WO2021247836A1 - Methods for targeting shp-2 to overcome resistance - Google Patents

Abstract

The present disclosure provides methods of treating cancer in a patient comprising administering a combination of a SHP-2 inhibitor, radiotherapy and an immune checkpoint inhibitor therapy. The radiation can increase SHP-2 expression in abscopal sites and triplecombination therapy modified the percentages of CD8+ cells, Tregs and TAMs, thereby enhancing the long-term adaptive antitumor immune response.

Description

DESCRIPTION

METHODS FOR TARGETING SHP-2 TO OVERCOME RESISTANCE

PRIORITY CLAIM

[0001] This application claims benefit of priority to U.S. Provisional Application Serial No. 63/034,021, filed June 3, 2020, the entire contents of which is hereby incorporated by reference.

BACKGROUND

1. Field

[0002] The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns methods of treating cancer with inhibition of SHP-2.

2. Description of Related Art

[0003] The study of immunology has led to breakthroughs in treating non-small cell lung cancer (NSCLC). The recent approval of an anti -PD 1 checkpoint drug for NSCLC has generated much interest in novel combination therapies that might provide further benefit for patients. Despite current advances with immunotherapy, the majority of solid tumors remain refractory to treatment due to the inhibitory nature of their stroma that mediates immune evasion. The stroma is rich with inhibitory cell populations such as regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), pro-tumor growth M2 macrophages, and cancer associated fibroblasts (CAFs). Therefore, there is a clear need to develop better treatments for NSCLC.

SUMMARY

[0004] In certain embodiments, the present disclosure provides a method of treating cancer in a subject comprising administering a Src homology phosphatase 2 (SHP-2) inhibitor, radiotherapy, and an effective amount of at least one immune checkpoint inhibitor to the subject. In some aspects, the subject has normal or low SHP-2 expression as compared to a control.

[0005] In some aspects, the radiotherapy is external-beam radiation, proton beam therapy, or brachytherapy. In particular aspects, the radiotherapy is an external-beam radiation therapy (XRT). [0006] In some aspects, the SHP-2 inhibitor is SHP-22, N0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC-87877, SPI-112, SPI-1 12Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11. In particular aspects, the SHP-2 inhibitor is SHP099.

[0007] In certain aspects, the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in the same composition. In particular aspects, the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in separate compositions, which may be administered simultaneously or the SHP-2 inhibitor may be administered before or after the immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor is administered to the subject after the radiotherapy. In other aspects, the immune checkpoint inhibitor is administered before the radiotherapy. In some aspects, the immune checkpoint inhibitor and radiotherapy are administered simultaneously. In some aspects, the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered simultaneously. In certain aspects, the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered within the same week. In some aspects, the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered within the same month. For example, the radiotherapy may be administered over the course of one week, two weeks, one month, or two months. The SHP-2 inhibitor and/or immune checkpoint inhibitor may be administered during that one week, two weeks, one month, or two months. In other aspects, the SHP-2 inhibitor and/or immune checkpoint inhibitor may be started before or after the radiotherapy, such as one day, two days, one week, two weeks, one month, two months, or three months before or after the radiotherapy. The SHP-2 inhibitor, radiotherapy, and/or immune checkpoint inhibitor may be administered daily, every two days, every three days, weekly, or monthly.

[0008] In some aspects, the method results in increased CD8⁺ T cells and decreased regulatory T cells. In certain aspects, the method results in reduced SHP-2 expression in Ml tumor associated macrophages. In specific aspects, the method results in decreased tumor- associated macrophages in tumors.

[0009] In particular aspects, the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some aspects, the at least one immune checkpoint inhibitor is anti -PD 1 therapy, anti- PDL1 therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy. In certain aspects, the at least one checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti -KIR antibody. In specific aspects, the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, EMP321, BMS-986016, or PBF-509. In some aspects, the method comprises administering more than one immune checkpoint inhibitor.

[0010] In certain aspects, the subject has been previously administered an immunotherapy, such as cell therapy or immune checkpoint inhibitor therapy. In some aspects, the subj ect was administered the immunotherapy at least one month prior to the current therapy, such as at least three months, six months, one year, or two years prior to the current therapy. In some aspects, the subject had low or no response to the immunotherapy.

[0011] In further aspects, the method further comprises an additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy comprises chemotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

[0012] In some aspects, the subject is a human. In certain aspects, the cancer is a lung cancer, a brain cancer, a breast cancer, a head and neck cancer, a cervical cancer, prostate cancer, a cancer of the eye, or a thyroid cancer.

[0013] In particular aspects, the SHP-2 inhibitor, radiotherapy, and/or immune checkpoint inhibitor are administered two or more times.

[0014] A further embodiment provides a method for sensitizing a subject to radiotherapy comprising administering a SHP-2 inhibitor to a subject determined to have normal or low SHP-2 expression. In some aspects, the subject is a human.

[0015] In some aspects, the method further comprises administering at least one immune checkpoint inhibitor. In certain aspects, the SHP-2 inhibitor is SHP-22, N0155, l-(4- (6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC- 87877, SPI-112, SPI-1 12Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter ofPTPNl 1. In some aspects, the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in the same composition. In other aspects, the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in separate compositions.

[0016] In certain aspects, the radiotherapy is external-beam radiation, proton beam therapy, or brachytherapy. In some aspects, the radiotherapy is an external-beam radiation therapy (XRT).

[0017] In particular aspects, the method results in increased CD8⁺ T cells and decreased regulatory T cells. In some aspects, the method results in reduced SHP-2 expression in Ml tumor associated macrophages. In certain aspects, the method results in decreased tumor- associated macrophages in tumors.

[0018] In some aspects, the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In specific aspects, the at least one immune checkpoint inhibitor is anti -PD 1 therapy, anti-PDLl therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy. In certain aspects, the at least one checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti -KIR antibody. In some aspects, the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, EMP321, BMS-986016, or PBF-509. In further aspects, the method comprises administering more than one immune checkpoint inhibitor.

[0019] In some aspects, the subject has been previously administered an immunotherapy, such as cell therapy or immune checkpoint inhibitor therapy. In certain aspects, the subject had low or no response to the immunotherapy.

[0020] In further aspects, the method further comprises an additional anti-cancer therapy. For example, the additional anti-cancer therapy comprises chemotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

[0021] A composition comprising an effective amount of a SHP-2 inhibitor and immune checkpoint inhibitor for use in the treatment of a disease or disorder in a subject determined to have normal or low SHP-2 expression. In some aspects, the SHP-2 inhibitor is SHP-22, N0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC-87877, SPI-112, SPI-1 12Me, Fumosorinone, demethylincisterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11. In particular aspects, the checkpoint inhibitor is selected from an inhibitor of CTLA- 4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In certain aspects, the immune checkpoint inhibitor is anti-PDl therapy, anti-PDLl therapy, anti-CTLA-4, anti- TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy. In some aspects, the at least one checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-Ll antibody, anti-PD- L2 antibody, anti-CTLA-4 antibody, and/or anti-KIR antibody. In some aspects, the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP- 224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, IMP321, BMS- 986016, or PBF-509.

[0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] FIGS. 1A-1E: Triple-combination therapy (radiation + a-PD-Ll + SHP099) inhibited tumor growth, improved survival and reduced lung metastases in a PD-1 resistant mouse model of lung cancer. (FIG. 1A) Mice (5 per group) were inoculated in the hind legs with 344SQ-R non-small cell lung cancer cells, with the right leg considered the primary tumor (and therefore irradiated) and the left leg the abscopal (unirradiated) tumor. Mice were treated with IgG (CTRL), radiation [RT] (12-Gy in 3 fractions), SHP099, a-PD-Ll, a-PD-Ll+ SHP099, RT + a-PD-Ll, and RT+ a-PD-Ll + SHP099 as shown. (FIG. IB) Tumor growth curves for irradiated sites indicated that triple therapy increased local control compared to every other group (*, P<0.05; **, P<0.01;***, P<0.001;****, P<0.0001). (FIG. 1C) Tumor growth curves for non-irradiated sites indicated that triple therapy led abscopal tumor control, while SHP099 alone was similar to SHP+XRT group (P=0.678). (FIG. ID) Proportions of mice surviving curves indicated that triple therapy extended mouse survival beyond day 60. (FIG. IE) Lung metastasis counts showed a decrease with the triple combination group. All experiments were done twice under the same schedule and with the same numbers of mice (5 per group) to confirm the results.

[0025] FIGS. 2A-2D: Radiation could decrease TAMs in irradiated tumors and increased TAMs in abscopal tumor, triple therapy can balance this effect. Tumors were harvested, processed, and analyzed by flow cytometry on day 21. (FIGS. 2A, 2B) Percentages of tumor- associated macrophages (TAMs) were analyzed at both primary (FIG. 2A) and abscopal (FIG. 2B) tumor sites. Radiation decreased the number of TAMs in the primary tumors (P=0.0424) and greatly increased TAMs at the abscopal tumors relative to control group (P=0.0292), but triple-combination therapy balance this influence for both primary (P=0.476) and abscopal (P=0.11) tumors relative to control group. (FIGS. 2C and 2D) Percentages of tumor-associated neutrophils (TANs) were not affected by any of the tested treatments at the primary tumor (FIG. 2C) and abscopal sites (FIG. 2D).

[0026] FIGS. 3A-3F: Triple therapy increased CD8⁺ T cells and decreased Tregs.

Tumors were harvested, processed, and analyzed by flow cytometry on day 21. (FIGS. 3A and 3B) In primary tumor (FIG. 3A), triple therapy significantly increased CD8⁺ relative to control group ( =0.0479) and seem to have an increase relative to radiation group (P= 0.169); in abscopal tumor (FIG. 3B), triple therapy significantly increased CD8⁺ relative to control group (P=0.0124) and abscopal tumor (P= 0.028). (FIG. 3C) In primary tumor, radiation increased the percentage of Tregs cells over the control (P=0.0009), but triple therapy decreased the percentage of Tregs relative to radiation group (P=0.0062). (FIGS. 3D, 3E and 3F) No differences were found among the various treatment groups in percentages of Tregs in abscopal tumor (FIG. 3D) and CD4⁺ lymphocytes at either the primary or abscopal tumor sites (FIGS. 3E and 3F).

[0027] FIGS. 4A-4B: SHP-2 are largely expressed in TAMs while radiation increased SHP-2 expression in abscopal tumor environment. Tumor-associated macrophages (TAMs) express the highest levels of SHP-2 and are increased by radiation. Day 21 (10 days post XRT), immune cells were isolated and phenotyped through flow cytometric analysis from either irradiated or unirradiated tumor (n = 3 per group). (FIG. 4A) SHP-2⁺ immune cell subgroup at the tumor site was highest in TAMs, followed by TANs, Tregs CD8⁺ and then CD4⁺ T cells. (FIG. 4B) Radiation increase the expression of SHP-2 in abscopal tumor, statically increased in TAMs (P=0.006) and seemingly in CD8⁺ ( =0.051), TANs (P= 0.177), Treg ( =0.2) and CD4⁺ ( =0.156).

[0028] FIGS. 5A-5C: SHP-2 is preferentially expressed in Ml TAMs and is further upregulated by radiation. (FIG. 5A) SHP-2 is more highly expressed in Ml TAMs than M2 TAMs. (FIG. 5B) Representative flow cytometry panels for SHP-2 expression after XRT in Ml TAMs. (FIG. 5C) XRT significantly increased SHP-2 expression in Ml TAMs in abscopal tumors (P=0.019) but not in primary tumors ( =0.084). Data are presented as means ± SD, with/¹ values derived from / tests. * <0.05, ** <0.01, ***/^><o.001.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] Radiotherapy traditionally has been used for local tumor control in the treatment of cancer. The recent discovery that radiotherapy can have anti-cancer effects on the immune system has led to the recognition of its ability to sensitize the tumor microenvironment to immunotherapy. However, radiation can also prompt adverse immunosuppressive effects that block aspects of systemic response at other tumor sites. SHP099 is a highly potent, selective, and orally bioavailable small-molecule SHP-2 inhibitor that stabilizes SHP-2 in an auto- inhibited conformation. It concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP-2 activity through an allosteric mechanism. The present studies showed that a SHP-2 inhibitor, such as SHP099, in combination with an immune checkpoint inhibitor enhanced immune-mediated responses to radiotherapy. The triple-combination therapy was tested in 129Sv/Ev mice with bilateral PD-1 resistant lung adenocarcinoma xenografts. Primary tumors were treated with stereotactic radiotherapy (36 Gy in three 12-Gy fractions), and abscopal tumors were monitored for a response. Triple-combination therapy significantly delayed local and abscopal tumor growth, improved survival rates, and reduced numbers of lung metastases. It was further found that radiation increased SHP-2 expression in abscopal sites and triple-combination therapy modified the percentages of CD8⁺ cells, Tregs and TAMs, thereby enhancing the long-term adaptive antitumor immune response. [0030] Specifically, the present studies found that radiotherapy could increase SHP-2 expression in TAMs, particularly in Ml TAMs. In addition, the novel triple-combination of XRT, a-PD-Ll (e.g., Durvalumab) and a-SHP-2 therapy had strong antitumor effects in the mouse model of anti-PDl -resistant NSCLC, and may be a therapeutic approach for anti-PDl- resistant NSCLC in patients.

[0031] Accordingly, in certain embodiments, the present disclosure provides methods for sensitizing patients to radiotherapy by the administration of a Src homology phosphatase 2 (SHP-2) inhibitor. The SHP-2 inhibition may be used to promote the abscopal effect and when administered in combination with an immune checkpoint inhibitor (e.g., anti-PDl, anti-PDLl or anti-CTLA-4) and radiotherapy (i.e., triple-combination therapy) cam modify the percentages of CD8⁺ cells, Tregs and TAMs, thereby enhancing the long-term adaptive antitumor immune response. Thus, methods are provided for SHP-2 inhibition in combination with an immune checkpoint inhibitor and radiotherapy to overcome resistance to immunotherapies and to increase CD8⁺ T cells and decrease Tregs in the tumor microenvironment, thus improving anti-tumor immune responses.

[0032] The present studies showed that radiation increased SHP-2 expression in tumor- associated macrophages (TAMs) and other immune cells in abscopal tumors and that the triple combination of the SHP-2 inhibitor, immunotherapy and radiotherapy promoted the abscopal effect and anti-tumor immune responses. Thus, in particular aspects, the present methods can be used to treat patients who are resistant to immunotherapy. In some embodiments, the present methods are used to treat patients with solid tumors that have been previously administered an immune checkpoint inhibitor therapy and had low or no response to the therapy.

I. Definitions

[0033] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0034] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more. [0001] The term “essentially” is to be understood that methods or compositions include only the specified steps or materials and those that do not materially affect the basic and novel characteristics of those methods and compositions.

[0002] As used herein, a composition or media that is “substantially free” of a specified substance or material contains < 30%, < 20%, < 15%, more preferably < 10%, even more preferably < 5%, or most preferably < 1% of the substance or material.

[0003] The terms “substantially” or “approximately” as used herein may be applied to modify any quantitative comparison, value, measurement, or other representation that could permissibly vary without resulting in a change in the basic function to which it is related.

[0004] The term “about” means, in general, within a standard deviation of the stated value as determined using a standard analytical technique for measuring the stated value. The terms can also be used by referring to plus or minus 5% of the stated value.

[0005] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0006] “Treatment” or “ treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease ( e.g ., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0007] “Prophylactically treating” includes: (1) reducing or mitigating the risk of developing the disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0008] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non limiting examples of human patients are adults, juveniles, infants and fetuses.

[0009] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “therapeutically effective amount” or “ pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.

[0010] As used herein, the term “IC₅₀” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[0011] An "anti-cancer" agent is capable of negatively affecting a cancer cell/tumor in a subject, for example, by promoting killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0012] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0013] “Pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with organic acids such as 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy- 2-ene-l -carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1 -carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, /J-chlorobenzenesul tonic acid, phenyl -substituted alkanoic acids, propionic acid, / oluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutyl acetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and A-methylglucamine. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

II. Methods of Treatment

[0014] Further provided herein are methods for treating or delaying progression of cancer in an individual comprising administering a SHP-2 inhibitor in combination with an immunotherapy, such as an immune checkpoint inhibitor. The subject may be further administered a radiotherapy.

[0015] Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer. In particular aspects, the cancer is non small cell lung cancer.

[0016] In some embodiments, a radiation therapy as disclosed herein is administered to treat a primary cancer. In some embodiments, while the radiation therapy may not be part of the main treatment for a cancer type, it may nonetheless be used to treat tumors that have spread to other parts of the body ( e.g ., metastatic tumors that have spread to the brain, spinal fluid, or testicles, or lung, etc.). The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; non-small cell lung cancer; renal cancer; renal cell carcinoma; clear cell renal cell carcinoma; lymphoma; blastoma; sarcoma; carcinoma, undifferentiated; meningioma; brain cancer; oropharyngeal cancer; nasopharyngeal cancer; biliary cancer; pheochromocytoma; pancreatic islet cell cancer; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal gland tumor; osteogenic sarcoma tumor; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; stomach cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer; colon cancer; rectal cancer; skin cancer; giant and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; papillary carcinoma; oral cancer; oropharyngeal cancer; nasopharyngeal cancer; respiratory cancer; urogenital cancer; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrointestinal cancer; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; lentigo maligna melanoma; acral lentiginous melanoma; nodular melanoma; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; an endocrine or neuroendocrine cancer or hematopoietic cancer; pinealoma, malignant; chordoma; central or peripheral nervous system tissue cancer; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; B-cell lymphoma; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; low grade/follicular non-Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; mantle cell lymphoma; Waldenstrom’s macroglobulinemia; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and/or hairy cell leukemia.

[0017] In some embodiments, the subject is a mammal, e.g., a primate, preferably a higher primate, e.g, a human (e.g., a patient having, or at risk of having, a disorder described herein). In one embodiment, the subject is in need of enhancing an immune response. In certain embodiments, the subject is, or is at risk of being, immunocompromised. For example, the subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy. Alternatively, or in combination, the subject is, or is at risk of being, immunocompromised as a result of an infection.

A. SHP-2 Inhibition

[0018] Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP- 2) (also known as tyrosine-protein phosphatase non-receptor type 11 (PTPN11), protein- tyrosine phosphatase ID (PTP-1D), or protein-tyrosine phosphatase 2C (PTP-2C)) is a non receptor phosphotyrosine phosphatase encoded by the PTPN11 gene. SHP-2 is widely expressed in most tissues and plays a regulatory role in various cell signaling events that are important for a diversity of cell functions, including mitogenic activation, metabolic control, transcription regulation, and cell migration.

[0019] In some aspects, the present methods and compositions comprise SHP-2 inhibition, such as with an allosteric SHP-2 inhibitor (e.g., SHP099), SHP-2 antibody, RNAi or SHP-2 siRNA that can decrease expression of SHP-2 or are directed to the PTPN11 transcript. The SHP-2 inhibitor is TN0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4- methylpiperidin-4-amine, NSC-1 17199, NSC-87877, SPI-112, SPI-1 12Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11. In some embodiments, the SHP-2 inhibitor is an allosteric inhibitor of SHP-2, e.g., a compound as described in US20170015680, US20170001975, or US20170204080, the entire contents of which are incorporated herein by reference. In another embodiment, the SHP-2 inhibitor is a compound described in WO 2016/203404, the entire contents of which are hereby incorporated herein by reference. The SHP-2 inhibitor may be an inhibitor as described in WO2019152454, incorporated herein by reference.

[0020] As used herein, a "disruption" of a gene refers to the elimination or reduction of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the disruption. Exemplary gene products include mRNA and protein products encoded by the gene. Disruption in some cases is transient or reversible and in other cases is permanent. Disruption in some cases is of a functional or full length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, gene activity or function, as opposed to expression, is disrupted. Gene disruption is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene silencing, knockdown, knockout, and/or gene disruption techniques, such as gene editing. Examples include antisense technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and/or complete absence of expression of a normal or "wild type" product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

[0021] For example, the disruption can be effected be sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of the gene or a portion thereof.

[0022] In some embodiments, gene disruption is achieved using antisense techniques, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozymes are used to selectively suppress or repress expression of the gene. siRNA technology is RNAi which employs a double-stranded RNA molecule having a sequence homologous with the nucleotide sequence of mRNA which is transcribed from the gene, and a sequence complementary with the nucleotide sequence. siRNA generally is homologous/complementary with one region of mRNA which is transcribed from the gene, or may be siRNA including a plurality of RNA molecules which are homologous/complementary with different regions. In some aspects, the siRNA is comprised in a polycistronic construct.

[0023] In some embodiments, the disruption is achieved using a DNA-targeting molecule, such as a DNA-binding protein or DNA-binding nucleic acid, or complex, compound, or composition, containing the same, which specifically binds to or hybridizes to the gene. In some embodiments, the DNA-targeting molecule comprises a DNA-binding domain, e.g., a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 2011/0301073.

[0024] In some aspects, these targeted chimeric nucleases or nuclease-containing complexes carry out precise genetic modifications by inducing targeted double-stranded breaks or single-stranded breaks, stimulating the cellular DNA-repair mechanisms, including error- prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), TALE nuclease (TALEN), and RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein, or a meganuclease.

[0025] The term “siRNA” (short interfering RNA) refers to short double stranded RNA complex, typically 19-28 base pairs in length. In other words, siRNA is a double-stranded nucleic acid molecule comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The complex often includes a 3 '-overhang. siRNA can be made using techniques known to one skilled in the art and a wide variety of siRNA is commercially available from suppliers such as Integrated DNA Technologies, Inc. (Coralville, Iowa).

[0026] The size of the RNAi loaded used herein may be less than 100 nucleotides in length, such as less than 75 nucleotides, particularly less than 50 nucleotides in length. For example, the RNA may have a length of about 10-100 nucleotides, such as 20-50 nucleotides, particularly 10-20, 15-25, 20-30, 25-35, 30-40, or 45-50 nucleotides.

[0027] The RNAi may be modified or non-modified. The RNAi may comprise an alteration of one or more nucleotides. Such alterations can include the addition of non nucleotide material, such as to the end(s) of the RNAi or internally (at one or more nucleotides of the RNA). In certain aspects, the RNAi molecule contains a 3'-hydroxyl group. Nucleotides in the RNAi molecules of the present disclosure can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural intemucleoside linkages. Additional modifications of siRNAs (e.g, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs. The RNAi may be conjugated or encapsulated for delivery, such as to lipids or nanoparticles.

[0028] Preferably, RNAi is capable of decreasing the expression of a protein by at least 10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and even more preferably by at least 75%, 80%, 90%, 95% or more.

[0029] The siRNA as used in the methods or compositions described herein may comprise a portion which is complementary to an mRNA sequence encoded by NCBI Reference Sequence for PTPN1. In an embodiment, the siRNA comprises a double-stranded portion (duplex). In an embodiment, the siRNA is 20-25 nucleotides in length. In an embodiment the siRNA comprises a 19-21 core RNA duplex with a one or 2 nucleotide 3' overhang on, independently, either one or both strands. In an embodiment, the overhang is UU. The siRNA can be 5' phosphorylated or not and may be modified with any of the known modifications in the art to improve efficacy and/or resistance to nuclease degradation. In a non limiting embodiment, the siRNA can be administered such that it is transfected into one or more cells. In one embodiment, a siRNA may comprise a double-stranded RNA comprising a first and second strand, wherein one strand of the RNA is 80, 85, 90, 95 or 100% complementary to a portion of an RNA transcript of a gene.

[0030] In one embodiment, a single strand component of a siRNA of the present disclosure is from 14 to 50 nucleotides in length. In another embodiment, a single strand component of a siRNA is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the present disclosure is 21 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the present disclosure is 22 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the present disclosure is 23 nucleotides in length. In one embodiment, a siRNA of the present disclosure is from 28 to 56 nucleotides in length.

B. Radiotherapy

[0031] Radiotherapy is the use of high-energy radiation from x-rays, gamma rays, neutrons, protons, and other sources to kill cancer cells and shrink tumors. Radiotherapy may also be called irradiation and radiation therapy. In some embodiments, radiotherapy is administered to a subject to modulate the tumor microenvironment.

[0032] In some embodiments, repeated administration of radiotherapy are administered to a subject to treat a cancer. X-rays, gamma rays, and charged particles are examples of types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external -beam radiation therapy (XRT)), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). In some embodiments, the radiotherapy is an XRT, and the radiotherapy may be administered to the subject at a low dosage ( e.g ., of about 0.2- 1.9 Gy per fraction for a total dose of about 2- 12Gy). The subject may receive a second anti-cancer therapy, such as an immunotherapy, in combination with the XRT (e.g., on the same day or within 1, 2, or 3 days) and SHP-2 inhibition.

[0033] Radiotherapy includes external-beam radiation therapy; internal radiation therapy (brachytherapy), and systemic radiation therapy. Types of external-beam radiation therapy include: Intensity-modulated radiation therapy (IMRT), Image-guided radiation therapy (IGRT), tomotherapy, stereotactic radiosurgery, stereotactic body radiation therapy, proton therapy, and other charged particle beams. Systemic radiation therapy uses radioactive substances, such as radioactive iodine or a radiolabeled monoclonal antibody, that travel in the blood and/or to tissues throughout the body to kill cancer cells.

[0034] Radiation therapy is a primary therapy for treating patients with various cancers such as, e.g ., inoperable localized non- small cell lung carcinoma. However, there is a high rate of local failure, and while it increases median survival, the therapy is often not curative. Standard radiation fractionation provides a daily dose on the order of 1.8-2Gy, to a final dose of 60-70Gy. By contrast, a Stereotactic Body Radiation Therapy (SBRT), also referred to as a stereotactic ablative radiotherapy (SABR), is a relatively novel technique in radiation therapy of lung carcinomas, delivering the total dose in 5 or fewer treatments of radiation (hypofractionation). Response rates in clinical trials suggest SBRT could be an important therapeutic advance. This approach may have significant relevance to the endogenous immune response, since lymphocytes are sensitive to even low radiation doses and are cleared rapidly from the radiation field. Standard fractionated radiation treatment may limit the effectiveness of the immune system by constantly removing tumor antigen-specific T cells at the target site. Thus, although standard fractionation has been shown to generate endogenous anti-tumor immune responses, SBRT hypofractionation may in some embodiments provide benefits when combined with an immunotherapy. In traditional external beam radiation therapy coupled with radiosensitizer administration, a beam of high energy X-rays, generated outside the patient by a linear accelerator, is delivered to a tumor. Most body tissue does not absorb or block X-rays, so they progress through the body, constantly releasing energy. When the cancer tumor is within the path of the X-ray, it receives some of that radiation; however, surrounding healthy tissue receives radiation as well. In order to limit the extent of collateral tissue damage, oncologists typically bombard the tumor area with the lowest level of effective radiation from many different points of entrance in an attempt to minimize damage to normal tissues. Even modem external beam radiation systems with improved real-time imaging of the patient anatomy will inevitably treat substantial normal tissue volumes when targeting the tumor.

[0035] Other energy sources, such as particle beams contain charged atomic particles. Particle beams have tremendous energy but also high mass and as such they slow down as they encounter body tissue. Particles can be controlled, for example, to release their energy at a specific point in the body. Particle beam therapy uses electrons, neutrons, heavy ions (such as protons, carbon ions and helium); and pi-mesons (also called pions).

[0036] Various radiotherapy methods that utilize g-rays, X-rays may be used. In some instances, it is anticipated that as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), or UV-irradiation may be used as a radiotherapy. In some embodiments, directed delivery of radioisotopes to tumor cells may also be used.

[0037] Radiation therapy may be stereotactic body radiotherapy, or SBRT. Stereotactic radiotherapy uses essentially the same approach as stereotactic radiosurgery to deliver radiation to the target tissue; however, stereotactic radiotherapy generally uses multiple small fractions of radiation as opposed to one large dose, but certain applications of SBRT may still be accomplished with a single fraction. Stereotactic body radiotherapy may be used to treat tumors, e.g, in the brain, lung, liver, pancreas, prostate, spine, as well as other parts of the body.

[0038] Radiotherapy may be used for curative, adjuvant, or palliative treatment. Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy ( e.g ., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X- Knife, TomoTherapy or Trilogy), Intensity -Modulated Radiation Therapy, particle therapy (e.g., proton therapy), brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy, Volumetric modulated arc therapy (VMAT), Virtual simulation, 3-dimensional conformal radiation therapy, and intensity-modulated radiation therapy, etc.

[0039] The radiotherapy may be administered in combination with a second anti-cancer therapy, such as an immunotherapy, e.g, an anti-CTLA-4 compound or antibody, an anti- 0X40 antibody, an anti-4- IBB antibody, or an anti -PD- 1 antibody. Anti-OX40 and anti -4- 1BB antibodies include agonist antibodies, and anti-PD-1 antibodies include antagonist antibodies, e.g, as described in WO2018150326. In some embodiments, the radiation therapy used is a SBRT. The cancer may be, e.g, an anti-PD-1 resistant cancer. Suitable examples of radiation therapy include, for example, external beam radiotherapy (EBRT or XRT) or teletherapy, brachytherapy or sealed source radiotherapy, or systemic radioisotope therapy or unsealed source radiotherapy. In some embodiments, an anti-PD-1 ABP (e.g, an antagonist antibody) is included in the combination. C. Immune Checkpoint Therapy

[0040] In some embodiments, the present methods comprise the combination of a radiotherapy and an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte- associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3 -di oxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

[0041] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g, International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0042] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art such as described in U.S. Patent Publication Nos. US20140294898, US2014022021, and US20110008369, all incorporated herein by reference.

[0043] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody e.g ., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g, an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO^®, is an anti-PD-1 antibody described in W02006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA^®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.

[0044] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA-4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA- 4, an inhibitory receptor for B7 molecules.

[0045] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g, a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. [0046] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: U.S. Patent No. 8,119,129; International Patent Publication Nos. WO 01/14424, WO 98/42752, and WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab); and U.S. Patent No. 6,207,156 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application Nos. W02001014424, and W02000037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0047] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g ., WO 01/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g, at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0048] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Patent Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins such as described in U.S. Patent No. 8,329,867, incorporated herein by reference.

D. Combination Therapies

[0049] In certain embodiments, the compositions and methods of the present embodiments involve radiation, SHP-2 inhibition and an immune checkpoint inhibitor in combination with at least one additional therapy. The additional therapy may be surgery (e.g, lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0050] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents ( e.g ., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0051] The triple combination therapy may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the SHP-2 inhibitor is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0052] Various combinations may be employed. For the example below SHP-2 inhibition, radiotherapy, and immune checkpoint inhibition is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A [0053] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.

1. Chemotherapy

[0054] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0055] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pan crati statin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics ( e.g ., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above_.,

2. Immunotherapy

[0056] Various immunotherapies are known that may be used in combination with a SHP-2 inhibitor as disclosed herein, including, e.g, anti -PD 1 antibodies or compounds, anti-PD-Ll antibodies or compounds, anti-CTLA-4 antibodies or compounds, 0X40 agonists, 4-1BB agonists, IDO inhibitors, Arginase inhibitors, anti-GITR antibodies or compounds, anti- LAG3 antibodies or compounds, anti-TIM3 antibodies or compounds, anti-TIGIT antibodies or compounds, and anti-MERTK antibodies or compounds, an oncolytic virus immunotherapy, intratumoral injections; immunotherapies targeting STING, NLRP3, TLR9, CPG, TLR4, TLR7/8, 0X40, 4- IBB, or MER-TK; an anti-CTLA-4, anti-PDl, anti-PDLl, or anti-CD40 immunotherapy; FLT-3-ligand immunotherapies, and/or cytokine immunotherapies including IL-2, IL-12, and IL-15. Additionally, the SHP-2 inhibition could be combined with cell therapies, such as T cells, NK cells, or dendritic cells that may be engineered to express a CAR or TCR.

[0057] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, /. e. , is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, gplOO, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pi 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MPM, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0058] Examples of immunotherapies include immune adjuvants, e.g ., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998; Christodoulides et ak, Microbiology, 144(Pt 11):3027-3037, 1998); cytokine therapy, e.g. , interferons a, b, and g, IL-1, GM-CSF, and TNF (Bukowski et ak, Clinical Cancer Res., 4(10):2337-2347, 1998; Davidson et ak, J. Immunother., 21(5):389-398, 1998; Hellstrand et ak, Acta Oncologica, 37(4):347-353, 1998); gene therapy, e.g. , TNF, IL-1, IL-2, and p53 (Qin et ak, Proc. Natl. Acad. Sci. USA, 95(24): 14411-14416, 1998; Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g. , anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hanibuchi et ak, Int. J. Cancer, 78(4):480-485, 1998; U.S. Patent 5,824,311).

[0059] In some embodiments, the immune therapy could be adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen- specific T cells or redirection of T cells through genetic engineering. Isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma. Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors.

[0060] In one embodiment, the present application provides for a combination therapy for the treatment of cancer wherein the combination therapy comprises adoptive T cell therapy. In one aspect, the adoptive T cell therapy comprises autologous and/or allogenic T cells. In another aspect, the autologous and/or allogenic T cells are targeted against tumor antigens.

III. Examples

[0061] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Characterization of Triple Combination Therapy

[0062] It was found that triple-combination therapy (radiation + a-PD-Ll (Durvalumab) + SHP099) inhibited tumor growth, improved survival and reduced lung metastases in a PD-1 resistant mouse model of lung cancer (FIG. 1). Mice (5 per group) were inoculated in the hind legs with 344SQ-R non-small cell lung cancer cells, with the right leg considered the primary tumor (and therefore irradiated) and the left leg the abscopal (unirradiated) tumor. Mice were treated with IgG (CTRL), radiation [RT] (12-Gy in 3 fractions), SHP099, a-PD-Ll, a-PD-Ll+ SHP099, RT + a-PD-Ll, and RT+ a-PD-Ll + SHP099 as shown in FIG. 1A. Tumor growth curves for irradiated sites indicated that triple therapy increased local control compared to every other group (*, P<0.05; **, P<0.01;***, P<0.001;****, P<0.0001). Tumor growth curves for non-irradiated sites indicated that triple therapy led abscopal tumor control, while SHP099 alone was similar to SHP+XRT group (P=0.678). Lung metastasis counts showed a decrease with the triple combination group. All experiments were done twice under the same schedule and with the same numbers of mice (5 per group) to confirm the results. The effect of the single agent vs the triple combination on primary tumor growth, secondary tumor growth, survival and lung metastasis all show that the triple combination works surprisingly well at decreasing tumor growth and metastasis as well as increasing survival as compared to any of the agents administered alone and also as compared to two of the three being administered alone (FIGS. 1B-1E).

[0063] It was further shown that radiation could decrease TAMs in irradiated tumors and increase TAMs in abscopal tumors, but the triple therapy balanced this effect (FIG. 2). Tumors were harvested, processed, and analyzed by flow cytometry on day 21. Percentages of tumor- associated macrophages (TAMs) were analyzed at both primary (FIG. 2A) and abscopal (FIG. 2B) tumor sites. Radiation decreased the number of TAMs in the primary tumors (P=0.0424) and greatly increased TAMs at the abscopal tumors relative to control group (P=0.0292), but triple-combination therapy balance this influence for both primary (P=0.476) and abscopal (P=0.11) tumors relative to control group. Percentages of tumor-associated neutrophils (TANs) were not affected by any of the tested treatments at the primary tumor (FIG. 2C) and abscopal sites (FIG. D).

[0064] The triple therapy also increased CD8⁺ T cells and decreased Tregs (FIG. 3). Tumors were harvested, processed, and analyzed by flow cytometry on day 21. In primary tumors, triple therapy significantly increased CD8⁺ relative to control group (P=0.0479) and seem to have an increase relative to radiation group (P= 0.169); in abscopal tumors, triple therapy significantly increased CD8+ relative to control group ( =0.0124) and abscopal tumor (P=0.028). In primary tumors, radiation increased the percentage of Tregs cells over the control (P=0.0009), but triple therapy decreased the percentage of Tregs relative to radiation group (P=0.0062). No differences were found among the various treatment groups in percentages of Tregs in abscopal tumor and CD4⁺ lymphocytes at either the primary or abscopal tumor sites. [0065] SHP-2 was largely expressed in TAMs while radiation increased SHP-2 expression in abscopal tumor environment (FIG. 4). Tumor-associated macrophages (TAMs) express the highest levels of SHP-2 and are increased by radiation. Day 21 (10 days post XRT), immune cells were isolated and phenotyped through flow cytometric analysis from either irradiated or unirradiated tumor (n = 3 per group). SHP-2⁺ immune cell subgroup at the tumor site was highest in TAMs, followed by TANs, Tregs CD8⁺ and then CD4⁺ T cells. Radiation increased the expression of SHP-2 in abscopal tumor, statically increased in TAMs (P=0.006) and seemingly in CD8⁺ ( =0.051), TANs ( =0.177), Treg ( =0.2) and CD4⁺ ( =0.156).

[0066] SHP-2 was preferentially expressed in Ml TAMs and was further upregulated by radiation (FIG. 5). SHP-2 was more highly expressed in Ml TAMs than M2 TAMs (FIG. 5A). Representative flow cytometry panels for SHP-2 expression after XRT in Ml TAMs. XRT showed significantly increased SHP-2 expression in Ml TAMs in abscopal tumors (P=0.019) but not in primary tumors (P=0.084).

* * * [0067] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998.

Bukowski et ah, Clinical Cancer Res., 4(10):2337-2347, 1998.

Christodoulides et ah, Microbiology, 144(Pt ll):3027-3037, 1998 Davidson et ah, J. Immunother., 21(5): 389-398, 1998.

Hanibuchi et ah, Int. J. Cancer, 78(4):480-485, 1998.

Hellstrand et ah, Acta Oncologica, 37(4):347-353, 1998.

Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject comprising administering a Src homology phosphatase 2 (SHP-2) inhibitor, radiotherapy, and an effective amount of at least one immune checkpoint inhibitor to the subject.

2. The method of claim 1, wherein the SHP-2 inhibitor is SHP-22, N0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC- 87877, SPI-112, SPI-112Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11.

3. The method of claim 1 or 2, wherein the SHP-2 inhibitor is SHP099.

4. The method of any of claims 1-3, wherein the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in the same composition.

5. The method of any of claims 1-4, wherein the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in separate compositions.

6. The method of any of claims 1-5, wherein the subject has normal or low SHP-2 expression as compared to a control.

7. The method of any of claim 1-6, wherein the radiotherapy is external -beam radiation, proton beam therapy, or brachytherapy.

8. The method of any of claims 1-6, wherein the radiotherapy is an external-beam radiation therapy (XRT).

9. The method of any of claims 1-8, wherein the immune checkpoint inhibitor is administered to the subject after the radiotherapy.

10. The method of any of claims 1-9, wherein the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered simultaneously.

11. The method of any of claims 1-9, wherein the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered within the same week.

12. The method of any of claims 1-9, wherein the SHP-2 inhibitor, immune checkpoint inhibitor, and radiotherapy are administered within the same month.

13. The method of any of claims 1-9, wherein the method results in increased CD8⁺ T cells and decreased regulatory T cells.

14. The method of any of claims 1-13, wherein the method results in reduced SHP-2 expression in Ml tumor associated macrophages.

15. The method of any of claims 1-14, wherein the method results in decreased tumor- associated macrophages in tumors.

16. The method of any of claims 1-15, wherein the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

17. The method of any of claims 1-16, wherein the at least one immune checkpoint inhibitor is anti -PD 1 therapy, anti-PDLl therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy.

18. The method of any of claims 1-17, wherein the at least one checkpoint inhibitor is an anti -PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti-KIR antibody.

19. The method of any of claims 1-17, wherein the at least one checkpoint inhibitor is an anti-PD-Ll antibody.

20. The method of any of claims 1-18, wherein the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, IMP321, BMS- 986016, or PBF-509.

21. The method of any of claims 1-178, wherein the at least one immune checkpoint inhibitor is durvalumab.

22. The method of any of claims 1-20, wherein the method comprises administering more than one immune checkpoint inhibitor.

23. The method of any of claims 1-22, wherein the subject has been previously administered an immunotherapy.

24. The method of claim 23, wherein the immunotherapy is cell therapy or immune checkpoint inhibitor therapy.

25. The method of claim 23 or 24, wherein the subject had low or no response to the immunotherapy.

26. The method of any of claims 1-25, wherein the method further comprises an additional anti-cancer therapy.

27. The method of claim 26, wherein the additional anti-cancer therapy comprises chemotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

28. The method of any of claims 1-27, wherein the subject is a human.

29. The method of any of claims 1-28, wherein the cancer is a lung cancer, a brain cancer, a breast cancer, a head and neck cancer, a cervical cancer, prostate cancer, a cancer of the eye, or a thyroid cancer.

30. The method of any of claims 1-29, wherein the SHP-2 inhibitor, radiotherapy, and/or immune checkpoint inhibitor are administered two or more times.

31. A method for sensitizing a subject to radiotherapy comprising administering a SHP-2 inhibitor to a subject determined to have normal or low SHP-2 expression.

32. The method of claim 31, further comprising administering at least one immune checkpoint inhibitor.

33. The method of claim 31 or 32, wherein the SHP-2 inhibitor is SHP-22, N0155, l-(4- (6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC-87877, SPI-112, SPI-112Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11.

34. The method of any of claims 31-33, wherein the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in the same composition.

35. The method of any of claims 31-34, wherein the SHP-2 inhibitor and at least one immune checkpoint inhibitor are administered in separate compositions.

36. The method of any of claims 31-35, wherein the radiotherapy is an external-beam radiation therapy (XRT).

37. The method of any of claims 31-36, wherein the method results in increased CD8⁺ T cells and decreased regulatory T cells.

38. The method of any of claims 31-37, wherein the method results in reduced SHP-2 expression in Ml tumor associated macrophages.

39. The method of any of claims 31-38, wherein the method results in decreased tumor- associated macrophages in tumors.

40. The method of any of claims 32-40, wherein the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

41. The method of any of claims 32-40, wherein the at least one immune checkpoint inhibitor is anti -PD 1 therapy, anti-PDLl therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy.

42. The method of any of claims 32-41, wherein the at least one checkpoint inhibitor is an anti -PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti-KIR antibody.

43. The method of any of claims 32-42, wherein the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, IMP321, BMS- 986016, or PBF-509.

44. The method of any of claims 32-43, wherein the method comprises administering more than one immune checkpoint inhibitor.

45. The method of any of claims 31-44, wherein the subject has been previously administered an immunotherapy.

46. The method of claim 45, wherein the immunotherapy is cell therapy or immune checkpoint inhibitor therapy.

47. The method of claim 45 or 46, wherein the subject had low or no response to the immunotherapy.

48. The method of any of claims 31-47, wherein the method further comprises an additional anti-cancer therapy.

49. The method of claim 48, wherein the additional anti-cancer therapy comprises chemotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

50. The method of any of claims 31-49, wherein the subject is a human.

51. A composition comprising an effective amount of a SHP-2 inhibitor and immune checkpoint inhibitor for use in the treatment of a disease or disorder in a subject determined to have normal or low SHP-2 expression as compared to a control.

52. The composition of claim 51, wherein the SHP-2 inhibitor is SHP-22, N0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC- 87877, SPI-112, SPI-112Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11.

53. The composition of claim 51 or 52, wherein the checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

54. The composition of any of claims 51-53, wherein the immune checkpoint inhibitor is anti -PD 1 therapy, anti-PDLl therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TEVB therapy, or anti -GITR therapy.

55. The composition of any of claims 51-54, wherein the at least one checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti-KIR antibody.

56. The composition of any of claims 51-55, wherein the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, IMP321, BMS- 986016, or PBF-509.

57. Use of a composition comprising an effective amount of a SHP-2 inhibitor and immune checkpoint inhibitor for the treatment of a disease or disorder in a subject determined to have normal or low SHP-2 expression as compared to a control.

58. The use of claim 57, wherein the SHP-2 inhibitor is SHP-22, N0155, l-(4-(6- bromonaphthalen-2-yl)thiazol-2-yl)-4-methylpiperidin-4-amine, NSC-1 17199, NSC- 87877, SPI-112, SPI-1 12Me, Fumosorinone, demethylinci sterol A3, 1 la-1, Cryptotanshinone, siRNA, shRNA, CRISPR/Cas9 or other gene expression disrupter of PTPN11.

59. The use of claim 57 or 58, wherein the checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.

60. The use of any of claims 57-59, wherein the immune checkpoint inhibitor is anti -PD 1 therapy, anti-PDLl therapy, anti-CTLA-4, anti-TIGIT therapy, anti-LAG3 therapy, anti-TIM3 therapy, or anti-GITR therapy.

61. The use of any of claims 57-60, wherein the at least one checkpoint inhibitor is an anti -PD-1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and/or anti-KIR antibody.

62. The use of any of claims 57-61, wherein the at least one immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, tremelimumab, ipilimumab, lirilumab AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A AMP-224, durvalumab, atezolizumab, alemtuzumab, avelumab, rHIgM12B7, IMP321, BMS- 986016, or PBF-509.