METHODS OF OVERCOMING RESISTANCE TO CHECKPOINT INHIBITOR THERAPIES

FIELD OF THE DISCLOSURE

The present disclosure relates to, in part, methods that are useful for detection and treatment of drug resistant cancer, and methods for developing new therapeutics against drug resistant cancers.

PRIORITY

This application claims the benefit of, and priority to, U.S. Provisional Application No. 63/425,085, filed November 14, 2022, the contents of each of which are hereby incorporated by reference in their entirety.

BACKGROUND

Drug resistance remains one of the biggest challenges in cancer therapy. Drug resistance is found across all types of cancer and all modes of treatment, including molecularly targeted therapy, immunotherapy, and chemotherapy. It is also common that a patient with advanced cancer receives a drug that is efficacious, but then weeks or months later the cancer recurs, and drug efficacy is lost or reduced. Unfortunately, few effective therapeutic options are available for patients having cancers that are resistant to the anti-checkpoint therapies. Developmentof resistance to checkpoint therapy appears to depend on genetic and immunological background of the host. For example, diverse types of effector T cells, which play a role in eliminating cancers, compete with Tregs in the tumor environment, which suppress not only the native anticancer immunity as well as the efficacy of immune checkpoint inhibitors. See, e.g., Gonzalez-Navajas et a/., The Impact of Tregs on the Anticancer Immunity and the Efficacy of Immune Checkpoint Inhibitor Therapies, Front. Immunol 2021 ; 12: 625783. Principe et al., Regulatory T-Cells as an Emerging Barrier to Immune Checkpoint Inhibition in Lung Cancer, Front Oncol. 2021 ; 11 : 684098; Marshall et a/., Tumors Establish Resistance to Immunotherapy by Regulating Treg Recruitment Via CCR4. J Immunother Cancer (2020) 8(2):e000764; Nishikawa and Sakaguchi, Regulatory T cells in cancer immunotherapy. Curr. Opin. Immunol. 2014; 27:1 ; Fares et al., Mechanisms of Resistance to Immune Checkpoint Blockade: Why Does Checkpoint Inhibitor Immunotherapy Not Work for All Patients?, Am Soc Clin Oncol Educ Book 2019; 39: 147-164. Likewise, CD4+CD25+ T regulatory cells contribute to resistance to anti-PD-1 therapeutics. Kamada et al., PD-1 + regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer, Proc. Natl. Acad. Sci. USA 2019; 116 (20) 9999-10008; Zuazo et al., Systemic CD4 Immunity as a Key Contributor to PD-L1/PD-1 Blockade Immunotherapy Efficacy, Front Immunol. 2020; 11 : 586907. Similarly, NK cells also appear to have a function in resistance to checkpoint inhibitors. Gemelli et al., Overcoming Resistance to Checkpoint Inhibitors: Natural Killer Cells in Non-Small Cell Lung Cancer, Front. Oncol., 2022; 12: 886440. Therefore, beter understanding the mechanisms of drug resistance in patients having greater proportions of Tregs in the tumor environment may provide guidance to future cancer treatment. Moreover, methods for developing new therapies for patients suffering from drug resistant cancer and methods for selecting appropriate drugs for patients having drug-resistant cancer are desired for improving outcomes in cancer patients.

Development of resistance to checkpoint therapy also appears to depend on genetic background of the tumors. For example, p53, which is the most frequently mutated tumor suppressor in human cancers, plays a role in both regulation of immunity and developing resistance to anti-checkpoint therapy. Shi and Jiang, A Different Facet of p53 Function: Regulation of Immunity and Inflammation During Tumor Development, Front Cell Dev B/o/ 2021 ; 9:762651 ; Wang eta/., Epithelial Mutant p53 Promotes Resistance to Anti-PD-1 -Mediated Oral Cancer Immunoprevention in Carcinogen-Induced Mouse Models, Cancers (Basel) 2021; 13(6): 1471 ; Sobol etal., Effect of adenoviral p53 (Ad-p53) tumor suppressor immune gene therapy on checkpoint inhibitor resistance and abscopal therapeutic efficacy. J. Clin. Oncol. 2017; 35: e14610; Chada et al., Tumor suppressor immune gene therapy to reverse immunotherapy resistance, Cancer Gene Ther 2021 ; doi: 10.1038/s41417-021 -00369-7; Cao et al., An unexpected role for p53 in regulating cancer cell-intrinsic PD-1 by acetylation, Sci Adv 2021 ;7(14):eabf4148. Therefore, better understanding the mechanisms of drug resistance in a p53 mutant background may provide guidance to future cancer treatment. Moreover, methods for developing new therapies for patients suffering from drug resistant cancer and methods for selecting appropriate drugs for patients having drug-resistant cancer are desired for improving outcomes in cancer patients.

SUMMARY

Accordingly, the present disclosure provides, in part, methods for selecting patients having a potent NK-cell mediated cytotoxicity, a Th1 - and/or M1 -dominant immune response, and/or weak activity of CD4- ^D25+T regulatory cells and/or patients suffering from a p53 mutant cancer, which is or is at risk of being checkpoint resistant, for cancer treatment, and methods for cancer treatment, based on, for instance, based on gene expression profiles of anti-PD-1 resistant cancers, he present disclosure also provides animal models suitable for testing an anti-cancer drug candidate for treating a p53 mutant cancer, which is or is at risk of being checkpoint resistant, and methods for making a pharmaceutical composition for treating cancer.

In aspects, the present disclosure relates to a method for selecting for a cancer treatment a patient having higher activity of Tregs compared to effector T cells in the tumor microenvironment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a patient having higher activity of Tregs compared to effector T cells in the tumor microenvironment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of determining treatment for a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1-dominant immune response, and/or weak activity of CD4- ^D25+T regulatory cells: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting for a cancer treatment in a subject for cancer treatment, wherein the subject exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1- dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1 -dominant immune response, and/or weak activity of CD4-HDD25+T regulatory cells, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, the presence of the potent NK-cell mediated cytotoxicity is determined based on measurement of one or more of the proportion of NKP46+ cells, the proportion of NKP46+CD69+ cells, extent of CD107a surface expression, extent of cytokine production (e.g. production of one or more of IFNy NFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8), and extent of lysis of target cells. In embodiments, the presence of the Th 1 - and/or M1 -dominant immune response compared to a control is determined based on one or more of the ratios of IgMJgG and/or IgEJgG antibodies, extent of cytokine production (e.g., production of one or more of IFNy, TNFa, LTa, IL- 17A, IL-6, IL-12, CXCR3, CCR5), surface expression of one or more markers (e.g., iNOS, CD80, CD86, CD64, CD16 and CD32, along with CD68 and/or CD11 b). In embodiments, the presence of the weak activity of CD4-HDD25+T regulatory cells compared to a control is determined based on one or more of the proportion of CD4+CD25+T regulatory cells in peripheral blood, extent of cytokine production (e.g., production of IL-2, IL-10 and TGF0), proliferation assay. In embodiments, the control is selected from a standard value or a sample from one or more normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof.

In aspects, the present disclosure relates to a method of determining treatment for a p53 mutant cancer in a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting a patient having a p53 mutant cancer for a cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a p53 mutant cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from patient that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is selected.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo- L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 , 2, 4-thiadiazol- 2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEM PERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101 , and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R- Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS- 741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI-3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)); and a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.)

In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from subject that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5- fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g, cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERU), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS- 663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K5 inhibitor including, but not limited to, PI-3065; PI 3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g, 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5- phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post- translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g, a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g, SGN- 75)), tumor myeloid-directed therapies (e.g, CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI- 3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, the one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is selected from Statl, Stat2, Tapi , Ifitm2, and Ifitm3.

In aspects, the present disclosure relates to a method of determining treatment for a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1 - and/or M1-dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting for a cancer treatment in a subject for cancer treatment, wherein the subject exhibits a potent NK-cell mediated cytotoxicity, a Th1 - and/or Mi- dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1 -dominant immune response, and/or weak activity of CD4-RDD25+ T regulatory cells, the method comprising: (a) contacting a cultured biological sample from a subject with I FNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of determining treatment for a p53 mutant cancer in a patient, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, the presence of the potent NK-cell mediated cytotoxicity is determined based on measurement of one or more of the proportion of NKP46+ cells, the proportion of NKP46CD69+ cells, extent of CD107a surface expression, extent of cytokine production (e.g. production of one or more of IFNy NFo, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8), and extent of lysis of target cells. In embodiments, the presence of the Th 1 - and/or M1 -dominant immune response compared to a control is determined based on one or more of the ratios of IgM :lgG and/or IgEJgG antibodies, extent of cytokine production (e.g., production of one or more of IFNy, TNFa, LTa, IL- 17A, IL-6, IL-12, CXCR3, CCR5), surface expression of one or more markers (e.g., iNOS, CD80, CD86, CD64, CD16 and CD32, along with CD68 and/or CD11 b). In embodiments, the presence of the weak activity of CD4+CD25+T regulatory cells compared to a control is determined based on one or more of the proportion of CD4+CD25+T regulatory cells in peripheral blood, extent of cytokine production (e.g., production of IL-2, IL-10 and TGFP), proliferation assay. In embodiments, the control is selected from a standard value or a sample from one or more normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof.

In aspects, the present disclosure relates to a method for selecting a patient having a p53 mutant cancer for a cancer treatment, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a p53 mutant cancer, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, the cultured biological sample from a subject is contacted with an interferon for less than about 7 hr, or less than about 6 hr, or less than about 5 hr, or less than about 4 hr, or less than about 3 hr, or less than about 2 hr, or less than about 1 hr. In embodiments, the cultured biological sample from a subject is contacted with an interferon for at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 30 minutes, or at least about 45 hr, or at least about 1 hr, or at least about 2 hr, or at least about 3 hr, or at least about 4 hr.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from patient that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is selected.

In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected. In embodiments, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS- 663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41 A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K5 inhibitor including, but not limited to, PI-3065; PI 3K y inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2.

In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from subject that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected. In embodiments, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5- phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post- translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN- 75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI- 3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected. In embodiments, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo- L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 , 2, 4-thiadiazol- 2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor {e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEM PERU), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBM 01 , and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R- Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS- 741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI-3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2.

In embodiments, the one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is selected from Statl, Stat2, Tapi , Ifitm2, and Ifitm3.

In embodiments, the biological sample comprises at least one tumor cell. In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof.

In embodiments, the evaluating informs classifying the patient into a high- or low-risk group. In embodiments, the high-risk classification comprises a high level of tumor cells having resistance to the cancer therapy with an ability to reduce or inhibit 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 having resistance to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In aspects, the present disclosure relates to a transgenic non-human animal comprising one or more p53 mutant tumor cells, wherein the tumor cells exhibit: (a) an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and/or (b) a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, when contacted with IFNy for less than about 8 hours.

In embodiments, the tumor cells are resistant to a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. In embodiments, the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is a human or humanized antibody. In embodiments, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), Cemiplimab (LIBTAYO), Atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (imfinzi).

In aspects, the present disclosure relates to a method of making a transgenic non-human animal comprising one or more p53 mutant cancer cells that are nonresponsive, resistant, or recalcitrance to a cancer therapy, wherein the cancer therapy has an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2, the method comprising: (a) injecting one or more parental p53 mutant cancer cells that are responsive to the cancer therapy in a non-human animal; (b) administering the 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 in a different non-human animal of the same species; and (e) repeating steps (b) to (d) three to ten more times. In aspects, the present disclosure relates to a method of making a transgenic non-human animal comprising one or more p53 mutant cancer cells that are nonresponsive, resistant, or recalcitrance to a cancer therapy, wherein the non-human animal exhibits a potent NK-cell mediated cytotoxicity, a Th1 - and/or M1 -dominant immune response, and/or weak activity of CD4- 25+ T regulatory cells, and wherein the cancer therapy has an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2, the method comprising: (a) injecting one or more parental p53 mutant cancer cells that are responsive to the cancer therapy in a non-human animal; (b) administering the 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 in a different non-human animal of the same species; and (e) repeating steps (b) to (d) three to ten more times.

In embodiments, steps (b) to (d) are repeated at least three times more. In embodiments, steps (b) to (d) are repeated at least four times more. In embodiments, steps (b) to (d) are repeated at least five times more. In embodiments, steps (b) to (d) are repeated less than eight times.

In embodiments, the cancer cells that survive the cancer therapy grow faster than the parental p53 mutant cancer cells in the presence of the cancer therapy that has an ability to reduce or inhibit 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 mouse belongs to BALB/c or C57BL/6 strain.

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

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A and FIG. 1 B illustrate the generation of anti-PD-1 -resistant MC38 tumors. FIG. 1A shows a schematic representation of the method used to generation of anti-PD-1-resistant MC38 tumors. The cells obtained after five rounds of selection are also referred to as MC38/AR herein. FIG. 1B shows a graph comparing the efficacy of an anti-PD-1 antibody (100 pg anti-PD-1 clone RMP1-14; BioXcell) in C57BL/6 mice harboring MC38 parental cells or PD-1 resistant MC38 cells obtained after third, fourth and fifth round of selection. FIG. 2A to FIG. 2E show the comparison of normalized baseline expression of the following genes in MC38/AR cells and MC38 parental cells: Statl (FIG. 2A), Stat2 (FIG. 2B), Tapi (FIG. 2C), Ifitm2 (FIG. 2D), and Ifitm3 (FIG. 2E). Gene expression was quantified using qRT-PCR, expression was normalized in comparison expression of the housekeeping control gene Rsp18 using the ddCt method, the relative expression in MC38 parental cells to 1 and plotted.

FIG. 3A to FIG. 3E show the comparison of normalized expression of the following genes in MC38/AR cells and MC38 parental cells in the presence of I FNy: Statl (FIG. 3A), Stat2 (FIG. 3B), Tapi (FIG. 3C), Ifitm2 (FIG. 3D), and Ifitm3 (FIG. 3E). Gene expression was quantified using qRT-PCR, expression was normalized in comparison expression of the housekeeping control gene Rsp18 using the ddCt method, the relative expression in MC38 parental cells to 1 and plotted.

DETAILED DESCRIPTION

The current disclosure is based, in part, the discovery of surprising upregulation in anti-PD-1 resistant cells of certain genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation in tumors in a host that shows a potent NK-cell mediated cytotoxicity, a Th 1 - and/or M1-dominant immune response, and weak activity of CD4-CD25+T regulatory cells. This is surprising, inter alia, because interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation are all involved in NK-cell mediated cytotoxicity, production of a Th1 - and/or M1 -dominant immune response, and weakening of CD4+CD25+ T regulatory cells, decreased susceptibility to cancer, as well as in increasing sensitivity of cancer cells to anti-PD-1 antibodies. Morel et al., EZH2 inhibition activates a dsRNA-STING- interferon stress axis that potentiates response to PD-1 checkpoint blockade in prostate cancer, Nat Cancer 2021 ; 2(4):444-456; Muller et al., Type I Interferons and Natural Killer Cell Regulation in Cancer, Front Immunol. 2017; 8: 304; Mizutani et al., Conditional IFNAR1 ablation reveals distinct requirements of type I IFN signaling for NK-cell maturation and tumor surveillance. Oncoimmunology 2012; 1 :1027-37; Bacher et al., Interferon-a suppresses cAMP to disarm human regulatory T cells, Cancer Res 2013; 73(18):5647-56; Longhi et al., Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly I C as adjuvant, J Exp Med. 2009; 206(7): 1589-602; Karachaliou et al., Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients, TherAdvMed Oncol. 10: 1758834017749748 (2018); Rafique et al., Immune Checkpoint Blockade and Interferon-a in Melanoma, Semin Oncol. 42(3): 436-447 (2015); Gemelli et al., Overcoming Resistance to Checkpoint Inhibitors: Natural Killer Cells in Non-Small Cell Lung Cancer, Front. Oncol., 2022; 12: 886440; Uehara et al., Intratumoral injection of IFN-0 induces chemokine production in melanoma and augments the therapeutic efficacy of anti-P D-L1 mAb, Biochemical and Biophysical Research Communications 490(2) 521 - 527 (2017) Gonzalez-Navajas et al., The Impact of Tregs on the Anticancer Immunity and the Efficacy of Immune Checkpoint Inhibitor Therapies, Front. Immunol 2021; 12: 625783. Principe eta/., Regulatory T-Cells as an Emerging Barrier to Immune Checkpoint Inhibition in Lung Cancer, Front Oncol. 2021 ; 11 : 684098; Marshall et al., Tumors Establish Resistance to Immunotherapy by Regulating Treg Recruitment Via CCR4. J Immunother Cancer (2020) 8(2):e000764; Nishikawa and Sakaguchi, Regulatory T cells in cancer immunotherapy. Curr. Opin. Immunol. 2014; 27:1; Fares et al., Mechanisms of Resistance to Immune Checkpoint Blockade: Why Does Checkpoint Inhibitor Immunotherapy Not Work for All Patients?, Am Soc Clin Oncol Educ Book 2019; 39: 147-164.

The current disclosure is also based, in part, the discovery of surprising dysregulation of certain genes associated with interferon response in p53-mutant tumors. This is surprising, inter alia, because p53 is known to have a role in the interferon response. Munoz-Fontela et al., Transcriptional role of p53 in interferon- mediated antiviral immunity, J Exp Med. 2008; 205(8): 1929-1938; Kim et al., Interferon-gamma induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells, Meeh Ageing Dev 2009; 130(3): 179-88; Thiem et al., IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression, J Exp Clin Cancer Res 2019; 38(1):397.

In addition, the data presented herein are also surprising, inter alia, in view of a publication disclosing an anti- PD-1 resistant tumor model, which reported that IFNy signaling, and antigen processing and presentation pathways were functional in both parental and resistant cell lines, instead, the activation of TGF0 and Notch signaling in anti-PD-1 resistant tumors. See Bernardo et al., An experimental model of anti-PD-1 resistance exhibits activation of TGF and Notch pathways and is sensitive to local mRNA immunotherapy, Oncoimmunology 2021 ; 10(1): 1881268. However, as noted in Example 3, prolonged treatment of cells with IFNy caused anomalous results as compared to shorter treatments. Without being bound by theory, it is that this may be caused by an indirect effect of IFNy exposure (e.g, negative feedback loops or the effects of cytokines induced by IFNy). For example, TGF activation, which is reported by Bernard et al., inhibits IFNy signaling Gabrielian et al., Effect of TGF-beta on interferon-gamma-induced HLA-DR expression in human retinal pigment epithelial cells, Invest Ophthalmol Vis Sci . 1994;35(13):4253-9; Park et al., TGF-beta 1 inhibition of IFN-gamma-induced signaling and Th1 gene expression in CD4+ T cells is Smad3 independent but MAP kinase dependent, Mol Immunol . 2007; 44(13):3283-90. Disclosed herein is an anti-PD-1 -resistant tumor model based murine colorectal carcinoma MC38 cells in C57BL/6 mice, which exbibit potent NK-cell mediated cytotoxicity, a Th1- and/or M1-dominant immune response, and weak activity of CD4+CD25+ T regulatory cells. Zhong et al., Comparison of the molecular and cellular phenotypes of common mouse syngeneic models with human tumors, BMC Genomics 2020; 21 (1 ):2. MC38 cells were by subjecting them to selection for surviving in view of repeated anti-PD-1 treatment (FIG. 1 A).

Methods of Determining a Cancer Treatment for a Patient; Methods of Selecting a Patient for a Cancer Treatment; and Methods of Treatment

In aspects, the present disclosure relates to a method of determining treatment for a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1-dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting for a cancer treatment in a subject for cancer treatment, wherein the subject exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1- dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1 -dominant immune response, and/or weak activity of CD4-HDD25+T regulatory cells, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, the presence of the potent NK-cell mediated cytotoxicity is determined based on measurement of one or more of the proportion of NKP46+ cells, the proportion of NKP46+CD69+ cells, extent of CD107a surface expression, extent of cytokine production (e.g. production of one or more of IFNy NFo, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8), and extent of lysis of target cells. In embodiments, the presence of the Th 1 - and/or M1 -dominant immune response compared to a control is determined based on one or more of the ratios of IgMJgG and/or IgEJgG antibodies, extent of cytokine production (e.g., production of one or more of IFNy, TNFa, LTa, IL- 17A, IL-6, IL-12, CXCR3, CCR5), surface expression of one or more markers (e.g., iNOS, CD80, CD86, CD64, CD16 and CD32, along with CD68 and/or CD11 b). In embodiments, the presence of the weak activity of CD4-HDD25+T regulatory cells compared to a control is determined based on one or more of the proportion of CD4+CD25+T regulatory cells in peripheral blood, extent of cytokine production (e.g., production of IL-2, IL-10 and TGF0), proliferation assay. In embodiments, the control is selected from a standard value or a sample from one or more normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof.

In embodiments, the presence of the potent NK-cell mediated cytotoxicity is determined based on measurement of one or more of the proportion of NKP46+ cells, the proportion of NKP46+CD69+ cells, extent of CD107a surface expression, extent of cytokine production (e.g. production of one or more of IFNy, TNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8), and extent of lysis of target cells. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by contacting the sample with an agent that specifically binds to a protein selected from NKP46, CD69, IFNy, TNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1 , CCL2, CCL3, CCL4, CCL5, and CXCL8. In embodiments, the agent that specifically binds to a protein selected from NKP46, CD69, IFNy, TNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8 is an antibody, a binding fragment thereof, an antibody-like molecule, or a binding fragment thereof. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins selected from protein selected from NKP46, CD69, IFNy, TNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1 , CCL2, CCL3, CCL4, CCL5, and CXCL8. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In embodiments, the presence of the Th1 - and/or M1-dominant immune response compared to a control is determined based on one or more of the ratios of IgMJgG and/or IgEJgG antibodies, extent of cytokine production (e.g., production of one or more of IFNy, TNFo, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5), surface expression of one or more markers (e.g., iNOS, CD80, CD86, CD64, CD16 and CD32, along with CD68 and/or CD11 b). In embodiments, the determination of a presence of the Th1- and/or M1-dominant immune response is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof. In embodiments, the determination of a presence of the Th1 - and/or M1 -dominant immune response is performed by contacting the sample with an agent that specifically binds to a protein selected from IgM, IgG, IgE, IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b. In embodiments, the agent that specifically binds to a protein selected from IgM, IgG, IgE, IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b is an antibody, a binding fragment thereof, an antibody-like molecule, or a binding fragment thereof. In embodiments, the determination of a presence of the Th1- and/or M1 -dominant immune response is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins selected from protein selected from IgM, IgG, IgE, IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In aspects, the present disclosure relates to a method of determining treatment for a p53 mutant cancer in a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting a patient having a p53 mutant cancer for a cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting a patient having a p53 mutant cancer for a cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a p53 mutant cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of determining treatment for a patient having higher activity of Tregs compared to effector T cells in the tumor microenvironment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting for a cancer treatment a patient having higher activity of Tregs compared to effector T cells in the tumor microenvironment, the method comprising:

(a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a patient having higher activity of Tregs compared to effector T cells in the tumor microenvironment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from patient that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when a lack of upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is selected.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo- L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.} and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 , 2, 4-thiadiazol- 2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEM PERU), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBM 01 , and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R- Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS- 741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI-3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from subject that is known to be sensitive to anti-PD- 1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5- fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS- 663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101 , and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K5 inhibitor including, but not limited to, PI-3065; PI 3K y inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2.

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected. Instead, in embodiments, a chemotherapy is selected, the chemotherapy is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.) and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5- phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post- translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN- 75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K5 inhibitor including, but not limited to, Pl- 3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)). In embodiments, the chemotherapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, the one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is selected from Statl, Stat2, Tapi , Ifitm2, and Ifitm3.

In aspects, the present disclosure relates to a method of determining treatment for a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th 1 - and/or M1-dominant immune response, and/or weak activity of CD4+CD25+T regulatory cells: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting for a cancer treatment in a subject for cancer treatment, wherein the subject exhibits a potent NK-cell mediated cytotoxicity, a Th1 - and/or Mi- dominant immune response, and/or weak activity of CD4CD25+T regulatory cells, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a cancer in a subject that exhibits a potent NK-cell mediated cytotoxicity, a Th1- and/or M1 -dominant immune response, and/or weak activity of CD4+CD25+ T regulatory cells, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, the presence of the potent NK-cell mediated cytotoxicity is determined based on measurement of one or more of the proportion of NKP46+ cells, the proportion of NKP46+CD69+ cells, extent of CD107a surface expression, extent of cytokine production (e.g. production of one or more of IFNyJNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8), and extent of lysis of target cells. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by contacting the sample with an agent that specifically binds to a protein selected from NKP46, CD69, IFNyJNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1 , CCL2, CCL3, CCL4, CCL5, and CXCL8. In embodiments, the agent that specifically binds to a protein selected from NKP46, CD69, IFNyJNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8 is an antibody, a binding fragment thereof, an antibody-like molecule, or a binding fragment thereof. In embodiments, the determination of a potent NK-cell mediated cytotoxicity is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins selected from protein selected from NKP46, CD69, IFNy NFa, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1 , CCL2, CCL3, CCL4, CCL5, and CXCL8. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In embodiments, the presence of the Th1 - and/or M1-dominant immune response compared to a control is determined based on one or more of the ratios of IgM: IgG and/or IgEJgG antibodies, extent of cytokine production (e.g., production of one or more of IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5), surface expression of one or more markers (e.g., iNOS, CD80, CD86, CD64, CD16 and CD32, along with CD68 and/or CD11 b). In embodiments, the determination of a presence of the Th1- and/or M1-dominant immune response is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof. In embodiments, the determination of a presence of the Th1 - and/or M1-dominant immune response is performed by contacting the sample with an agent that specifically binds to a protein selected from IgM, IgG, IgE, IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b. In embodiments, the agent that specifically binds to a protein selected from IgM, IgG, IgE, IFNy, TNFa, LTa, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b is an antibody, a binding fragment thereof, an antibody-like molecule, or a binding fragment thereof. In embodiments, the determination of a presence of the Th1- and/or M1 -dominant immune response is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins selected from protein selected from IgM, IgG, IgE, I FNy, TNFo, LTo, IL-17A, IL-6, IL-12, CXCR3, CCR5, iNOS, CD80, CD86, CD64, CD16 and CD32, CD68, and CD11 b. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In embodiments, the presence of the weak activity of CD4+CD25+ T regulatory cells compared to a control is determined based on one or more of the proportion of CD4+CD25+ T regulatory cells in peripheral blood, extent of cytokine production (e.g., production of IL-2, IL-10 and TGF ), proliferation assay. In embodiments, the control is selected from a standard value or a sample from one or more normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof. In embodiments, the determination of a weak activity of CD4+CD25+ T regulatory cells is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof. In embodiments, the determination of a weak activity of CD4- D25+T regulatory cells is performed by contacting the sample with an agent that specifically binds to a protein selected from CD4, CD25, IL-2, IL-10 and TGF . In embodiments, the agent that specifically binds to a protein selected from CD4, CD25, IL-2, IL-10 and TGF0 is an antibody, a binding fragment thereof, an antibody-like molecule, or a binding fragment thereof. In embodiments, the determination of a weak activity of CD4-HDD25+T regulatory cells is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins selected from protein selected from CD4, CD25, IL-2, IL-10 and TGF . In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In aspects, the present disclosure relates to a method of determining treatment for a p53 mutant cancer in a patient, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method for selecting a patient having a p53 mutant cancer for a cancer treatment, the method comprising: (a) contacting a cultured biological sample from a subject with IFNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen process! ng/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In aspects, the present disclosure relates to a method of treating a p53 mutant cancer, the method comprising: (a) contacting a cultured biological sample from a subject with I FNy for less than about 8 hours; (b) evaluating the sample for the presence, absence, or level of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and (c) selecting a cancer therapy based on the evaluation of step (b).

In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, when an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from patient that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is selected. In embodiments, an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is selected.

In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a healthy tissue, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected, wherein the therapy that is selected for administration is selected from an antimetabolite chemotherapeutic (e.g, 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5- phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post- translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741 , CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, M EDI 6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN- 75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI- 3065; PI3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)), optionally wherein the therapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2. In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to another biological sample from subject that is known to be sensitive to anti-PD-1 therapy, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2 is not selected, wherein the therapy that is selected for administration is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS-663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1 R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1 R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41 A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K6 inhibitor including, but not limited to, PI-3065; PI 3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)), optionally wherein the therapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, when a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is observed compared to a prior biological sample obtained from the subject, a cancer monotherapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is not selected, wherein the therapy that is selected for administration is selected from an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and a protein translation inhibitor (e.g., silvestrol and omacetaxine), a ribosome biogenesis inhibitor (e.g., diazaborine, lamotrigine and ribozinoindoles), an inhibitor of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX-3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), a topoisomerase inhibitor (e.g., Camptothecins, indenoisoquinoline, phenanthridines, indolocarbazoles, indotecan, indimitecan, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, etoposide and teniposide), a checkpoint inhibitor (e.g., CTLA-4 inhibitors including, but not limited to, ipilimumab (YERVOY); PD-1 inhibitors including, but not limited to, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Dostarlimab (JEMPERLI), and Cemiplimab (LIBTAYO); PD-L1 inhibitors including, but not limited to, and atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (IMFINZI); and LAG-3 inhibitors including, but not limited to, Relatlimab), an agonist of an immune stimulator or co-stimulator (e.g., a CD40 agonist including, but not limited to, MEDI5083, CP-870,893 (Selicrelumab), APX005M (Sotigalimab), lucatumumab, mitazalimab (ADC-1013), ChiLob7/4, CDX-1140, and SEA-CD40; a 4-1 BB agonist including, but not limited to, urelumab (BMS- 663513) and utomilumab (PF-05082566); a CD30 agonist including, but not limited to, brentuximab; a GITR agonist including, but not limited to, MEDI1873, TRX518 and MK-4166; an 0X40 agonist including, but not limited to, MEDI6383, ABBV-368, GSK3174998, MOXR0916 (vonlerolizumab), MEDI6469, MEDI0562, INCAGN01949, IBI101, and BMS-986178, 9B12; an LIGHT agonist including, but not limited to, LIGHT-VTP and SAR252067), and a CD70 agonist (e.g., SGN-75)), tumor myeloid-directed therapies (e.g., CSF1R inhibitor including, but not limited to, BLZ945, GW2580, PLX3397 and the CSF1R-Fc-CD40L protein; CCL2 inhibitor including, but not limited to, PF-04136309, RS504393, AZD2423, BMS-741672, BMS-813160, CCX140, Cenicriviroc, Plozalizumab and Carlumab); the recepteur d’origine nantais (RON) receptor tyrosine kinase inhibitor including, but not limited to, IMC-41 A10, Narnatumab, Zt/f2, Zt/g4 and PCM5B14; CD20 inhibitor including, but not limited to, Rituximab, Ofatumumab, Ublituximab, and Ocaratuzumab; BTK inhibitor including, but not limited to, Ibrutinib; SYK inhibitor including, but not limited to, TAK-659, Fostamatinib, and Entospletinib; PI3K5 inhibitor including, but not limited to, PI-3065; PI 3Ky inhibitor including, but not limited to, TG100-115 and AS605240) and cellular therapies (e.g., tumor-infiltrating lymphocytes (or TIL) therapy, Natural Killer cell (NK cell) therapy, and CAR T-cell therapy including, but not limited to, Axicabtagene ciloleucel (YESCARTA), Brexucabtagene autoleucel (TECARTUS), Ciltacabtagene autoleucel (CARVYKTI), Idecabtagene vicleucel (ABECMA), Lisocabtagene maraleucel (BREYANZI), and Tisagenlecleucel (KYRMRIAH)), optionally wherein the therapy is selected in combination with a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

In embodiments, the one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation is selected from Statl, Stat2, Tapi, Ifitm2, and Ifitm3.

In embodiments, the cultured biological sample from a subject is contacted with an interferon for less than about 7 hr, or less than about 6 hr, or less than about 5 hr, or less than about 4 hr, or less than about 3 hr, or less than about 2 hr, or less than about 1 hr. In embodiments, the cultured biological sample from a subject is contacted with an interferon for at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 30 minutes, or at least about 45 hr, or at least about 1 hr, or at least about 2 hr, or at least about 3 hr, or at least about 4 hr.

In embodiments, the biological sample is a fresh tissue sample, frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In embodiments, the biological sample is a biopsy sample. In embodiments, the biopsy sample is selected from endoscopic biopsy, bone marrow biopsy, endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), needle biopsy (e.g., fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, X-ray-assisted biopsy, computerized tomography (CT)-assisted biopsy, magnetic resonance imaging (MRI)-assisted biopsy and ultrasound-assisted biopsy), skin biopsy (e.g., shave biopsy, punch biopsy, and incisional biopsy) and surgical biopsy.

In embodiments, the biological sample comprises a body fluid selected from blood, plasma, serum, lacrimal fluid, tears, bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluids, secretions, aspirate, scraping, and/or excretions and/or cells therefrom. In embodiments, the biological sample comprises a washing or lavage selected from a ductal lavage or bronchoalveolar lavage, and/or cells therefrom.

In embodiments, the biological sample is obtained by a technique selected from scrapes, swabs, and biopsy. In embodiments, the biological sample is obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or surgical instrumentation.

In embodiments, the biological sample comprises at least one tumor cell. In embodiments, the tumor is selected from Hodgkin's and non-Hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular nonHodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small noncleaved cell NHL; bulky 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 myeloblastic leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), Meigs’ syndrome cancer; renal carcinoma; colorectal cancer; and adrenal cancer.

In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescent staining, ELISA, and fluorescent activating cell sorting (FACS) or a combination thereof.

In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins induced by the Jak/Stat pathway. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins involved in interferon responsiveness. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins selected from Statl, Stat2, Tapi, Ifitm2, and Ifitm3.

In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of nucleic acids that encodes one or more proteins involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acid that encode one or more proteins induced by the Jak/Stat pathway. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acid that encode one or more proteins involved in interferon responsiveness. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acid encoding one or more proteins selected from Statl , Stat2, Tapi , Ifitm2, and Ifitm3. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or probe.

In embodiments, the evaluating informs classifying the patient into a high or low risk group. In embodiments, the high-risk classification comprises a high level of tumor cells having resistance to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. In embodiments, low risk classification comprises a low level of tumor cells having resistance to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. In embodiments, the low risk or high- risk classification is indicative of withholding of a neoadjuvant therapy. In embodiments, the low risk or high- risk classification is indicative of withholding of an adjuvant therapy. In embodiments, the evaluating is predictive of a positive response to and/or benefit from the cancer treatment. In embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from the cancer treatment. In embodiments, the evaluating is predictive of a positive response to and/or benefit from neoadjuvant chemotherapy or a nonresponsiveness to and/or lack of benefit from neoadjuvant chemotherapy. In embodiments, the evaluating is predictive of a positive response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy. I n embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from neoadjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from neoadjuvant chemotherapy. In embodiments, the evaluating is predictive of a negative or neutral response to and/or benefit from adjuvant chemotherapy or a non-responsiveness to and/or lack of benefit from adjuvant chemotherapy. In embodiments, the evaluating informs administration or withholding of the cancer treatment. In embodiments, the evaluating informs administration of neoadjuvant therapy. In embodiments, the evaluating informs administration of adjuvant therapy. In embodiments, the evaluating informs withholding of neoadjuvant therapy. In embodiments, the evaluating informs withholding of adjuvant therapy.

In embodiments, the neoadjuvant therapy and/or the adjuvant therapy is selected from a chemotherapeutic agent, a cytotoxic agent, a checkpoint inhibitor, an antimetabolite chemotherapeutic (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the neoadjuvant therapy and/or the adjuvant therapy is selected from a protein translation inhibitor (e. g . , a modulator of assembly and/or function of ribosomal complex, a modulator of expression and/or function of tRNA, a modulator of synthesis and/or uptake of amino acids, a modulator of post-translational modification (e.g., decoration of the translated protein with carbohydrates), a modulator of protein degradation, and a modulator of protein transport (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through the ER/Golgi network, etc.), etc.) or topoisomerase inhibitors. In embodiments, the neoadjuvant therapy and/or the adjuvant therapy is selected from a protein translation inhibitor (e.g., silvestrol and omacetaxine) ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozinoindoles), inhibitors of rRNA and/or tRNA synthesis (e.g., quarfloxin (CX- 3543) and CX-5461), an inhibitor of synthesis of amino acids (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1 ,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), an inhibitor of uptake of amino acids (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), a modulator of post-translational modification (e.g., glycosylation inhibitor tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein transport (e.g., cyclosporin A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin).

Transgenic non-human animal Models for Testing Cancer Therapy

In aspects, the present disclosure relates to a transgenic non-human animal comprising one or more tumor cells, wherein the tumor cells exhibit: (a) an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and/or (b) a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, when contacted with IFNy for less than about 8 hours.

In embodiments, the tumor cells are resistant to a cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. In embodiments, the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2 is an antibody. In embodiments, the antibody is a human or humanized antibody. In embodiments, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), Cemiplimab (LIBTAYO), Atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (imfinzi). In embodiments, the one or more tumor cells exhibit: (a) an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and/or (b) a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, when contacted with IFNy for less than about 8 hours. In embodiments, the one or more tumor cells demonstrate an upregulation of one or more proteins involved in interferon responsiveness. In embodiments, the one or more tumor cells demonstrate an upregulation of one or more genes associated with cellular response to IFNy.

In embodiments, the transgenic non-human animal is a rodent. In embodiments, the rodent is a mouse. In embodiments, the mouse belongs to BALB/c or C57BL/6 strain.

In embodiments, the one or more cancer cells are colorectal carcinoma cells. In embodiments, the one or more cancer cells are p53 mutant and/or SMAD4 mutant and/or Kras+. In embodiments, the one or more cancer cells are derived from MC38 cells or a derivative thereof.

In aspects, the present disclosure relates to a method of making a transgenic non-human animal comprising one or more p53 mutant cancer cells that are nonresponsive, resistant, or recalcitrance to a cancer therapy, wherein the cancer therapy has an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2, the method comprising: (a) injecting one or more parental p53 mutant cancer cells that are responsive to the cancer therapy in a non-human animal; (b) administering the 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 in a different non-human animal of the same species; and (e) repeating steps (b) to (d) three to ten more times. method of making a transgenic non-human animal comprising one or more p53 mutant cancer cells that are nonresponsive, resistant, or recalcitrance to a cancer therapy, wherein the non-human animal exhibits a potent NK-cell mediated cytotoxicity, a Th1 - and/or M1-dominant immune response, and/or weak activity of CD4- ^D25+ T regulatory cells, and wherein the cancer therapy has an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2, the method comprising: (a) injecting one or more parental p53 mutant cancer cells that are responsive to the cancer therapy in a non-human animal; (b) administering the 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 in a different non-human animal of the same species; and (e) repeating steps (b) to (d) three to ten more times. In embodiments, steps (b) to (d) are repeated at least three times more. In embodiments, steps (b) to (d) are repeated at least four times more. In embodiments, steps (b) to (d) are repeated at least five times more. In embodiments, steps (b) to (d) are repeated less than eight times.

In embodiments, the cancer cells that survive the cancer therapy grow faster than the parental p53 mutant cancer cells in the presence of the cancer therapy that has an ability to reduce or inhibit 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 mouse belongs to BALB/c or C57BL/6 strain.

In embodiments, the one or more p53 mutant cancer cells are colorectal carcinoma cells. In embodiments, the one or more p53 mutant cancer cells are SMAD4 mutant and/or Kras+. In embodiments, the one or more cancer cells are derived from MC38 cells or a derivative thereof.

In embodiments, the cancer therapy that has the ability reduce or inhibit function and/or activity of PD-1 , PD- L1 and/or PD-L2 is an antibody. In embodiments, the antibody is a human or humanized antibody. In embodiments, the antibody is a human or humanized antibody. In embodiments, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011 , CURE TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), Cemiplimab (LIBTAYO), Atezolizumab (TECENTRIQ), Avelumab (BAVENCIO), and Durvalumab (imfinzi). In embodiments, the cancer therapy is capable of inhibiting the growth of tumor when administered to a transgenic non-human animal transgenic non-human animal harboring a parental cancer cell tumor compared to an untreated transgenic non-human animal harboring a parental cancer cell tumor.

In embodiments, the tumor cells that survive the cancer therapy exhibit: (a) an upregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation; and/or (b) a lack of significant upregulation and/or a downregulation of one or more genes involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, when contacted with IFNy for less than about 8 hours. In embodiments, the one or more tumor cells demonstrate an upregulation of one or more genes associated with cellular response to IFNy and/or type I IFN signaling pathway.

In aspects, the present disclosure relates to a transgenic animal made according to the method of any of the embodiments disclosed herein. The transgenic non-human animal can be any animal that is known to be useful for mimicking the human cancer. In embodiments, the transgenic non-human animal may 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 mouse belongs to BALB/c or C57BL/6 strain. Such mice can be purchased from different suppliers, e.g., from Charles River Laboratories. In embodiments, the transgenic non-human animal is a transgenic non-human animal (without limitation, e.g., a transgenic mouse). In embodiments, the transgenic non-human animal is a transgenic mouse. In embodiments, the one or more genetic change that causes spontaneous tumors and/or inducible tumors. In embodiments, the transgenic mouse has a heterozygous mutation in Trp53 gene.

In embodiments, the formation of tumor in the transgenic non-human animal (e.g., mouse) is caused by a gene knock-out of one or more genes, optionally, the gene knock-out of one or more genes is inducible. In embodiments, the knock-out of one or more genes carried out using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the knock-out of one or more genes is associated with an upregulation of one or more genes in the cells that have the knock-out of one or more genes. In embodiments, the knock-out of one or more genes is associated with a downregulation of one or more genes in the cells that have knock-out of one or more genes. In embodiments, the upregulation and the downregulation of one or more genes are independently optionally inducible. In embodiments, the upregulation and/or the downregulation is caused by placing one or more sequences (without limitation, e.g., an RNAi construct, a Cre recombinase construct and a gene knock-in construct) under the control of a promoter that controls expression of one or more genes.

In embodiments, the transgenic non-human animal (e.g., mouse) is cancer prone. In embodiments, the transgenic non-human animal (e.g., mouse) has mutations that will cause rampant chromosomal instability. Such cancer-prone non-human animals are disclosed by Artandi et al., Telomere dysfunction promotes nonreciprocal translocations and epithelial cancers in mice, Nature 2000; 406(6796): 641-645; Zhu et al., Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations, Cell 2002; 109(7): 811-821 ; Olive et al., Mutant p53 Gain of Function in Two Mouse Models of Li-Fraumeni Syndrome, Ce//2004;119(6): 847-860; Lang et al., Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome, Ce// 2004; 119(6): 861-872; and Hingorani et al., Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice, Cancer Cell 2005; 7(5): 469-483, which are hereby incorporated by reference in their entirety. Transgenic non-human animal (e.g., mouse) are disclosed in U.S. Patent Nos. 4,736,866; 5,175,383; 5,491 ,283; 5,569,824; 5,907,079; 6,610,905; 6,639,121 ; 7,728,189; 8,722,964; 9,185,890; 9,820,476, which are hereby incorporated by reference in their entirety.

In embodiments, the formation of tumor in the transgenic non-human animal (e.g., mouse) is caused by a knock-in of one or more tumor-causing-genes, optionally, the knock-in of one or more tumor-causing-genes is inducible. Such tumor-causing-genes are well-known in the art, and in embodiments include known oncogenes selected from c-Myc, HRAS ^G12V or Kras ^G12D and dominant negative p53 mutants. In embodiments, the knock-in of one or more tumor-causing-genes carried out using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the knock-in of one or more tumor-causing-genes is associated with an upregulation of one or more genes in the cells that have the knock-in of one or more tumor-causing-genes. In embodiments, the knock-in of one or more tumor-causing-genes is associated with a downregulation of one or more genes in the cells that have knock-in of one or more tumor-causing-genes. In embodiments, the upregulation and the downregulation of one or more genes are independently optionally inducible. In embodiments, the upregulation and/or the downregulation is caused by placing one or more sequences (without limitation, e.g., an RNAi construct, a Cre recombinase construct and a further gene knock-in construct) under the control of a promoter that controls expression of one or more genes.

In embodiments, the formation of tumor in the transgenic non-human animal (e.g., mouse) is caused by a chromosomal translocation, optionally, the chromosomal translocation is inducible. In embodiments, the chromosomal translocation carried out using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the chromosomal translocation is associated with an upregulation of one or more genes in the cells that have the chromosomal translocation. In embodiments, the chromosomal translocation is associated with a downregulation of one or more genes in the cells that have chromosomal translocation. In embodiments, the upregulation and the downregulation of one or more genes are independently optionally inducible. In embodiments, the upregulation and/or the downregulation is caused by placing one or more sequences (without limitation, e.g., an RNAi construct, a Cre recombinase construct and a gene knock-in construct) under the control of a promoter that controls expression of one or more genes.

In embodiments, the formation of tumor in the transgenic non-human animal (e.g., mouse) is caused by a chromosomal inversion, optionally, the chromosomal inversion is inducible. In embodiments, the chromosomal inversion carried out using cre-loxP, CRISPR/Cas9, or the like, or a combination thereof. In embodiments, the chromosomal inversion is associated with an upregulation of one or more genes in the cells that have the chromosomal inversion. In embodiments, the chromosomal inversion is associated with a downregulation of one or more genes in the cells that have chromosomal inversion. In embodiments, the upregulation and the downregulation of one or more genes are independently optionally inducible. In embodiments, the upregulation and/or the downregulation is caused by placing one or more sequences (without limitation, e.g., an RNAi construct, a Cre recombinase construct and a gene knock-in construct) under the control of a promoter that controls expression of one or more genes.

Methods of Testing Cancer Therapy and Making a Pharmaceutical Composition for Treating Cancer

In aspects, the present disclosure relates to a method for testing an anti-cancer 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 the method of any of the embodiments disclosed herein;

(b) administering the 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 aspects, the present disclosure relates to a method for making 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 the method of any of the embodiments disclosed herein; (b) administering the anti-cancer drug candidate to the transgenic non-human animal, and

(c) selecting an anti-cancer drug that is effective in slowing or inhibiting cancer growth in the transgenic non- human animal; and (d) formulating the anti-cancer drug or candidate for administration to a human patient. In embodiments, the anti-cancer drug candidate is selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

Formulations

The cancer therapy (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, 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, which are hereby incorporated by reference in their entirety.

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

Further, any cancer therapy (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can 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 can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically 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 agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

In embodiments, the compositions described herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).

Administration, Dosing, and Treatment Regimens

The present disclosure includes the described cancer therapy (and/or additional agents) in various formulations. Any cancer therapy (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for 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. Patent 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), incorporated herein by reference. Where necessary, the formulations comprising the cancer therapy (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.

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

In one embodiment, any cancer therapy (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In embodiments, the administering is effected orally or by parenteral injection. In most instances, administration results in the release of any agent described herein into the bloodstream.

Any cancer therapy (and/or additional agents) described herein can be administered orally. Such cancer therapy (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.

In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In one embodiment, for instance in the treatment of cancer, the cancer therapy (and/or additional agents) are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node. In embodiments, for instance in the treatment of cancer, the cancer therapy (and/or additional agents) are 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 sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

The dosage of any cancer therapy (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion. Any cancer therapy described herein, can be administered 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), concurrently with, or subsequent 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 after) the administration of an additional agent, to a subject in need thereof. In embodiments any cancer therapy and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours 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 can 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. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the 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 disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

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

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

In embodiments, a suitable dosage of the cancer therapy (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight ,or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1 .1 mg/kg, about 1 .2 mg/kg, about 1.3 mg/kg, about 1 .4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg body weight, inclusive of all values and ranges therebetween.

In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989). Any cancer therapy (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent 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 herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

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

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

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

As used herein, the phrase “potent NK-cell mediated cytotoxicity” in a subject means that the extent of cytotoxic activity of NK cells in the subject is more than average cytotoxic activity of NK cells in control subjects. The control subjects may be normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof. The extent of cytotoxic activity of NK cells in the subject may be measured using an appropriate assay including, but not limited to, an assay measuring the proportion of NKP46+ cells in peripheral blood, the proportion of NKP46+CD69+ cells in peripheral blood, extent of CD107a surface expression, extent of cytokine production (e.g., production of IFNy NFo, granulocyte macrophage colony-stimulating factor (GM-CSF), CCL1 , CCL2, CCL3, CCL4, CCL5, and CXCL8), extent of ability to induce lysis of target cells, and a combination thereof. The appropriate assays include, e.g., assays that use one or more agents that specifically binds to one or more proteins expressed by NK cells and/or one or more agents that specifically binds to nucleic acid encoding one or more proteins expressed by NK cells (e.g., one or more ELISA-based, a flowcytometry-based, a RT-PCR-based, a real-time cell electronic sensing (RT-CES) system-based assay). Somanchi et al., A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry, PLoS One 2015; 10(10): e0141074; Park et al., Evaluation of NK Cell Function by Flowcytometric Measurement and Impedance Based Assay Using Real-Time Cell Electronic Sensing System, Biomed Res Int. 2013; 2013: 210726.

As used herein, the term “weak activity of CD4+CD25+ T regulatory cells” in a subject means that the extent of activity of CD4- 3D25+ T regulatory cells in the subject is more than average activity of CD4-H3D25+ T regulatory cells in control subjects. The control subjects may be normal subjects, subjects suffering from cancer, subjects suffering from cancer relapse, or a combination thereof. The activity of CD4+CD25+ T regulatory cells compared to a control may be measured using an appropriate assay including, but not limited to, an assay measuring the proportion of CD4+CD25+T regulatory cells in peripheral blood, extent of cytokine production (e.g., production of IL-2, IL-10 and TGF|3), proliferation assay. The appropriate assays include, e.g., assays that use one or more agents that specifically binds to one or more proteins expressed by NK cells and/or one or more agents that specifically binds to nucleic acid encoding one or more proteins expressed by NK cells (e.g., one or more ELISA-based, a flowcytometry-based, a RT-PCR-based). Antony and Restifo CD4-HDD25+ T Regulatory Cells, Immunotherapy of Cancer, and lnterleukin-2, J Immunother. 2005; 28(2): 120-128; Thornton and Shevach, CD4+CD25+ lmmunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production, J. Exp. Med. 188: 287-296; Chattopadhyay et al., Effect of CD4- D25+ and CD4+CD25’ T Regulatory Cells on the Generation of Cytolytic T Cell Response to a Self but Human Tumor-Associated Epitope In Vitro, J Immunol 2006; 176 (2) 984-990.

EXAMPLES

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

Example 1: Generation of Anti-PD-1 -Resistant MC38 Tumors

Murine colorectal carcinoma MC38 cell were subjected to selection for surviving the anti-PD-1 treatment to generate anti-PD-1- antibody resistant MC38 cells. The method used to generation of the anti-PD-1 -resistant MC38 tumor cells is illustrated in FIG. 1A. Briefly, wild-type (WT) MC38 colorectal carcinoma cells were acquired from the National Cancer Institute (NCI), and were cultured in IMDM media, with 10% FBS, antibiotic/antimycotic, and gentamycin (all GIBCO), in an incubator at 37 °C with 5% CO2. Cell lines in active culture were tested monthly using the VENOR GEM Mycoplasma Detection Kit (Sigma).

500,000 MC38 cells were inoculated on the hind flank of C57BL/6 mice (Jackson Laboratories), and when tumors became palpable, mice where either treated with vehicle (PBS) or anti-PD-1 (100 mg of clone RMP1- 14 on days 0, 3, and 6 via intraperitoneal injection (IP); BioXCell). Tumor growth was measured over time and after approximately 20 days following the first treatment, tumors from anti-PD-1 -treated mice were isolated (indicating round 1), dissociated using collagenase (StemCell Technologies), washed in 1 x PBS, and plated in culture media (1 ^st round MC38/AR cells), these cells were passaged a minimum of 2 times and were then used to inoculate new C57BL/6 mice. Again, another course of vehicle or anti-PD-1 was given to the animals, tumor measurements were taken over time, and tumors were isolated approximately 20 days after treatment, from anti-PD-1-non-responding animals (indicating round 2; 2 ^nd round MC38/AR cells). This in vivo anti-PD-1 selective pressure was performed for a total of 5 rounds until none of the mice responded to anti-PD-1 therapy. These isolated tumors are referred to as ‘5 ^th round’ and represent the tumors cells used to characterize the MC38 CPI acquired resistance model (MC38/AR) (FIG. 1A).

The efficacy of anti-PD-1 antibody in MC38 cell- or MC38/AR cell-allografts was compared. Briefly, C57BL/6 mice were inoculated in rear flanks with MC38 parental cells or MC38/AR cells obtained after 3, 4 or 5 rounds of selection. When the starting tumor volume (STV) reached 80-100 mm ³ , mice harboring treatments were initiated. Half of the mice harboring MC38 parental cell tumors were treated with vehicle only control on days 0, 3, and 6. The remaining mice harboring MC38 parental cell tumors and mice harboring tumors of MC38/AR cells after 3, 4 or 5 rounds of selection (3 ^rd , 4 ^th or 5 ^th round MC38/AR cells) were given a series of intraperitoneal injections of 100 pg anti-PD-1 (clone RMP1-14; BioXcell) on days 0, 3, and 6. Tumor volumes were measured on day 20 and plotted. As shown in FIG. 1 B, the growth of MC38 parental cell tumors in mice treated with the anti-PD-1 antibody was retarded to about 43% compared to the growth of MC38 parental cell tumors in mice that were treated with vehicle only control. This response was in line with a typically observed response to anti-PD-1 therapy. In contrast, the growth of 3 ^rd or 4 ^th round MC38/AR cells tumors showed very little tumor retardation compared to the growth of the MC38 parental cell tumors in mice that were treated with vehicle only control (FIG. 1B). Interestingly, the growth of 5 ^th round MC38/AR cell tumors showed a slight stimulation in tumor growth compared to the growth of the MC38 parental cell tumors in mice that were treated with vehicle only control (FIG. 1B).

These results demonstrate, inter alia, that the anti-PD-1 resistance of anti-PD-1 resistant cells was developed after therapy (acquired resistance) in immune competent mice. These results further demonstrate, inter alia, that anti-PD-1 therapy slightly stimulated the growth of the PD-1 resistant cells disclosed herein.

Example 2: Analysis of Expression of Genes Associated with Interferon Responsiveness, Jak/Stat Signaling, and Antigen Processing/Presentation in Response to Interferon Gamma in Resistant Cells PD-1 Resistant Cells

The expression of genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, including Statl , Stat2, Tapi, Ifitm2, and Ifitm3, was studied in the 5 ^th round MC38/AR cells compared to the parental MC38 cells.

Briefly, cells from vehicle treated mice harboring parental MC38 cells and four 5 ^th round MC38/AR tumors were cultured overnight in vitro. Cell culture supernatant was removed and RLT lysis buffer (Qiagen) prepared with 5% p-mercaptoethanol was added directly to the cells. Following lysis, lysates were homogenized with the Qiagen QIASHREDDER, and RNA was harvested using Qiagen RNeasy columns including on-column DNasel digestion. 1 g of RNA was then reverse transcribed using Origene First Strand cDNA synthesis reagents. cDNA was diluted further with nuclease-free water and qPCR was performed at a series of genes, in triplicate, and SYBR Green signal was assessed on the BioRad CFX Opus 96 and CFX Touch 96. Mouse validated gene primer sequences from Origene were used, and included mouse Statl, Stat2, Tapi , Ifitm2, Ifitm3, and the house-keeping control Rps18. Fold-change in gene expression at baseline was calculated using the ddCT method where the first WT tumor sample was set to 1 . Each additional gene was compared to this sample and the Rps18 house-keeping control. The results are shown in FIG. 2A to FIG. 2E. As shown, compared to MC38 parental cells, MC38/AR cells showed an increased expression of Statl (FIG. 2A), Stat2 (FIG. 2B), Tapi (FIG. 2C), Ifitm2 (FIG. 2D), and Ifitm3 (FIG. 2E).

These results demonstrate that Statl , Stat2, Tapi , Ifitm2, and Ifitm3 gene, which are involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation are overexpressed in the anti-PD- 1 resistant MC38/AR cells. These results also indicate, inter alia, that the overexpression of genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, including Statl , Stat2, Tapi , Ifitm2, and Ifitm3 in a p53 mutant cancer, indicates a development of resistance, a lack of response, or recalcitrance to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2. These results also indicate, inter alia, that the lack of overexpression of genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, including Statl , Stat2, Tapi , Ifitm2, and Ifitm3 in a p53 mutant cancer, indicates sensitivity to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

Example 3: Dysregulation of Genes Associated with Interferon Responsiveness, Jak/Stat Signaling, and Antigen Processing/Presentation in Response to Interferon Gamma in Resistant Cells PD-1 Resistant Cells in Response to Interferon Gamma Stimulation

Since the genes associated with interferon responsiveness were overexpressed in the anti-PD-1 resistant MC38/AR cells compared to the parental MC38 cells, the effect of interferon gamma (I FNy) on the expression of these genes was studied. Cells from vehicle treated mice harboring parental MC38 cells and four 5 ^th round MC38/AR tumors were treated +/- 20 ng/mL mouse I FNy (R&D Systems) for various durations and gene expression was analyzed (data not shown). It was found that the gene expression profiles following shorter (e.g., 8 hours or less) and extended exposure (e.g., overnight exposure or longer) were not consistent with each other (data not shown). Without being bound by theory, it is believed that this may be caused by an indirect effect of I FNy exposure (e.g, negative feedback loops or the effects of cytokines induced by I FNy). Therefore, a shorter I FNy exposure was used.

Cells from vehicle treated mice harboring parental MC38 cells and four 5 ^th round MC38/AR tumors were treated +/- 20 ng/mL mouse I FNy (R&D Systems) for 3 hours. After 3 hours, cell culture supernatant was removed and RLT lysis buffer (Qiagen) prepared with 5% P-mercaptoethanol was added directly to the cells. Following lysis, lysates were homogenized with the Qiagen QIASHREDDER, and RNA was harvested using Qiagen RNeasy columns including on-column DNasel digestion. 1 pg of RNA was then reverse transcribed using Origene First Strand cDNA synthesis reagents. cDNA was diluted further with nuclease-free water and qPCR was performed at a series of genes, in triplicate, and SYBR Green signal was assessed on the BioRad CFX Opus 96 and CFX Touch 96. Mouse validated gene primer sequences from Origene were used, and included mouse Statl , Stat2, Tapi , Ifitm2, Ifitm3, and the house-keeping control Rps18. Fold-change in IFNy responsiveness was calculated using the ddCT method where each IFNy treated tumor samples was normalized to its representative IFNy untreated samples. The results are shown in FIG. 3A to FIG. 3E. As shown, upon treatment with IFNy, compared to MC38 parental cells, MC38/AR cells showed a decreased expression of Statl (FIG. 3A), Stat2 (FIG. 3B), Tapi (FIG. 3C), and Ifitm3 (FIG. 3E). Ifitm2 showed a very modest increase in expression upon IFNy treatment in MC38/AR cells compared to MC38 parental cells (FIG. 3D).

These results suggest, inter alia, while these genes are normally induced by stimuli such as IFNy, a paradoxical dysregulation of Statl , Stat2, Tapi , Ifitm2, and Ifitm3 genes that are involved in interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation was observed. Interestingly, at baseline, these genes exhibit a transcriptional activation in absence of I FNy the anti-PD-1 resistant MC38/AR cells compared to the parental MC38 cells (See FIG 2A to FIG 2E). However, these results indicate. Inter alia, that these genes showed an impaired IFNy-responsiveness in MC38/AR cells compared to the parental MC38 cells (See FIG 3A to FIG 3E).

These results also indicate, inter alia, that impaired IFNy-responsiveness (e.g., a lack of overexpression) of genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, including Statl, Stat2, Tapi , Ifitm2, and Ifitm3 in a p53 mutant cancer, indicates a development of resistance, a lack of response, or recalcitrance to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1 , PD-L1 and/or PD-L2. These results also indicate, inter alia, that IFNy-responsiveness (e.g., overexpression) of genes associated with interferon responsiveness, Jak/Stat signaling, and antigen processing/presentation, including Statl , Stat2, Tapi , Ifitm2, and Ifitm3 in a p53 mutant cancer, indicates sensitivity to the cancer therapy with an ability to reduce or inhibit function and/or activity of PD-1, PD-L1 and/or PD-L2.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

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

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

EQUIVALENTS

While the invention 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 invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention 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, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.