CN116829953A

CN116829953A - Methods of determining resistance to checkpoint inhibitor therapy

Info

Publication number: CN116829953A
Application number: CN202180092719.0A
Authority: CN
Inventors: T·施赖伯; G·弗罗姆; S·达西瓦
Original assignee: Shattuck Labs Inc
Current assignee: Shattuck Labs Inc
Priority date: 2020-12-03
Filing date: 2021-12-03
Publication date: 2023-09-29
Also published as: CN116847866A

Abstract

The present disclosure relates to animal models, methods for screening and testing anti-cancer drug candidates, and methods for treating cancer, evaluating the efficacy of cancer treatment, and selecting patients for cancer therapy.

Description

Methods of determining resistance to checkpoint inhibitor therapy

Priority

The application requires U.S. provisional application No. 63/121,083 filed on 12/3/2020; U.S. provisional application No. 63/173,090, filed on 4/9 of 2021; U.S. provisional application No. 63/215,735 filed on 28 th month 6 of 2021; the rights and priority of U.S. provisional application No. 63/276,066 filed on 5 of 11 of 2021, the contents of each of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates in part to methods useful for detecting and treating drug-resistant cancers, as well as methods of developing new therapies for drug-resistant cancers.

Description of electronically submitted text files

The contents of the electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable format copy of the sequence listing (file name: SHK-038pc1_st25; date of creation: 2021, 12, 1, day; size 1,571 bytes).

Background

Drug resistance remains one of the biggest challenges of cancer therapy. Drug resistance exists for all types of cancers and for all modes of treatment, including molecular targeted therapies, immunotherapies and chemotherapies. In addition, cancer treatment is often practiced empirically, and many patients with chemotherapy-resistant disease receive multiple cycles of generally toxic therapy before significant lack of efficacy. The unpredictable response to a particular therapy is a major obstacle to improving outcome in cancer patients. In some patients, the onset of effective therapy is delayed due to unpredictable responses. In addition, it is also common for patients with advanced cancer to receive drugs that help shrink their tumors, but after weeks or months the cancer recurs and the drugs no longer function. Unfortunately, for some patients with cancers that are resistant to checkpoint therapy, few treatment options are available. Thus, resistance, whether pre-treatment (intrinsic or primary) or post-treatment (acquired) is responsible for most cancer recurrences, which are one of the leading causes of death. Thus, there is a need for better understanding of the mechanism of drug resistance, providing guidance for future cancer treatments. Furthermore, in order to improve the outcome of cancer patients, methods of developing new therapies for patients with drug resistant cancers and methods of selecting appropriate drugs for patients with drug resistant cancers are needed.

Disclosure of Invention

Thus, the present disclosure provides, in part, methods of selecting patients for cancer treatment, and methods of cancer treatment based on, for example, gene expression profiles based on anti-PD-1 resistant cancers. The present disclosure also provides animal models suitable for testing anti-cancer drug candidates, as well as methods of preparing pharmaceutical compositions for treating cancer.

In one aspect, the present disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing the presence, absence or level of one or more genes associated with a gene-ontology (GO) pathway in a sample, the GO pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes: and (c) selecting a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 based on the evaluation of step (b). In embodiments, the mice belong to the BALB/C or C57BL/6 strains. In embodiments, the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (keyruda), pilutilize bezumab (pimelizumab) (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimip Li Shan anti (Cemiplimab) (libaio), atuzumab (Atezolizumab) (TECENTRIQ), avistuzumab (Avelumab) (bavendio), and duvalumab (imfinzi).

In one aspect, the present disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing the presence, absence or level of one or more genes associated with a gene-ontology (GO) pathway in a sample, the GO pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes; and (c) selecting a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, the mice belong to the BALB/C or C57BL/6 strains. In embodiments, the cancer therapy capable of inhibiting the ability and/or activity of PD-1, PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimiput Li Shan antibody (libaiyo), avilamab (TECENTRIQ), avermectin (BAVENCIO), and dulluzumab (imfinzi).

In one aspect, the present disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing the presence, absence or level of one or more genes associated with a gene-ontology (GO) pathway in a sample, the GO pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes; and (c) selecting a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan (topotecan), etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine), ribosome biosynthesis inhibitors (e.g., diazaborine, lamotrigine and ribozinoindole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxin (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitor sulfasalazine, erastin or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators and protein transport modulators (e.g., cyclosporin A, fendilin, panbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiropirone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147 and cotransin); and (d) administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; and optionally administering the therapy selected in steps (c) (ii) and (c) (iii). In embodiments, the mice belong to the BALB/C or C57BL/6 strains. In embodiments, the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimiput Li Shan antibody (libayo), atuzumab (TECENTRIQ), avermectin (bavenciio) and dimvaluzumab (imfinzi).

In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer treatment capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from the subject. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with a cellular response to ifnγ is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with a cellular response to ifnγ is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with a cellular response to ifnγ, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with a type I IFN signaling pathway as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with a type I IFN signaling pathway as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample. In embodiments, the biological sample is a biopsy sample. In embodiments, the biological sample comprises a body fluid selected from the group consisting of blood, plasma, serum, tears, lacrimal fluid, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, body fluids containing cells, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scrape, bone marrow samples, tissue biopsy samples, surgical samples, stool, other body fluids, secretions and/or excretions, and/or cells therefrom.

In embodiments, the biological sample comprises at least one tumor cell.

In embodiments, the evaluation is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with the Gene Ontology (GO) pathway identified herein.

In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with the gene body (GO) pathway identified herein.

In embodiments, the assessment informs the classification of the patient as a high risk group or a low risk group. In embodiments, the high risk classification comprises a high level of tumor cells that are resistant to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, the low risk classification comprises a low level of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In one aspect, the present disclosure relates to a transgenic non-human animal comprising one or more tumor cells, wherein the tumor cells have: (a) Upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (b) down-regulation of one or more genes associated with a gene body (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes. In embodiments, the transgenic non-human animal is transgenic. In embodiments, the tumor cells are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the transgenic non-human animal is a rodent. In embodiments, the rodent is a mouse. In embodiments, the mice belong to the BALB/C or C57BL/6 strains.

In one aspect, the present disclosure relates to a method for testing an anticancer drug candidate, the method comprising: (a) Providing a transgenic non-human animal of any of the embodiments disclosed herein: (b) Administering an anti-cancer drug candidate to the transgenic non-human animal, and (c) evaluating whether the anti-cancer drug candidate is effective in slowing or inhibiting cancer growth in the transgenic non-human animal. In one aspect, the present disclosure relates to an anticancer drug candidate selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

In one aspect, the present disclosure relates to a method of preparing a pharmaceutical composition for treating cancer, the method comprising: (a) Providing a transgenic non-human animal of any of the embodiments disclosed herein; (b) Administering an anti-cancer drug candidate to the transgenic non-human animal, and (c) selecting an anti-cancer drug effective to slow or inhibit the growth of cancer in the transgenic non-human animal; and (d) formulating the anticancer drug or drug candidate for administration to a human patient. In one aspect, the present disclosure relates to an anticancer drug candidate selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

In embodiments, upregulation is compared to healthy tissue. In embodiments, up-regulation is compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, up-regulation is compared to a prior biological sample obtained from the subject. In embodiments, downregulation is compared to healthy tissue. In embodiments, down-regulation is compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation is compared to a prior biological sample obtained from the subject.

In embodiments, the anti-checkpoint agent is an antibody selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimipro Li Shan anti (libayo), alemtuzumab (TECENTRIQ), avistuzumab (bavenciio), and dulluzumab (imfinzi).

In one aspect, the present disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azapirone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.).

In one aspect, the present disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azapirone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.). In one aspect, the present disclosure relates to a method of treating cancer, the method comprising (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.; and (d) administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; and optionally administering the therapy selected in steps (c) (ii) and (c) (iii).

In one aspect, the present disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing activation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras by a biological sample; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azapirone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.).

In one aspect, the invention relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing activation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras by a biological sample; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azapirone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.).

In one aspect, the present disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing activation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras by a biological sample; and (c) selecting a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.; and (d) administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; and optionally administering the therapy selected in steps (c) (ii) and (c) (iii).

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering a cancer therapy selected from the group consisting of: an antimetabolite chemotherapeutic agent, a topoisomerase inhibitor, an inhibitor of protein translation, an inhibitor of ribosomal biogenesis, an inhibitor of rRNA and/or tRNA synthesis, an inhibitor of amino acid uptake, a modulator of post-translational modification, a modulator of protein degradation, a modulator of protein transport, a topoisomerase inhibitor, wherein the subject has received or is receiving an anti-cancer treatment capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, and wherein the subject has developed a lack of response, resistance, or non-compliance to the cancer treatment capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2; and wherein the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is upregulated in at least one tumor cell in the subject as compared to healthy tissue, a prior biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (b) Evaluating an anti-tumor response of an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring a decrease in tumor size in a subject; (c) If no decrease in tumor size is observed, administering a Trim7 modulator and/or a proteasome inhibitor; (d) Re-evaluating an anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring tumor reduction in the subject; and (e) if a decrease in tumor size is observed, stopping administration of Trim7 modulator. In embodiments, the proteasome inhibitor is selected from bortezomib (bortezomib), carfilzomib (carfilzomib), ixazomib (ixazomib), predzomib (oprozomib), delazomib (delanzomib), and marizomib (marizomib).

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (b) The anti-tumor response with anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 was evaluated using the following steps: (i) obtaining a biological sample from a subject; (ii) Assessing overexpression and/or activation of TRIM7 in a biological sample; (c) If overexpression and/or activation of TRIM7 is observed, administering TRIM7 modulators and/or proteasome inhibitors; (d) The anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is re-evaluated using the following steps: (i) obtaining a biological sample from a subject; (ii) Assessing overexpression and/or activation of TRIM7 in a biological sample; and (e) stopping the administration of TRIM7 modulator if overexpression and/or activation of TRIM7 is not observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, iferum Sha Zuomi, obrenzomib, delazomib, and malizomib.

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (b) The overexpression and/or activation of TRIM7 was evaluated using the following steps: (i) obtaining a biological sample from a subject; and (ii) assessing overexpression and/or activation of TRIM7 in the biological sample; (c) If overexpression and/or activation of TRIM7 is observed, administering TRIM7 modulators and/or proteasome inhibitors; (d) Re-evaluation of overexpression and/or activation of TRIM7 using the following steps: (i) obtaining a biological sample from a subject; (ii) Assessing overexpression and/or activation of TRIM7 in a biological sample; and (e) stopping the administration of TRIM7 modulator if overexpression and/or activation of TRIM7 is not observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, iferum Sha Zuomi, obrenzomib, delazomib, and malizomib.

In embodiments, the Trim7 modulator is a Trim7 inhibitor. In embodiments, trim7 modulators are selected from the group consisting of small interfering RNAs (siRNA), short hairpin RNAs (shRNA), micrornas (miRNA), antisense RNAs, guide RNAs (gRNA), small molecules, antibodies, peptides, and peptidomimetics. In embodiments, small interfering RNAs (siRNA), short hairpin RNAs (shRNA), micrornas (miRNA), antisense RNAs or guide RNAs (gRNA) inhibit Trim7 protein production. In embodiments, the peptide mimetic mimics the target of Trim7 and thereby inhibits Trim7 activity.

In embodiments, the Trim7 modulator is a mitogen-activated and stress-activated kinase 1 (MSK 1) inhibitor, wherein the MSK1 inhibitor modulates Trim7 via inhibiting a downstream effect of MSK 1. In embodiments, the MSK1 inhibitor is selected from the group consisting of Ro 31-8220, SB-747651A and H89. In an embodiment, the MSK1 inhibitor is SB-747651A.

Any aspect disclosed herein may be combined with any other aspect.

Brief description of the drawings

The patent or application document contains at least one drawing in color. The patent office will provide copies of this patent or patent application publication with color drawings at the request and payment of the necessary fee.

FIGS. 1A and 1B show the generation of anti-PD-1 resistant CT26 tumors. FIG. 1A shows a schematic representation of a method for producing an anti-PD-1 resistant CT26 tumor. Briefly, BALB/C mice were inoculated with 500,000 murine colon carcinoma CT26 cells when the average tumor volume reached 80-100mm ³ On day 0, mice were treated with anti-PD-1 (clone RMP1-14; bioXcell) antibodies. Tumors were excised, and surviving tumor cells were isolated and cultured in vitro. These cells are called "firstA round of "," first generation "or" F1 generation "cells. The first generation cells were inoculated into BALB/C mice and isolated after another course of treatment against PD-1 "second round", "second generation" or "F2 generation" cells. After two additional rounds (4 total rounds) of anti-PD-1 selection, "round 4", "4 th generation" or "F4 generation" cells were isolated. FIG. 1B shows a graph comparing the efficacy of anti-PD-1 antibodies (100. Mu.g of anti-PD-1 clone RMP1-14; bioXcell) in BALB/C mice carrying CT26 parental cells and PD-1 resistant cells. BALB/C mice were vaccinated in the posterior flank with CT26 parental cells and PD-1 resistant fourth generation cells. When the initial tumor volume (STV) reaches 80-100mm ³ At this time, mice were randomized into the following two treatment groups: (1) vehicle (PBS) and (2) anti-PD-1 antibody. On days 0, 3 and 6, mice were given a series of intraperitoneal injections of vehicle or 100. Mu.g of anti-PD-1 (clone RMP1-14; bioXcell). Tumor volumes were measured at the indicated days.

FIGS. 2A-2D show transcriptome analysis using RNA-seq against PD-1 resistant cell lines. Fig. 2A (upper panel) shows Principal Component Analysis (PCA) of spatially separated samples based on transcriptome expression. FIG. 2A (bottom panel) shows the Differential Expressed Genes (DEG) determined between the groups (parent and 2 nd, parent and 4 th, 2 nd and 4 th) and plotted in a heat map. Hierarchical clustering was performed to rank the genes on each row, dividing the genes into 2 major clusters in each comparison, with one subset of gene expression being lower in one dataset (blue) and higher in the other dataset (red). FIG. 2B shows genes that were up-or down-regulated in each dataset. Genes were entered into PANTHER to determine the Gene Ontology (GO) associated with each gene set. The gene set is shown with the relevant p-value. FIG. 2C (upper panel) shows a Venn diagram of gene expression overlap between all data sets. FIG. 2C (bottom panel) shows transcripts per million parts of selected genes (TPM; normalized expression data), demonstrating higher gene baseline expression associated with PD-L1, antigen processing/presentation, protein translation, ER transport; in some data sets, higher than others. FIG. 2D shows transcripts per million of selected genes (TPM; normalized expression data), demonstrating higher baseline expression of genes associated with PD-L1, antigen processing/presentation, protein translation, ER transport; in some data sets, it is preferable to others.

FIG. 3 shows that there is an inconsistency in gene expression and cell surface protein expression in PD-L1/2MHC class I and B2M. Parental and fourth generation anti-PD-1 resistant cells were harvested from the culture and analyzed for surface expression of PD-L1, PD-L2, MHC class I and β2 microglobulin (B2M) by flow cytometry. Gating is plotted as shown, the percentage of cells in each gating is shown above each graph, the MFI (mean fluorescence intensity) of each marker is shown to the right of each percentage.

FIGS. 4A and 4B show transcriptome analysis of anti-PD-1 resistant cell lines. Fig. 4A shows a comparison of second and fourth generation anti-PD-1 resistant cell lines with b16.F10, b16.F10 is a murine melanoma tumor that was used as a model for anti-PD-1 "primary resistance" because these tumors did not respond to anti-PD-1 therapy. Cells were cultured in ifnγ for 24 hours to assess in vitro reactivity. This mimics how tumor cells react in vivo because immune cells infiltrate and secrete effector cytokines (e.g., ifnγ). FIG. 4A (left panel) shows DEG identified between untreated and IFNγ treated parent CT 26. Wherein 338 genes have available data from other datasets; and these values are shown in the other columns. Log2 fold changes are plotted in a heat map and genes are hierarchically clustered based on parental CT 26. Genes are divided into 3 major clusters. Fig. 4A (right) shows the GO path associated with DEG. The relevant genes were entered into PANTHER to identify pathways associated with deregulated genes. FIG. 4B shows transcripts per million of selected genes (TPM; normalized expression data) demonstrating that CT26 anti-PD-1 resistant cells down-regulate these genes when these cells are challenged with IFNγ despite their baseline over-activation by type I and type II interferons.

Fig. 5A-5D show contradictory deregulation of certain genes associated with acquired resistance against PD-1. Genes encoding CD274 (fig. 5A) and B2m (fig. 5B) that were overexpressed in fourth generation anti-PD-1 resistant cells were overexpressed in wild-type CT26 cells, but were inhibited in fourth generation anti-PD-1 resistant cells in the presence of ifnγ. On the other hand, the genes encoding Trim7 (fig. 5C) and Lrg1 (fig. 5D) that were suppressed in fourth generation anti-PD-1 resistant cells were suppressed in wild-type CT26 cells, but were overexpressed in fourth generation anti-PD-1 resistant cells that were responsive to ifnγ. The second generation anti-PD-1 resistant cells showed an intermediate phenotype.

FIGS. 6A and 6F show the identification of driver genes involved in resistance to PD-1 acquisition and the functional pathways affected by the driver genes. FIG. 6A, panels 1-4, illustrate a method for identifying driver genes. FIG. 6A (panel 1) shows that in the presence of IFNγ, the fourth generation anti-PD-1 resistant cells had 1,999 genes down-regulated and 3607 genes up-regulated compared to CT26 cells. FIG. 6A (panel 2) shows that 1,060 genes were down-regulated in fourth generation anti-PD-1 resistant cells but up-regulated in CT26 cells. FIG. 6A (panel 3) shows 688 genes down-regulated in fourth generation anti-PD-1 resistant cells compared to CT26 cells, as revealed by ranking according to responsiveness to IFNγ. FIG. 6A (panel 4) shows 70 genes up-regulated in vivo in fourth generation anti-PD-1 resistant cells compared to CT26 cells. FIG. 6B shows the Gene Ontology (GO) pathway associated with the genes identified using the method of FIG. 6A. FIG. 6C shows functional pathways affected by the TRIM protein family. FIG. 6D shows the functional pathways by which Elk1 and c-Jun function. FIG. 6E shows functional linkages between Lrg1, B2m and Arg1 and other genes. FIG. 6F shows the expression levels of Elk1 in tumor and surrounding normal tissues in a cancer genomic profile (TCGA) cancer genomic program.

Fig. 7A to 7D show that Stat1 (fig. 7A), stat2 (fig. 7B), irf1 (fig. 7C) and Tap1 (fig. 7D) genes overexpressed in response to ifnγ are overexpressed in fourth-generation anti-PD-1 resistant cells and inhibited in the presence of ifnγ in fourth-generation anti-PD-1 resistant cells. The second generation anti-PD-1 resistant cells showed an intermediate phenotype.

FIGS. 8A and 8B show pathway analysis of differentially expressed genes, which resulted in the identification of Ras and Rap1 signaling pathways. FIG. 8A shows the use of the WEB-based GEne SeT analysis kit (WebGestalt) to identify pathways using the first 1,000 genes from FIG. 6A (panel 2). Fig. 8B shows a Volcano plot of the data presented in fig. 8A. Fig. 8C shows RAS signaling pathways. Oval shapes were used to show fusion with Raf/Mek/Erk signaling. Fig. 8D shows RAP1 signaling pathway. Oval shapes were used to show fusion with Raf/Mek/Erk signaling.

Fig. 9A-9C show that the ccs 5 (RANTES) (fig. 9A), cxcl10 (IP-10) (fig. 9B), and Ifnb1 (fig. 9C) genes that are overexpressed in response to ifnγ are overexpressed in fourth-generation anti-PD-1 resistant cells and inhibited in the presence of ifnγ in fourth-generation anti-PD-1 resistant cells. The second generation anti-PD-1 resistant cells showed an intermediate phenotype.

Detailed Description

The present disclosure is based in part on the surprising discovery that upregulation of one or more genes associated with the following Gene Ontology (GO) functions is associated with resistance (acquired or primary resistance) to anti-PD-1 therapy: upregulation of IκB kinase/NFκB signaling, type I IFN signaling pathways, upregulation of IFNα production, upregulation of defensive responses, upregulation of IFNβ production, and regulation of inflammatory responses. This is surprising, inter alia, because these pathways are known to be associated with the sensitivity of cancer cells to anti-PD-1 antibodies. Karachaliou et al, interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients, ther Adv Med Oncol.10:1758834017749748 (2018); rafique et al Immune Checkpoint Blockade and Interferon- αin Melanoma, semin Oncol.42 (3): 436-447 (2015); uehara et al Intratumoral injection of IFN-beta induces chemokine production in melanoma and augments the therapeutic efficacy of anti-PD-L1 mAb, biochemical and Biophysical Research Communications 490 (2) 521-527 (2017). Thus, these results establish biomarkers associated with availability and primary resistance to anti-PD-1 therapies.

In embodiments, these results establish biomarkers associated with acquired resistance and primary resistance to PD-1 therapy. Thus, based on these biomarkers, in embodiments disclosed herein, treatment with anti-PD-1 therapy may be selected for a patient based on evaluating a biological sample from the patient for the presence, absence, or level of genes associated with one or more of the gene-body (GO) pathways disclosed herein in the sample. For example, in embodiments, the observed up-or down-regulation of one or more genes associated with the various GO functions disclosed herein can be used to diagnose resistance (acquired or primary resistance) to anti-PD-1 therapy.

Single cell RNA sequencing of tumors that have acquired resistance to PD-1 inhibitory antibody production as disclosed herein demonstrate progressive acquisition of the hyperactive phenotype of transcription. Specifically, the number of up-regulated transcripts is greater in the PD-1-acquired resistant tumor than in the parent PD-1 antibody-sensitive tumor. This observation suggests, without wishing to be bound by theory, that acquired resistance is an active process in which tumor cells that up-regulate genes in certain pathways gain survival advantages over those that do not up-regulate or down-regulate overall transcriptional activity. Thus, in embodiments, tumors that have acquired resistance to checkpoint inhibitors (including PD-1 or PD-L1 blockers) may have increased sensitivity to certain classes of chemotherapy that target transcriptionally active cells. Thus, in some aspects, tumors with acquired resistance to checkpoint inhibitors may be treated with chemotherapy categories targeting transcriptionally active cells. In embodiments, the chemotherapeutic class includes antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, etc.) or topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the chemotherapy categories may be used as neoadjuvant or adjuvant therapy.

Furthermore, the data presented herein demonstrate that while acquired resistant tumors are characterized by an overactive transcriptional phenotype, many up-regulated transcripts are not accompanied by an increase in the expression of the corresponding proteins. For example, genes associated with CD274 (PD-L1), β2 macroglobulin, and other transcripts related to interferon sensitivity and antigen presentation are upregulated in acquired resistant tumors, but the amount of PD-L1 or β2 macroglobulin expression in acquired resistant tumors is not correspondingly increased. Taken together, these findings indicate, inter alia, that acquired resistant tumors are attempting to up-regulate a number of key genes that will drive increased sensitivity to anti-tumor immune responses, but that acquired defects in one or more post-transcriptional proteins have disrupted the response. For example, an increase in PD-L1 mRNA levels would be expected to translate into an increase in PD-L1 protein levels. Thus, in embodiments, modulators comprising one or more of the following processes may be helpful because defects in post-translational processes are incorporated to create a synthetic lethal phenotype in a population of cancer cells that are resistant to PD-1 or PD-L1 blockers: protein translation (e.g., assembly and/or function of ribosomal complexes, sufficient expression and/or function of tRNA, sufficient synthesis and/or uptake of amino acids, etc.), post-translational modification (e.g., modification of the translated protein with carbohydrates important for function or prevention of degradation) or transport mechanisms (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through ER/Golgi network, etc.). In embodiments, the class of chemotherapy may be used as neoadjuvant therapy or adjuvant therapy.

A method of determining a cancer treatment for a patient; a method of selecting a patient for cancer treatment; and methods of treatment

Accordingly, in one aspect, the present disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing the presence, absence or level of one or more genes associated with a gene-ontology (GO) pathway in a sample, the GO pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes; and (c) selecting a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 based on the evaluation of step (b).

In one aspect, the present disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing the presence, absence or level of one or more genes associated with a gene-ontology (GO) pathway in a sample, the GO pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes; and (c) selecting a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azomycin, HUN, 527293, 147 and 7293); and (d) administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; and optionally administering the therapy selected in steps (c) (ii) and (c) (iii).

In non-limiting embodiments, administration of a therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or L2 is continued if no up-regulation of genes associated with GO pathways selected from the group consisting of positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, regulation of ikb kinase/nfkb signaling, positive regulation of type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing by endogenous peptides via MHC class I, up-regulation of genes associated with GO pathways presented are observed, and/or if no down-regulation of genes associated with GO pathways selected from the group consisting of phospholipid efflux, fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA replication and ATP biosynthesis processes are observed.

In non-limiting embodiments, if up-regulation of a gene associated with a GO pathway selected from the group consisting of positive regulation of a cell cycle process, regulation of G1/S transition, regulation of cell division, regulation of cell proliferation, regulation of iκb kinase/nfκb signaling, positive regulation of type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defense response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and up-regulation of a gene associated with an endogenous peptide via antigen processing of MHC class I, a GO pathway of presentation, and/or if down-regulation of a gene associated with a GO pathway selected from the group consisting of phospholipid efflux, fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translational protein, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation extension, DNA-dependent DNA replication and ATP biosynthesis process is observed, continuing administration of a therapy capable of inhibiting function of PD-1, PD-L1 and/or PD-L2 and/or a complement of cancer is/or wherein the cancer is treated by administration of the therapy is selected from the group consisting of cancer: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azathiophanone, asmine, HUN, CAM-7293, and 147).

In non-limiting embodiments, if up-regulation of a gene associated with a GO pathway selected from the group consisting of positive regulation of a cell cycle process, regulation of G1/S transition, regulation of cell division, regulation of cell proliferation, regulation of iκb kinase/nfκb signaling, positive regulation of type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of a defensive response, positive regulation of ifnβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing and presentation, and up-regulation of an endogenous peptide via antigen processing of MHC class I, presentation of GO pathway, and/or if down-regulation of a gene associated with a GO pathway selected from the group consisting of phospholipid efflux, fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent cotranslations protein, ribosomal small subunit assembly, phospholipid efflux, translation regulation, mitochondrial respiratory chain complex I, mitochondrial translation extension, DNA-dependent DNA replication and ATP biosynthesis process is observed, then the administration of a therapy capable of inhibiting PD-1, PD-L1 and/or PD-L2 and/or a cancer therapy wherein the activity is selected from cancer is administered is not continued: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azathiophanone, asmine, HUN, CAM-7293, and 147).

In embodiments, upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue. In embodiments, upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the lack of upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue. In embodiments, the lack of upregulation of one or more genes associated with the GO pathway set forth in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample obtained from the subject.

In embodiments, downregulation of one or more genes associated with the GO pathway listed in (ii) as compared to healthy tissue is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, downregulation of one or more genes associated with the GO pathway listed in (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 compared to healthy tissue. In embodiments, the lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the lack of up-regulation of one or more genes associated with the GO pathway is indicative of response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample from a subject, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, the lack of up-regulation of one or more genes associated with the GO pathway is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, the lack of upregulation of one or more genes associated with the GO pathway compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from the group consisting of (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I.

In embodiments, upregulation of one or more genes associated with a GO pathway, as compared to a prior biological sample from a subject, is indicative of resistance, lack of response, or noncompliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, upregulation of one or more genes associated with a GO pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) upregulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, upregulation of one or more genes associated with a GO pathway, compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) a type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling, and antigen processing, and (e) presentation of endogenous peptides via MHC class I.

In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from the subject. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to ifnγ, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with upregulation of cell cycle processes is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with upregulation of the cell cycle process is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with upregulation of the cell cycle process as compared to the standard is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with upregulation of cell cycle processes as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with upregulation of the cell cycle process is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with upregulation of the cell cycle process as compared to the standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, upregulation of one or more genes associated with modulation of G1/S transition is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with modulation of G1/S transition is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with modulation of G1/S transition, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with modulation of G1/S shift as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulation of G1/S shift is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with modulation of G1/S shift as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with modulation of cell division is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with modulation of cell division is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with modulation of cell division is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with modulation of cell division is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from the subject. In embodiments, the lack of upregulation of one or more genes associated with modulation of cell division is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with modulation of cell division as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with modulation of cell proliferation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with modulation of cellular proliferation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with modulation of cellular proliferation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with modulation of cellular proliferation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, the lack of upregulation of one or more genes associated with modulation of cellular proliferation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with modulation of cellular proliferation, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling as compared to a prior biological sample from the subject is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling as compared to a standard is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with upregulation of iκb kinase/nfκb signaling as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, upregulation of one or more genes associated with modulation of an innate immune response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with modulation of an innate immune response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with modulation of an innate immune response, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of up-regulation of one or more genes associated with the modulation of an innate immune response as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulation of an innate immune response is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be susceptible to treatment with anti-PD-1. In embodiments, the lack of upregulation of one or more genes associated with modulation of an innate immune response, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing/presentation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing/presentation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing/presentation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing/presentation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from the subject. In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing/presentation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing/presentation as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, upregulation of one or more genes associated with antigen processing/presentation of endogenous peptides via MHC class I is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with antigen processing/presentation of endogenous peptides via MHC class I is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with antigen processing/presentation of endogenous peptide via MHC class I is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with antigen processing/presentation of endogenous peptides via MHC class I is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, the lack of upregulation of one or more genes associated with antigen processing/presentation of endogenous peptides via MHC class I is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with antigen processing/presentation of endogenous peptides via MHC class I is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a standard.

In embodiments, upregulation of one or more genes associated with upregulation of ifnα production, as compared to a prior biological sample from the subject, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, upregulation of one or more genes associated with upregulation of ifnα production is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with upregulation of ifnα production, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with upregulation of ifnα production as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with upregulation of ifnα production is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with upregulation of ifnα production as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with upregulation of a defensive response as compared to a prior biological sample from a subject is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, upregulation of one or more genes associated with upregulation of a defensive response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with upregulation of the defensive response as compared to a standard is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with upregulation of a defensive response as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with upregulation of a defensive response is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with upregulation of a defensive response as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, upregulation of one or more genes associated with upregulation of ifnβ production, as compared to a prior biological sample from the subject, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, upregulation of one or more genes associated with upregulation of IFNbeta production is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with upregulation of IFNbeta production, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes associated with upregulation of ifnβ production as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with upregulation of IFNbeta production is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with upregulation of IFNbeta production as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, upregulation of one or more genes associated with modulation of an inflammatory response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, upregulation of one or more genes associated with modulation of an inflammatory response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of one or more genes associated with modulation of an inflammatory response is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of upregulation of one or more genes associated with modulation of an inflammatory response is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, the lack of upregulation of one or more genes associated with modulation of an inflammatory response is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the lack of upregulation of one or more genes associated with modulation of an inflammatory response, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with phospholipid efflux is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a previous biological sample from a subject. In embodiments, downregulation of one or more genes associated with phospholipid efflux is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with phospholipid efflux is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a standard.

In embodiments, a lack of down-regulation of one or more genes associated with phospholipid efflux as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with phospholipid efflux is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with phospholipid efflux as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, down-regulation of one or more genes associated with negative regulation of fibrinolysis as compared to a prior biological sample from a subject is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, downregulation of one or more genes associated with negative regulation of fibrinolysis is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with negative regulation of fibrinolysis, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a lack of down-regulation of one or more genes associated with negative regulation of fibrinolysis compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with negative regulation of fibrinolysis is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with negative regulation of fibrinolysis compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with chylomicron assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with chylomicron assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with chylomicron assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, the lack of down-regulation of one or more genes associated with chylomicron assembly as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with chylomicron assembly is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with chylomicron assembly as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with plasma membrane repair is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with plasma membrane repair is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with plasma membrane repair, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a lack of down-regulation of one or more genes associated with plasma membrane repair as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with plasma membrane repair is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be susceptible to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with plasma membrane repair, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with the membrane-targeted SRP-dependent cotranslations protein is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with the membrane-targeted SRP-dependent co-translational proteins is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with the membrane-targeted SRP-dependent cotranslations protein is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, a lack of down-regulation of one or more genes associated with the membrane-targeted SRP-dependent co-translated protein as compared to a prior biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with the membrane-targeted SRP-dependent co-translational proteins is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with the membrane-targeted SRP-dependent co-translated protein as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with ribosomal small subunit assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a previous biological sample from a subject. In embodiments, downregulation of one or more genes associated with ribosomal small subunit assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with ribosomal small subunit assembly is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, a lack of down-regulation of one or more genes associated with ribosomal small subunit assembly as compared to a previous biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with ribosomal small subunit assembly is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with ribosomal small subunit assembly as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, down-regulation of one or more genes associated with translational regulation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with translational regulation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with translational regulation, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a lack of down-regulation of one or more genes associated with translational regulation as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with translational regulation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with translational regulation, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I as compared to a previous biological sample from a subject is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I as compared to a standard is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial respiratory chain complex I as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial respiratory chain complex I is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial respiratory chain complex I as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation as compared to a standard is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial translation elongation as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial translation elongation is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with mitochondrial translation elongation compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, a lack of down-regulation of one or more genes associated with DNA-dependent DNA replication as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with DNA-dependent DNA replication is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes associated with DNA-dependent DNA replication as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, down-regulation of one or more genes associated with ATP biosynthesis process is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from a subject. In embodiments, downregulation of one or more genes associated with ATP biosynthesis process is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes associated with the ATP biosynthesis process is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a standard.

In embodiments, a lack of down-regulation of one or more genes associated with an ATP biosynthesis process as compared to a prior biological sample from a subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a lack of down-regulation of one or more genes associated with an ATP biosynthesis process is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be susceptible to treatment with anti-PD-1. In embodiments, a lack of down-regulation of one or more genes associated with an ATP biosynthesis process as compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 may be indicated, for example, when the expression of one or more of Rpl41, rps15, and Rps8 is high and/or the expression of one or more of Cd274, B2M, tap1, tap2, casp1, and Gasta3 is low. In embodiments, for example, cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 may not be indicated when one or more of Rpl41, rps15, and Rps8 is low and/or one or more of Cd274, B2M, tap1, tap2, casp1, and Gasta3 is high. In embodiments, patients characterized by low expression of one or more of Rpl41, rps15, and Rps8 and/or high expression of one or more of Cd274, B2M, tap1, tap2, casp1, and Gasta3 may benefit from adjuvant or neoadjuvant therapy that eliminates PD-1 non-responsive cells.

In embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample. In embodiments, the biological sample is a biopsy sample. In embodiments, the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy.

In embodiments, the biological sample comprises a sample selected from the group consisting of blood, plasma, serum, tears, lacrimal fluid, bone marrow, blood cells, ascites, tissue or fine needle biopsies, body fluids containing cells, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, stool, lymph, gynecological fluid, skin swabs, vaginal swabs, oral swabs, nasal swabs, irrigation or lavage fluids such as catheter lavage or bronchoalveolar lavage, aspirates, scrapes, bone marrow samples, tissue biopsies, surgical samples, stool, other body fluids, secretions and/or excretions, and/or cells derived therefrom. In embodiments, the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing, and biopsy. In embodiments, the biological sample is obtained by using a brush, (cotton) swab, spatula, washing/cleaning solution, a needle biopsy device, puncturing the cavity with a needle or surgical instrument.

In embodiments, the biological sample comprises at least one tumor cell. In embodiments, the tumor is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cell NHL, large tumor disease (bulk disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia (Waldenstrom's Macroglobulinemia), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia (chronic myeloblastic leukemia). In some embodiments, the cancer is basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, digestive system cancer, endometrial cancer, ocular carcinoma, glioblastoma, oral cavity carcinoma, small-cell carcinoma, glioblastoma, oral cavity carcinoma, small-size carcinoma, oral cavity glioblastoma, oral cavity carcinoma, small-cell carcinoma, cervical carcinoma, carcinoma of the like, oral cancer and pharyngeal cancer); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine cancer or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), as well as abnormal vascular proliferation associated with zebra-nevi, oedema (e.g., brain tumor-associated), meigs's syndrome cancer), renal cancer, colorectal cancer, and adrenal cancer.

In embodiments, the sample is evaluated by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes.

In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a gene-ontology (GO) pathway selected from (a) cellular responses to ifnγ, (B) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a cellular response to ifnγ. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a type I IFN signaling pathway. In embodiments, the sample is evaluated by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with upregulation of ifnα production. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with the positive regulation of a defensive response. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with upregulation of IFNbeta production. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with modulation of an inflammatory response.

In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a gene body (GO) pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes.

In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a gene body (GO) pathway selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing and presentation, (c) a type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling, and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a cellular response to ifnγ. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a type I IFN signaling pathway. In embodiments, the sample is evaluated by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with upregulation of ifnα production. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with positive regulation of a defensive response. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with upregulation of IFNbeta production. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with modulation of an inflammatory response. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

In embodiments, the assessment informs the classification of the patient as a high risk group or a low risk group. In embodiments, the high risk classification includes high levels of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the low risk classification includes low levels of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, a low risk or high risk classification indicates cessation of neoadjuvant therapy. In embodiments, a low risk or high risk classification indicates cessation of adjuvant therapy. In embodiments, the evaluation predicts a positive response to and/or benefits from a cancer treatment. In embodiments, the evaluation predicts a negative or neutral response to and/or benefits from cancer treatment. In embodiments, the evaluation predicts a positive response to and/or benefit from neoadjuvant chemotherapy, or does not respond to and/or cannot benefit from neoadjuvant chemotherapy. In embodiments, the evaluation predicts a positive response to and/or benefit from adjuvant chemotherapy, or does not respond to or and/or cannot benefit from adjuvant chemotherapy. In embodiments, the evaluation predicts a negative or neutral response to and/or benefit from neoadjuvant chemotherapy or does not respond to and/or cannot benefit from neoadjuvant chemotherapy. In embodiments, the evaluation predicts a negative or neutral response to and/or benefit from adjuvant chemotherapy, or does not respond to and/or cannot benefit from adjuvant chemotherapy. In embodiments, the evaluation informs the administration or cessation of cancer therapy. In embodiments, the assessment informs the administration of the neoadjuvant therapy. In embodiments, the assessment informs the administration of the adjuvant therapy. In embodiments, the assessment informs cessation of neoadjuvant therapy. In embodiments, the assessment informs cessation of adjuvant therapy. In embodiments, the neoadjuvant and/or adjuvant therapy is a chemotherapeutic agent. In embodiments, the neoadjuvant therapy and/or adjuvant therapy is a cytotoxic agent. In embodiments, the neoadjuvant therapy and/or adjuvant therapy is a checkpoint inhibitor.

In embodiments, the anti-cancer drug candidate is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

In embodiments, the class of chemotherapy includes antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, etc.) or topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.).

In embodiments, chemotherapy is selected from protein translation (e.g., ribosome complex assembly and/or function, sufficient expression and/or function of tRNA, sufficient synthesis and/or uptake of amino acids, etc.), post-translational modification (e.g., modification of the translated protein with carbohydrates important for function or prevention of degradation), or transport mechanisms (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through ER/Golgi network, etc.) may be helpful, also because of defects in the binding post-translational processes, to generate synthetic lethal phenotypes in cancer cell populations that are resistant to PD-1 or PD-L1 blockers. The class of chemotherapy includes antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, etc.) or topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the neoadjuvant and/or adjuvant therapy is selected from the group consisting of chemotherapeutic agents, cytotoxic agents, checkpoint inhibitors, antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the neoadjuvant and/or adjuvant therapy is selected from protein translation inhibitors (e.g., ribosome complex assembly and/or function modulators, tRNA expression and/or function modulators, amino acid synthesis and/or uptake modulators, post-translational modification modulators (e.g., modification of translated proteins with carbohydrates), protein degradation modulators, and protein transport modulators (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through the ER/golgi network, etc.), or topoisomerase inhibitors.

In embodiments, the neoadjuvant and/or adjuvant therapy is selected from protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azathiophanone, asmine, HUN, CAM-7293, and 147).

In embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample. In embodiments, the biological sample is a biopsy sample. In embodiments, the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy. In embodiments, the biological sample comprises a bodily fluid selected from the group consisting of blood, plasma, serum, tears, lacrimal fluid, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, fecal matter, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scrape, bone marrow samples, tissue biopsy samples, surgical samples, stool, other bodily fluids, secretions and/or excretions, and/or cells therefrom. In embodiments, the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing, and biopsy. In embodiments, the biological sample is obtained by using a brush, (cotton) swab, spatula, washing/cleaning solution, a needle biopsy device, puncturing the cavity with a needle or surgical instrument. In embodiments, the biological sample comprises at least one tumor cell.

In embodiments, the tumor is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-lytic NHL, megaly disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia in some embodiments, cancer is basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and central nervous system cancer, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye cancer, head and neck carcinoma, gastric cancer (including gastrointestinal carcinoma), glioblastoma, liver cancer, hepatoma, epithelial tumors, kidney or renal carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, and lung squamous carcinoma), melanoma, oral cavity carcinoma, cervical carcinoma, carcinoma of the tongue, carcinoma of the oral cavity, carcinoma of the respiratory system, carcinoma of the human skin, carcinoma of the human tumor, carcinoma of the human skin, carcinoma of the cervical carcinoma, carcinoma of the head, carcinoma of the head, carcinoma, cancer of head, carcinoma, cancer of cervical carcinoma, cancer carcinoma, laryngeal carcinoma, cancer, cancer, including hodgkin's lymphoma and non-hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), abnormal vascular proliferation associated with zematous hamartoma, oedema (e.g., associated with brain tumor), cancer of the migraines syndrome, renal cancer, colorectal cancer, and adrenal cancer.

In embodiments, the evaluation is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a gene-ontology (GO) pathway selected from (I) positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes.

In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a gene-ontology (GO) pathway selected from (a) cellular responses to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling, and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a cellular response to ifnγ. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a type I IFN signaling pathway.

In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a gene body (GO) pathway selected from the group consisting of: (i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or (ii) phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a cellular response to ifnγ. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a type I IFN signaling pathway. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

In embodiments, the assessment informs the classification of the patient as a high risk group or a low risk group. In embodiments, the high risk classification includes high levels of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the low risk classification includes low levels of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, a low risk classification indicates cessation of cancer therapy. In embodiments, a high risk classification indicates administration of a cancer therapy.

In one aspect, the present disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1, and/or (ii) genes selected from RPL41, RPS15, RPS8, TRIM7, and LRG 1; and (c) selecting a cancer therapy based on the evaluation of step (b).

In one aspect, the invention relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy based on the evaluation of step (b).

In one aspect, the invention relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase) inhibitors bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitors NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitors sulfasalazine, erastin, or sorafenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, nalquin, triline, spirone, thiomerone, asylvanil, HUN-7293, and topoisomerase inhibitors, 147, or the like).

In one aspect, the present disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the expression of the biological sample for: (i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and (c) selecting a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.; and (d) administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In non-limiting embodiments, if no up-regulation of overexpression of a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is observed, and/or no down-regulation of a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is observed, then administration of a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is continued.

In non-limiting embodiments, if up-regulation of overexpression of a gene selected from CD274, B2M, STAT, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is observed, and/or down-regulation of a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is observed, then administration of a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is continued, wherein supplemental administration of a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase) inhibitors bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitors NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitors sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, naltrexone, quinine, triptyline, spirone, thiomerone, asylum, HUN, track, 7293, and 147).

In non-limiting embodiments, if an up-regulation of overexpression of a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is observed; and/or downregulation of a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG1 is observed, then administration of a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is not continued, wherein administration of a cancer therapy selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azathiophanone, asmine, HUN, CAM-7293, and 147).

In embodiments, when the biological sample comprises at least one tumor cell and the gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM and KRT1 is not up-regulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample obtained from a patient known to be sensitive to anti-PD-1 therapy; and/or selecting a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 when the gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG1 is not down-regulated in the at least one tumor cell as compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

In embodiments, when the biological sample comprises at least one tumor cell and the gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is up-regulated in the at least one tumor cell as compared to healthy tissue, a previous biological sample obtained from a subject, or another biological sample obtained from a patient known to be sensitive to anti-PD-1 therapy; and/or when the gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG1 is down-regulated in the at least one tumor cell as compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy, the cancer therapy is selected from: antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosome biosynthesis inhibitors, rRNA and/or tRNA synthesis inhibitors, amino acid uptake inhibitors, post-translational modification regulators, protein degradation regulators, protein transport regulators, topoisomerase inhibitors.

In embodiments, upregulation of one or more genes listed in (b) (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue. In embodiments, downregulation of one or more of the genes listed in (b) (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

In embodiments, upregulation of one or more genes listed in (b) (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, downregulation of one or more genes listed in (b) (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

In embodiments, upregulation of one or more genes listed in (b) (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject. In embodiments, downregulation of one or more genes listed in (b) (ii) is indicative of lack of response, development of resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the lack of upregulation of one or more genes listed in (b) (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue. In embodiments, a lack of down-regulation of one or more genes listed in (b) (ii) compared to healthy tissue is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, the lack of upregulation of one or more genes listed in (b) (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of down-regulation of one or more genes listed in (b) (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

In embodiments, the lack of upregulation of one or more genes listed in (b) (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject. In embodiments, the lack of down-regulation of one or more genes listed in (b) (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample. In embodiments, the biological sample is a biopsy sample. In embodiments, the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy. In embodiments, the biological sample comprises a bodily fluid selected from the group consisting of blood, plasma, serum, tears, lacrimal fluid, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, fecal matter, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scrape, bone marrow samples, tissue biopsy samples, surgical samples, stool, other bodily fluids, secretions and/or excretions, and/or cells therefrom. In embodiments, the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing, and biopsy. In embodiments, the biological sample is obtained by using a brush, (cotton) swab, spatula, washing/cleaning solution, a needle biopsy device, puncturing the cavity with a needle or surgical instrument.

In embodiments, the biological sample comprises at least one tumor cell. In embodiments, the tumor is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-lytic NHL, megaly disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and central nervous system cancers, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric (including gastrointestinal tract carcinoma), glioblastoma, liver carcinoma, intraepithelial tumors, kidney or kidney carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma and squamous carcinoma), melanoma, myeloblastoma, neuroblastoma, oral cavity carcinoma (lip carcinoma, oral cavity carcinoma, tongue carcinoma, and testicular carcinoma), carcinoma of the respiratory system, carcinoma of the uterus, carcinoma of the human skin, carcinoma of the stomach, carcinoma of the stomach (including gastrointestinal tract, stomach, gastrointestinal carcinoma, gastrointestinal carcinoma, and carcinoma, gastrointestinal carcinoma, cancer, gastrointestinal carcinoma, stomach cancer, carcinoma, lung cancer, carcinoma, liver, carcinoma, liver, cancer liver, cancer, lung cancer, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), as well as abnormal vascular proliferation associated with zebra-like hamartoma, oedema (e.g., associated with brain tumors), megles syndrome cancer, renal cancer, colorectal cancer and adrenal cancer.

In embodiments, the evaluation is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof. In embodiments, the assessment is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes listed in (b) (i) and/or (b) (ii). In embodiments, agents that specifically bind to one or more proteins include antibodies, antibody-like molecules, or fragments thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with the genes listed in (b) (i) and/or (b) (ii). In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

In one aspect, the invention relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) Assessing activation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras by a biological sample; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of: (i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase) inhibitors bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitors NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitors sulfasalazine, erastin, or sorafenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, nalquin, triline, spirone, thiomerone, asylvanil, HUN-7293, and topoisomerase inhibitors, 147, or the like).

In embodiments, when the biological sample comprises at least one tumor cell and the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is not upregulated in the at least one tumor cell, a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is selected as compared to healthy tissue, a previous biological sample obtained from a subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

In embodiments, when the biological sample comprises at least one tumor cell and the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is up-regulated in the at least one tumor cell as compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy, the cancer therapy is selected from the following: antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosome biosynthesis inhibitors, rRNA and/or tRNA synthesis inhibitors, amino acid uptake inhibitors, post-translational modification regulators, protein degradation regulators, protein transport regulators, topoisomerase inhibitors.

In embodiments, upregulation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of a lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue. In embodiments, upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from a subject.

In embodiments, the lack of upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras as compared to healthy tissue is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the lack of upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, a lack of up-regulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras as compared to a prior biological sample obtained from a subject is indicative of progression of a lack of response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

In embodiments, the evaluation is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras. In embodiments, agents that specifically bind to one or more proteins include antibodies, antibody-like molecules, or fragments thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

Trim7 is a member of a large family that contains about 80 different ternary motif proteins (ternary motif-containing (Trim)). The protein family contains 80 TRIM protein members in humans. In embodiments, trim family activation can be determined by controlling the IFN response genes (i.e., IRF1/IRF3/IRF7, JAK/STAT, and NFkB) to regulate the innate immune response. In embodiments, trim family members have a C-terminal SPRY domain (e.g., in Trim6 and Trim 7). In embodiments, trim7 is determined based on induction of proliferation, EMT, and acquisition of chemotherapy-resistant phenotypes.

In embodiments, trim7 pathway activation can be determined using E3 ubiquitin ligase activity. In embodiments, trim7 pathway activation can be determined by c-Jun/AP1 activation of Ras-Raf-MEK-ERK signaling. In embodiments, trim7 pathway activation may be determined by determining protein ubiquitination.

In embodiments, trim7 pathway activation may be determined using ubiquitination and stabilization of the AP1 co-activator RACO-1 and/or AP 1-mediated increase in gene expression. The characteristics of AP 1-mediated gene expression are well known in the art.

In embodiments, trim7 pathway activation can be determined based on K63-linked ubiquitination of target proteins, including proteins involved in cell proliferation and innate immune responses. In embodiments, trim7 pathway activation may be determined based on Trim7 phosphorylation, K63-linked ubiquitination, and/or protein levels of an AP-1 co-activator (referred to as RACO-1). In embodiments, trim7 pathway activation may be determined based on the level or activity of STING (interferon gene stimulus, MITA, ERIS, MPYS, TMEM 173). In embodiments, trim7 pathway activation may be determined by ubiquitination of the K48 linkage of STING. In embodiments, trim7 pathway activation may be determined by up-regulation of IFNb, IP-10, and Rantes. In embodiments, trim7 pathway activation may be determined based on activation of the proteins shown in fig. 6C.

Mitogen-activated protein kinase 8 interacting protein 1 (Mapk 8ip 1) is also known as c-Jun amino-terminal kinase interacting protein 1. In embodiments, mapk8ip1 pathway activation may be determined based on an assay of functional polyprotein complexes in different components of the JNK pathway, including RAC1 or RAC2, MAP3K11/MLK3 or MAP3K7/TAK1, MAP2K7/MKK7, mapk8/JNK1 and/or Mapk9/JNK2. In embodiments, mapk8ip1 pathway activation may be determined based on the level and/or sensitivity of apoptosis-induced apoptosis.

In embodiments, ETS-like-1 protein (Elk 1) pathway activation can be determined based on activation of Ras-Raf-MEK-ERK signaling as is well known in the art. In embodiments, elk1 pathway activation can be determined based on Elk1 phosphorylation. In embodiments, elkl pathway activation can be determined based on activation of Elkl target genes as is well known in the art (see, e.g., odrowaz and Sharrocks, ELK1 Uses Different DNA Binding Modes to Regulate Functionally Distinct Classes of Target Genes, PLOS Genetics 8 (5): e1002694 (2012).

In embodiments, leucine-rich alpha-2-glycoprotein 1 (Lrg 1) pathway activation may be determined based on induction of tgfβ and/or SMAD1/5/8 signaling.

In embodiments, ras pathway activation can be determined based on activation of the proteins shown in FIG. 8C.

In embodiments, rap1 pathway activation can be determined based on activation of the proteins shown in fig. 8D.

In embodiments, arginase 1 (Arg 1) pathway activation may be determined based on an assay for arginine hydrolysis to ornithine and urea. In embodiments, arginase 1 (Arg 1) pathway activation may be determined based on inhibiting Tumor Infiltrating Lymphocytes (TILs). In embodiments, arginase 1 (Arg 1) pathway activation may be determined based on levels or synthesis of polyamines (via L-ornithine).

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering a cancer therapy selected from the group consisting of: antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosomal biogenesis inhibitors, rRNA and/or tRNA synthesis inhibitors, amino acid uptake inhibitors, post-translational modification regulators, protein degradation regulators, protein transport regulators, topoisomerase inhibitors, wherein the subject has received or is receiving an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, and wherein the subject has developed a lack of response, resistance or non-compliance with a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (b) Evaluating an anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring a decrease in tumor size in a subject; (c) If no decrease in tumor size is observed, administering a Trim7 modulator and/or a proteasome inhibitor; (d) Re-evaluating an anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring the decrease in tumor size of the subject; and (e) if a decrease in tumor size is observed, stopping administration of Trim7 modulator. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, iferum Sha Zuomi, obrenzomib, delazomib, and malizomib.

In one aspect, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; (b) The anti-tumor response of an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is evaluated using the following steps: (i) obtaining a biological sample from a subject; (ii) Assessing TRIM7 overexpression and/or activation of a biological sample; (c) If overexpression and/or activation of TRIM7 is observed, administering TRIM7 modulators and/or proteasome inhibitors; (d) The anti-tumor response of an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is re-evaluated using the following steps: (i) obtaining a biological sample from a subject; (ii) Assessing overexpression and/or activation of TRIM7 in a biological sample; and (e) if overexpression and/or activation of TRIM7 is not observed, stopping administration of TRIM7 modulator. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, iferum Sha Zuomi, obrenzomib, delazomib, and malizomib.

In embodiments, wherein the Trim7 inhibitor is a small molecule or peptide inhibitor that binds to Trim7 protein at or near a protein segment selected from the group consisting of MAAVGPRTGPGTGAEALALAAEL (SEQ ID NO: 1), AATRAPPFPLPCP (SEQ ID NO: 2), HGSQAAAARAAAARCG (SEQ ID NO: 3) and NVSLKTFVLKGMLKKFKEDLRGELEKEEKV (SEQ ID NO: 4).

In embodiments, the Trim7 modulator is a mitogen and stress activated kinase 1 (MSK 1) inhibitor, wherein the MSK1 inhibitor modulates Trim7 via inhibiting a downstream effect of MSK 1. In embodiments, the MSK1 inhibitor is selected from the group consisting of Ro 31-8220, SB-747651A and H89. In an embodiment, the MSK1 inhibitor is SB-747651A.

In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, iferum Sha Zuomi, obrenzomib, delazomib, and malizomib.

In embodiments, the evaluation is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by Trim7 pathway. In embodiments, the agent that specifically binds to one or more proteins includes an antibody, an antibody-like molecule, or a fragment thereof. In embodiments, the evaluation is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with Trim7 pathway. In embodiments, the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

In embodiments, the evaluation is performed by measuring E3 ubiquitin ligase activity. In embodiments, the evaluation is performed by measuring protein ubiquitination and/or K48 linked ubiquitination of the interferon gene Stimulators (STING) and/or the AP-1 coactivator RACO-1. In embodiments, evaluation is performed by measuring c-Jun/AP1 activation via Ras-Raf-MEK-ERK signaling and/or AP1 mediated increase in gene expression. In embodiments, the evaluation is performed by measuring ubiquitination and stabilization of the AP1 coactivator RACO-1. In embodiments, the evaluation is performed by measuring K63-linked ubiquitination of target proteins, including proteins involved in cell proliferation and innate immune responses. In embodiments, the evaluation is performed by measuring Trim7 phosphorylation, K63-linked ubiquitination and/or protein levels of the AP-1 coactivator RACO-1. In embodiments, by measuring IFN beta, IP-10 and/or Rantes up-regulation to evaluate.

In embodiments, the method further comprises administering an anti-checkpoint agent. In embodiments, the anti-checkpoint agent is an antibody selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimipro Li Shan anti (libayo), alemtuzumab (TECENTRIQ), avistuzumab (bavenciio), and dulluzumab (imfinzi). In embodiments, the pharmaceutical composition comprising the cancer therapy and the anti-checkpoint agent is administered simultaneously or contemporaneously. In embodiments, the pharmaceutical composition comprising cancer therapy is administered after administration of the anti-checkpoint agent. In embodiments, the pharmaceutical composition comprising the cancer therapy is administered prior to administration of the anti-checkpoint agent.

Transgenic non-human animal models for testing cancer therapies

In one aspect, the present disclosure relates to a transgenic non-human animal comprising one or more tumor cells, wherein the tumor cells have: (a) Upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (b) down-regulation of one or more genes associated with a gene body (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes. In embodiments, the transgenic non-human animal is transgenic.

In embodiments, the tumor cells are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2. In embodiments, the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimiput Li Shan antibody (libayo), atuzumab (TECENTRIQ), avermectin (bavenciio) and dimvaluzumab (imfinzi). In embodiments, the one or more tumor cells have: (a) Upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (b) down-regulation of one or more genes associated with a gene-body (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes. In embodiments, one or more tumor cells have upregulation of one or more genes associated with a cellular response to ifnγ. In embodiments, one or more tumor cells have upregulation of one or more genes associated with a type I IFN signaling pathway.

The transgenic non-human animal may be any animal known to be useful for mimicking human cancer. In embodiments, the transgenic non-human animal can be a pig, cow, dog, cat, horse, donkey, goat, sheep, llama, or non-human primate (e.g., chimpanzee). In embodiments, the transgenic non-human animal is a mammal. In embodiments, the transgenic non-human animal may be a rodent, such as a rat, mouse, hamster, rabbit, or guinea pig. In a preferred embodiment, the transgenic non-human animal is a mouse. In a preferred embodiment, the transgenic non-human animal is a rat. In embodiments, the mice belong to the BALB/C or C57BL/6 strains. Such mice may be purchased from different suppliers, such as Charles River Laboratories. In embodiments, the transgenic non-human animal is a transgenic non-human animal (but not limited to, e.g., a transgenic mouse). In embodiments, the transgenic non-human animal that is transgenic is a transgenic mouse. In embodiments, one or more genetic alterations cause spontaneous tumors and/or induced tumors.

In embodiments, tumor formation in a transgenic non-human animal (e.g., mouse) that is transgenic is caused by gene knockout of one or more genes, optionally, gene knockout of one or more genes is inducible. In embodiments, the knockout of one or more genes is performed using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the knockout of one or more genes is associated with upregulation of one or more genes in the cell from which the one or more genes were knocked out. In embodiments, the knockout of one or more genes is associated with downregulation of one or more genes in the cell from which the one or more genes were knocked out. In embodiments, up-and down-regulation of one or more genes is independently optionally inducible. In embodiments, up-regulation and/or down-regulation is caused by placing one or more sequences (but not limited to, e.g., RNAi constructs, cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

In embodiments, tumor formation in a transgenic non-human animal (e.g., mouse) is caused by the knock-in of one or more tumorigenic genes, optionally, the knock-in of one or more tumorigenic genes is inducible. Such tumorigenic genes are well known in the art and include in embodiments selected from c-Myc, HRAS ^G12V Or Kras ^G12D And known oncogenes of dominant negative p53 mutants. In embodiments, the knock-in of one or more tumorigenic genes is performed using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the knock-in of one or more tumorigenic genes is associated with the upregulation of one or more genes in the cell with the knock-in of one or more tumorigenic genes. In embodiments, the knock-in of one or more tumorigenic genes is associated with down-regulation of one or more genes in cells with knock-in of one or more tumorigenic genes. In embodiments, up-and down-regulation of one or more genes is independently optionally inducible. In embodiments, up-regulation and/or down-regulation is caused by placing one or more sequences (but not limited to, e.g., an RNAi construct, a Cre recombinase construct, and an additional gene knock-in construct) under the control of a promoter that controls the expression of one or more genes.

In embodiments, tumor formation in a transgenic non-human animal (e.g., mouse) is caused by chromosomal translocation, optionally, the chromosomal translocation is inducible. In embodiments, chromosomal translocation is performed using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, chromosomal translocation is associated with upregulation of one or more genes in a cell having chromosomal translocation. In embodiments, a chromosomal translocation is associated with downregulation of one or more genes in a cell having the chromosomal translocation. In embodiments, up-and down-regulation of one or more genes is independently optionally inducible. In embodiments, up-regulation and/or down-regulation is caused by placing one or more sequences (but not limited to, e.g., RNAi constructs, cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

In embodiments, tumor formation in a transgenic non-human animal (e.g., mouse) is caused by chromosomal inversion, optionally, the chromosomal inversion is inducible. In embodiments, chromosomal inversion is performed using cre-loxP, CRISPR/Cas9, or the like, or combinations thereof. In embodiments, chromosomal inversion is associated with upregulation of one or more genes in a cell having a chromosomal inversion. In embodiments, chromosomal inversion is associated with downregulation of one or more genes in a cell having the chromosomal inversion. In embodiments, up-and down-regulation of one or more genes is independently optionally inducible. In embodiments, up-regulation and/or down-regulation is caused by placing one or more sequences (but not limited to, e.g., RNAi constructs, cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

In any of the embodiments disclosed herein, the transgenic mouse carries a knock-in construct that causes up-regulation of one or more genes associated with a gene-body (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I. In any of the embodiments disclosed herein, the transgenic mouse contains a knock-in construct that causes down-regulation of one or more genes associated with a gene-body (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes.

In any of the embodiments disclosed herein, the transgenic mice have a promoter that controls expression of one or more genes, causing up-regulation of the one or more genes and/or down-regulation of the one or more genes, e.g., via placing the RNAi construct, cre recombinase, and/or gene knock-in construct under the control of the promoter. In embodiments, the promoter is a strong promoter. In embodiments, the promoter is an inducible promoter that allows for controlled and independent up-and/or down-regulation of one or more genes. In embodiments, the promoter is selected from the group consisting of a doxycycline-inducible promoter, a tamoxifen-inducible promoter, and a cumate-inducible promoter. In embodiments, the promoter is a promoter that may be a tissue specific promoter. In embodiments, the tissue specific promoter is selected from the group consisting of a Mouse Mammary Tumor Virus (MMTV) or Whey Acidic Protein (WAP) promoter for expression in the mammary gland, a mouse pro-melanoidin- α (POMC) promoter for central nervous system (hippocampal) specific expression, a human tail-box 2 (CDX 2) promoter for colon epithelial specific expression, a mouse SRY-box gene 2 (SRY-box containing gene, sox 2) promoter for ectodermal cell specific expression, a gamma-glutamyl transferase 1 (Ggt 1) promoter for kidney epithelial specific expression, an albumin promoter/enhancer (Alb) for liver (hepatocyte) specific expression, a human surfactant lung-associated protein C (SFTPC) promoter for lung (endodermal) specific expression, a pancreas and duodenum homeobox 1 (pdx 1) promoter for pancreatic epithelial specific expression, a rat intestinal II promoter for pancreatic cell specific expression, and a mouse-protein-binding protein (fag) 5-rich protein (mg) or a mouse lipocalin-binding protein (mg) of 5.

Methods for generating transgenic mouse models in Cancer studies are well known in the art (Walrath et al Genetically Engineered Mouse Models in Cancer Research, adv Cancer Res.106:113-164 (2010)). These methods generally involve constructing a targeting vector containing a modified sequence to be introduced into mouse embryonic stem cells. The genetically altered embryonic stem cells are then inserted into mice. The blastocysts were implanted into the uterus of female mice, eventually producing chimeric offspring. The heterozygous mice are then crossed to provide mice that are homozygous for the knockout gene and/or the knock-in gene. Alternative methods of gene inactivation include the CRISPR/Cas9 system (Beil-Wagner et al (2016) Sci Rep., 6:21377). Methods for introducing modified promoter sequences are known in the art and include the use of targeting vectors to be introduced into animal embryonic stem cells as described above. The knocked-out embryonic stem cells are then inserted into the blasts of the animals to produce offspring, which are then crossed to produce homozygous mice. Alternatively, the method of promoter modification in the genome may comprise a CRISPR/Cas9 system. In addition to modification of the animal genome, protein production can also be achieved in transgenic non-human animals by reliance on viral or non-viral expression vectors.

Some methods include injecting a vector containing a nucleic acid construct that directs the desired change in mice. In embodiments, the vector is a DNA vector. In embodiments, the vector is a viral vector. The vector then reaches the desired tissue and induces the desired gene changes in the mice.

Expression vectors that allow expression of transgenes in transgenic non-human animals, such as mice, are known. Useful mammalian expression vectors have been described in the prior art and such expression systems are commercially available from different manufacturers. Useful expression systems include plasmid or viral vector based systems such as pLIVE (Minis Bio), LENTI-Smart (InvivoGen), genScript expression vectors, pAdVAntage (Promega), viraPower lentivirus, adenovirus expression systems (Invitrogen) and adeno-associated virus expression systems (Cell Biolabs).

Suitable mammalian expression vectors typically comprise a promoter functionally linked to nucleic acids encoding one or more genes that are up-regulated and/or down-regulated. The promoter sequence must be compact and ensure strong expression. Suitable promoters include, but are not limited to, the cytomegalovirus promoter (CMV), the Spleen Focus Forming Virus (SFFV) U3 promoter, and the adenovirus major late promoter (Ad MLP), the promoters discussed above. As an optional component, the mammalian expression vector may include suitable enhancer elements to increase the expression level. Examples include the SV40 early gene enhancer (Dijkema et al (1985) EMBO J.4:761) and the Long Terminal Repeat (LTR) enhancer of Rous sarcoma virus (Gorman et al (1982 b) Proc. Natl. Acad. Sci.79:6777). The expression vector also optionally comprises transcription termination and polyadenylation sequences to improve expression of genes encoding up-and/or down-regulated one or more genes. Suitable transcription terminators and polyadenylation signals may, for example, be derived from SV40 (Sambrook et al (1989), molecular Cloning: ALaboratory Manual). Any other element known in the art to support efficient expression may be added to the expression vector, such as the woodchuck hepatitis post-transcriptional regulatory element (wPRE). In embodiments, the carrier may be a carrier that can be administered to an animal by injection. In embodiments, the mammalian expression vector is a pLIVE expression vector (Minis Bio).

In embodiments, the vector is an expression vector, optionally it is a viral expression vector. Viral vectors typically comprise a viral genome in which a portion of the native sequence has been deleted in order to introduce a heterologous polynucleotide without disrupting the infectivity of the virus. Due to the specific interaction between the viral components and host cell receptors, viral vectors are well suited for efficient transfer of genes into target cells. Suitable viral vectors for facilitating gene transfer into mammalian cells or organisms are well known in the art and may be derived from different types of viruses, such as retrovirus, adenovirus, adeno-associated virus (AAV), orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus or alphavirus. For a summary of the different viral vector systems, see Nienhuis et al, therapeutics, vol.16: viruses and Bone Marrow, N.S. young (ed.), 353-414 (1993).

In embodiments, the vector is an adeno-associated virus (AVV) vector, such as an AAV vector selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or a chimeric AAV derived therefrom, that would be more suitable for efficient transduction in target tissue (Wu et al, 2006,Mol Therapy 14:316-27; bowles et al, 2012,Mol Therapy 20:443-455). After transfection, AAV elicits only a slight immune response (if any) in the host. Furthermore, AAV vectors can also be effective in entering terminally differentiated cells from blood as compared to other vector systems. Thus, in embodiments, AAV is highly suitable for gene transfer methods. In embodiments, AAV serotype 6 and AAV serotype 9 are used for transduction in mice.

Recombinant viral vectors can be produced according to standard techniques. For example, recombinant adenovirus or adeno-associated viral vectors can be propagated in human 293 cells (which provide trans E1A and E1B functions) to titers ranging from 107-1013 viral particles/mL. The viral vector may be desalted by a gel filtration method such as agarose column and purified by subsequent filtration before it is applied in vivo. Purification reduces potential deleterious effects on the subject to whom the vector is administered. The administered virus is substantially free of wild-type and replication competent viruses. The purity of the virus can be demonstrated by suitable methods, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining. This applies to AAV and adenovirus vectors.

Transduction of the vector into the transgenic non-human animal may be achieved by systemic application, for example by intravenous (including hydrodynamic tail vein injection), intra-arterial or intraperitoneal delivery of the vector. In a preferred embodiment, the vector is administered systemically.

Method of preparing transgenic non-human animal models for testing cancer therapies

In one aspect, the present disclosure relates to a method of making a transgenic non-human animal comprising one or more cancer cells that are non-responsive, resistant, or non-compliant to a cancer therapy, wherein the cancer therapy is capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, the method comprising: (a) Injecting one or more parental cancer cells in the non-human animal that are responsive to the cancer therapy; (b) administering a cancer therapy to the non-human animal; (c) isolating cancer cells that survive the cancer therapy; (d) Injecting cancer cells that survive the cancer therapy into a different non-human animal of the same species; and (e) repeating steps (b) through (d) two to ten more times.

In embodiments, steps (b) through (d) are repeated at least once. In embodiments, steps (b) through (d) are repeated at least twice more. In embodiments, steps (b) through (d) are repeated at least three more times. In embodiments, steps (b) through (d) are repeated less than five times. In embodiments, the transgenic non-human animal is a rodent. In embodiments, the rodent is a mouse.

In embodiments, the mice belong to the BALB/C or C57BL/6 strains. In embodiments, the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimiput Li Shan antibody (libaiyo), avilamab (TECENTRIQ), avermectin (BAVENCIO), and dulluzumab (imfinzi).

In embodiments, when the cancer therapy is administered to a transgenic non-human animal carrying a parent cancer cell tumor, the cancer therapy is capable of inhibiting the growth of the tumor as compared to an untreated transgenic non-human animal carrying the parent cancer cell tumor. In embodiments, tumor cells that survive cancer therapy have: (a) Upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I; and/or (b) down-regulation of one or more genes associated with a gene body (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes.

In embodiments, one or more tumor cells have upregulation of one or more genes associated with a cellular response to ifnγ. In embodiments, one or more tumor cells have upregulation of one or more genes associated with a type I IFN signaling pathway.

In one aspect, the disclosure relates to transgenic animals made according to the methods of any of the embodiments disclosed herein.

Methods of testing cancer therapies and preparing pharmaceutical compositions for treating cancer

In one aspect, the present disclosure relates to a method for testing an anticancer drug candidate, the method comprising: (a) Providing a transgenic non-human animal of any of the embodiments disclosed herein, or a transgenic non-human animal made according to any of the embodiments disclosed herein; (b) Administering an anti-cancer drug candidate to the transgenic non-human animal, and (c) evaluating whether the anti-cancer drug candidate is effective to slow or inhibit cancer growth in the transgenic non-human animal. In one aspect, the present disclosure relates to an anticancer drug candidate selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

In one aspect, the present disclosure relates to a method of preparing a pharmaceutical composition for treating cancer, the method comprising: (a) Providing a transgenic non-human animal of any of the embodiments disclosed herein, or a transgenic non-human animal made according to any of the embodiments disclosed herein; (b) Administering an anti-cancer drug candidate to the transgenic non-human animal; and (c) selecting an anti-cancer agent effective to slow or inhibit the growth of cancer in the transgenic non-human animal; and (d) formulating the anticancer drug or candidate drug for administration to a human patient. In embodiments, the anti-cancer drug candidate is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

Formulations

The cancer therapies (and/or additional agents) described herein may have sufficiently basic functional groups that can react with inorganic or organic acids; or a carboxyl group, which may be reacted with an inorganic or organic base to form a pharmaceutically acceptable salt. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids as is well known in the art. Such salts include pharmaceutically acceptable salts listed, for example, in the following documents: journal of Pharmaceutical Science,66,2-19 (1977) and The Handbook of Pharmaceutical Salts; properties, selection, and use.P.H.Stahl and C.G.Wermuth (eds.), verlag, zurich (Switzerland) 2002, the entire contents of which are incorporated herein by reference.

In embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

Furthermore, any of the cancer therapies (and/or additional agents) described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or excipient. Such compositions may optionally comprise an appropriate amount of pharmaceutically acceptable excipients to provide a suitable form of administration. The pharmaceutical excipients may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients may be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can also be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline solutions as well as aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

In embodiments, the compositions described herein are resuspended in saline buffer (including but not limited to TBS, PBS, etc.).

Administration, dosing and treatment regimens

The present disclosure includes cancer therapies (and/or additional agents) in the various formulations described. Any cancer therapy (and/or additional agents) described herein may take the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets (capsules), capsules, liquid-containing capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other suitable use. DNA or RNA constructs encoding the protein sequences may also be used. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso r. Gennaro eds.,19th ed. 1995), which is incorporated herein by reference.

Formulations comprising cancer therapy (and/or additional agents) may also comprise a solubilizing agent, if desired. Moreover, the agent may be delivered using suitable vehicles or delivery devices known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device. The composition for administration may optionally include a local anesthetic, such as lidocaine, to reduce pain at the injection site.

Formulations comprising the cancer therapies (and/or additional agents) of the present disclosure may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods typically include the step of combining the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Generally, formulations are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into dosage forms (e.g., wet or dry granulation, powder mixtures, etc., and then tableting using conventional methods known in the art) for the desired formulation.

In one embodiment, any cancer therapy (and/or additional agents) described herein is formulated according to conventional procedures into a composition suitable for the mode of administration described herein.

Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or external, in particular for application to the ear, nose, eye or skin. In embodiments, administration is achieved by oral or parenteral injection. In most cases, administration results in the release of any of the agents described herein into the blood stream.

Any of the cancer therapies (and/or additional agents) described herein may be administered orally. Such cancer therapies (and/or additional agents) may also be administered by any other convenient route, such as by intravenous infusion or bolus injection, by absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with another bioactive agent. Administration may be systemic or local. Various delivery systems are known, for example encapsulated in liposomes, microparticles, microcapsules, capsules, etc., and can be used for administration.

In particular embodiments, it may be desirable to administer locally to an area in need of treatment. In one embodiment, for example in the treatment of cancer, cancer therapy (and/or additional agents) is administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix, and/or blood vessels surrounding and/or feeding tumor cells, including, for example, tumor vasculature, tumor infiltrating lymphocytes, fibroblast reticulocytes, endothelial Progenitor Cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of extracellular matrix (ECM), dendritic cells, antigen presenting cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located near the tumor) or lymph nodes and/or targeted to the tumor microenvironment or lymph nodes. In embodiments, for example in the treatment of cancer, the cancer therapy (and/or additional agents) is administered intratumorally.

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of a sterile solid composition (e.g., a lyophilized composition) which may be dissolved or suspended in a sterile injection medium immediately prior to use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage and dosing regimen of any cancer therapy (and/or additional agents) described herein may depend on various parameters, including, but not limited to, the disease being treated, the general health of the subject, and the discretion of the administering physician. Any of the cancer treatments described herein can be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of another agent. In embodiments, any of the cancer therapies and additional agents described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart to 2 hours apart, 2 hours apart to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

The dosage of any cancer therapy (and/or additional agents) described herein may depend on several factors, including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Furthermore, pharmacogenomic (genotype impact on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic drug) information about a particular subject may affect the dose used. Furthermore, the exact individual dosage may be adjusted to some degree depending on a variety of factors, including the particular combination of agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage may be expected.

For administration of any of the cancer therapies (and/or additional agents) described herein by parenteral injection, the dosage may be about 0.1mg to about 250mg per day, about 1mg to about 20mg per day, or about 3mg to about 5mg per day. Generally, when administered orally or parenterally, dosages of any of the agents described herein can be from about 0.1mg to about 1500mg per day, or from about 0.5mg to about 10mg per day, or from about 0.5mg to about 5mg per day, or from about 200 to about 1,200mg per day (e.g., about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1,000mg, about 1,100mg, about 1,200mg per day).

In embodiments, administration of the cancer therapies (and/or additional agents) described herein is by parenteral injection at a dose of about 0.1mg to about 1500mg per treatment, or about 0.5mg to about 10mg per treatment, or about 0.5mg to about 5mg per treatment, or about 200 to about 1,200mg per treatment (e.g., about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1,000mg, about 1,100mg, about 1,200mg per treatment).

In embodiments, a suitable dose of the cancer therapy (and/or the additional agent) is in the range of about 0.01mg/kg to about 100mg/kg body weight or about 0.01mg/kg to about 10mg/kg body weight of the subject, e.g., about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.08mg/kg, about 0.09mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 1mg/kg, about 1.07 mg/kg, about 1.2mg/kg, about 1.3mg/kg, about 4mg/kg, about 1.5mg, about 5mg/kg, about 1.5mg, about 5mg/kg, about 0.6mg/kg, about 1.1mg/kg, about 0.5mg/kg, about 0.5mg, about 0.6mg/kg, about 1.1mg/kg, about 0mg/kg, about 0.5mg, about 0mg, about 0.5mg, about 0mg and about 0mg/kg, about 0.1.5 mg, about 0mg and about 0 mg.

In another embodiment, delivery may be in vesicles, particularly liposomes (see Langer,1990,Science 249:1527-1533; treat et al Liposomes in therapy of Infectious Disease and Cancer, lopez-Berestein and Fidler (ed.), lists, new York, pp.353-365 (1989).

Any of the cancer therapies (and/or additional agents) described herein may be administered by controlled or sustained release means or by delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in the following patents: U.S. Pat. nos. 3,845,770;3,916,899;3,536,809;3,598,123;4,008,719;5,674,533;5,059,595;5,591,767;5,120,548;5,073,543;5,639,476;5,354,556; and 5,733,556, each of which is incorporated by reference herein in its entirety. Such dosage forms may be used to provide controlled or sustained release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymeric matrices, gels, osmotic membranes, osmotic systems, multi-layered coatings, microparticles, liposomes, microspheres, or combinations thereof to provide the desired release profile in varying proportions. Controlled or sustained release of the active ingredient may be stimulated by a variety of conditions including, but not limited to, a change in pH, a change in temperature, a light stimulus of an appropriate wavelength, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, langer and Wise (eds.), CRC pres., boca Raton, florida (1974), controlled Drug Bioavailability, drug Product Design and Performance, smolen and Ball (eds.), wiley, new York (1984), ranger and Peppas,1983, J.macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al, 1985,Science 228:190;During et al, 1989, ann. Neurol.25:351; howard et al, 1989, J.Neurosurg. 71:105).

In another embodiment, the controlled release system may be placed in proximity to the target area to be treated, thus requiring only a portion of the systemic dose (see, e.g., goodson, medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). Other controlled release systems discussed in the review of Langer,1990,Science 249:1527-1533) may be used.

Administration of any cancer therapy (and/or additional agents) described herein may independently be one to four times per day or one to four times per month or one to six times per year or once every two, three, four, or five years. Administration may last one day or one month, two months, three months, six months, one year, two years, three years, and may even last the subject's lifetime.

The dosage regimen utilizing any of the cancer therapies (and/or additional agents) described herein can be selected in accordance with a variety of factors, including the type, species, age, weight, sex, and medical condition of the subject; the severity of the condition to be treated; route of administration; renal or hepatic function in a subject; pharmaceutical genomics of individuals; and the specific compounds of the invention used. Any of the cancer therapies (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three, or four times per day. Furthermore, any of the cancer therapies (and/or additional agents) described herein may be administered continuously throughout the dosage regimen rather than intermittently.

Examples

The examples herein are provided to illustrate the advantages and benefits of the present disclosure and to further assist one of ordinary skill in the art in preparing or using cells that are resistant to anti-PD-1, anti-PD-L1, and/or anti-PD-L2 therapies. The embodiments herein are also presented in order to more fully illustrate the preferred aspects of the present disclosure. These examples should in no way be construed as limiting the scope of the present disclosure, which is defined by the appended claims. Embodiments may include or incorporate any of the variations, aspects or implementations of the disclosure described above. The variations, aspects, or embodiments described above may also each include or incorporate variations of any or all other variations, aspects, or embodiments of the disclosure.

Example 1: generation of anti-PD-1 resistant CT26 tumors

Murine colon cancer CT26 cells were selected to survive anti-PD-1 treatment to generate anti-PD-1 antibody resistant CT26 cells. The method for generating anti-PD-1 resistant CT26 tumor cells is shown in fig. 1A. Briefly, BALB/C mice were obtained from Jackson Laboratory and after several days of adaptation, 500,000 murine colon cancer CT26 cells were inoculated on the posterior flank of the mice. When the average tumor volume reaches 80-100mm ³ On day 0 (indicated), mice were given a series of intraperitoneal injections of 100. Mu.g of anti-PD-1 (clone RMP1-14; bioXcell) on days 0, 3 and 6. Tumors were resected from mice that did not respond to anti-PD-1 therapy approximately 10-14 days after initial treatment. Tumors were dissociated using collagenase (StemCell Technologies), washed in 1x PBS, and plated in IMDM medium supplemented with 10% fetal bovine serum, 1% glutimax, and 1% antibiotic-antifungal agent (all from GIBCO). Cells were passaged at least 5 times and then inoculated into new recipient mice as described above (round 2). Likewise, when the tumor reaches 80-100mm ³ At this time, another anti-PD-1 course of treatment was initiated: 100 μg of anti-PD-1 (clone RMP1-14; bioXcell) was injected intraperitoneally on days 0, 3 and 6. This procedure was repeated for a total of 4 rounds, at which time none of the treated mice responded to anti-PD-1 therapy. The cell lines generated after 2 rounds of anti-PD-1 selection are referred to throughout this disclosure as "second round", "second generation" or "F2 generation". The cell lines generated after 4 rounds of anti-PD-1 selection are referred to as "round 4", "generation 4" or "generation F4".

The efficacy of anti-PD-1 antibodies in CT26 cells or anti-PD-1 antibody resistant CT26 cell-allografts was compared. Briefly, BALB/C mice were vaccinated in the posterior flank with CT26 parental cells and PD-1 resistant fourth generation cells. When the initial tumor volume (STV) reaches 80-100mm ³ At this time, mice were randomized into the following two treatment groups: (1) vehicle (PBS) and (2) anti-PD-1 antibody. On days 0, 3 and 6, mice were treatedA series of intraperitoneal injections of vehicle or 100. Mu.g of anti-PD-1 (clone RMP1-14; bioXcell) were administered. Tumor volumes were measured on the indicated days and plotted as a function of time. As shown in fig. 1B, the growth of CT26 parental cell tumors was significantly retarded when treated with anti-PD-1 antibodies as compared to vehicle-only controls (compare the gray solid line to the black solid line in fig. 1B). In contrast, PD-1 resistant cells showed little, if any, inhibition of tumors. These results demonstrate, inter alia, that anti-PD-1 resistance (acquired resistance) against PD-1 resistant cells occurs after therapy in immunocompetent mice.

Thus, anti-PD-1 antibody resistant cells were developed. In addition, a mouse cell model containing anti-PD-1 resistant cells was developed. Thus, the mouse models disclosed herein can be used to test anti-cancer drug candidates by administering them to mice bearing anti-PD-1 antibody resistant CT26 allografts, and evaluating whether the anti-cancer drug candidates are effective in slowing or inhibiting the growth of cancer. Anticancer or candidate agents effective in slowing or inhibiting the growth of cancer may be formulated for administration to human patients.

Example 2: transcriptome analysis using RNA-seq against PD-1 resistant cell lines

Three different parental CT26 cells (ATCC; experimental replicates), two tumors isolated independently from "second round" mice, four tumors isolated independently from "fourth round" mice (all "biological replicates") with or without 20ng/mL mouse IFNγ (Biolegend) at 37deg.C/5% CO ₂ Incubate for 24 hours. The following day, RNA was isolated from cells using Qiagen RNeasy reagent, including QIASHREDDER homogenate and on-column DNaseI digestion, according to manufacturer's instructions. RNA-seq and data analysis were performed on the isolated RNA. Briefly, a sequencing library was generated and sequenced on ILLUMINA HISEQ (2X 150 paired end reads), targeting each sample>20×10 ⁶ And reading. The sequences were adjusted using trimmate v.0.36 and mapped to the mouse (Mus musculus) GRCm38 reference genome using STAR ALIGNER v.2.5.2 b. Unique gene hit counts were calculated using featureouts from the subspecad software package v.1.5.2. Counting bits onlyUnique reads within the exon regions. Differential gene expression was determined using DESeq2 and p-value and log2 fold changes were generated using Wald test; log2 fold change >1 and adjusted p value<0.05 as a cutoff for significance.

As shown in fig. 2A (top panel), principal Component Analysis (PCA) illustrates that samples can be spatially separated based on transcriptome expression. Differentially Expressed Genes (DEG) were determined between each set (parent and 2 nd, parent and 4 th, 2 nd and 4 th) and plotted in a heat map. As shown in fig. 2A (lower panel), hierarchical clustering was performed to rank the genes on each row, dividing the genes into 2 major clusters in each comparison, with a subset of gene expression lower in one dataset (blue) and higher in the other dataset (red). The genes were then analyzed using the panher application to determine the Gene Ontology (GO) associated with each gene set. The gene set is shown with the relevant p-value. Up-regulated or down-regulated genes were determined in each dataset. As shown in fig. 2B, genes associated with the following GO were up-regulated in second generation cells compared to the parental CT26 cells: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, and regulation of cell proliferation. Compared to the parental CT26 cells, genes related to the following GO were down-regulated in the second generation cells: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, and plasma membrane repair (fig. 2B). Further, as shown in fig. 2B, the genes associated with the following GO were up-regulated in fourth generation cells compared to the parental CT26 cells: upregulation of IκB kinase/NFκB signaling, type I IFN signaling pathways, cellular responses to IFNγ, regulation of innate immune responses. This is surprising, inter alia, because it is well known that cellular responses to ifnγ are involved in sensitivity against PD-1 and other immunotherapeutic agents. Compared to the parental CT26 cells, the genes associated with the following GO were down-regulated in fourth generation cells: membrane-targeting SRP-dependent co-translational proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation (fig. 2B). Interestingly, genes associated with the following GO were up-regulated in fourth generation cells compared to second generation cells: cellular responses to ifnγ, positive regulation of ikb kinase/nfkb signaling, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I (fig. 2B). Compared to the second generation cells, the genes associated with the following GO were down-regulated in the fourth generation cells: mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA dependent DNA replication and ATP biosynthesis process (fig. 2B). A venn plot of gene expression overlap between all data sets was created. As shown in fig. 2C (upper panel), DEG shows that there is significant overlap between fourth-generation cells and second-generation cells. FIG. 2A (bottom panel) shows the transcripts per million at selected genes (TPM; normalized expression data), demonstrating higher gene baseline expression associated with PD-L1, antigen processing/presentation, protein translation, ER transport; in some data sets, higher than others. As shown in FIG. 2C (bottom panel), the genes Cd274, B2M, tap1, tap2, casp1 and Gasta3 showed increasing expression from CT26 parent cells to second and fourth generation cells. Furthermore, the genes Rpl41, rps15 and Rps8 showed a gradual decrease in expression from the CT26 parent cell to the second and to the fourth generation cells (fig. 2C (lower panel)). As shown in FIG. 2D, genes Stat1, stat2, irf, ltbr and Pvr showed increasing expression from CT26 parent cells to second and fourth generation cells.

These results in particular identify biomarkers associated with acquired resistance to anti-PD-1 therapy. Such biomarkers may be used to identify patients who may benefit from anti-PD-1 therapy or who do not benefit from anti-PD-1 therapy. Thus, based on these biomarkers, a patient may be selected for treatment with anti-PD-1 therapy based on evaluating a biological sample from the patient for the presence, absence, or level of genes associated with one or more gene-body (GO) pathways disclosed herein in the sample. For example, cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 may be indicated when the expression of one or more of Rpl41, rps15, and Rps8 is high and/or the expression of one or more of Cd274, B2m, tap1, tap2, casp1, and Gasta3 is low. In contrast, for example, when the expression of one or more of Rpl41, rps15, and Rps8 is low and/or the expression of one or more of Cd274, B2m, tap1, tap2, casp1, and Gasta3 is high, a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 may not be indicated. Patients characterized by low expression of one or more of Rpl41, rps15, and Rps8 and/or high expression of one or more of Cd274, B2m, tap1, tap2, casp1, and Gasta3 may benefit from adjuvant or neoadjuvant therapy that eliminates PD-1-unresponsive cells.

Example 3: cell surface expression of PD-L1, PD-L2, MHC class I and beta 2 microglobulin in anti-PD-1 resistance CT26

Surface expression of PD-L1, PD-L2, MHC class I and beta 2 microglobulin (B2 m) was studied as Cd274 (PD-L1), beta 2 microglobulin (B2 m) and Tap1 and Tap2 genes, which may be involved in the processing and transport of major histocompatibility complex class I-related antigens to the endoplasmic reticulum, were gradually down-regulated from CT26 parental cells to second-generation cells and to fourth-generation cells. Briefly, parental and second and fourth generation cells were harvested from culture and analyzed by flow cytometry for surface expression of PD-L1, PD-L2, MHC class I and β2 microglobulin (B2M). Gating is plotted as shown, the percentage of cells in each gating is shown above each graph, the MFI (mean fluorescence intensity) of each marker to the right of each percentage. As shown in FIG. 3, there was no down-regulation of surface expression of PD-L1, MHC class I and β2 microglobulin (B2M) in fourth generation anti-PD-1 resistant cells compared to the parental cells. On the other hand, the surface expression of PD-L2 is down-regulated in fourth generation anti-PD-1 resistant cells compared to the parental cells. These data indicate that inconsistent surface expressed gene expression and cell surface protein expression are observed in PD-L1/2MHC class I and B2M as compared to RNA expression.

Example 4: transcriptome analysis of anti-PD-1 resistant cell lines and primary resistant anti-PD-1 resistant cell lines

Next, transcriptome profiles of second and fourth generation anti-PD-1 resistant cell lines were studied by comparison with each other and with anti-PD-1 primary resistant cell lines. B16.f10 murine melanoma tumor cell lines were used as an anti-PD-1 "primary resistance" model, as these tumors did not respond to anti-PD-1 therapies. Three different parental CT26 cells (ATCC; "experimental replication"), independent from "second round" miceTwo tumors isolated immediately, four tumors isolated independently from "fourth round" mice (both "biological replicates"), and two different flasks of parental b16.f10 cells (ATCC; "experimental replicates") with or without 20ng/mL mouse ifnγ (Biolegend) at 37 ℃/5% CO ₂ Incubate for 24 hours to assess in vitro reactivity. This mimics how tumor cells react in vivo because immune cells infiltrate and secrete effector cytokines such as ifnγ. The following day, RNA was isolated from cells using Qiagen RNeasy reagent, including QIASHREDDER homogenate and on-column DNaseI digestion, according to manufacturer's instructions. RNA-seq and data analysis were performed on the isolated RNA. Briefly, a sequencing library was generated and sequenced on ILLUMINA HISEQ (2X 150 paired end reads), targeting each sample >20×10 ⁶ And (5) reading. The sequence was adjusted using trimmate v.0.36 and mapped to the mouse GRCm38 reference genome using STAR ALIGNER v.2.5.2b. Unique gene hit counts were calculated using featureouts from the subspecad software package v.1.5.2. Only unique reads within the exon regions were counted. Differential gene expression was determined using DESeq2 and p-value and log2 fold changes were generated using Wald test; log2 fold change>1 and adjusted p value<0.05 as a cutoff for significance. DEG was identified between untreated and ifnγ -treated parental CT 26. As shown in fig. 4A (left panel), log2 fold changes are plotted in a heat map and genes are hierarchically clustered based on parent CT 26. Wherein 338 genes have available data from other datasets; and these values are shown in the other columns (fig. 4A (left panel)). Genes were divided into 3 major clusters (fig. 4A (left panel)). The relevant gene is input into panHER to identify the pathway associated with the deregulated gene and to identify the GO pathway associated with DEG. As shown in fig. 4A (right panel), compared to the parental CT26 cells and b16.f10 cells, up-regulation of the expression of genes associated with the following GO pathway was enriched in both the second and fourth generation cells: l-phenylalanine catabolic processes, phospholipid efflux, tyrosine catabolic processes, upregulation of transcription by RNA polymerase II promoters in response to acidic pH, and drug output. Thus, up-regulation of genes associated with one or more of these GO pathways may be associated with acquired resistance to anti-PD-1 therapy And (5) correlation. Furthermore, downregulation of expression of genes associated with the following GO pathways was observed in fourth generation cells compared to parental CT26 cells and b16.f10 cells: protection from NK cell mediated cytotoxicity, IGS15 protein conjugation, antigen processing/presentation via MHC class I, MHC protein complex assembly, and cytosol to ER trafficking (fig. 4A (right panel)). Thus, down-regulation of one or more of these GO pathways may be associated with acquired resistance to anti-PD-1 therapy. As shown in fig. 4A (right panel), downregulation of expression of genes associated with the following GO pathways was enriched in fourth generation cells and b16.F10 cells compared to the parental CT26 cells: upregulation of IκB kinase/NFκB signaling, type I IFN signaling pathways, upregulation of IFNα production, upregulation of defensive responses, upregulation of IFNβ production, and regulation of inflammatory responses. Thus, down-regulation of one or more of these GO pathways may be associated with resistance to anti-PD-1 therapy. FIG. 4B shows the transcripts per million at selected genes (TPM; normalized expression data). As shown in fig. 4B, CT26 anti-PD-1 resistant cells down-regulated these genes when they were challenged with ifnγ, although these cells had baseline overactivation of type I and type II interferons. This is particularly surprising because it is well known that upregulation of iκb kinase/nfκb signaling, type I IFN signaling pathways, upregulation of ifnα production, upregulation of defensive responses, upregulation of ifnβ production, and regulation of inflammatory responses all involve sensitivity to anti-PD-1 and other immunotherapeutic agents.

These results establish, inter alia, biomarkers related to acquired resistance to anti-PD-1 therapy, and biomarkers related to resistance to anti-PD-1 therapy (acquired or primary resistance). Such biomarkers may be used to identify patients who may benefit from anti-PD-1 therapy or who do not benefit from anti-PD-1 therapy. Thus, based on these biomarkers, a patient may be selected for treatment with anti-PD-1 therapy based on evaluating a biological sample from the patient for the presence, absence, or level of genes associated with one or more gene-body (GO) pathways disclosed herein in the sample. For example, when Trim7 and/or Ank3 expression is high and/or Tap2 and/or Casp1 expression is low, cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 may be indicated. In contrast, for example, when Trim7 and/or Ank3 expression is high and/or Tap2 and/or Caspl expression is low, no cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is indicated. Patients characterized by high Trim7 and/or Ank3 expression and/or low Tap2 and/or Caspl expression may benefit from adjuvant or neoadjuvant therapy that eliminates PD-1 non-responsive cells.

Example 5: contradictory deregulation of genes differentially regulated in anti-PD-1 resistant cells compared to parental cells

Next, the effect of ifnγ on the expression of certain genes differentially expressed in fourth generation anti-PD-1 resistant cells compared to wild type CT26 cells was investigated. Briefly, parental CT26 cells, second generation anti-PD-1 resistant cells and fourth generation anti-PD-1 resistant cells were treated with or without 20ng/mL mouse IFNγ (Biolegend) at 37deg.C/5% CO ₂ Incubate for 24 hours. The following day, RNA was isolated from cells using Qiagen RNeasy reagent, including QIASHREDDER homogenate and on-column DNaseI digestion, according to manufacturer's instructions. RNA-seq and data analysis were performed on the isolated RNA. Briefly, a sequencing library was generated and sequenced on ILLUMINA HISEQ (2X 150 paired end reads), targeting each sample>20×10 ⁶ And reading. The regulatory sequences were mapped to the mouse GRCm38 reference genome using trimmate v.0.36 and using STAR ALIGNER v.2.5.2b. Unique gene hit counts were calculated using featureouts from the subspecid package v.1.5.2. Only unique reads falling within the exon regions were counted. Differential gene expression was determined using DESeq2 and p-value and log2 fold changes were generated using Wald test; iog2 fold change >1 and adjusted p value<0.05 as a cutoff for significance.

To understand the effect of ifnγ, each million transcripts (TPM; standardized expression data) of representative genes that were overexpressed (Cd 274 and B2 m) or suppressed (Trim 7 and Lrg 1) were analyzed. As expected, expression of Cd274 (PD-L1) increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5A (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Cd274 in wild-type CT26 cells was increased (compare closed circle data in fig. 5A (left and right panels)). Paradoxically, the expression in response to ifnγ, cd274 (PD-L1) decreased from wild-type CT26 cells to second-generation anti-PD-1 resistant cells and to fourth-generation anti-PD-1 resistant cells (fig. 5A (right panel)).

As expected, B2M expression was also increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5B (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of B2M in wild-type CT26 cells was increased (compare closed circle data in fig. 5B (left and right panels)). Paradoxically, expression in response to ifnγ, B2M decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5B (right panel)).

As expected, trim7 expression decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5C (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Trim7 in wild-type CT26 cells was increased (compare closed circle data in fig. 5C (left and right panels)). Paradoxically, expression of Trim7 in response to ifnγ decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5C (right panel)).

Similarly, expression of Lrg1 decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5D (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Lrg1 in wild-type CT26 cells was increased (compare closed circle data in fig. 5D (left and right panels)). Paradoxically, expression in response to ifnγ, lrg1 decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 5D (right panel)).

These results in particular indicate that when grown in the presence or absence of ifnγ, the Cd274, B2m, trim7, lrg1 and other genes are paradoxically deregulated compared to the parental cancer cells.

Example 6: identification of resistance driver genes

Since deregulation is observed across multiple genes, not to be bound by theory, it is hypothesized that contradictory deregulation is a functional outcome of acquired resistance against PD-1 agents. To identify driver genes involved in the observed contradictory disorders, the analysis disclosed in FIG. 6A was performed. Briefly, a Differentially Expressed Gene (DEG) in fourth generation anti-PD-1 resistant cells when grown in the presence of IFNγ was identified as compared to the parental CT26 cells. As shown in FIG. 6A (panel 1), in the presence of IFNγ, the fourth generation anti-PD-1 resistant cells had 1,999 genes down-regulated and 3607 genes up-regulated compared to CT26 cells. From these genes, those were identified that were down-regulated in fourth generation anti-PD-1 resistant cells but up-regulated in CT26 cells. As shown in fig. 6A (panel 2), this standard was used to reduce to 1,060 genes, which were down-regulated in fourth generation anti-PD-1 resistant cells but up-regulated in CT26 cells, with a further reduction in these genes based on responsiveness to ifnγ. As shown in fig. 6A (panel 3), the fourth generation anti-PD-1 resistant cells showed 688 genes down-regulated compared to CT26 cells according to the response rank to ifnγ. These genes include Lrg1, spry2, arg1, trim8, trim2, mapk8ip1, trim7, trim6, etc. (fig. 6A (panel 3)).

Then, to further reduce these genes, parental CT26 cells, second generation anti-PD-1 resistant cells, and fourth generation anti-PD-1 resistant cells were cultured in mice. RNA was isolated from tumors and analyzed for RNA-seq and data. From 688 genes (FIG. 6A (panel 3)). Genes that are also up-regulated in vivo were identified. As shown in FIG. 6A (panel 4), there were 70 genes up-regulated in fourth generation anti-PD-1 resistant cells in vivo, including Krt8, mapk8ip1, arg1 and Lrg1, compared to CT26 cells. FIG. 6B shows the Gene Ontology (GO) pathway enriched in at least two steps in FIG. 6A. This analysis determined that proteins of the TRIM family (especially TRIM 7), mapk8ip1, elk1, lrg1, arg1 are some driving factors. FIG. 6C shows functional pathways affected by TRIM family proteins. FIG. 6D shows the functional pathways by which Elk1 and c-Jun function. FIG. 6E shows functional linkages between Lrg1, B2m and Arg1 and other genes. FIG. 6F shows the expression levels of Elk1 in tumor and surrounding normal tissues in a cancer genomic profile (TCGA) cancer genomic program.

Example 7: contradictory deregulation of additional differential regulatory genes

The transcripts per million (TPM; normalized expression data) of other representative genes overexpressed (Stat 1, stat2, irf1 and Tap 1) were analyzed. Expression of Stat1 increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells to fourth generation anti-PD-1 resistant cells (fig. 7A (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Stat1 in wild-type CT26 cells was increased (compare closed circle data in fig. 7A (left and right panels)). Paradoxically, expression in response to ifnγ, stat1 decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7A (right panel)).

Expression of Stat2 was also increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells to fourth generation anti-PD-1 resistant cells (fig. 7B (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Stat2 in wild-type CT26 cells was increased (compare closed circle data in fig. 7B (left and right panels)). Paradoxically, expression in response to ifnγ, stat2 decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7B (right panel)).

Similarly, irf1 expression increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7C (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Irf1 in wild-type CT26 cells was increased (compare closed circle data in fig. 7C (left and right panels)). Paradoxically, expression of Irf1 in response to ifnγ decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7C (right panel)).

As expected, tap1 expression increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7D (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Tap1 in wild-type CT26 cells was increased (compare closed circle data in fig. 7D (left and right panels)). Paradoxically, expression of Tap1 in response to ifnγ decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 7D (right panel)).

Example 8; pathway analysis of differential regulatory genes

Pathway analysis was performed using the WEB-based GEne SeT analysis kit (webgelstat). Briefly, the first 1,000 genes down-regulated in fourth generation anti-PD-1 resistant cells but up-regulated in CT26 cells (see fig. 6A (panel 2)) were ranked based on fold-differences in expression between fourth generation anti-PD-1 resistant cells and parent CT-26 samples. These genes and ranks were input to webgelstat to identify the enrichment/de-enrichment pathways associated with these genes using a gene set enrichment analysis (gene set enrichment analysis, GSEA) and the kyoto gene and genome encyclopedia (Kyoto Encyclopedia of Genes and Genomes, KEGG) pathway function database. Statistical significance was performed using a Benjamini-Hochberg false discovery rate (false discovery rate, FDR). This analysis shows that the following pathways are overactive in fourth generation anti-PD-1 resistant cells compared to parental CT-26 cells: circadian rhythm induction (circardian entrainment), cAMP signaling pathway, cholinergic synapses, melanogenesis, rap1 signaling pathway, human cytomegalovirus infection, human immunodeficiency virus 1 infection, glutamatergic synapses, pathways in cancer and Ras signaling pathway (FIG. 8A). In fourth generation anti-PD-1 resistant cells the following pathways are inhibited compared to the parental CT-26 cells: systemic lupus erythematosus, micrornas in cancer, hepatocellular carcinoma, tuberculosis, and protein processing in the endoplasmic reticulum. To visualize the enrichment scores and significance, volcanic platforms (volcano plat) were prepared based on the data presented in fig. 8A. Fig. 8B shows a volcanic plot (volcano plot). As shown in fig. 8B, although circadian induction is the furthest "point" to the right, the pathways for cancer and Ras signaling are highest (i.e., have the most pronounced FDR values). Although not very significant (all FDR >.05, which typically occurs in genomics noise), the Ras/Rap1 signaling pathway was identified in this unbiased analysis. FIG. 8C shows RAS signaling pathways illustrating fusion with Raf/Mek/Erk signaling. FIG. 8D shows the RAP1 signaling pathway, illustrating fusion with Raf/Mek/Erk signaling.

Example 9: contradictory disorders of Ccl5 (RANTES), cxcl10 (IP-10) and Ifnb1

Next, each million transcripts (TPM; standardized expression data) of Ccl5 (RANTES), cxcl10 (IP-10) and Ifnb1 were analyzed. These genes are induced by Trim7 activation.

Ccl5 (RANTES) and Cxcl10 (IP-10) belong to genes regulated by IFNγ. Promoters that regulate Ccl5 (RANTES) and Cxcl10 (IP-10) contain binding sites for STAT1 dimer and/or ISGF 3. As expected, expression of Ccl5 (RANTES) increased from wild-type CT26 cells to second-generation anti-PD-1 resistant cells and fourth-generation anti-PD-1 resistant cells (fig. 9A (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Ccl5 (RANTES) in wild-type CT26 cells was increased (compare closed circle data in fig. 9A (left and right panels)). Paradoxically, expression in response to ifnγ, ccl5 (RANTES) decreased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 9A (right panel)).

As expected, cxcl10 (IP-10) expression increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 9B (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Cxcl10 (IP-10) in wild-type CT26 cells was increased (compare closed circle data in fig. 9B (left and right panels)). Paradoxically, the expression of ifnγ, cxcl10 (IP-10) was reduced from wild-type CT26 cells to second generation anti-PD-1 resistant cells and to fourth generation anti-PD-1 resistant cells (fig. 9B (right panel)).

Expression of Ifnb1 was also increased from wild-type CT26 cells to second generation anti-PD-1 resistant cells to fourth generation anti-PD-1 resistant cells (fig. 9C (left panel)). When wild-type CT26 cells were induced with ifnγ, increased expression of Ifnb1 in wild-type CT26 cells was increased (compare closed circle data in fig. 9C (left and right panels)). Paradoxically, the expression of ifnγ, ifnb1 was reduced from wild-type CT26 cells to second-generation anti-PD-1 resistant cells and to fourth-generation anti-PD-1 resistant cells (fig. 9C (right panel)).

These results illustrate, inter alia, that acquired resistance against PD-1 is associated with contradictory regulation of Trim7 regulatory genes, such as Ccl5 (RANTES), cxcl10 (IP-10) and Ifnb 1. Thus, these results indicate that anti-PD-1 resistant tumors can be treated with Trim7 modulators, e.g., trim7 inhibitors.

Incorporated by reference

All patents and publications mentioned herein are incorporated by reference in their entirety.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior application.

As used herein, all headings are for organizational purposes only and are not meant to limit the disclosure in any way. The content of any individual part may equally apply to all parts.

Equivalent solution

While the application has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically disclosed herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

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Claims

1. A method of determining a cancer treatment for a patient, the method comprising:

(a) Obtaining a biological sample from a subject;

(b) Assessing the presence, absence or level of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of:

(i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via mhc I class; and/or

(ii) Phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated protein, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes;

and

(c) Selecting a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 based on the evaluation of step (b).

2. A method of selecting a patient for cancer treatment, the method comprising:

(a) Obtaining a biological sample from a subject;

and

(c) Cancer therapies are selected that are capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

3. A method of treating cancer, the method comprising:

(a) Obtaining a biological sample from a subject;

(i) Positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or

and

(c) Selecting a cancer therapy selected from the group consisting of:

(i) An agent capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2;

(ii) Antimetabolite chemotherapeutics (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine, 6-diazo-5-oxo-L-norleucine (DON), diazoserine, and acitretin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and

(iii) Protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindoxine (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.; and

(d) Administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, and optionally administering the therapy selected in steps (c) (ii) and (c) (iii).

4. The method of any one of claims 1 to 3, wherein upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

5. The method of any one of claims 1-4, wherein upregulation of one or more genes associated with the GO pathway set forth in (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

6. The method of any one of claims 1-5, wherein upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of progression of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

7. The method of any one of claims 1 to 3, wherein the lack of upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to healthy tissue.

8. The method of any one of claims 1-3 or 7, wherein the lack of upregulation of one or more genes associated with the GO pathway set forth in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

9. The method of any one of claims 1-3, 7, or 8, wherein the lack of upregulation of one or more genes associated with the GO pathway listed in (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

10. The method of any one of claims 1 to 6, wherein downregulation of one or more genes associated with the GO pathway listed in (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

11. The method of any one of claims 1 to 6 or 10, wherein down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

12. The method of any one of claims 1-6, 10, or 11, wherein down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of development of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

13. The method of any one of claims 1-3 or 7-9, wherein a lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

14. The method of any one of claims 1 to 3, 7 to 9 or 13, wherein the lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

15. The method of any one of claims 1-3, 7-9, 13, or 14, wherein the lack of down-regulation of one or more genes associated with the GO pathway listed in (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

16. The method of any one of claims 1-3, 7-9, or 13-15, wherein lack of up-regulation of one or more genes associated with a GO pathway compared to a previous biological sample from the subject is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) a type I IFN signaling pathway, (d) positive regulation and antigen processing of iκb kinase/nfκb signaling, and (e) presentation of endogenous peptides via MHCI class.

17. The method of any one of claims 1 to 3, 7 to 9, or 13 to 16, wherein the lack of up-regulation of one or more genes associated with the GO pathway is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy, wherein the GO pathway is selected from (a) a cellular response to ifny, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of ikb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I.

18. The method of any one of claims 1 to 3, 7 to 9, or 13 to 17, wherein a lack of up-regulation of one or more genes associated with the GO pathway compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I.

19. The method of any one of claims 1-6 or 10-12, wherein upregulation of one or more genes associated with a GO pathway compared to a prior biological sample from the subject is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, wherein the GO pathway is selected from the group consisting of (a) a cellular response to ifny, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of ikb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via mhc class.

20. The method of any one of claims 1 to 6, 10 to 12 or 19, wherein upregulation of one or more genes associated with a GO pathway is indicative of resistance, lack of response or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy, wherein the GO pathway is selected from (a) cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHCI class.

21. The method of any one of claims 1 to 6, 10 to 12, 19 or 20, wherein upregulation of one or more genes associated with a GO pathway compared to a standard is indicative of resistance, lack of response or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, wherein the GO pathway is selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via MHC class I.

22. The method of any one of claims 1-3, 7-9, or 13-18, wherein the lack of up-regulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample from the subject.

23. The method of any one of claims 1 to 3, 7 to 9, 13 to 18, or 22, wherein the lack of up-regulation of one or more genes associated with a cellular response to ifnγ is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy.

24. The method of any one of claims 1 to 3, 7 to 9, 13 to 18, 22 or 23, wherein the lack of upregulation of one or more genes associated with a cellular response to ifnγ, as compared to a standard, is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2.

25. The method of any one of claims 1-6, 10-12, or 19-21, wherein upregulation of one or more genes associated with a cellular response to ifnγ is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a previous biological sample from the subject.

26. The method of any one of claims 1 to 6, 10 to 12, 19 to 21 or 25, wherein upregulation of one or more genes associated with a cellular response to ifnγ is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a patient known to be sensitive to anti-PD-1 therapy.

27. The method of any one of claims 1 to 6, 10 to 12, 19 to 21, 25 or 26, wherein upregulation of one or more genes associated with a cellular response to ifnγ, as compared to a standard, is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

28. The method of any one of claims 1-6, 10-12, 19-21, or 25-27, wherein upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from the subject.

29. The method of any one of claims 1 to 6, 10 to 12, 19 to 21, or 25 to 28, wherein upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a patient known to be sensitive to anti-PD-1 therapy.

30. The method of any one of claims 1-6, 10-12, 19-21, or 25-29, wherein upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of resistance, lack of response, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a standard.

31. The method of any one of claims 1-3, 7-9, 13-18, or 22-24, wherein the lack of upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a prior biological sample from the subject.

32. The method of any one of claims 1 to 3, 7 to 9, 13 to 18, 22 to 24, or 31, wherein the lack of upregulation of one or more genes associated with a type I IFN signaling pathway is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 as compared to a patient known to be sensitive to anti-PD-1 therapy.

33. The method of any one of claims 1-3, 7-9, 13-18, 22-24, 31, or 32, wherein a lack of up-regulation of one or more genes associated with a type I IFN signaling pathway compared to a standard is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

34. The method of any one of claims 1 to 33, wherein the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample.

35. The method of any one of claims 1 to 34, wherein the biological sample is a biopsy sample.

36. The method of claim 35, wherein the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy.

37. The method of any one of claims 1 to 34, wherein the biological sample comprises a bodily fluid selected from the group consisting of blood, plasma, serum, tears, tear water, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scratch, bone marrow sample, tissue biopsy sample, surgical sample, stool, other bodily fluids, secretions and/or excretions, and/or cells therefrom.

38. The method according to any one of claims 1 to 37, wherein the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing and biopsy.

39. The method of claim 38, wherein the biological sample is obtained by using a brush, (cotton) swab, spatula, flush/wash solution, needle biopsy device, puncture cavity with a needle or surgical instrument.

40. The method of any one of claims 34 to 39, wherein the biological sample comprises at least one tumor cell.

41. The method of claim 40, wherein the tumor is selected from the group consisting of hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-lytic cell NHL, megaly disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and central nervous system cancer, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric (including gastrointestinal tract carcinoma), glioblastoma, liver carcinoma, hepatoma, intraepithelial tumors, kidney or kidney carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, and squamous cell carcinoma), melanoma, myeloblastoma, neuroblastoma, oral cavity carcinoma, oral cavity, tongue carcinoma, biliary tract carcinoma, carcinoma of the oral cavity, carcinoma of the tongue, carcinoma of the human skin, carcinoma of the human skin, carcinoma, such as the stomach, carcinoma, stomach, gastric cancer, such as, and carcinoma, cancer, such as the stomach, and cancer, liver cancer, cancer, including hodgkin's lymphoma and non-hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), abnormal vascular proliferation associated with zematous hamartoma, oedema (e.g., associated with brain tumor), cancer of the migraines syndrome, renal cancer, colorectal cancer, and adrenal cancer.

42. The method of any one of claims 1 to 41, wherein the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

43. The method of any one of claims 1 to 42, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more proteins encoded by one or more genes associated with a gene body (GO) pathway selected from the group consisting of:

(ii) Phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes.

44. The method of claim 43, wherein the assessment is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a gene-ontology (GO) pathway selected from (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation and antigen processing of iκb kinase/nfκb signaling, and (e) presentation of endogenous peptide via MHC class I.

45. The method of claim 43 or claim 44, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a cellular response to ifnγ.

46. The method of any one of claims 43-45, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a type I IFN signaling pathway.

47. The method of any one of claims 1 to 42, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more nucleic acids of one or more genes associated with a gene body (GO) pathway selected from the group consisting of:

48. The method of claim 47, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a gene-body (GO) pathway selected from the group consisting of (a) a cellular response to ifnγ, (B) negative regulation of antigen processing/presentation, (c) type I IFN signaling pathway, (d) positive regulation of iκb kinase/nfκb signaling and antigen processing, and (e) presentation of endogenous peptide via mhc class I.

49. The method of claim 47 or claim 48, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a cellular response to ifnγ.

50. The method of any one of claims 47-49, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a type I IFN signaling pathway.

51. The method of any one of claims 47-50, wherein the agent that specifically binds one or more nucleic acids is a nucleic acid primer or probe.

52. The method of any one of claims 1 to 51, wherein the assessment informs classification of the patient as a high risk group or a low risk group.

53. The method of claim 52, wherein the high risk classification comprises a high level of tumor cells that are resistant to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

54. The method of claim 52, wherein low risk classification comprises low levels of tumor cells that are resistant to cancer therapies capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

55. The method of any one of claims 52 to 54, wherein a low risk classification or a high risk classification indicates cessation of neoadjuvant therapy.

56. The method of any one of claims 52-54, wherein a low risk classification or a high risk classification indicates cessation of adjuvant therapy.

57. The method of any one of claims 52 to 54, wherein the evaluation predicts a positive response to and/or benefits from the cancer treatment.

58. The method of any one of claims 52 to 54, wherein the assessment predicts a negative or neutral response to and/or benefit from the cancer treatment.

59. The method of any one of claims 52 to 54, wherein the assessment predicts a positive response to neoadjuvant chemotherapy and/or benefits from neoadjuvant chemotherapy or anergy to neoadjuvant chemotherapy and/or lack of benefit from neoadjuvant chemotherapy.

60. The method of any one of claims 52 to 54, wherein the assessment predicts a positive response to adjuvant chemotherapy and/or benefits from adjuvant chemotherapy or anergy to adjuvant chemotherapy and/or lack of benefit from adjuvant chemotherapy.

61. The method of any one of claims 52 to 54, wherein the assessment predicts a negative or neutral response to neoadjuvant chemotherapy and/or benefits from neoadjuvant chemotherapy or anergy to neoadjuvant chemotherapy and/or lack of benefit from neoadjuvant chemotherapy.

62. The method of any one of claims 52 to 54, wherein the assessment predicts a negative or neutral response to adjuvant chemotherapy and/or benefits from adjuvant chemotherapy or anergy to adjuvant chemotherapy and/or lack of benefit from adjuvant chemotherapy.

63. The method of any one of claims 52-54, wherein the assessment informs administration or cessation of the cancer treatment.

64. The method of any one of claims 52 to 54, wherein the assessment informs administration of neoadjuvant therapy.

65. The method of any one of claims 52 to 54, wherein the assessment informs administration of adjuvant therapy.

66. The method of any one of claims 52 to 54, wherein the assessment informs cessation of neoadjuvant therapy.

67. The method of any one of claims 52 to 54, wherein the assessment informs cessation of adjuvant therapy.

68. The method of any one of claims 55-67, wherein the neoadjuvant and/or the adjuvant is selected from a chemotherapeutic agent, a cytotoxic agent, a checkpoint inhibitor, an antimetabolite chemotherapeutic agent (e.g., 5-fluorouracil, methotrexate, capecitabine, azacytidine), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.).

69. The method of any one of claims 55 to 68, wherein the neoadjuvant and/or the adjuvant is selected from a protein translation inhibitor (e.g., a ribosome complex assembly and/or function modulator, a tRNA expression and/or function modulator, an amino acid synthesis and/or uptake modulator, a post-translational modification modulator (e.g., modification of a translated protein with a carbohydrate), a protein degradation modulator, and a protein transport modulator (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through the ER/golgi network, etc.), or a topoisomerase inhibitor.

70. The method of claim 69, wherein the neoadjuvant and/or the adjuvant is selected from a protein translation inhibitor (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or sorafenib), post-translational modification modulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation modulators, and protein transport modulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azathiophanone, asmine, HUN, CAM-7293, and 147).

71. A transgenic non-human animal comprising one or more tumor cells, wherein the tumor cells have:

(a) Upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: positive regulation of cell cycle processes, regulation of G1/S transitions, regulation of cell division, regulation of cell proliferation, positive regulation of ikb kinase/nfkb signaling, type I IFN signaling pathway, cellular response to ifnγ, positive regulation of ifnα production, positive regulation of defensive response, positive regulation of ifnβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing/presentation, and antigen processing/presentation of endogenous peptides via MHC class I; and/or

(b) Downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the group consisting of: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, membrane-targeting SRP-dependent co-translated proteins, ribosomal small subunit assembly, phospholipid efflux, translational regulation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis processes.

72. The transgenic non-human animal of claim 71, wherein the tumor cell is resistant to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

73. The transgenic non-human animal of claim 72, wherein the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is an antibody.

74. The transgenic non-human animal of any one of claims 71-73, wherein the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keyruda), pilizumab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimapr Li Shan antibody (libayo), alemtuzumab (TECENTRIQ), avermectin (bavendio), and dimvaluzumab (imfinzi).

75. The transgenic non-human animal of any one of claims 71-74, wherein the one or more tumor cells have:

76. The transgenic non-human animal of claim 75, wherein the one or more tumor cells have upregulation of one or more genes associated with a cellular response to ifnγ.

77. The transgenic non-human animal of claim 75 or claim 76, wherein the one or more tumor cells have upregulation of one or more genes associated with a type I IFN signaling pathway.

78. The transgenic non-human animal of any one of claims 71-77, wherein the transgenic non-human animal is a rodent.

79. The transgenic non-human animal of claim 78, wherein the rodent is a mouse.

80. The transgenic non-human animal of claim 79, wherein the mouse belongs to the BALB/C or C57BL/6 strain.

81. A method of making a transgenic non-human animal comprising one or more cancer cells that are non-responsive, resistant, or non-compliant to a cancer therapy, wherein the cancer therapy is capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, the method comprising:

(a) Injecting one or more parental cancer cells in a non-human animal that are responsive to the cancer therapy;

(b) Administering the cancer therapy to a non-human animal;

(c) Isolating cancer cells that survive the cancer therapy;

(d) Injecting cancer cells that survive the cancer therapy into different non-human animals of the same species; and

(e) Repeating steps (b) to (d) two to more than ten times.

82. The method of claim 81, wherein steps (b) through (d) are repeated at least one additional time.

83. The method of claim 81, wherein steps (b) through (d) are repeated at least two additional times.

84. The method of claim 81, wherein steps (b) through (d) are repeated at least three additional times.

85. The method of claim 81, wherein steps (b) through (d) are repeated less than five times.

86. The method of any one of claims 81-85, wherein the transgenic non-human animal is a rodent.

87. The method of claim 86, wherein the rodent is a mouse.

88. The method of claim 87, wherein the mouse belongs to the BALB/C or C57BL/6 strain.

89. The method of any one of claims 81-88, wherein the cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is an antibody.

90. The method of claim 89, wherein the antibody is selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (keytuda), pildamab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cimipro Li Shan antibody (libayo), atuzumab (TECENTRIQ), avermectin (bavenciio), and dimvaluzumab (imfinzi).

91. The method of any one of claims 81-90, wherein the cancer therapy is capable of inhibiting tumor growth when administered to a transgenic non-human animal carrying a parent cancer cell tumor as compared to an untreated transgenic non-human animal carrying a parent cancer cell tumor.

92. The method of claims 81-91, wherein tumor cells that survive the cancer therapy have:

93. The method of claim 92, wherein the one or more tumor cells have upregulation of one or more genes associated with a cellular response to ifnγ and/or a type I IFN signaling pathway.

94. A transgenic animal prepared according to the method of any one of claims 81 to 93.

95. A method of testing an anticancer drug candidate, the method comprising:

(a) Providing a transgenic non-human animal of any one of claims 71 to 80 or a transgenic non-human animal prepared according to the method of any one of claims 81 to 94;

(b) Administering an anti-cancer drug candidate to a transgenic non-human animal, and

(c) Evaluating whether the anti-cancer drug candidate is effective in slowing or inhibiting cancer growth in the transgenic non-human animal.

96. A method of preparing a pharmaceutical composition for treating cancer, the method comprising:

(c) Selecting an anti-cancer agent effective to slow or inhibit the growth of cancer in the transgenic non-human animal; and

(d) An anticancer drug or drug candidate is formulated for administration to a human patient.

97. The method of claim 95 or claim 96, wherein said anti-cancer drug candidate is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

98. A method of determining a cancer treatment for a patient, the method comprising:

(a) Obtaining a biological sample from a subject;

(b) The following expression of the biological samples was evaluated:

(i) Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT 1; and/or

(ii) Genes selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and

(c) Selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from the group consisting of:

(iii) Protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitor sulfasalazine, erastin, or larfenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, quiniline, triptyline, spirone, thiomerone, azapirone, azomycin, HUN, 147, 7293, and topoisomerase inhibitors, etc.).

99. A method of selecting a patient for cancer treatment, the method comprising:

(a) Obtaining a biological sample from a subject;

(b) The following expression of the biological samples was evaluated:

(ii) Genes selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1;

and

(iii) Protein translation inhibitors (e.g., silversrol and homoharringtonine); ribosomal biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozinndole), rRNA and/or tRNA synthesis inhibitors (e.g., fluquindox (CX-3543) and CX-5461), amino acid synthesis inhibitors (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase) inhibitors bis-2- (5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethylsulfide (BPTES), PAGDH inhibitors NCT-503), amino acid uptake inhibitors (e.g., SLC7a11 inhibitors sulfasalazine, erastin, or sorafenib), post-translational modification regulators (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), protein degradation regulators, and protein transport regulators (e.g., cyclosporin a, fendiline, pandazole, paroxetine, parthenolide, nalquin, triline, spirone, thiomerone, asylvanil, HUN-7293, and topoisomerase inhibitors, 147, or the like).

100. A method of treating cancer, the method comprising:

(a) Obtaining a biological sample from a subject;

(b) The following expression of the biological samples was evaluated:

(ii) Genes selected from RPL41, RPS15, RPS8, TRIM7 and LRG 1; and

(c) Selecting a cancer therapy selected from the group consisting of:

(d) Administering a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2; and optionally administering the therapy selected in steps (c) (ii) and (c) (iii).

101. The method of any one of claims 98-100, wherein when the biological sample comprises at least one tumor cell, a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2 is selected, and

genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6 and KRT1 are not up-regulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy; and/or

Genes selected from RPL41, RPS15, RPS8, TRIM7 and LRG1 are not down-regulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

102. The method of any one of claims 98-100, wherein when the biological sample comprises at least one tumor cell, and

Genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP2, CASP1, IRF, LTBR, PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6 and KRT1 are up-regulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy; and/or

When a gene selected from RPL41, RPS15, RPS8, TRIM7 and LRG1 is down-regulated in the at least one tumor cell, the cancer therapy is selected from the group consisting of:

antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosome biosynthesis inhibitors, rRNA and/or tRNA synthesis inhibitors, amino acid uptake inhibitors, post-translational modification regulators, protein degradation regulators, protein transport regulators, topoisomerase inhibitors.

103. The method of any one of claims 98 to 102, wherein upregulation of one or more genes listed in (b) (i) is indicative of lack of response, resistance, or noncompliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

104. The method of any one of claims 98 to 103, wherein downregulation of one or more genes listed in (b) (ii) is indicative of lack of response, resistance, or noncompliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

105. The method of any one of claims 98-104, wherein upregulation of one or more genes listed in (b) (i) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

106. The method of any one of claims 98-104, wherein down-regulation of one or more genes listed in (b) (ii) is indicative of lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

107. The method of any one of claims 98-106, wherein upregulation of one or more genes listed in (b) (i) is indicative of lack of response, development of resistance or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample obtained from the subject.

108. The method of any one of claims 98-107, wherein down-regulation of one or more genes listed in (b) (ii) is indicative of lack of response, development of resistance or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2, as compared to a prior biological sample obtained from the subject.

109. The method of any one of claims 98 to 108, wherein a lack of upregulation of one or more genes listed in (b) (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

110. The method of any one of claims 98 to 109, wherein a lack of down-regulation of one or more genes listed in (b) (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 compared to healthy tissue.

111. The method of any one of claims 98-110, wherein a lack of up-regulation of one or more genes listed in (b) (i) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

112. The method of any one of claims 98-111, wherein a lack of down-regulation of one or more genes listed in (b) (ii) is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

113. The method of any one of claims 98-112, wherein a lack of up-regulation of one or more genes listed in (b) (i) is indicative of progression of a lack of response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

114. The method of any one of claims 98-113, wherein a lack of down-regulation of one or more genes listed in (b) (ii) is indicative of progression of a lack of response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

115. The method of any one of claims 98-114, wherein the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample.

116. The method of any one of claims 98-115, wherein the biological sample is a biopsy sample, optionally wherein the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy.

117. The method of any one of claims 98 to 116, wherein the biological sample comprises a bodily fluid selected from the group consisting of blood, plasma, serum, tears, tear water, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scratch, bone marrow sample, tissue biopsy sample, surgical sample, stool, other bodily fluids, secretions and/or excretions, and/or cells therefrom.

118. The method of any one of claims 98 to 117, wherein the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing, and biopsy, optionally wherein the biological sample is obtained by using a brush, (cotton) swab, spatula, flush/wash, needle biopsy device, puncture cavity with a needle or surgical instrument.

119. The method of any one of claims 115-118, wherein the biological sample comprises at least one tumor cell.

120. The method of claim 101, 102 or 119, wherein the tumor is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-lytic cell NHL, megaly disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and central nervous system cancer, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric (including gastrointestinal tract carcinoma), glioblastoma, liver carcinoma, hepatoma, intraepithelial tumors, kidney or kidney carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, and squamous cell carcinoma), melanoma, myeloblastoma, neuroblastoma, oral cavity carcinoma, oral cavity, tongue carcinoma, biliary tract carcinoma, carcinoma of the oral cavity, carcinoma of the tongue, carcinoma of the human skin, carcinoma of the human skin, carcinoma, such as the stomach, carcinoma, stomach, gastric cancer, such as, and carcinoma, cancer, such as the stomach, and cancer, liver cancer, cancer, including hodgkin's lymphoma and non-hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), abnormal vascular proliferation associated with zematous hamartoma, oedema (e.g., associated with brain tumor), cancer of the migraines syndrome, renal cancer, colorectal cancer, and adrenal cancer.

121. The method of any one of claims 98 to 120, wherein the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

122. The method of any one of claims 98-121, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes listed in (b) (i) and/or (b) (ii), optionally wherein the agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or binding fragment thereof.

123. The method of any one of claims 98 to 122, wherein the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with the genes listed in (b) (i) and/or (b) (ii).

124. The method of claim 123, wherein the agent that specifically binds one or more nucleic acids is a nucleic acid primer or probe.

125. A method of determining a cancer treatment for a patient, the method comprising:

(a) Obtaining a biological sample from a subject;

(b) Assessing activation of a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras by a biological sample; and

126. A method of selecting a patient for cancer treatment, the method comprising:

(a) Obtaining a biological sample from a subject;

127. A method of treating cancer, the method comprising:

(a) Obtaining a biological sample from a subject;

(c) Selecting a cancer therapy selected from the group consisting of:

128. The method of any one of claims 125-127, wherein when the biological sample comprises at least one tumor cell, and

in the absence of upregulation of the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras in the at least one tumor cell, a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 is selected as compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

129. The method of any one of claims 125-127, wherein when the biological sample comprises at least one tumor cell, and

when the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1 and Ras is up-regulated in the at least one tumor cell, the cancer therapy is selected from the group consisting of:

Antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosomal biogenesis inhibitors, inhibitors of rRNA and/or tRNA synthesis, inhibitors of amino acid uptake, modulators of post-translational modification, modulators of protein degradation, modulators of protein transport, and topoisomerase inhibitors.

130. The method of any one of claims 125-129, wherein upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of lack of response, resistance, or noncompliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to healthy tissue.

131. The method of any one of claims 125-130, wherein upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of lack of response, resistance, or noncompliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

132. The method of any one of claims 125-131, wherein upregulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of lack of response, resistance, or progression of non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

133. The method of any one of claims 125-132, wherein a lack of up-regulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras as compared to healthy tissue is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2.

134. The method of any one of claims 125-133, wherein a lack of up-regulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of a response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

135. The method of any one of claims 125-134, wherein a lack of up-regulation of a pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is indicative of progression of a lack of response to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2, as compared to a prior biological sample obtained from the subject.

136. The method of any one of claims 125-135, wherein the biological sample is a fresh tissue sample, a frozen tumor tissue sample, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue sample.

137. The method of any one of claims 125-136, wherein the biological sample is a biopsy sample.

138. The method of claim 137, wherein the biopsy sample is selected from the group consisting of an endoscopic biopsy, a bone marrow biopsy, an endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), a needle biopsy (e.g., fine needle aspiration, needle penetration biopsy, vacuum assisted biopsy, X-ray assisted biopsy, computed Tomography (CT) assisted biopsy, magnetic Resonance Imaging (MRI) assisted biopsy, and ultrasound assisted biopsy), a skin biopsy (e.g., scratch biopsy, puncture biopsy, and cut biopsy), and a surgical biopsy.

139. The method of any one of claims 125-138, wherein the biological sample comprises a bodily fluid selected from the group consisting of blood, plasma, serum, tears, tear water, bone marrow, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing bodily fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation or lavage fluid such as catheter lavage or bronchoalveolar lavage, aspirate, scratch, bone marrow sample, tissue biopsy sample, surgical sample, stool, other bodily fluids, secretions and/or excretions, and/or cells therefrom.

140. The method of any one of claims 125-139, wherein the biological sample is obtained by a technique selected from the group consisting of scraping, swabbing, and biopsy.

141. The method of claim 140, wherein the biological sample is obtained by using a brush, (cotton) swab, spatula, flush/wash solution, needle biopsy device, puncture cavity with a needle or surgical instrument.

142. The method of any one of claims 136 to 141, wherein the biological sample comprises at least one tumor cell.

143. The method of claim 126, 127 or 142, wherein the tumor is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-lytic cell NHL, megaly disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, or chronic granulocytic leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and central nervous system cancer, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal carcinoma, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric (including gastrointestinal tract carcinoma), glioblastoma, liver carcinoma, hepatoma, intraepithelial tumors, kidney or kidney carcinoma, laryngeal carcinoma, leukemia, liver carcinoma, lung carcinoma (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, and squamous cell carcinoma), melanoma, myeloblastoma, neuroblastoma, oral cavity carcinoma, oral cavity, tongue carcinoma, biliary tract carcinoma, carcinoma of the oral cavity, carcinoma of the tongue, carcinoma of the human skin, carcinoma of the human skin, carcinoma, such as the stomach, carcinoma, stomach, gastric cancer, such as, and carcinoma, cancer, such as the stomach, and cancer, liver cancer, cancer, including hodgkin's lymphoma and non-hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic cellular NHL, giant tumor disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic granulocytic leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative diseases (PTLD), abnormal vascular proliferation associated with zematous hamartoma, oedema (e.g., associated with brain tumor), cancer of the migraines syndrome, renal cancer, colorectal cancer, and adrenal cancer.

144. The method of any one of claims 125-143, wherein the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

145. The method of any one of claims 125-144, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more proteins encoded by a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras.

146. The method of claim 145, wherein the agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof.

147. The method of any one of claims 125-146, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more nucleic acids of one or more genes associated with a pathway selected from the group consisting of Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras.

148. The method of claim 147, wherein the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

149. A method of treating cancer in a subject in need thereof, the method comprising administering a cancer therapy selected from the group consisting of: antimetabolite chemotherapeutics, topoisomerase inhibitors, protein translation inhibitors, ribosomal biogenesis inhibitors, rRNA and/or tRNA synthesis inhibitors, amino acid uptake inhibitors, post-translational modification regulators, protein degradation regulators, protein transport regulators, topoisomerase inhibitors, wherein

The subject has received or is receiving an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2;

wherein the subject has developed a lack of response, resistance, or non-compliance to a cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1, and/or PD-L2; and is also provided with

Wherein the pathway selected from Mapk8ip1, trim7, elk1, lrg1, arg1, rap1, and Ras is upregulated in at least one tumor cell in the subject, as compared to healthy tissue, a prior biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

150. A method of treating cancer in a subject in need thereof, the method comprising:

(a) Administering an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2;

(b) Evaluating an anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring a decrease in tumor size in a subject;

(c) If no decrease in tumor size is observed, administering a Trim7 modulator and/or a proteasome inhibitor;

(d) Re-evaluating an anti-tumor response with an anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 by monitoring tumor reduction in the subject; and

(e) If a decrease in tumor size is observed, administration of Trim7 modulator is stopped.

151. A method of treating cancer in a subject in need thereof, the method comprising:

(b) The anti-tumor response with anti-cancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 was evaluated using the following steps:

(i) Obtaining a biological sample from a subject;

(ii) Assessing overexpression and/or activation of TRIM7 in a biological sample;

(c) If overexpression and/or activation of TRIM7 is observed, administering TRIM7 modulators and/or proteasome inhibitors;

(d) Antitumor response with anticancer therapy capable of inhibiting the function and/or activity of PD-1, PD-L1 and/or PD-L2 was re-evaluated using the following steps:

(i) Obtaining a biological sample from a subject;

and

(e) If overexpression and/or activation of TRIM7 is not observed, administration of TRIM7 modulator is stopped.

152. A method of treating cancer in a subject in need thereof, the method comprising:

(b) The overexpression and/or activation of TRIM7 was evaluated using the following steps:

(i) Obtaining a biological sample from a subject; and

(d) Re-evaluation of overexpression and/or activation of TRIM7 using the following steps:

(i) Obtaining a biological sample from a subject;

and

153. The method of any one of claims 150 to 152, wherein the Trim7 modulator is a Trim7 inhibitor.

154. The method of any one of claims 150 to 153, wherein the Trim7 modulator is selected from the group consisting of small interfering RNAs (sirnas), short hairpin RNAs (shrnas), micrornas (mirnas), antisense RNAs, guide RNAs (grnas), small molecules, antibodies, peptides and peptidomimetics.

155. The method of claim 154, wherein the small interfering RNA (siRNA), short hairpin RNA (shRNA), microrna (miRNA), antisense RNA, or guide RNA (gRNA) inhibits Trim7 protein production.

156. The method of claim 154, wherein the peptide mimetic mimics a target of Trim7, thereby inhibiting Trim7 activity.

157. The method of claim 153, wherein the Trim7 inhibitor is a small molecule or peptide inhibitor that binds to Trim7 protein at or near a protein segment selected from the group consisting of MAAVGPRTGPGTGAEALALAAEL (SEQ ID NO: 1), AATRAPPFPLPCP (SEQ ID NO: 2), HGSQAAAARAAAARCG (SEQ ID NO: 3) and NVSLKTFVLKGMLKKFKEDLRGELEKEEKV (SEQ ID NO: 4).

158. The method of any one of claims 150 to 152, wherein the Trim7 modulator is a mitogen and stress activated kinase 1 (MSK 1) inhibitor, wherein the MSK1 inhibitor modulates Trim7 via inhibition of downstream effects of MSK 1.

159. The method of claim 158, wherein the MSK1 inhibitor is selected from Ro31-8220, SB-747651a and H89.

160. The method of claim 159, wherein the MSK1 inhibitor is SB-747651a.

161. The method according to any one of claims 150 to 160, wherein the proteasome inhibitor is selected from bortezomib, carfilzomib, i Sha Zuomi, osprezomib, delazomib, and malizomib.

162. The method of any one of claims 151-161, wherein the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, intracellular western blotting, immunofluorescent staining, ELISA, and Fluorescence Activated Cell Sorting (FACS), or a combination thereof.

163. The method of any one of claims 151-162, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more proteins encoded by the Trim7 pathway.

164. The method of claim 163, wherein the agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof.

165. The method of any one of claims 151-164, wherein the evaluating is performed by contacting the sample with an agent that specifically binds one or more nucleic acids of one or more genes associated with Trim7 pathway.

166. The method of claim 165, wherein the agent that specifically binds to one or more nucleic acids is a nucleic acid primer or probe.

167. The method of any one of claims 151-166, wherein the evaluation is performed by determining E3 ubiquitin ligase activity.

168. The method according to any one of claims 151 to 167, wherein said evaluation is performed by determining the protein ubiquitination and/or K48 linked ubiquitination of the interferon gene Stimulators (STING) and/or the AP-1 coactivator RACO-1.

169. The method of any one of claims 151-168, wherein the evaluation is performed by assaying for c-Jun/AP1 activation via Ras-Raf-MEK-ERK signaling and/or an increase in AP 1-mediated gene expression.

170. The method according to any one of claims 151 to 169, wherein said evaluating is performed by determining ubiquitination and stabilization of the AP1 co-activator RACO-1.

171. The method of any one of claims 151-170, wherein the evaluation is performed by determining K63-linked ubiquitination of a target protein, comprising a protein involved in cell proliferation and an innate immune response.

172. The method according to any one of claims 151 to 171, wherein said evaluation is performed by determining Trim7 phosphorylation, K63-linked ubiquitination and/or protein levels of the AP-1 co-activator RACO-1.

173. The method of any one of claims 151-172, wherein the evaluation is performed by measuring upregulation of IFN beta, IP-10, and/or Rantes.