 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1, as described in U.S. Patent No. 6,362,325. In one embodiment, the anti-CD137 antibody is U.S. The antibody is one of the antibodies described in the patent number (e.g., 53A2). In one embodiment, the anti-CD137 antibody is one of the antibodies described in U.S. Patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E1). In one embodiment, the antibody is Pfizer's PF-05082566 (PF-2566). In another embodiment, the anti-CD137 antibody useful for the present invention cross-competes with the anti-CD137 antibody described herein. In one embodiment, the anti-CD137 antibody binds to the same epitope as the anti-CD137 antibody described herein. In another embodiment, the anti-CD137 antibody useful for the present invention includes the six CDRs of the anti-CD137 antibody described herein.

[0327] Anti-KIR antibody A particular aspect of the present invention relates to a method for treating a subject suffering from a tumor with a high TMB status, comprising administering an immunotherapy agent to the subject, wherein the immunotherapy agent comprises an anti-KIR antibody or its antigen-binding moiety. The method further comprises measuring the TMB status of a biological sample obtained from the subject. Furthermore, the present invention intends to administer an anti-KIR antibody or its antigen-binding moiety to a subject identified as suitable for such treatment, for example, based on the measurement of high TMB.

[0328] Antibodies that specifically bind to KIRs block the interaction between killer cell immunoglobulin-like receptors (KIRs) on NK cells and their ligands. Blocking these receptors promotes NK cell activation and potentially facilitates the destruction of tumor cells by the latter. Examples of anti-KIR antibodies are described in international publications WO2014 / 055648, WO2005 / 00316, WO2005 / 009465, WO2006 / 072625, WO2006 / 072626, WO2007 / 042573, WO2008 / 084106, WO2010 / 065939, WO2012 / 071411 and WO / 2012 / 160448.

[0329] One anti-KIR antibody useful in the present invention is lirirumab (also known as BMS-986015, IPH2102, or the S241P variant of 1-7F9), first described in International Publication WO2008 / 084106. A further anti-KIR antibody useful in the present invention is 1-7F9 (also known as IPH2101), first described in International Publication WO2006 / 003179. In one embodiment, the anti-KIR antibody for the composition of the present invention cross-competes with lirirumab or I-7F9 for binding to KIR. In another embodiment, the anti-KIR antibody binds to the same epitope as lirirumab or I-7F9. In yet another embodiment, the anti-KIR antibody comprises six CDRs of lirirumab or I-7F9.

[0330] Anti-GITR antibody A particular aspect of the present invention relates to a method for treating a subject suffering from a tumor with a high TMB status, comprising administering an immunotherapy agent to the subject, wherein the immunotherapy agent comprises an anti-GITR antibody or its antigen-binding moiety. The method further comprises measuring the TMB status of a biological sample obtained from the subject. Furthermore, the present invention intends to administer an anti-GITR antibody or its antigen-binding moiety to a subject identified as suitable for such treatment, for example, based on the measurement of high TMB.

[0331] Anti-GITR antibodies can be any anti-GITR antibodies that specifically bind to human GITR targets and activate glucocorticoid-induced tumor necrosis factor receptor (GITR). GITR is a member of the TNF receptor superfamily expressed on the surface of multiple types of immune cells, including regulatory T cells, effector T cells, B cells, natural killer (NK) cells, and activated dendritic cells ("anti-GITR agonist antibodies"). Specifically, GITR activation enhances the proliferation and function of effector T cells and neutralizes the suppression by activated T regulatory cells. In addition, GITR activation promotes anti-tumor immunity by increasing the activity of other immune cells such as NK cells, antigen-presenting cells, and B cells. Examples of anti-GITR antibodies are described in International Publications WO / 2015 / 031667, WO / 2015 / 184,099, WO / 2015 / 026,684, WO11 / 028683 and WO / 2006 / 105021, U.S. Publications 7,812,135 and 8,388,967, and U.S. Publications 2009 / 0136494, 2014 / 0220002, 2013 / 0183321 and 2014 / 0348841.

[0332] In one embodiment, a useful anti-GITR antibody in the present invention is TRX518 (e.g., described in Schaer et al. Curr Opin Immunol. (2012) Apr; 24(2): 217-224 and WO / 2006 / 105021). In another embodiment, the anti-GITR antibody is selected from MK4166, MK1248 and antibodies described in WO11 / 028683 and US 8,709,424, for example, antibodies comprising a VH chain containing SEQ ID NO: 104 and a VL chain containing SEQ ID NO: 105 (where SEQ ID NOs are those of WO11 / 028683 or US 8,709,424). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as described in WO2015 / 031667, for example, an antibody comprising VH CDR 1-3 containing SEQ ID NOs. 31, 71, and 63 of WO2015 / 031667, and VL CDR 1-3 containing SEQ ID NOs. 5, 14, and 30 of WO2015 / 031667, respectively. In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as described in WO2015 / 184099, for example, an antibody Hum231#1 or Hum231#2, or their CDRs, or their derivatives (e.g., pab1967, pab1975, or pab1979). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody described in JP2008278814, WO09 / 009116, WO2013 / 039954, US20140072566, US20140072565, US20140065152 or WO2015 / 026684, or INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (MedImmune). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody described in PCT / US2015 / 033991 (e.g., an antibody containing the variable region of 28F3, 18E10, or 19D3). For example, the anti-GITR antibody may be an antibody containing the following VH chain, VL chain, or their CDR:

[0333] VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVS (Sequence ID 1), and

[0334] VL: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIK (Sequence ID 2); or

[0335] VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSS (Sequence ID 3), and

[0336] VL: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (Sequence ID 4); or

[0337] VH: VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSS (Sequence ID 5), and

[0338] VL: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (Sequence ID 6).

[0339] In a particular embodiment, an antibody comprising the above-mentioned VH and VL chain pair, or their CDRs, comprises the heavy chain constant region of either a wild-type or mutant, e.g., effectorless, IgG1 isotype. In one embodiment, an anti-GITR antibody comprises the following heavy chain and light chain amino acid sequences:

[0340] Heavy chain: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMD VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 7), and

[0341] Light chain: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 8), or

[0342] Heavy chain: qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwyegsnkyyadsvkgrftisrdnskntlylqmnslraedtavyycarggsmvrgdyyygmdv wgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepks cdkthtcppcpapeaegapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpssiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg (SEQ ID NO: 9), and

[0343] Light chain: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 10).

[0344] In certain embodiments, the anti-GITR antibody cross-competes with the anti-GITR antibodies described herein, e.g., TRX518, MK4166, or antibodies containing the amino acid sequences of the VH and VL domains described herein. In some embodiments, the anti-GITR antibody binds to the same epitope as the anti-GITR antibodies described herein, e.g., TRX518, MK4166, or antibodies containing the amino acid sequences of the VH and VL domains described herein. In certain embodiments, the anti-GITR antibody comprises six CDRs of TRX518, MK4166, or six CDRs of an antibody containing the amino acid sequences of the VH and VL domains described herein.

[0345] Further antibodies In one aspect, the immunotherapy comprises an anti-TGFβ antibody. In a particular aspect, the anti-TGFβ antibody is the anti-TGFβ antibody described in International Publication No. WO / 2009 / 073533.

[0346] In one embodiment, the immunotherapy comprises an anti-IL-10 antibody. In a particular embodiment, the anti-IL-10 antibody is the anti-IL-10 antibody described in International Publication No. WO / 2009 / 073533.

[0347] In some other embodiments, the immunotherapy includes an anti-B7-H4 antibody. In certain embodiments, the anti-B7-H4 antibody is the anti-B7-H4 antibody described in International Publication No. WO / 2009 / 073533.

[0348] In certain embodiments, the immunotherapy comprises an anti-Fas ligand antibody. In certain embodiments, the anti-Fas ligand antibody is the anti-Fas ligand antibody described in International Publication No. WO / 2009 / 073533.

[0349] In one embodiment, the immunotherapy comprises an anti-CXCR4 antibody. In a particular embodiment, the anti-CXCR4 antibody is an anti-CXCR4 antibody described in International Publication No. 2014 / 0322208 (e.g., urocuplumab (BMS-936564)).

[0350] In one embodiment, the immunotherapy comprises an anti-mesoserine antibody. In a particular embodiment, the anti-mesoserine antibody is the anti-mesoserine antibody described in U.S. Patent No. 8,399,623.

[0351] In one embodiment, the immunotherapy comprises an anti-HER2 antibody. In a particular embodiment, the anti-HER2 antibody is Herceptin (U.S. Patent No. 5,821,337), trastuzumab, or ado-trastuzumab emtansine (Kadcyla, e.g., WO / 2001 / 000244).

[0352] In one embodiment, the immunotherapy comprises an anti-CD27 antibody. In one embodiment, the anti-CD27 antibody is varlilumab (also known as “CDX-1127” and “1F5”), a human IgG1 antibody that is an agonist against human CD27, as described in U.S. Patent No. 9,169,325.

[0353] In one embodiment, the immunotherapy comprises an anti-CD73 antibody. In a particular embodiment, the anti-CD73 antibody is CD73.4.IgG2C219S.IgG1.1f.

[0354] In one embodiment, the immunotherapy comprises an anti-MICA antibody. As used herein, the anti-MICA antibody is an antibody or its antigen-binding fragment that specifically binds to MHC class I polypeptide-associated sequence A. In one embodiment, the anti-MICA antibody binds to MICB in addition to MICA. In one embodiment, the anti-MICA antibody inhibits the cleavage of membrane-bound MICA and the release of soluble MICA. In a particular embodiment, the anti-MICA antibody is the anti-MICA antibody described in U.S. Patent Publication 2014 / 004112 A1, 2016 / 046716 A1, or 2017 / 022275 A1.

[0355] In one embodiment, the immunotherapy comprises an anti-TIM3 antibody. As used herein, the anti-TIM3 antibody is an antibody or its antigen-binding fragment that specifically binds to T-cell immunoglobulin and mucin domain-containing TIM3, also known as hepatitis A virus cell receptor 2 (HAVCR2). In one embodiment, the anti-TIM3 antibody can stimulate an immune response, such as an antigen-specific T-cell response. In one embodiment, the anti-TIM3 antibody binds to soluble or membrane-bound human or cynomolgus monkey TIM3. In a particular embodiment, the anti-TIM3 antibody is the anti-TIM3 antibody described in International Publication No. WO / 2018 / 013818, which is incorporated herein by reference in its entirety.

[0356] In certain embodiments, further anticancer therapy is administered concurrently with, or after, or concurrently with and after, the administration of an anti-PD-1 antibody (or anti-PD-L1 antibody) and an anti-CTLA-4 antibody. In some embodiments, further anticancer therapy is administered concurrently with, or after, the administration of an anti-PD-1 antibody (or anti-PD-L1 antibody) and an anti-CTLA-4 antibody. In some embodiments, further anticancer therapy is administered after, or concurrently with and after, the administration of an anti-PD-1 antibody (or anti-PD-L1 antibody) and an anti-CTLA-4 antibody. In other embodiments, further anticancer therapy is administered between the administration of the anti-PD-1 antibody (or anti-PD-L1 antibody) and the anti-CTLA-4 antibody. In certain embodiments, the further anticancer therapy agent, the anti-PD-1 antibody (or anti-PD-L1 antibody) and / or the anti-CTLA-4 antibody are combined into a single formulation. In other embodiments, further anticancer therapies, anti-PD-1 antibodies (or anti-PD-L1 antibodies) and / or anti-CTLA-4 antibodies are separate formulations.

[0357] Pharmaceutical composition and dosage The therapeutic agent of the present invention may consist of a composition, for example, a pharmaceutical composition comprising an antibody and / or cytokine and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, and the like. Preferably, carriers for compositions containing antibodies are suitable for intravenous, intramuscular, subcutaneous, non-enteral, spinal, or epithelial administration (e.g., by injection or infusion), while carriers for compositions containing antibodies and / or cytokines are suitable for non-enteral administration, such as oral administration. In one embodiment, subcutaneous injection is based on Halozyme Therapeutics’ ENHANZE® drug delivery technology (see U.S. Patent No. 7,767,429, the entirety of which is incorporated herein by reference). ENHANZE® uses antibody co-formulations with recombinant human hyaluronidase enzyme (rHuPH20), which eliminates conventional limitations on the amount of biological agents and drugs that can be delivered subcutaneously by the extracellular matrix (see U.S. Patent No. 7,767,429). The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Accordingly, in some embodiments, the pharmaceutical compositions of the present invention may further comprise recombinant human hyaluronidase enzyme, for example, rHuPH20.

[0358] In some embodiments, an anti-PD-1 antibody or anti-PD-L1 antibody is administered in a fixed dose together with an anti-CTLA-4 antibody in a single composition. In some embodiments, the anti-PD-1 antibody is administered in a fixed dose together with an anti-CTLA-4 antibody. In some embodiments, the anti-PD-L1 antibody is administered in a fixed dose together with an 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 1:140 The ratios are approximately 1:160, 1:180, 1:200, 200:1, 180:1, 160:1, 140:1, 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1 (mg).

[0359] In nivolumab monotherapy, doses of up to 10 mg / kg every two weeks were achieved without reaching the maximum tolerated dose (MTD). However, significant toxicity reported in other clinical trials of checkpoint inhibitors and anti-angiogenic agents (see, e.g., Johnson et al., 2013; Rini et al., 2011) supports the selection of nivolumab doses of 10 mg / kg or less.

[0360] Treatment is continued as long as a clinical benefit is observed, until unacceptable toxicity or disease progression occurs. Nevertheless, in certain embodiments, the dose of anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody administered is significantly lower than the approved dose, i.e., below the therapeutic dose of the drug. Anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody may be administered at the dose that produces the best efficacy as a monotherapy in clinical trials, for example, approximately 3 mg / kg of nivolumab administered once every three weeks (Topalian et al., 2012a; Topalian et al., 2012), or at significantly lower doses, i.e., below the therapeutic dose.

[0361] Dosage and frequency of administration vary depending on the half-life of the antibody in the subject. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are generally administered over a long period at relatively long intervals. Some patients continue treatment due to their life expectancy. In therapeutic applications, relatively high doses are often required at relatively short intervals until the progression of the disease slows or stops, or preferably until the patient shows partial or complete improvement of disease symptoms. After that, the patient may be administered a prophylactic regimen.

[0362] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as not to be excessively toxic to the patient and so as to be effective in obtaining an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration. The selected dose level may vary depending on the activity of the particular composition of the present invention used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and other factors well known in the medical field. The compositions of the present invention may be administered by one or more routes of administration using one or more of the various methods well known in the art. As will be understood by those skilled in the art, the route of administration and / or method may vary depending on the desired result.

[0363] kit Kits comprising (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody for therapeutic use are also within the scope of the present invention. Kits generally include labels and instructions for use indicating the intended use of the kit contents. The term labels include documentary or recorded material that is attached to or included with the kit or otherwise incorporated into the kit. Accordingly, the present invention provides a kit for the treatment of subjects suffering from tumors derived from NSCLC, the kit comprising: (a) an anti-PD-1 antibody or an anti-PD-L1 antibody in a dose ranging from 0.1 to 10 mg per kg of body weight; (b) an anti-CTLA-4 antibody in a dose ranging from 0.1 to 10 mg per kg of body weight; and (c) instructions for the use of (a) an anti-PD-1 antibody or an anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody in the method described herein. In one embodiment, the present invention provides a kit for treating subjects suffering from tumors derived from NSCLC, the kit comprising: (a) an anti-PD-1 antibody in a dose range of 200 mg to 800 mg or an anti-PD-L1 antibody in a dose range of 200 mg to 1800 mg; (b) an anti-CTLA-4 antibody in a dose range of 10 mg to 800 mg; and (c) instructions for the use of (a) the anti-PD-1 antibody or the anti-PD-L1 antibody and (b) the anti-CTLA-4 antibody in the method described herein.

[0364] In certain preferred embodiments for treating human patients, the kit comprises an anti-human PD-1 antibody as described herein, for example, nivolumab or pembrolizumab. In certain preferred embodiments for treating human patients, the kit comprises an anti-human PD-L1 antibody as described herein, for example, atezolizumab, durvalumab or avelumab. In certain preferred embodiments for treating human patients, the kit comprises an anti-human CTLA-4 antibody as described herein, for example, ipilimumab, tremelimumab, MK-1308 or AGEN-1884.

[0365] In one embodiment, the kit further comprises a cytokine or a variant thereof. In a particular embodiment, the kit comprises (a) an anti-PD-1 antibody or an anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody, and (c) a CD122 agonist.

[0366] In one embodiment, the kit further comprises the comprehensive genome profiling assay described herein. In another embodiment, the kit comprises FOUNDATIONONE® CDX (商標) The kit further includes a genome profiling assay. In one embodiment, the kit further includes instructions for administering (a) an anti-PD-1 antibody or anti-PD-L1 antibody and (b) an anti-CTLA-4 antibody to a subject identified as having a high TMB state, e.g., a high TMB state of at least about 10 mutations per megabase of the genome tested, according to the method described herein. In another embodiment, the kit further includes instructions for administering (a) an anti-PD-1 antibody or anti-PD-L1 antibody, (b) an anti-CTLA-4 antibody and (c) a cytokine, e.g., a CD122 agonist, to a subject identified as having a high TMB state, e.g., a high TMB state of at least about 10 mutations per megabase of the genome tested, according to the method described herein.

[0367] All references cited above, as well as all references cited herein, are incorporated herein by their entirety by citation.

[0368] The following embodiments are provided for illustrative purposes only and should not be construed as limiting. [Examples]

[0369] Examples Example 1: Combination therapy of nivolumab and ipilimumab in patients with high tumor mutational burden in non-small cell lung cancer. Nivolumab plus ipilimumab demonstrated promising efficacy in a Phase 1 NSCLC trial, with tumor mutational burden (TMB) emerging as a potentially useful biomarker. This trial is an open-label, multipart Phase 3 clinical trial of first-line nivolumab and nivolumab-based combinations in a biomarker-selected NSCLC population. We report Part 1 results regarding the co-primary endpoint of progression-free survival (PFS) with nivolumab plus ipilimumab combination therapy versus chemotherapy in patients with high TMB (10 or more mutations per megabase). This trial will also continue with the primary endpoint of overall survival in PD-L1-selected patients.

[0370] Patients were either not receiving chemotherapy, had stage IV NSCLC, or had relapsed NSCLC. Patients with tumor PD-L1 expression of 1% or higher were randomized in a 1:1:1 ratio to nivolumab plus ipilimumab, nivolumab only, or chemotherapy. Patients with tumor PD-L1 expression of less than 1% were randomized in a 1:1:1 ratio to nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy only. TMB is FOUNDATIONONE® CDX (商標) The measurement was performed using [a specific method / tool].

[0371] In patients with high TMB (10 or more mutations per megabase), PFS was significantly longer with nivolumab plus ipilimumab compared to chemotherapy (HR, 0.58; 97.5% CI, 0.41–0.81; P=0.0002); the 1-year PFS rates were 43% and 13%, respectively, and the median PFS (95% CI) was 7.2 months (5.5–13.2 months) and 5.5 months (4.4–5.8 months), respectively. The objective response rates were 45.3% and 26.9%, respectively. The benefit of nivolumab plus ipilimumab over chemotherapy was nearly consistent within subgroups, including subgroups with PD-L1 expression rates of ≥1% and <1%. The incidence of grade 3 to 4 treatment-related adverse events was 31% and 36%, respectively.

[0372] In NSCLC patients with TMB ≥ 10 mutations / Mb, regardless of PD-L1 expression, the combination of nivolumab and ipilimumab as the first-line treatment significantly improved progression-free survival (PFS) compared to chemotherapy. These results validate the efficacy of nivolumab and ipilimumab in NSCLC and the role of TMB as a biomarker for patient selection.

[0373] Patient selection Fresh or archival tumor biopsy specimens obtained within six months prior to registration (in cases where the patient had not received any systemic anticancer therapy) were tested for PD-L1 in the central laboratory using an anti-PD-L1 antibody (28-8 antibody) (Hanna, N., et al. J Oncol Pract 13:832-7 (2017)).

[0374] This trial included adult patients with stage IV / recurrent squamous or non-squamous NSCLC histologically confirmed to contain PD-L1, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (Oken MM, et al. Am J Clin Oncol 5:649-55 (1982)), and no prior systemic chemotherapy as first-line therapy for advanced or metastatic disease. See Figure 1. All patients underwent imaging for brain metastasis screening. Patients with known EGFR mutations or ALK translocations sensitive to targeted therapy, autoimmune diseases, or untreated central nervous system metastases were excluded. Patients with central nervous system metastases were eligible if they had received appropriate treatment and had returned to neurological baseline at least two weeks prior to randomization.

[0375] Further inclusion and exclusion criteria included a history of adjuvant or neoadjuvant chemotherapy for locally advanced lesions, or a history of curative chemoradiotherapy, up to six months prior to enrollment. Prior palliative radiotherapy for non-central nervous system lesions had to be completed at least two weeks prior to randomization. Patients had either discontinued glucocorticoids or been receiving a steady or tapered dose of 10 mg or less of prednisolone (or equivalent) per day at least two weeks prior to randomization.

[0376] Clinical trial design and procedures This clinical trial was a multipart Phase 3 study designed to compare and evaluate different nivolumab-based regimens with chemotherapy in different patient populations. Patients with tumor PD-L1 expression levels of ≥1% and <1% were enrolled simultaneously at the same center for 16 months (Figure 2). Patients with PD-L1 expression levels of ≥1% were randomly stratified (1:1:1) based on tumor tissue (squamous cell type vs. non-squamous cell type NSCLC) to receive (i) nivolumab 3 mg / kg every two weeks and ipilimumab 1 mg / kg every six weeks, (ii) platinum-based chemotherapy every three weeks for up to four cycles based on histological evidence, or (iii) nivolumab 240 mg every two weeks. Patients with PD-L1 expression levels less than 1% were randomly stratified (1:1:1) based on tumor tissue to receive (i) nivolumab 3 mg / kg every two weeks and ipilimumab 1 mg / kg every six weeks, (ii) a histologically-based platinum-based drug every three weeks for up to four cycles, or (iii) nivolumab 360 mg and a histologically-based platinum-based drug every three weeks for up to four cycles. Non-squamous NSCLC patients with stable disease or a response after four cycles of chemotherapy or nivolumab chemotherapy could continue maintenance therapy with pemetrexed or pemetrexed and nivolumab. All treatments were continued until disease progression, unacceptable toxicity, or completion of the protocol (up to two years for immunotherapy). Crossover between treatment groups within the study was not permitted.

[0377] Of the 2,877 patients enrolled in Part 1 of the trial, 1,739 were randomized. Of the 1,138 patients who were not randomized, 909 became unmeeting of the trial criteria (common reasons including the identification of EGFR / ALK mutations, decreased ECOG PS, untreated brain metastases, and unassessable PD-L1 expression), 88 patients withdrew their consent, 40 patients died, 33 patients experienced adverse events (unrelated to the investigational drug), 6 patients became unfollowable, and 62 patients were excluded for other reasons.

[0378] As shown in Tables 16 and 17, baseline characteristics were similar across all randomized and TMB-evaluable patients, and were well-balanced across treatment groups.

[0379] [Table 22]

[0380] [Table 23]

[0381] Tumor gene mutational load analysis TMB is a validated assay using FOUNDATIONONE® CDX, employing next-generation sequencing to detect substitutions, insertions, deletions (indels), and copy number changes in 324 genes and selective gene rearrangements. (商標)This was evaluated in preserved or fresh formalin-fixed paraffin-embedded tumor samples (Ettinger, DS, et al. J Natl Compr Canc Netw, 15:504-35 (2017)). An independent report has demonstrated agreement between TMB estimated from whole exome sequencing (WES) and TMB estimated from targeted next-generation sequencing (NGS). See Szustakowski J., et al. Evaluation of tumor mutation burden as a biomarker for immune checkpoint inhibitor efficacy: A calibration study of whole exome sequencing with FoundationOne®. Presented at the American Association for Cancer Research 2018 Annual Meeting; 2018; Chicago, Illinois; Zehir A, et al. Nat Med 2017;23:703-713; Rizvi H., et al., J Clin Oncol 2018;36:633-41. TMB was calculated according to a previously defined method. Reck, M., et al., N Engl J Med, 375:1823-33 (2016). In summary, TMB was defined as the number of somatic cells, codings, substitutions, and short indels per megabase of the genome examined. All substitutions and indels in the coding regions of target genes, including synonymous mutations, were filtered for both oncogenic driver events according to COSMIC and germ cell status according to the dbSNP and ExAC databases, in addition to a private database of rare germ cell events compiled in the Foundation Medicine clinical cohort. Further filtering was also performed based on a computational assessment of germ cell status using the SGZ (somatic-germ-zygosity) tool. Aguiar, PN, et al., ESMO Open, 2:e000200 (2017).

[0382] As shown in Table 18, of all randomized patients (N=1739), 1649 (95%) had tumor samples for TMB evaluation, and 1004 (58%) had valid TMB data for TMB-based efficacy analysis.

[0383] [Table 24]

[0384] Of the TMB-evaluable patients in all treatment groups, 444 (44%) had TMB ≥ 10 mutations / Mb, of which 139 were randomized to nivolumab + ipilimumab and 160 to chemotherapy. As shown in Table 19, baseline characteristics between the two treatment groups were balanced, including the distribution of PD-L1 expression. In the TMB-evaluable population, there was no correlation between TMB and PD-L1 expression. See Figures 7A and 7B.

[0385] [Table 25]

[0386] During a minimum follow-up period of 11.2 months, 17.7% and 5.6% of patients treated with nivolumab plus ipilimumab and chemotherapy, respectively, continued treatment. See Table 20.

[0387] [Table 26]

[0388] Of the patients assigned to chemotherapy, 28.1% received subsequent immunotherapy. See Table 21.

[0389] [Table 27]

[0390] The median treatment duration was 4.2 months (range: 0.03 to 24.0+) for nivolumab plus ipilimumab combination therapy and 2.6 months (range: 0.03 to 22.1+) for chemotherapy. The median number of administrations of nivolumab (every 2 weeks) and ipilimumab (every 6 weeks) received as combination therapy was 9 (range: 1 to 53) and 3 (range: 1 to 18), respectively.

[0391] In patients with high TMB (≥10 mutations / Mb), 24.2% in the nivolumab + ipilimumab group and 3.1% in the chemotherapy group were continuing treatment at the time of database lock. The most common reasons for discontinuing treatment were disease progression (37.8% and 47.2%, respectively), investigational drug toxicity (25.9% and 8.8%, respectively), and completion of necessary treatment in patients in the chemotherapy group (26.4% in the nivolumab + ipilimumab group compared to 0%).

[0392] Endpoints and evaluation In Part 1 of this clinical trial, two primary endpoints were established. The first primary endpoint was progression-free survival (PFS) as assessed by blinded, independent central review of nivolumab + ipilimumab versus chemotherapy in the patient population selected for TMB. Based on previous findings (Ramalingam SS, et al. Tumor mutation burden (TMB) as a biomarker for clinical benefit from dual immune checkpoint blockade with nivolumab (nivo) + ipilimumab (ipi) in first-line (1L) non-small cell lung cancer (NSCLC): identification of TMB cutoff from CheckMate 568. Presented at the American Association for Cancer Research 2018 Annual Meeting; 2018; Chicago, Illinois.), a predefined TMB cutoff of ≥10 mutations / Mb was selected for the pre-planned analysis of the primary endpoint. The second primary endpoint was overall survival (OS) in a patient population selected by PD-L1, compared to nivolumab plus ipilimumab versus chemotherapy.

[0393] As shown in Table 22, secondary endpoints in patients selected for TMB include PFS with nivolumab versus chemotherapy in patients with TMB ≥ 13 mutations / Mb and ≥ 1% PD-L1 expression, and OS with nivolumab + ipilimumab versus platinum combination chemotherapy in patients with TMB ≥ 10 mutations / Mb.

[0394] [Table 28]

[0395] The ≥13 mutations / Mb TMB cut-off, a secondary endpoint of PFS for nivolumab vs chemotherapy, was based on an analysis of prior trials, including a bridging trial that converted whole exome sequencing data to FoundationOne® CDX (商標) data. See Carbone, et al. N Engl J Med 2017;376:2415-26;Szustakowski et al. Evaluation of tumor mutation burden as a biomarker for immune checkpoint inhibitor efficacy: A calibration study of whole exome sequencing with FoundationOne®In: American Association for Cancer Research 2018 Annual Meeting. Chicago, Illinois; 2018. Overall response rate (ORR), response duration, and safety were exploratory endpoints. Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. PD-L1 was determined as described above. Labeling:PD-L1 IHC 28-8 pharmDx. Dako North America, 2016. (Accessed October 20, 2016, accessdata.fda.gov / cdrh_docs / pdf15 / P150027c.pdf.).

[0396] TMB was defined as the number of somatic, coding, base substitutions, and short insertions / deletions (indels) per megabase of the genome examined and was determined using the FoundationOne® CDX (商標) assay. For example, the FoundationOne® CDX (商標)See Foundation Medicine, 2018 (last accessed February 8, 2018, foundationmedicine.com / genomic-testing / foundation-one-cdx); Chalmers et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med 2017;9:34; and Sun JX, He Y, Sanford E, et al. The number of mutations after applying various filters was divided by the counted region (0.8 Mb) to calculate the number of mutations / Mb.

[0397] In patients with TMB ≥ 10 mutations / Mb, with PFS as the primary endpoint in the comparison between nivolumab + ipilimumab and chemotherapy, a two-sided log-rank test estimated a favorable hazard ratio of 0.66 for nivolumab + ipilimumab versus chemotherapy with 80% power, based on a sample size of 265 patients with approximately 221 death or disease progression events. Hazard ratios for PFS with relevant two-sided confidence intervals were estimated using an unstratified Cox proportional hazards model with treatment group as a single covariate. Multivariate analysis was performed in patients with TMB ≥ 10 mutations / Mb to evaluate the impact of known prognostic baseline factors on PFS. For primary and secondary comparisons specified in hierarchical hypothesis tests for patients selected by TMB, corresponding two-sided 97.5% CI hazard ratio estimates were calculated (see Table 22 above). For all other estimates, two-sided 95% confidence intervals (CIs) were calculated, which should not be used to infer differences in treatment efficacy. Survival curves were estimated using the Kaplan-Meier method.

[0398] In conclusion, this trial met its primary endpoint, and these results could establish two new standard therapies for advanced NSCLC. First, all untreated NSCLC patients should undergo TMB testing, as TMB has proven to play an important and independent role as a biomarker. Second, this trial presented nivolumab plus ipilimumab combination therapy as a new first-line therapy option for patients with high TMB ≥ 10 mutations / Mb. These results provide a more personalized approach to lung cancer treatment by combining effective first-line chemotherapy and immunotherapy while maintaining effective second-line therapy options. Using TMB as a predictive biomarker for NSCLC patients provides an example of precision medicine, tailoring treatment to patients who are most likely to benefit from combination immunotherapy.

[0399] All randomized patients In all randomized patients (with or without PD-L1 expression), nivolumab plus ipilimumab versus chemotherapy improved progression-free survival (PFS) (hazard ratio [HR], 0.83; 95% CI, 0.72–0.96), with a 1-year PFS rate of 31% versus 17%. The median PFS was 4.9 months (95% CI, 4.1–5.6) with nivolumab plus ipilimumab and 5.5 months (95% CI, 4.6–5.6) with chemotherapy. Similar benefits were observed in TMB-evaluated patients with nivolumab plus ipilimumab versus chemotherapy (HR, 0.82; 95% CI, 0.68–0.99), with a 1-year PFS rate of 32% versus 15%. The median PFS was 4.9 months (95% CI, 3.7–5.7) and 5.5 months (95% CI, 4.6–5.6), respectively. See Figures 4A and 4B.

[0400] Patients with high TMB (≥10 mutations / Mb) vs. patients with low TMB Analysis of the primary endpoint in patients with high TMB (≥10 mutations / Mb) showed a significant improvement in PFS with nivolumab plus ipilimumab versus chemotherapy (HR, 0.58; 97.5% CI, 0.41–0.81; P=0.0002), with 1-year PFS rates of 43% versus 13% (chemotherapy), and median PFS of 7.2 months (95% CI, 5.5–13.2) and 5.5 months (95% CI, 4.4–5.8), respectively. See Figure 4A. In a pre-specified multivariate analysis of PFS in patients with TMB ≥ 10 mutations / Mb, the efficacy of nivolumab + ipilimumab versus chemotherapy, adjusted for baseline PD-L1 expression level (≥ 1%, < 1%), sex, tumor histology (squamous, non-squamous), and ECOG PS (0, ≥ 1), was consistent with the primary PFS analysis (HR, 0.57; 95% CI, 0.40–0.80, multivariate Cox model P=0.0002). In patients with TMB < 10 mutations / Mb, no improvement in PFS was observed with nivolumab + ipilimumab versus chemotherapy (HR, 1.07; 95% CI, 0.84–1.35); median PFS was 3.2 months (95% CI, 2.7–4.3) with nivolumab + ipilimumab and 5.5 months (95% CI, 4.3–5.6) with chemotherapy. See Figure 5.

[0401] The response rate was 45.3% with nivolumab plus ipilimumab and 26.9% with chemotherapy (Table 23). Eisenhauer, EA, et al. Eur J Cancer, 45:228-47 (2009). The percentage of patients who maintained a response after one year was 68% with nivolumab plus ipilimumab and 25% with chemotherapy (Figure 4B).

[0402] [Table 29]

[0403] Selected subgroups in patients with high TMB (≥10 mutations / Mb) Subgroup analyses by PD-L1 status showed that nivolumab plus ipilimumab combination therapy improved PFS compared to chemotherapy in patients with PD-L1 expression levels of ≥1% and <1%. See Figures 6A and 6B. In patients with both squamous and non-squamous cell tumor histological types, nivolumab plus ipilimumab combination therapy resulted in improved PFS compared to chemotherapy. See Figures 6C and 6D. In most other subgroups of patients with TMB ≥ 10 mutations / Mb, nivolumab plus ipilimumab combination therapy improved PFS compared to chemotherapy. See Figure 6E.

[0404] Nivolumab monotherapy The secondary endpoint of the trial was the efficacy of nivolumab (n=79) versus chemotherapy (n=71) in patients with TMB ≥ 13 mutations / Mb and PD-L1 expression rate ≥ 1% (patients with PD-L1 expression rate < 1% were not eligible for nivolumab). In this patient group, no improvement in PFS was observed with nivolumab (HR, 0.95; 97.5% CI, 0.61, 1.48; P=0.7776). The median PFS was 4.2 months (95% CI, 2.7–8.3) with nivolumab and 5.6 months (95% CI, 4.5–7.0) with chemotherapy. See Figure 7.

[0405] In patients with TMB ≥ 10 mutations / Mb and PD-L1 expression rate ≥ 1%, the median PFS was 7.1 months (95% CI, 5.5–13.5) with nivolumab plus ipilimumab combination therapy, compared to 4.2 months (95% CI, 2.6–8.3) with nivolumab monotherapy. See Figure 8.

[0406] The results of this trial demonstrate that in patients with advanced NSCLC and TMB ≥ 10 mutations / Mb, first-line therapy with nivolumab plus ipilimumab is associated with improved PFS compared to chemotherapy. The effect of the immunotherapy combination was sustained, with a 1-year progression-free rate of 43% (13% in the chemotherapy group) and 68% of responders (25% in the chemotherapy group) maintaining their response at 1 year. The efficacy of nivolumab plus ipilimumab was observed in patients with squamous and non-squamous histological subtypes with PD-L1 expression of ≥ 1% and < 1%, and was consistent in most other subgroups. In all randomized patients, the combination of nivolumab plus ipilimumab improved PFS compared to chemotherapy, and TMB ≥ 10 mutations / Mb was an effective biomarker. In particular, the effect of nivolumab plus ipilimumab combination therapy was enhanced in patients with high TMB, but no effect compared to chemotherapy was observed in patients with low TMB (< 10 mutations / Mb). Furthermore, in patients with TMB ≥ 10 mutations / Mb, nivolumab plus ipilimumab combination therapy showed improved efficacy compared to nivolumab monotherapy, highlighting the importance of dual immune checkpoint inhibition in NSCLC patients with TMB ≥ 10 mutations / Mb. This trial is continuing to evaluate the primary endpoint of overall survival (OS) in patients selected based on PD-L1.

[0407] This clinical trial demonstrates that TMB and PD-L1 expression are independent biomarkers. In patients with high TMB, the benefits of nivolumab plus ipilimumab were similar to chemotherapy in patients with tumor PD-L1 expression of ≥1% and those with <1%. Therefore, nivolumab plus ipilimumab represents a novel and effective treatment regimen for patients with TMB ≥ 10 mutations / Mb, regardless of PD-L1 expression.

[0408] The safety of nivolumab plus ipilimumab was consistent with previously reported data on first-line therapy for NSCLC. Previous trials evaluated various nivolumab plus ipilimumab dosing regimens in eight cohorts, demonstrating that a regimen of 3 mg / kg nivolumab every two weeks plus 1 mg / kg ipilimumab every six weeks was well-tolerated and effective. (Hellmann, MD, et al. Lancet Oncol, 18:31-41 (2017)). These findings were confirmed in our large-scale international trial, and no new safety signals were observed with this combination therapy. The rates of treatment-related selective adverse events and treatment-related discontinuation were slightly higher compared to nivolumab monotherapy, and the combination therapy was well-tolerated with a low incidence of selective adverse events.

[0409] The incidence of treatment-related adverse events leading to discontinuation was higher with nivolumab plus ipilimumab than with chemotherapy. This is thought to be partly related to the longer duration of treatment with nivolumab plus ipilimumab and the resulting longer progression-free survival (PFS).

[0410] Significant questions remain regarding the role of immunotherapy / combined immunotherapy versus combined immunotherapy / chemotherapy, the optimal sequencing of treatments, whether TMB can identify patients who can benefit from combined immunotherapy / chemotherapy, and whether it can identify the optimal TMB cutoff for PD-1 / L1 monotherapy. Given that the results of this trial validated the clinical utility of TMB as an important and independent biomarker, multidisciplinary efforts are needed to ensure sufficient tumor tissue availability and acceptable treatment change time for testing. The 58% TMB result rate reported in this trial was primarily due to the limited availability of sufficient quantity or quality tumor samples, resulting in limited tissue requests for biomarker analysis as part of the trial. In clinical practice, when the intention of TMB testing is known in advance and sufficient quantity and quality tumor samples can be collected and submitted, successful TMB determination can be expected in 80%–95% of patients undergoing testing. 24 CheckMate 817 (NCT02869789) prospectively evaluates the feasibility of TMB testing in patients with advanced NSCLC and TMB ≥ 10 mutations / Mb as first-line therapy with nivolumab plus ipilimumab, and may help identify gaps and opportunities in education to optimize the feasibility of TMB testing. Furthermore, TMB is a reliable and reproducible biomarker, and simultaneously provides comprehensive genomic profiling through next-generation sequencing of multiple potentially therapeutically effective oncogenes. Therefore, TMB testing, utilizing already established techniques, provides widely applicable and clinically important information in a single test, guiding the management of NSCLC in first-line therapy.

[0411] Treatment after progression and overall survival follow-up Nivolumab treatment or nivolumab plus ipilimumab combination treatment was approved for continued administration after disease progression if it demonstrated clinical utility as assessed by the principal investigator and the patient remained well-tolerated. After discontinuation of the investigational drug treatment, patients were followed up on overall survival every three months via in-person or telephone contact.

[0412] Example 2: Nivolumab + Ipilimumab combination therapy in non-small cell lung cancer with PD-L1 expression less than 1% The inventors report the results of a Phase 3 clinical trial of Example 1, with the efficacy and safety of nivolumab plus ipilimumab combination therapy and nivolumab plus chemotherapy as the primary endpoints in patients with PD-L1 expression of less than 1%. Recent studies have shown that adding anti-PD-(L)1 therapy to chemotherapy improves outcomes compared to chemotherapy alone. However, it was observed that there was little benefit in non-squamous NSCLC patients with PD-L1 expression of less than 1% (PFS HR: 0.75 and 0.77).

[0413] Patients were those with stage IV or relapsed NSCLC who had not received chemotherapy. Patients with tumor PD-L1 expression of ≥1% were randomized in a 1:1:1 ratio to nivolumab + ipilimumab, nivolumab + chemotherapy, or chemotherapy, while patients with tumor PD-L1 expression of <1% were randomized in a 1:1:1 ratio to nivolumab + ipilimumab, nivolumab + chemotherapy, or chemotherapy (Figure 1). TMB is FOUNDATIONONE® CDX (商標) The determination was made using [the specified method]. Secondary endpoints of this trial include measuring progression-free survival in patients with tumor PD-L1 expression less than 1% after treatment with nivolumab plus chemotherapy compared to chemotherapy alone, measuring overall survival in the PD-L1 selective population treated with nivolumab plus ipilimumab compared to chemotherapy alone, and measuring progression-free survival in the TMB selective population treated with nivolumab plus ipilimumab compared to chemotherapy alone.

[0414] In this clinical trial, a total of 550 patients were identified as having PD-L1 expression of less than 1%. Of these, 177 received nivolumab plus chemotherapy, 187 received nivolumab plus ipilimumab, and 186 received chemotherapy alone. Table 24 shows the baseline characteristics of patients with tumor PD-L1 expression of less than 1%.

[0415] [Table 30]

[0416] result In patients with tumor PD-L1 expression <1% who received nivolumab plus chemotherapy, the 1-year progression-free survival (PFS) rate was 26%, compared to 14% in patients treated with chemotherapy alone (Figure 9A). The overall response rate in patients treated with nivolumab plus chemotherapy was 36.7%, compared to 23.1% in patients treated with chemotherapy alone (Figure 9B). The duration of response (DOR) in patients treated with nivolumab plus chemotherapy was approximately 28% at 1 year, compared to approximately 24% in patients treated with chemotherapy alone (Figure 9C). Furthermore, the overall response rate (ORR) in patients treated with nivolumab plus ipilimumab was approximately 25.1%, and the median DOR was approximately 17.97 months (95% CI: 12.2, NR) (data not shown).

[0417] Analysis of the patient population revealed that when comparing patient responses to nivolumab plus chemotherapy versus chemotherapy alone, patients with non-squamous NSCLC had a lower unstratified hazard ratio (HR, 0.68) than patients with squamous NSCLC (0.92) (Figure 9D). Furthermore, patients identified as having a high TMB (≥10 mutations / Mb) had a lower unstratified HR (0.56) than patients with a low TMB (<10 mutations / Mb) (0.87) (Figure 9D).

[0418] Subsequently, patients were stratified based on their TMB status. Among patients with high TMB (≥10 mutations / Mb) with tumor PD-L1 expression <1%, the 1-year PFS rate was approximately 45% in the nivolumab + ipilimumab group, approximately 27% in the nivolumab + chemotherapy group, and approximately 8% in the chemotherapy alone group (Figure 10A). The median PFS was 7.7 months in patients treated with nivolumab + ipilimumab, 6.2 months in patients treated with nivolumab + chemotherapy, and 5.3 months in patients treated with chemotherapy alone (Figure 10A).

[0419] Conversely, in patients with low TMB (<10 mutations / Mb) tumor PD-L1 expression of 1% or more, the 1-year PFS after either nivolumab plus ipilimumab or nivolumab plus chemotherapy was approximately 18%, while the 1-year PFS after chemotherapy alone was approximately 16% (Figure 10B). The median PFS was 3.1 months in the nivolumab plus ipilimumab group and 4.7 months in the nivolumab plus chemotherapy or chemotherapy alone group (Figure 10B).

[0420] The duration of response (DOR) was also measured for each treatment group. In patients with high TMB (tumor PD-L1 expression <1%), the nivolumab + ipilimumab treatment group showed a 1-year DOR rate of approximately 93%, while the nivolumab + chemotherapy treatment group showed a 1-year DOR rate of approximately 33% (Figure 10C). In the group treated with chemotherapy alone, 1-year survival was not achieved (Figure 10C). The median DOR was 7.4 months in the nivolumab + chemotherapy treatment group and 4.4 months in the chemotherapy alone group (Figure 10C). The nivolumab + ipilimumab treatment group did not reach a median DOR (Figure 10C). The response rates for these treatment groups were 60.5% for nivolumab + chemotherapy, approximately 36.8% for nivolumab + ipilimumab, and approximately 20.8% for chemotherapy alone (data not shown). This difference was considerably larger in low-tumor-myelopathy (TMB) patients with tumor PD-L1 expression less than 1%, who showed an ORR of 27.8% after nivolumab plus chemotherapy and 22.0% after chemotherapy alone (data not shown).

[0421] safety Treatment-related adverse events (TRAEs) are summarized in Table 25 and Figure 11. There were 4 treatment-related deaths in the nivolumab + chemotherapy group, 3 in the nivolumab + ipilimumab group, and 6 in the chemotherapy group. Treatment-related adverse events in the chemotherapy group were similar to those in the nivolumab + chemotherapy group and were consistent with previous reports (Figure 11).

[0422] [Table 31]

[0423] The PFS HR for nivolumab plus chemotherapy versus chemotherapy alone was observed to be 0.74 (95% CI: 0.58, 0.94; NSQ PFS HR = 0.68, 95% CI: 0.51, 0.90) in patients with PD-L1 expression <1%, which was consistent with other clinical trials of PD-(L)1 + chemotherapy combinations. TMB testing is clinically relevant in selecting patients for immunotherapy + immunotherapy and immunotherapy + chemotherapy. In the comparison between nivolumab plus chemotherapy and chemotherapy alone, the PFS benefit is greater in patients with high TMB (≥10 mutations / Mb) and PD-L1 expression <1%. In patients with low TMB (<10 mutations / Mb) and PD-L1 expression <1%, there is no PFS benefit from immunotherapy + immunotherapy or immunotherapy + chemotherapy. Furthermore, there are few Grade 3 / 4 TRAEs in which the safety profiles of immunotherapy + immunotherapy and immunotherapy + chemotherapy are potentially favorable.

[0424] All publications, patents, and patent applications described herein are included by reference to the same extent as when each individual publication, patent, or patent application is specifically and individually indicated as being included by reference.

[0425] This application claims the benefit of priority under U.S. Provisional Application No. 62 / 650,845, filed March 30, 2018, and U.S. Provisional Application No. 62 / 671,906, filed May 15, 2018, and incorporates the entire contents of those applications herein by reference.

Claims

1. A pharmaceutical composition comprising nivolumab for use in combination with ipilimumab in the treatment of patients with tumors originating from non-small cell lung cancer (NSCLC), Here, the tumor has less than 1% PD-L1 expression. Here, the tumor has a tumor mutational load (TMB) state of at least about 10 mutations per megabase of the gene examined. Here, the TMB state is determined by genome profiling, and 9. ここでゲノムプロファイルDASH、HARSH3、SHAR2、SHORE、SHR1020 LOY2)、SYS10、SYSYS4、SYSY2、SYS10、S J2、HYD2、HYS4、HYH3、DYH1 9. SYSYS11 SYSYSYSYSYSYSYSYSYS FAMOUS、HARSH、HARSH、SHASH、SHARE S、SUB、SH1、SHKH4、SHHHH、SHHHH、0 CHA4(S1739)、SYS(SYS3)、KS1 2.001.0043.00.00.00.00.00.00.00.00.00 4、HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 、SY1、SYS1、SYS3、SYS1、SYSY2、SYS1 4、H11、CH05S、SHH3H、SHYS 、SAR3、SHAY、SHAY、SHAY、SAR13、S DASH、SHYS、SHYSH、SHYS1、SHHH S1(S123)、S11S300S3)、SHAS 、HY1、HYS、HYS、HYSHY、SYSYS 131. SHARE(プロモーターのみ) SHA11 CHA11, SH77, DASH, SH1, SH1, SH1 11、SHY、SH2、SHY、SHYS3、SHYS1 10、DY124、CH4、DICH(501)、SH13 2 、SHK、SHY2、SHYS、SHYS3、SH3 14、DY20、SYS、DICH2、SHYS、3 CHEEKS1、SHEY1、SHEYKHY、SHEY10、 HYD3、CHYSH、HYD88、SHY2、SHYS 114、DY10、SHY2、CHS6、CHY23 3. THIS IS YOUR LIFE DA1、DY13、CH33、CH2、CH33 Z、DY2K(GLOVE3)、DY2、DYCH、SYS31、S 22、SHA2、SHA1、DHAS1、CHEN4、DEN 、HYWH(LOVE2) 、HYSY22、HYS2Y1Y、S 22、SH53、SHASH1、CH274(SH001)、0 3、CHSH、HH1S、SHYS、SHHYS、SHH1A pharmaceutical composition comprising 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, PRKC1, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, 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, MAPK4, NTRK1, PTPN11, SOX10, XPO1, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2C, ESR1, FUBP1, IRF2, MED12, PALB2, RAF1, SPTA1, BCOR, CEBP, EZH2, GABRA6, IRF4, MEF2B, PARK2, RANBP2, SRC, BCORL1, CHD2, FAM46C, GATA1, IRS2, MEN1, PAX5, RARA, STAG2, BLM, CHD, FANCA, GATA2, JAK1, MET, PBRM1, RB1, and STAT3.,

2. The pharmaceutical composition according to claim 1, wherein the TMB state is measured in a biological sample obtained from the subject before administration.

3. The pharmaceutical composition according to claim 1 or 2, wherein the TMB state is determined by sequencing nucleic acids in the tumor and identifying genomic modifications in the sequenced nucleic acids.

4. Genome modification, (i) One or more nonsynonymous mutations; (ii) One or more missense mutations; (iii) One or more modifications selected from the group consisting of base pair substitutions, base pair insertions, base pair deletions, copy number changes (CNAs), gene rearrangements, and any combination thereof; or Any combination of (iv)(i) through (iii), Includes one or more somatic mutations selected from, The pharmaceutical composition according to claim 3.

5. The TMB status of the tumor is determined by the following genes: ABL1, BRAF, CHEK1, FANCC, GATA3, JAK2, MITF, PDCD1LG2 (PD-L2), RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDGFRA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4 (C17orf39), KAT6A (MYST3), MRE11A, 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, AMER1 (FAM123B), C11orf30 (EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT (only the promoter), APC, CARD11, CTNN1A, FBXW7, GNAS, KEAP1, MYC, PIK3R1, SDHA, TET2, AR, CBF2, CTNNB1, FGF10, GPR124, KEL, MYCL (MYCL1), PIK3R2, SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC, TNFIP3, ARFRP1, CCND1, CYLD, FGF19, GRM3, KLHL6, MYD88, PMS2, SDHD, TNFR14, ARID1A, CCND2, DAX1, FGF23, GSK3B, KMT2A (MLL), NF1, POLD1, SETD2, TOP1, ARID1B, CCND3, DDR2, FGF3, H3F3A, KMT2C (MLL3), NF2, POLE, SF3B1, TOP2A, ARID2, CCNE1, DICER1, FGF4, HGF, KMT2D (MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274 (PD-L1), DNMT3A, FGF6, HNF1A, KRAS, NFKBIA, PRDM1,SMAD2, TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMO1, NKX2-1, PREX2, SMAD3 , TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B1, LRP1B, NOTCH1, PRKAR1A, SMAD4, T SHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U 2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMARCB1, VE GFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, C DK4, EPHA7, FLCN, IGF1R, MAP2K1 (MEK1), NRAS, PTCH1, SNCAIP, WISP3, AXL, CDK6, EPHB1, FLT1, IGF2, MAP2K2 (MEK2), NSD1, PTEN, SOCS1, WT1, BAP1, CDK 8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPO1, BARD1, CDKN1A , ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKN1B, ERBB 4, FOXL2, IL7R, MCL1, NTRK3, RAC1, SOX9, ZNF217, BCL2L1, CDKN2A, ERG, FOX P1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS 2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2C, ESR1, FUBP1, IRF2, MED1 2. At least approximately 10 mutations, at least approximately 11 mutations, at least approximately 12 mutations per megabase of the examined genome, as measured by a genome profile consisting of 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, and STAT3.A pharmaceutical composition according to any one of claims 1 to 4, comprising 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.

6. The pharmaceutical composition according to any one of claims 2 to 5, wherein the biological sample comprises a tumor tissue biopsy, a liquid biopsy, blood, serum, plasma, exoRNA, circulating tumor cells, ctDNA, cfDNA, or any combination thereof.

7. The TMB status is, (i) Genome sequencing, (ii) Exome sequencing, (iii) Genome profiling, or Any combination of (iv)(i) through (iii) A pharmaceutical composition according to any one of claims 1 to 6, determined by...

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein nivolumab is administered once every two, three, or four weeks in a body weight-based dose ranging from 0.1 mg to 20.0 mg per kg of body weight, or in a constant 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.

9. Nivolumab is (i) Administer once every three weeks at a dose of 2 mg per kg of body weight; (ii) Administer once every two weeks at a dose of 3 mg per kg of body weight; (iii) It is administered once every two weeks at a constant dose of approximately 200 mg; (iv) Administered once every two weeks at a constant dose of approximately 240 mg; or (v) The pharmaceutical composition according to any one of claims 1 to 8, administered once every four weeks in a constant dose of approximately 480 mg.

10. Ipilimumab is administered once every two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks in a body weight-based dose ranging from 0.1 mg to 20.0 mg per kg of body weight, or in a constant 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. A pharmaceutical composition according to any one of claims 1 to 9.

11. Ipilimumab is (i) Administer once every six weeks at a dose of 1 mg per kg of body weight; (ii) Administer once every four weeks at a dose of 1 mg per kg of body weight; or (iii) Administered in a constant dose of at least approximately 80 mg, A pharmaceutical composition according to any one of claims 1 to 10.

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