Methods of modulating antigenicity to enhance recognition by t-cells

Abstract

To provide methods for increasing tumor antigenicity by increasing the level of expression of phosphoneoantigens in cells (e.g., restoring the expression of a phosphoneoantigen on the surface of a cancer cell); and also to provide methods for enhancing the recognition of phosphoneoantigens by immune cells.SOLUTION: Provided is a method comprising the step for contacting a cancer cell with a therapeutic agent, specifically ML095, which decreases the activity of a cancer-associated phosphatase in the cancer cell, specifically alkaline phosphatase, acid phosphatase, and protein serine / threonine phosphatase.SELECTED DRAWING: None

Description

[Background technology]

[0001] 2. Background of the Invention Cancer is caused by the accumulation of genetic and epigenetic alterations that lead to uncontrolled cell proliferation. These oncogenic lesions deregulate important intracellular molecular signaling pathways, thereby resulting in malignant cell behavior. Advances in our knowledge of the oncogenic lesions that induce cancer have led to the development of small molecules that can interfere with oncogenic signaling pathways. However, the genetic instability of cancer can also lead to highly heterogeneous clonal diversity that causes some cancer cells to contain genetic lesions that confer resistance to treatment and make responses to therapy transient.

[0002] Immunotherapy, which harnesses the endogenous immune response, has been used to treat cancer. Treatments that enhance the ability of immune cells to selectively target cancer cells have shown clinical responses against multiple tumor types.

[0003] Cytotoxic T lymphocytes (CTLs) are components of our adaptive immune system that function in a coordinated manner to control and eliminate external threats, e.g., viruses and bacteria, as well as internal threats, e.g., cellular transformation events that may result in cancer. CTLs are a major subpopulation of T cells that have killing functions and have evolved to recognize and target cells with potentially harmful properties, such as viruses or cancer. These immune cells are potent and specific. CTLs can kill cells that express low levels of antigens (e.g., 1-10 antigen molecules) on their cell surface and can discriminate between antigens among millions of potentially similar molecules.

[0004] The recognition of tumor cells by the immune system depends on the presence of unique tumor antigens that are recognized by immune cells (e.g., CTLs). A wide variety of cancer antigens have been identified and are typically classified according to whether they contain sequence changes, whether they are derived from non-mutated overexpressed antigens, whether they are developmental antigens, and / or whether they are derived from changes from post-translational modifications. Tumor antigenicity is an important limiting factor that prevents immune responses from successfully targeting cancer. Furthermore, cancers can evolve to limit their antigenicity and thereby become resistant to immunotherapy. Thus, there is a need for methods to preserve or enhance tumor antigenicity to allow the recognition of tumor cells by the immune system. Summary of the Invention

[0005] Summary of the Invention The present inventors have discovered that phosphoneoantigens are an important class of tumor antigens present on the surface of some cancer cells, and that these phosphoneoantigens allow selective recognition and targeting of cancer cells by immune cells. Phosphine-neoantigens refer to any phosphorylated tumor antigen (e.g., phosphorylated peptides or proteins on the surface of cells) that do not occur on the same type of non-cancerous cell. However, some cancers increase the expression of phosphatases to hide these phosphoneoantigens and avoid detection by immune cells. Thus, the present invention provides methods and compositions that increase tumor antigenicity by increasing the level of expression of phosphoneoantigens in cells (e.g., restoring expression of phosphoneoantigens on the surface of cancer cells). The present invention also provides methods and compositions that enhance the recognition of phosphoneoantigens by immune cells.

[0006] In a first aspect, the invention features a method of increasing expression of phosphoneoantigens in cancer cells, the method comprising contacting the cancer cells with a therapeutic agent that reduces activity of a cancer-associated phosphatase in the cancer cells. In some embodiments, the cancer cells are cells in a subject having cancer, and contacting the cancer cells with the therapeutic agent is performed by administering the therapeutic agent to the subject.

[0007] In another aspect, the invention features a method of treating cancer or reducing the likelihood of developing cancer in a subject in need thereof, the method including administering to the subject a therapeutic agent that reduces activity of a cancer-associated phosphatase in cells of the cancer.

[0008] In some embodiments, administration of a therapeutic agent increases expression of phosphoneoantigens in cells of the cancer. In some embodiments, expression of phosphoneoantigens is increased by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% compared to cancer cells of the same type that were not contacted with the therapeutic agent. In some embodiments, the level of phosphoneoantigens is restored to the level of cancer cells that do not express cancer-associated phosphatases.

[0009] In some embodiments, administration of a therapeutic agent reduces the activity of a cancer-associated phosphatase in cells of the cancer, hi some embodiments, the activity of the cancer-associated phosphatase is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% compared to cancer cells of the same type that have not been contacted with the therapeutic agent. The activity of a cancer-associated phosphatase can be measured by a decrease in enzymatic activity (e.g., the activity of a cancer-associated phosphatase is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%), a decrease in the level of a phosphorylated substrate of the phosphatase (e.g., the level of a phosphorylated substrate, e.g., a phosphoneoantigen associated with the phosphatase, is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%), or a decrease in the level of a phosphorylated substrate of the phosphatase (e.g., the level of a phosphorylated substrate, e.g., a phosphoneoantigen associated with the phosphatase, is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%). %, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000%), or the level of expression of a phosphatase (e.g., the level of expression of a phosphatase is increased by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000%).

[0010] In some embodiments, the subject has previously been determined to have increased cancer-associated phosphatase activity in cells of the cancer (e.g., at least a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% increased activity level).

[0011] In some embodiments, the therapeutic agent is an inhibitor (e.g., a small molecule inhibitor) of a cancer-associated phosphatase. In some embodiments, the phosphatase inhibitor is selected from ML095, thiophosphate, Lp-bromotetramisole, tetramisole, levamisole, 5804079, L-phenylalanine, 1-paphthyl phosphate, MLS-0315848, MLS0390945, or MLS-0315687 (CID-715454).

[0012] In some embodiments, the method further comprises contacting the cancer cells with an immune checkpoint modulating agent. In some embodiments, when the cancer cells are cells in a subject having cancer, contacting the cancer cells with the immune checkpoint modulating agent is performed by administering the immune checkpoint modulating agent to the subject. In some embodiments, increasing expression of phosphoneoantigens in the cancer cells (e.g., by inhibiting cancer-associated phosphatases) enhances the immune response to the cancer cells when the immune checkpoint modulating agent is administered (e.g., enhances the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of immune checkpoint modulator required to achieve a therapeutic effect (e.g., reducing the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% or more).

[0013] In some embodiments, the immune checkpoint modulating agent is an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-NKG2A antibody, an anti-LAG-3 antibody, an anti-cMet antibody, an anti-CTLA-4 / PD-1 bispecific antibody, an anti-TIM-3 / PD-1 bispecific antibody, an anti-PD1 / LAG3 bispecific antibody, an anti-PD-1 / cMet bispecific antibody, or an antigen-binding fragment thereof.

[0014] In some embodiments, the immune checkpoint modulator is pembrolizumab, nivolumab, atelizumab, spartalizumab, camrelizumab, pidilizumab, cemiplimab, tislelizumab, JS001, MEDI0680, BCD-100, JNJ-63723283, CBT-501, TSR-042, MGA012, PF-06801591, KN035, LZM009, XmAb20717, AGEN2034, Sym021, AK105, AK104, HLX10, CS1003, STI-1110, MGD013, MCLA-134, AM001, or EDI5752.

[0015] In some embodiments, the method further comprises contacting the cancer cells with an immunomodulatory cytokine. The immunomodulatory cytokine can be selected from interferons (e.g., IFNα, IFNβ or IFNγ), interleukins (e.g., IL-2, IL-12 or IL-15) or synthetic mimics of naturally occurring cytokines (e.g., Neo-2 / 15, which mimics IL-2 and IL-15 activity). In some embodiments, when the cancer cells are cells in a subject with cancer, contacting the cancer cells with an immunomodulatory cytokine is performed by administering the immunomodulatory cytokine to the subject. In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) enhances the immune response to the cancer cells when an immunomodulatory cytokine is administered (e.g., enhances the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of immunomodulatory cytokine required to achieve a therapeutic effect (e.g., reduces the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more).

[0016] In some embodiments, the method further comprises contacting the cancer cell with an agent that inhibits myeloid-derived suppressor cells.The agent that inhibits myeloid-derived suppressor cells (MDSCs) can be selected from any agent that inhibits myeloid-derived suppressor cells known to those skilled in the art, such as indoleamine 2,3-dioxygenase (IDO) inhibitor, anti-VEGF antibody, nitric oxide synthetase (NOS) inhibitor, CSF-1R inhibitor, arginase inhibitor, or multikinase inhibitor.In some embodiments, when the cancer cell is a cell in a subject with cancer, contacting the cancer cell with an agent that inhibits myeloid-derived suppressor cells is carried out by administering the agent that inhibits myeloid-derived suppressor cells to the subject. In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) enhances the immune response to the cancer cells when an agent that inhibits myeloid-derived suppressor cells is administered (e.g., enhances the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of an agent that inhibits myeloid-derived suppressor cells required to achieve a therapeutic effect (e.g., reducing the dose by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more).

[0017] In some embodiments, the method further comprises vaccinating the subject with a phosphoneoantigen and, optionally, a suitable adjuvant (e.g., administering to the subject a phosphoneoantigen and, optionally, a suitable adjuvant in an amount sufficient to induce an immune response to the phosphoneoantigen). In some embodiments, increasing expression of the phosphoneoantigen in the cancer cells (e.g., by inhibiting cancer-associated phosphatases) enhances the immune response to the cancer cells upon vaccination with the phosphoneoantigen (e.g., enhances the immune response by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of vaccination with phosphoneoantigens required to achieve a therapeutic effect (e.g., reducing the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% or more).

[0018] In some embodiments, the method further comprises administering to the subject T cells genetically modified to express a T cell receptor (TCR), where the TCR specifically binds to a phosphoneoantigen. In some embodiments, increasing expression of the phosphoneoantigen in the cancer cells (e.g., by inhibiting a cancer-associated phosphatase) enhances the immune response to the cancer cells when the genetically modified T cells are administered (e.g., enhances the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of engineered T cells required to achieve a therapeutic effect (e.g., reducing the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more).

[0019] In some embodiments, the method further comprises contacting the cancer cells with an anti-proliferative agent. In some embodiments, when the cancer cells are cells within a subject having cancer, contacting the cancer cells with the anti-proliferative agent is performed by administering the anti-proliferative agent to the subject.

[0020] In some embodiments, the antiproliferative agent is MK-2206, ON013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platin, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacytidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin. , docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, or vinorelbine.

[0021] In some embodiments, the cancer cells have been previously determined to have increased cancer-associated phosphatase activity (e.g., 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% or more activity compared to the same type of wild-type cell). The activity of a cancer-associated phosphatase can be measured by a decrease in enzymatic activity (e.g., the activity of a cancer-associated phosphatase is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%), a decrease in the level of a phosphorylated substrate of the phosphatase (e.g., the level of a phosphorylated substrate, e.g., a phosphoneoantigen associated with the phosphatase, is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%), or a decrease in the level of a phosphorylated substrate of the phosphatase (e.g., the level of a phosphorylated substrate, e.g., a phosphoneoantigen associated with the phosphatase, is reduced by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000%). %, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000%), or the level of expression of a phosphatase (e.g., the level of expression of a phosphatase is increased by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000%).

[0022] In another aspect, the invention features a method of identifying a subject at risk of developing cancer, the method including determining the activity of a cancer-associated phosphatase in the subject's cells, wherein an increase in cancer-associated phosphatase activity (e.g., a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase compared to the same type of wild-type cell) indicates an increased risk of developing cancer.

[0023] In some embodiments, if a subject is determined to have an elevated risk of developing cancer, the subject is administered a therapeutic agent that reduces the activity of cancer-associated phosphatases in cells of the cancer.

[0024] In some embodiments, the therapeutic agent is an inhibitor (e.g., a small molecule inhibitor) of cancer-associated phosphatase. In some embodiments, the phosphatase inhibitor is selected from ML095, thiophosphate, Lp-bromotetramisole, tetramisole, levamisole, 5804079, L-phenylalanine, 1-pafutylphosphate, MLS-0315848, MLS0390945, or MLS-0315687 (CID-715454).

[0025] In some embodiments, if a subject is determined to have an elevated risk of developing cancer, the subject is vaccinated with a phosphoneoantigen and an appropriate adjuvant.

[0026] In another aspect, the invention features a method of producing a nucleic acid encoding a TCR that binds a phosphoneoantigen. The method typically includes: (a) immunizing a non-human mammal with a phosphoneoantigen, said immunization optionally further comprising administering an adjuvant; (b) isolating from the mammal of step (a) a cell that expresses a TCR that binds to a phosphoneoantigen antigen; and (c) isolating from the cells of step (b) a nucleic acid encoding a TCR that binds to a phosphoneoantigen antigen. Includes.

[0027] In some embodiments, the method further comprises expressing a nucleic acid encoding a TCR that binds a phosphoneoantigen in a host cell, thereby producing a TCR that binds a phosphoneoantigen.

[0028] In another aspect, the invention features a pharmaceutical composition including a T cell, where the T cell is genetically modified to express a TCR, and the TCR specifically binds to a phosphoneoantigen.

[0029] In another aspect, the invention features a method of treating cancer in a subject in need thereof, the method includes administering to the subject a T cell genetically modified to express a TCR, where the TCR specifically binds to a phosphoneoantigen.

[0030] In another aspect, the invention features a method of treating cancer or reducing the likelihood of developing cancer in a subject in need thereof, the method including vaccinating the subject with a phosphoneoantigen and, optionally, a suitable adjuvant.

[0031] In some embodiments of any of the above aspects, the cancer-associated phosphatase is alkaline phosphatase. In some embodiments, the alkaline phosphatase is selected from placental alkaline phosphatase (ALPP), placental-like alkaline phosphatase (ALPPL2), intestinal alkaline phosphatase (ALPI) or tissue-nonspecific alkaline phosphatase (ALPL).

[0032] In some embodiments of any of the above aspects, the cancer-associated phosphatase is an acid phosphatase. In some embodiments, the acid phosphatase is prostatic acid phosphatase (ACPP).

[0033] In some embodiments of any of the above aspects, the cancer-associated phosphatase is a protein serine / threonine phosphatase (PSP).

[0034] In some embodiments of any of the above aspects, the increase in phosphoneoantigens is associated with increased activity of a cancer-associated phosphatase (e.g., increased activity or increased expression of a cancer-associated phosphatase).

[0035] In some embodiments of any of the above aspects, the cancer is acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer. , ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma. [Brief description of the drawings]

[0036] [Figure 1A] Figures 1A-C are a collection of images showing the expression of ALPP in healthy and cancerous tissues. Figure 1A shows protein expression in 35 healthy tissues as defined by the Human Protein Atlas (http: / / www.proteinatlas.org / ). [Figure 1B] Figures 1A-C are a collection of images showing expression of ALPP in healthy and cancerous tissues, and Figure 1B shows RNA expression in the FANTOM5 series of healthy tissue samples. [Figure 1C] Figure 1A-C are a collection of images showing the expression of ALPP in healthy and cancerous tissues, and Figure 1C shows the RNA expression of ALPP in 30 cancer types obtained from cBioPortal for Cancer Genomics (http: / / www.cbioportal.org). [Figure 2A]Figures 2A-C are a collection of images showing ALPPL2 expression in healthy and cancerous tissues. Figure 2A shows protein expression in 35 healthy tissues as defined by the Human Protein Atlas (http: / / www.proteinatlas.org / ). [Figure 2B] Figures 2A-C are a collection of images showing ALPPL2 expression in healthy and cancerous tissues, and Figure 2B shows RNA expression in the FANTOM5 series of healthy tissue samples. [Figure 2C] Figures 2A-C are a collection of images showing the expression of ALPPL2 in healthy and cancerous tissues, and Figure 2C shows the RNA expression of ALPPL2 in 30 cancer types obtained from cBioPortal for Cancer Genomics (http: / / www.cbioportal.org). [Figure 3A] Figures 3A-C are a collection of images showing the expression of ALPI in healthy and cancerous tissues. Figure 3A shows protein expression in 35 healthy tissues as defined by the Human Protein Atlas (http: / / www.proteinatlas.org / ). [Figure 3B] Figures 3A-C are a collection of images showing ALPI expression in healthy and cancerous tissues, and Figure 3B shows RNA expression in the FANTOM5 series of healthy tissue samples. [Figure 3C] Figures 3A-C are a collection of images showing the expression of ALPI in healthy and cancerous tissues, and Figure 3C shows the RNA expression of ALPI in 30 cancer types obtained from cBioPortal for Cancer Genomics (http: / / www.cbioportal.org). [Figure 4A] Figures 4A-C are a collection of images showing ACPP expression in healthy and cancerous tissues: Figure 4A shows protein expression in 35 healthy tissues as defined by the Human Protein Atlas (http: / / www.proteinatlas.org / ). [Figure 4B]Figures 4A-C are a collection of images showing ACPP expression in healthy and cancerous tissues, and Figure 4B shows RNA expression in the FANTOM5 series of healthy tissue samples. [Figure 4C] Figures 4A-C are a collection of images showing the expression of ACPP in healthy and cancerous tissues, and Figure 4C shows the RNA expression of ACPP in 30 cancer types obtained from the cBioPortal for Cancer Genomics (http: / / www.cbioportal.org), showing high prostate expression. [Diagram 5] Image collection showing lentivector constructs for ALPP, ALPPL2 and luciferase expression. The ALPP (left) and luciferase (right) vectors are tagged with GFP, and the ALPPL2 vector (center) is tagged with mCherry. [Figure 6] A collection of images showing labeling of lymphoblastoid cell lines (LCL) with ALPP, ALPPL2 or luciferase. Purified LCL-ALPP, LCL-ALPPL2 or LCL-GL cells were imaged by microscopy 3 weeks after transduction. Top panel: LCL-ALPP lines were consistently green (GFP); middle panel: LCL-ALPPL2 lines were consistently red (mCherry); bottom panel: LCL-GL lines were consistently green (GFP). [Figure 7A] Figures 7A-B show that ALPP or ALPPL2 expression inhibits specific CD8+ T cell responses to autologous LCL cells. LCL cells pre-labeled with violet dye were co-cultured (at a ratio of 1:1) with either LCL-ALPP, LCL-ALPPL2 or LCL-GL in the presence (10:1) or absence (0:1) of autologous specific CD8+ T cells. After 16 hours, cells were collected and FACS was used to determine the ratio between parental LCL and either LCL-ALPP, LCL-ALPPL2 or LCL-GL. Figure 7A is a collection of flow cytometry traces showing the ratio of LCL-ALPP, LCL-ALPPL2 or LCL-GL to parental LCL. [Figure 7B]Figures 7A-B show that ALPP or ALPPL2 expression inhibits specific CD8+ T cell responses to autologous LCL cells. LCL cells pre-labeled with violet dye were co-cultured (at a ratio of 1:1) with either LCL-ALPP, LCL-ALPPL2 or LCL-GL in the presence (10:1) or absence (0:1) of autologous specific CD8+ T cells. After 16 hours, cells were collected and FACS was used to determine the ratio between parental LCL and either LCL-ALPP, LCL-ALPPL2 or LCL-GL. Figure 7B is a graph corresponding to the flow cytometry data in Figure 7A. [Figure 8A] 8A-B are a collection of Western blots showing confirmation of ALPP or ALPPL2 expression in lentiviral modified H2009 and A431 haploidentical cell lines. ALPP and ALPPL2 expression was confirmed by Western blot in the H2009 (FIG. 8A) cell line. [Figure 8B] Figures 8A-B are a collection of Western blots showing confirmation of ALPP or ALPPL2 expression in lentiviral modified H2009 and A431 haploidentical cell lines. ALPP and ALPPL2 expression was confirmed by Western blot in A431 (Figure 8B) cell line. For A431: C1 and C3-C6 are single clones of A431ALPP KO; C7 and C9-C11 are single clones of A431ALPPL2 KO; C13 is a single clone of scrambled knockout. [Figure 9A] 9A is a collection of images showing the use of NBT-BCIP reaction assays to confirm ALPP or ALPPL2 expression in lentivirus-modified H2009 (FIG. 9A) haploidentical cell lines. Cells positive for either ALPP or ALPPL2 stain dark brown. Here, clone C6 was used for A431ALPP KO, clone C9 was used for A431ALPPL2 KO, and clone C13 was used for A431 scramble. [Figure 9B]9B is a collection of images showing the use of NBT-BCIP reaction assays to confirm ALPP or ALPPL2 expression in lentivirus-modified A431 (FIG. 9B) haploidentical cell lines. Cells positive for either ALPP or ALPPL2 stain dark brown. Here, clone C6 was used for A431ALPP KO, clone C9 was used for A431ALPPL2 KO, and clone C13 was used for A431 scramble. [Figure 10A] Figure 10A-B is a collection of graphs showing that ALPP or ALPPL2 expression inhibits killing of HLA-matched haploidentical targets by specific T cells. In 96-well plates, parental tumor cell lines pre-labeled with violet dye were seeded together with lentivirus-modified cell lines (at a ratio of 1:1) in the presence (CD8:LCL=1:1, 10:1) or absence (CD8:LCL=0:1) of autologous specific CD8 T cells. After 16 hours, cells were tested by FACS for calculation of the ratio between lentivirus-modified cell lines and parental lines. Statistical results are shown for H2009 (Figure 10A). A normalized ratio (y-axis) equal to 1 indicates no preferential killing for either tumor cell line, a normalized ratio greater than 1 indicates more killing of the parental line, and a normalized ratio less than 1 indicates less killing of the parental line. All results were normalized to the condition in which no specific T cells were present. * indicates p<0.05. [Figure 10B]Figure 10A-B is a collection of graphs showing that ALPP or ALPPL2 expression inhibits killing of HLA-matched haploidentical targets by specific T cells. In 96-well plates, parental tumor cell lines pre-labeled with violet dye were seeded together with lentivirus-modified cell lines (at a ratio of 1:1) in the presence (CD8:LCL=1:1, 10:1) or absence (CD8:LCL=0:1) of autologous specific CD8 T cells. After 16 hours, cells were tested by FACS for calculation of the ratio between lentivirus-modified cell lines and parental lines. Statistical results are shown for A431 (Figure 10B). A normalized ratio (y-axis) equal to 1 indicates no preferential killing for either tumor cell line, a normalized ratio greater than 1 indicates more killing of the parental line, and a normalized ratio less than 1 indicates less killing of the parental line. All results were normalized to the condition in which no specific T cells were present. * indicates p<0.05. [Figure 11A] Figures 11A-B show that ML095 restores specific CD8+ T cell killing of H2009ALPP OE. Figure 11A is a collection of FACS traces. [Figure 11B] Figures 11A-B show that ML095 restores specific CD8+ T cell killing of H2009ALPP OE. Figure 11B is a graph corresponding to Figure 11A. [Figure 12A] Figures 12A-B show that ML095 restores specific CD8+ T cell killing of H2009ALPP2 OE. Figure 12A is a collection of FACS traces. [Figure 12B] Figures 12A-B show that ML095 restores specific CD8+ T cell killing of H2009ALPP2 OE. Figure 12B is a graph corresponding to Figure 12A. [Figure 13A] Figures 13A-B show that ML095 increases specific CD8+ T cell killing of A431 compared to A431ALPP KO. Figure 13A is a collection of FACS traces. [Figure 13B]Figures 13A-B show that ML095 increases specific CD8+ T cell killing of A431 compared to A431ALPP KO. Figure 13B is a graph corresponding to Figure 13A. [Figure 14A] Figures 14A-B show that ML095 increases specific CD8+ T cell killing of A431 compared to A431ALPP2 KO. Figure 14A is a collection of FACS traces. [Figure 14B] Figures 14A-B show that ML095 increases specific CD8+ T cell killing of A431 compared to A431ALPP2 KO. Figure 14B is a graph corresponding to Figure 14A. [Figure 15A] Figures 15A-B show that ML095 does not affect specific CD8+ T cell killing of A431 compared to A431 scrambled KO. Figure 15A is a collection of FACS traces. [Figure 15B] Figures 15A-B show that ML095 does not affect specific CD8+ T cell killing of A431 compared to A431 scrambled KO. Figure 15B is a graph corresponding to Figure 15A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] definition In order to facilitate understanding of the present invention, certain terms are defined below. Terms defined herein have the meanings commonly understood by those skilled in the art in the field related to the present invention. Terms such as "a", "an" and "the" are not intended to represent only a single item, but encompass a superclass of specific examples that may be used for description. The terminology used herein is used to describe specific embodiments of the present invention, but their use does not limit the present invention, except as described in the claims.

[0038] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0039] As used herein, any value provided in a range includes both the upper and lower boundaries and any value included within those upper and lower boundaries.

[0040] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a phosphatase inhibitor, a phosphoneoantigen, or any therapeutic agent described herein) to a subject by any effective route. Exemplary routes of administration are described herein below.

[0041] As used herein, the term "inhibitor" refers to an agent (e.g., a small molecule or an antibody) that reduces or inhibits enzyme activity. Inhibitors may reduce enzyme activity by directly binding to the enzyme, by blocking the enzyme binding site, or by altering the enzyme's conformation. Inhibitors may reduce enzyme activity by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100% or more. Inhibitors may also completely block or inhibit enzyme activity. The activity of inhibitors may be concentration-dependent or independent.

[0042] As used herein, the term "antibody" refers to a molecule that specifically binds to or immunologically reacts with a particular antigen and contains at least a variable domain of a heavy chain, usually containing at least the variable domains of the heavy and light chains of an immunoglobulin. Antibodies and antigen-binding fragments, variants or derivatives thereof include polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, heteroconjugate antibodies (e.g., bi-, tri- and tetraspecific antibodies, diabodies, triabodies and tetrabodies), single domain antibodies (sdAbs), epitope-binding fragments such as Fab, Fab' and F(ab') 2 , Fd, Fv, single chain Fv (scFv), rIgG, single chain antibody, disulfide-linked Fv (sdFv), V L or V HThese include, but are not limited to, fragments comprising any of the domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. The antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) refers to both intact molecules and antibody fragments (e.g., Fab and F(ab')) capable of specifically binding to a target protein. 2 It is meant to include both Fab and F(ab') fragments. 2 The fragment lacks the Fc fragment of an intact antibody.

[0043] The term "antigen-binding fragment" as used herein refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full-length antibody. Antibody fragments include Fab, F(ab') 2 , scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) V L , V H , C L and C H (ii) a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, called F(ab') 2 Fragment; (iii) V H and C H (iv) a V fragment of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V domain; H and VL domain, sdAb (Ward et al., Nature 341: 544-546, 1989); (vi) V H (vii) an sdAb fragment consisting of the V domain; H or V L (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, optionally connected by a synthetic linker. In addition, the two domains of the Fv fragment, the V L and V H are encoded by separate genes, they can be synthesized by recombinant methods L and V H The regions may be connected by a linker that allows them to be produced as a single protein chain paired to form a monovalent molecule (known as a single-chain Fv (scFv)). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain instances, by chemical peptide synthesis procedures known in the art.

[0044] As used herein, the term "cell type" refers to a group of cells that share a statistically classifiable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial tissue, nervous tissue, connective tissue or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel or other structure and / or region within an organism.

[0045] As used herein, "combination therapy" or "administration in combination" means that two or more different agents or treatments are administered as part of a defined treatment plan for a particular disease or condition. The treatment plan defines the dose and administration period of each agent so that the effects of the individual agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent, and the agents may be formulated together. In other embodiments, the two or more agents are not formulated together, but are administered in a sequential manner as part of a defined plan. In some embodiments, the administration of the two or more agents or treatments in combination is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments may be partially additive, wholly additive, or more than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes, for example, a first therapeutic agent of the combination may be administered by intravenous injection and a second therapeutic agent of the combination may be administered orally.

[0046] As used herein, the terms "effective amount", "therapeutically effective amount" and "sufficient amount" of a composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector, cell or any therapeutic agent described herein refer to an amount sufficient to produce a beneficial or desired result, including an effect at the cellular level, tissue level or clinical result, when administered to a subject, including a mammal (e.g., human), and thus "effective amount" or its synonyms depend on the context in which it is applied. For example, when treating cancer, it is the amount of the composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector, cell or therapeutic agent sufficient to achieve a treatment response compared to the response obtained without administering the composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector, cell or therapeutic agent. The amount of the individual compositions described herein that corresponds to such an amount will vary depending on various factors, such as the individual agent, pharmaceutical formulation, route of administration, type of disease or disorder, characteristics of the subject (e.g., age, sex, weight) or host to be treated, etc., but may be conventionally determined by one of ordinary skill in the art. Also, as used herein, a "therapeutically effective amount" of a composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector, cell or therapeutic agent of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector, cell or therapeutic agent of the present disclosure can be easily determined by a person skilled in the art by conventional methods known in the art. Dosage regimens can be adjusted to provide optimal therapeutic responses.

[0047] As used herein, the terms "increase" and "decrease" refer to a change in the function, expression or activity of the measurement being greater or less, respectively, relative to the reference.For example, after administration of a phosphatase inhibitor in the methods described herein, the amount of the measurement marker described herein (e.g., phosphoneoantigen level and / or phosphatase activity and / or expression level) can increase or decrease by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more in the subject relative to the amount of the marker before administration.Generally, the measurement is measured at a time after administration when the administration produces the effect referred to, for example, at least 6 hours, 12 hours, 24 hours, 1 week, 1 month, 3 months or 6 months after the treatment regimen is started.

[0048] As used herein, a "pharmaceutical composition" or "pharmaceutical preparation" is a composition or preparation that has pharmacological activity or other direct effect in the mitigation, treatment or prevention of disease and / or is a finished dosage form or formulation thereof and is indicated for use in a subject (e.g., use in a human subject).

[0049] As used herein, the term "pharmacologically acceptable" refers to compounds, substances, compositions and / or dosage forms suitable for contact with the tissues of a subject, e.g., a mammal (e.g., a human), consistent with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic response and other significant complications.

[0050] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, e.g., a human). A subject treated according to the methods described herein may be one diagnosed with a particular condition or one at risk for developing such a condition. Diagnosis may be performed by any method or technique known in the art. One skilled in the art will understand that a subject treated according to the present disclosure may be one that has been subjected to standard testing, or may be one that has been identified as at risk without testing due to the presence of one or more risk factors associated with the disease or condition.

[0051] "Treatment" and "treating" as used herein refer to the medical management of a subject intended to improve, reverse, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment aimed at improving a disease, pathological condition, or disorder), causal treatment (treatment directed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to relieve symptoms), preventive treatment (treatment aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder), and supportive treatment (treatment used to supplement another treatment). Treatment also includes reduction in the magnitude of a disease or condition, whether detectable or undetectable, prevention of the spread of a disease or condition, delaying or slowing the progression of a disease or condition, reversal or alleviation of a disease or condition, and remission (whether partial or total). "Ameliorating" or "alleviating" a disease or condition means reducing the magnitude and / or undesirable clinical manifestations of the disease, disorder or condition and / or slowing or extending the time course of its progression compared to the magnitude or time course in the absence of treatment. "Treatment" also means extending life span compared to expected life span if not treated. Those in need of treatment include those already with the condition or disease, as well as those susceptible to the condition or disorder or those in which the condition or disorder is to be prevented.

[0052] As used herein, the term "therapeutic agent" refers to any agent administered to a subject or used in a cell that produces a therapeutic effect as described herein. The therapeutic agent may include any composition, small molecule, peptide, vaccine, antibody, vector construct, viral vector or cell of the present disclosure. In a preferred embodiment, the therapeutic agent is administered in an amount that produces a beneficial or desired result in the subject compared to a control. One or more therapeutic agents described herein (e.g., phosphatase inhibitors, immune checkpoint regulators, immunomodulatory cytokines, agents that inhibit myeloid-derived suppressor cells, antiproliferative agents, phosphoneoantigen vaccines or CAR-T) may be administered as monotherapy or in combination as described herein.

[0053] As used herein, the term "cancer" refers to a condition characterized by unregulated or abnormal cell growth. The terms "cancer cell," "tumor cell," and "tumor" refer to an abnormal cell, mass or population of cells resulting from excessive division, which may be malignant or benign, as well as pre-cancerous and cancerous, of all cells and tissues.

[0054] As used herein, the term "activation" refers to the response of immune cells to perceived damage. When immune cells are activated, they proliferate, secrete proinflammatory cytokines, differentiate, present antigens, become more polarized, more phagocytic and cytotoxic. Factors that stimulate the activation of immune cells include proinflammatory cytokines, pathogens, and non-self antigen presentation (e.g., pathogen-derived antigens presented by dendritic cells, macrophages, or B cells).

[0055] As used herein, the term "modulating an immune response" refers to any change in the cells of the immune system or any change in the activity of cells involved in the immune response. Such regulation or modulation includes an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur in the immune system. Cells involved in the immune response include, but are not limited to, T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, ILCs, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils. In some instances, "modulating" an immune response means that the immune response is stimulated or enhanced, and in other instances, "modulating" an immune response means that the immune system is suppressed.

[0056] As used herein, the term "T cell" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T cell receptor (TCR) on the cell surface. There are various subsets of T cells, each with a distinct function (e.g., effector T cells and memory T cells).

[0057] As used herein, the term "helper T cells" (Th cells) refers to a subset of T cells that help other white blood cells in immunological processes, including maturation of B cells to plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. The cells become activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or support active immune responses. The cells can differentiate into one of a variety of subtypes (e.g., Th1, Th2, Th3, Th17, or TH9) that secrete different cytokines that promote different types of immune responses.

[0058] As used herein, the term "regulatory T cells" (Treg) refers to a subpopulation of immunosuppressive T cells that express the biomarkers CD4, FOXP3 and CD25. Treg regulate the immune system, maintain tolerance to self-antigens, prevent autoimmune diseases, and suppress anti-tumor immune responses. Treg are believed to suppress tumor immunity, thus interfering with the body's innate ability to control the growth of cancerous cells. Treg exert their immunosuppressive effects through IL-2 / IL-2 receptor-dependent and CTLA-4-dependent mechanisms, as well as by the production of inhibitory cytokines (e.g., IL-10 and TGF-beta).

[0059] As used herein, the term "phosphoneoantigen" refers to any phosphorylated tumor antigen (e.g., a phosphorylated peptide or protein on the surface of a cell) that does not occur in non-cancerous cells of the same type. Phosphineonoantigens can result from deregulated oncogenic cell signaling processes, e.g., increased expression of cancer-associated kinases in cancer cells compared to non-cancerous cells of the same type.

[0060] As used herein, the term "cancer-associated phosphatase" refers to any phosphatase that is selectively expressed or overexpressed in cancer cells compared to the same wild-type cell. Expression of cancer-associated phosphatase can occur as a result of genetic mutations associated with cancer. Cancer-associated phosphatases can be phosphatases that are typically only expressed during development (e.g., in fetal placental cells) and reactivated by genetic mutations associated with cancer. Cancer-associated phosphatases as described herein include alkaline phosphatases (e.g., ALPP, ALPP-L2 and ALPL), acid phosphatases (e.g., ACPP) and protein serine / threonine phosphatases (PSPs), as described herein.

[0061] As used herein, the term "immune checkpoint modulator" refers to an immunotherapeutic agent used to modulate the immune system in the treatment of cancer. Immune checkpoint modulators (e.g., checkpoint inhibitors) are agents such as i) antibodies that directly block inhibitory pathways in T cells or NK cells (e.g., PD-1 targeting antibodies, such as nivolumab and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) antibodies that directly activate stimulatory pathways in T cells or NK cells (e.g., antibodies targeting OX40, GITR, or 4-1BB), iii) agents that block inhibitory pathways in immune cells. or agents such as antibodies that eliminate inhibitory immune cell populations via antibody-dependent cellular cytotoxicity (e.g., CTLA-4 targeting antibodies, e.g., ipilimumab, antibodies targeting VISTA, and antibodies targeting PD-L2, Gr1, or Ly6G); and iv) agents such as antibodies that directly block inhibitory pathways in cancer cells or enhance cytotoxicity against cancer cells via antibody-dependent cellular cytotoxicity (e.g., rituximab, antibodies targeting PD-L1, and antibodies targeting B7-H3, B7-H4, Gal-9, or MUC1). Exemplary immune checkpoint modulating agents are provided herein.

[0062] As used herein, the term "immunomodulatory cytokine" refers to a small protein involved in cell signaling. Immunomodulatory cytokines can be produced and secreted by immune cells, such as T cells, B cells, macrophages and mast cells, and can include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors. Exemplary immunomodulatory cytokines are provided herein.

[0063] As used herein, the term "agents that inhibit myeloid-derived suppressor cells" refers to any therapeutic agent that inhibits the function of myeloid-derived suppressor cells in a subject, for example, inactivates myeloid-derived suppressor cells, differentiates myeloid-derived suppressor cells into mature cells (fully differentiated myeloid-derived suppressor cells are less immunosuppressive than early myeloid cells), blocks the development of myeloid-derived suppressor cells, or reduces the number of myeloid-derived suppressor cells. Agents that inhibit myeloid-derived suppressor cells are known to those of skill in the art and may include small molecule inhibitors of myeloid-derived suppressor cells, antibodies, or therapeutic nucleic acids. Agents that inhibit myeloid-derived suppressor cells include, but are not limited to, indoleamine 2,3-dioxygenase (IDO) inhibitors, anti-VEGF antibodies, nitric oxide synthetase (NOS) inhibitors, CSF-1R inhibitors, arginase inhibitors, or multikinase inhibitors. Other exemplary agents that inhibit myeloid-derived suppressor cells are provided in Wesolowski et al., J Immunother Cancer. 1: 10 (2013), which is incorporated herein by reference in its entirety.

[0064] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

[0065] Detailed Description of the Invention The inventors herein describe methods and compositions for increasing tumor antigenicity by increasing the level of expression of phosphoneoantigens within cells (e.g., restoring expression of phosphoneoantigens on the surface of cancer cells). The present invention also provides methods and compositions for enhancing recognition of phosphoneoantigens by immune cells. Below, the inventors also describe methods and compositions for the treatment of cancer by increasing tumor antigenicity and / or enhancing recognition of phosphoneoantigens by immune cells.

[0066] Phosphononeoantigens Genetic mutations that accumulate in cancer include both driver mutations that change oncogenic signal pathways and passenger mutations that do not contribute to cancer. Although these passenger mutations do not cause oncogenesis, some of them may become recognized by the immune system as neoantigens. Neoantigens can be recognized by the immune system, and thus tumor-specific neoantigens can enable targeted cell killing of tumor cells by the immune system.

[0067] In addition to changes in protein residues, neoantigens can be generated through changes in post-translational modifications of proteins. For example, genetic mutations that accumulate in cancers can result in phosphoneoantigens (e.g., cancer-specific phosphorylation of proteins on the surface of cancer cells) that can be recognized by the immune system.

[0068] Phosphoneoantigens are a subset of phosphorylated tumor antigens that do not occur in nature and arise from deregulated, oncogenic cell signaling processes. This aberrant phosphorylation generates "altered selves" and, when the phosphoproteins are degraded, the peptide fragments retain the phosphate group, indicating that they are abnormal.

[0069] Secreted phosphatases can remove phosphate groups from peptides. The present invention is based on the discovery that many cancers evolve to "turn on" (increase their expression or activity) cancer-associated phosphatases, such as placental phosphatases, and strip phosphopeptides of their phosphate groups, thereby reducing overall tumor antigenicity.

[0070] Inhibition of cancer-associated phosphatase activity therefore prevents the destruction of phosphoneoantigens and allows the immune response to detect cancer cells. Specific inhibitors of these enzymes restore phosphorylated tumor antigens, enhance tumor recognition by T cells, and extend the activity of cancer immunotherapy agents, such as those described herein (e.g., checkpoint regulators, immunomodulatory cytokines, or agents that inhibit myeloid-derived suppressor cells).

[0071] Cancer-associated phosphatases The cancer-associated phosphatase described herein includes any phosphatase that is selectively expressed or overexpressed in cancer cells compared to the same wild-type cell. The expression of cancer-associated phosphatase can occur as a result of genetic mutation associated with cancer. The cancer-associated phosphatase can be a phosphatase that is typically only expressed during development (e.g., in fetal placental cells) and reactivated by genetic mutation associated with cancer. The expression of cancer-associated phosphatase can result in dephosphorylation of phosphoneoantigens, thereby reducing the tumor antigenicity of the cancer cell.

[0072] Alkaline phosphatase Exemplary cancer-associated phosphatases useful in the present invention include alkaline phosphatases. There are at least four distinct but related human alkaline phosphatases: intestinal (ALPI), placental (ALPP), placental-like (ALPPL2) and tissue-nonspecific (ALPL) (mainly expressed in liver, bone and kidney). Alkaline phosphatases contain zinc and magnesium ions geometrically coordinated with the substrate phosphate monoester moiety that mediates hydrolysis. Due to this mechanism, they have a very wide range of catalytic activity towards phosphate groups bound by both nucleic acids and proteins. Due to their high catalytic activity, they are widely used in biomedical research to dephosphorylate proteins and nucleic acids, and are linked to antibodies as detection agents in immunoassays, such as ELISA and immunohistochemistry.

[0073] Alkaline phosphatase, placental type (ALPP; gene name: ALPP; UniProt P05187) shows highly restricted expression, being expressed primarily in the placenta. Although the exact biological role of this enzyme remains to be elucidated, biochemically, ALPP is the most thermostable alkaline phosphatase and is relatively resistant to chemical inhibition. Expression of ALPP in healthy and cancerous tissues is provided in Figure 1A-C.

[0074] Alkaline phosphatase, placenta-like 2 (ALPP-L2; gene name: ALPPL2; UniProt P10696) shows 97.3% sequence homology with ALPP and exhibits identical catalytic and biological activity as ALPP. Expression of ALPP-L2 in healthy and cancerous tissues is provided in Figure 2A-C.

[0075] ALPP and ALPP-L2 enzymes are not known to be involved in any physiological processes outside the placenta. Therefore, targeting these enzymes may provide greater safety and tolerance compared to existing immune tolerance mechanisms, such as PD-1 / PD-L1 and regulatory T cell-mediated tolerance, which are known to be involved in cancer immune escape. Inhibition of either pathway is known to be associated with severe autoimmune-like toxicity, and therefore these processes are believed to have a significant impact on self-tolerance.

[0076] Alkaline phosphatase, intestine (ALPI; gene name: ALPI; UniProt P09923) encodes a digestive brush border enzyme. The enzyme is highly expressed in the intestine and is also upregulated in colorectal, renal and liver cancer. Expression of ALPI in healthy and cancerous tissues is provided in Figure 3A-C.

[0077] Acid phosphatase Other useful cancer-associated phosphatases include acid phosphatases. There are multiple acid phosphatase isoenzymes that vary by tissue type, including ACP1, ACP2, ACPP (i.e., ACP3), ACP5, ACP6 and ACPT.

[0078] Prostatic acid phosphatase (ACPP, also called PAP or PSAP) is expressed exclusively in prostate tissue. Prostate cancer also shows very high ACPP expression. The expression of ACPP in healthy and cancerous tissues is provided in Figure 4A-C.

[0079] Protein serine / threonine phosphatases Cancer-related phosphatases are also useful, including but not limited to protein serine / threonine phosphatases (PSPs). PSPs are phosphoprotein phosphatases that act on phosphorylated serine / threonine residues. There are several known groups of PSPs, including PPP1 (α, β, γ1, γ2), PPP2 (previously 2A), PPP3 (previously 2b, also known as calcineurin), PPP2C, PPP4, PPP5 and PPP6.

[0080] Inhibitors of cancer-associated phosphatases The inhibitors of cancer-associated phosphatases described herein typically reduce or inhibit the expression, function or signaling of phosphatases to treat cancer (e.g., by increasing the antigenicity of cancer cells). In preferred embodiments, the inhibitors of cancer-associated phosphatases are specific inhibitors of the cancer-associated phosphatases associated with the cancer to be treated.

[0081] The inhibitor of cancer-associated phosphatase can be selected from many different modes.The inhibitor of cancer-associated phosphatase can be a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule, e.g., an mRNA, or a hybrid DNA-RNA molecule), a polypeptide (e.g., an antibody molecule, e.g., an antibody or its antigen-binding fragment), a nuclease (e.g., Cas9, TALEN, or ZFN), or a small molecule (e.g., a small molecule antagonist of cancer-associated phosphatase).The inhibitor of cancer-associated phosphatase can also be a viral vector that expresses an inhibitor of cancer-associated phosphatase or a cell that is infected with a viral vector.

[0082] low molecule In a preferred embodiment, the inhibitor of cancer-associated phosphatase is a small molecule inhibitor, more preferably a small molecule inhibitor selective for cancer-associated phosphatase (e.g., ALPP, ALPP-L2, ALPI, ALPL, ACPP or PSP). Exemplary small molecule inhibitors of cancer-associated phosphatases include ML095, thiophosphate, Lp-bromotetramisole, tetramisole, levamisole, 5804079, L-phenylalanine, 1-pafutylphosphate, MLS-0315848, MLS0390945 and MLS-0315687 (CID-715454). Table 1 shows the inhibition constants (Ki or IC) of these exemplary phosphatase inhibitors. 50 ) are provided ("+" indicates that inhibitory activity was observed but the Ki or IC50 was not quantified).

[0083] [Table 1]

[0084] Further small molecule inhibitors of cancer-associated phosphatases are known to those skilled in the art. Small molecule inhibitors of cancer-associated phosphatases can also be identified through screening based on their ability to reduce or inhibit the activity or expression of cancer-associated phosphatases. Small molecules include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, synthetic polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heteroorganic and organometallic compounds) that usually have a molecular weight of less than about 5,000 grams per mole, such as organic or inorganic compounds that have a molecular weight of less than about 2,000 grams per mole, such as organic or inorganic compounds that have a molecular weight of less than about 1,000 grams per mole, such as organic or inorganic compounds that have a molecular weight of less than about 500 grams per mole, and the salts, esters and other pharmacologic acceptable forms of such compounds.

[0085] Pharmaceutical compositions comprising small molecule inhibitors of cancer-associated phosphatases can be formulated for the treatment of the cancers described herein.

[0086] antibody The inhibitor of a cancer-associated phosphatase can be an antibody or an antigen-binding fragment thereof. For example, the inhibitor of a cancer-associated phosphatase described herein is an antibody that binds to the cancer-associated phosphatase and reduces or blocks the activity and / or function of the cancer-associated phosphatase by blocking the binding between the cancer-associated phosphatase and a binding partner.

[0087] nucleic acid In some embodiments, the inhibitor of a cancer-associated phosphatase is an inhibitory RNA molecule, e.g., acting via the RNA interference (RNAi) pathway. The inhibitory RNA molecule may reduce the expression level (e.g., protein level or mRNA level) of the cancer-associated phosphatase. For example, the inhibitory RNA molecule includes short interfering RNA, short hairpin RNA, and / or microRNA that target the cancer-associated phosphatase. siRNA is a double-stranded RNA molecule typically having a length of about 19-25 base pairs. shRNA is an RNA molecule that includes a hairpin turn that reduces the expression of a target gene through RNAi. shRNA can be delivered to cells in the form of a plasmid, e.g., a viral or bacterial vector, e.g., by transfection, electroporation, or transduction. MicroRNA is a non-coding RNA molecule typically having a length of about 22 nucleotides. miRNA binds to a target site on an mRNA molecule and silences the mRNA, e.g., by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. In some embodiments, the inhibitory RNA molecule reduces the level and / or activity of a negative or positive functional regulator, while in other embodiments, the inhibitory RNA molecule reduces the level and / or activity of an inhibitor of a positive functional regulator.

[0088] Inhibitory RNA molecules can be modified to include, for example, modified nucleotides, such as 2'-fluoro, 2'-o-methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, 2'-deoxyuridine. Without being bound by theory, it is believed that certain modifications can increase nuclease resistance and / or serum stability or decrease immunogenicity.

[0089] In some embodiments, the inhibitory RNA molecule reduces the level and / or activity or function of cancer-associated phosphatase.In other embodiments, the inhibitory RNA molecule increases the degradation of cancer-associated phosphatase and / or reduces the stability of cancer-associated phosphatase.The inhibitory RNA molecule can be chemically synthesized or in vitro transcribed.

[0090] The production and use of inhibitory therapeutics based on non-coding RNA, such as ribozymes, RNAse P, siRNA and miRNA, is also known in the art, as described, for example, in Sioud, RNA Therapeutics: Function, Design, and Delivery (Method in Molecular Biology). Humana Press 2010.

[0091] Viral Vectors The cancer-associated phosphatase inhibitor can be delivered by a viral vector (e.g., a viral vector expressing the cancer-associated phosphatase inhibitor). The viral vector can be administered (e.g., injected) directly into a tumor to treat the cancer.

[0092] Viral genomes provide a rich source of vectors that can be used for efficient delivery of exogenous genes to mammalian cells.Viral genomes are particularly useful vectors in gene delivery, because the polynucleotides contained in their genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction.These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35 and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpes viruses) and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, avian C virus, mammalian C, B, D viruses, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology (Third Edition) Lippincott-Raven, Philadelphia, 1996).Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.

[0093] Combination therapy The inhibitor of cancer-associated phosphatase described herein can be administered in combination with a second therapeutic agent for the treatment of cancer.In some embodiments, the second therapeutic agent is selected based on tumor type, tumor tissue of origin, tumor stage, or mutation in genes expressed by tumor.

[0094] Immune checkpoint modulators In a preferred embodiment of the present invention, the inhibitor of cancer-associated phosphatase is administered in combination with immune checkpoint regulator.In some embodiments, the immune checkpoint inhibitor is administered in combination with the inhibitor of cancer-associated phosphatase at a lower dose than when administered in the absence of the inhibitor of cancer-associated phosphatase (e.g., at 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or less than when administered in the absence of the inhibitor of cancer-associated phosphatase).In some embodiments, the therapeutic activity of the immune checkpoint regulator is enhanced when administered in combination with the inhibitor of cancer-associated phosphatase.

[0095] Immune checkpoint modulators (also known as checkpoint inhibitors) include agents such as i) antibodies that directly block inhibitory pathways in T cells or NK cells (e.g., PD-1 targeting antibodies, e.g., nivolumab and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) antibodies that directly activate stimulatory pathways in T cells or NK cells (e.g., antibodies targeting OX40, GITR, or 4-1BB), and iii) agents that block inhibitory pathways in immune cells. There are at least four types of agents: antibodies that block or eliminate inhibitory immune cell populations by antibody-dependent cytotoxicity (e.g., CTLA-4 targeting antibodies, e.g., ipilimumab, antibodies targeting VISTA, and antibodies targeting PD-L2, Gr1, or Ly6G); and iv) agents that directly block inhibitory pathways in cancer cells or enhance cytotoxicity against cancer cells by antibody-dependent cytotoxicity (e.g., rituximab, antibodies targeting PD-L1, and antibodies targeting B7-H3, B7-H4, Gal-9, or MUC1). Such agents described herein can be designed and manufactured, for example, by conventional methods known in the art (e.g., Templeton, Gene and Cell Therapy, 2015; Green and Sambrook, Molecular Cloning, 2012).

[0096] Exemplary useful immune checkpoint inhibitors include an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-NKG2A antibody, an anti-LAG-3 antibody, an anti-cMet antibody, an anti-CTLA-4 / PD-1 bispecific antibody, an anti-TIM-3 / PD-1 bispecific antibody, an anti-PD1 / LAG3 bispecific antibody, an anti-PD-1 / cMet bispecific antibody, or an antigen-binding fragment thereof.

[0097] Further exemplary immune checkpoint inhibitors include pembrolizumab, nivolumab, atelizumab, spartalizumab, camrelizumab, pidilizumab, cemiplimab, tislelizumab, JS001, MEDI0680, BCD-100, JNJ-63723283, CBT-501, TSR-042, MGA012, PF-06801591, KN035, LZM009, XmAb20717, AGEN2034, Sym021, AK105, AK104, HLX10, CS1003, STI-1110, MGD013, MCLA-134, AM001 or EDI5752.

[0098] Immunomodulatory cytokines In a preferred embodiment, the inhibitor of cancer-associated phosphatase is administered in combination with immunomodulatory cytokine.For example, when the immunomodulatory cytokine is administered in combination with the inhibitor of cancer-associated phosphatase, it is administered at a lower dose than when it is administered in the absence of the inhibitor of cancer-associated phosphatase (for example, it is administered at a dose of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or less than when it is administered in the absence of the inhibitor of cancer-associated phosphatase).In another example, the therapeutic activity of the immunomodulatory cytokine is enhanced when it is administered in combination with the inhibitor of cancer-associated phosphatase.

[0099] Immunomodulatory cytokines are small proteins involved in cell signaling. They can be produced and secreted by immune cells, such as T cells, B cells, macrophages and mast cells, and include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors.

[0100] The immunomodulatory cytokines described herein include anti-inflammatory cytokines that are produced or secreted by immune cells to reduce inflammation. Immune cells that produce and secrete anti-inflammatory cytokines include T cells (e.g., Th cells), macrophages, B cells and mast cells. Anti-inflammatory cytokines include IL4, IL-10, IL-11, IL-13, interferon alpha (IFNα) and transforming growth factor beta (TGFβ).

[0101] Further immunomodulatory cytokines include small chemokines that can induce directed chemotaxis between adjacent cells. Exemplary classes of such chemokines include CC chemokines, CXC chemokines, C chemokines, and CXC3 chemokines. Chemokines can regulate the migration and homing of immune cells, including the migration and homing of monocytes, macrophages, T cells, mast cells, eosinophils, and neutrophils. Chemokines responsible for immune cell migration include CCL19, CCL21, CCL14, CCL20, CCL25, CCL27, CXCL12, CXCL13, CCR9, CCR10, and CXCR5. Chemokines that can direct the migration of inflammatory leukocytes to sites of inflammation or injury include CCL2, CCL3, CCL5, CXCL1, CXCL2, and CXCL8.

[0102] Preferably, such immunomodulatory cytokines are selected from interleukins or interferons, such as IL-2, IL-12, IL-15, IFNα, IFNβ, IFNγ or synthetic mimics of naturally occurring cytokines (e.g., Neo-2 / 15, which mimics IL-2 and IL-15 activity).

[0103] Agents that inhibit myeloid-derived suppressor cells (MDSCs) In a preferred embodiment, the inhibitor of cancer-associated phosphatase is administered in combination with the agent that inhibits myeloid-derived suppressor cells.For example, when the agent that inhibits myeloid-derived suppressor cells is administered in combination with the inhibitor of cancer-associated phosphatase, it is administered at a lower dose than when it is administered in the absence of the inhibitor of cancer-associated phosphatase (for example, it is administered at a dose of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or less than when it is administered in the absence of the inhibitor of cancer-associated phosphatase).In another example, the therapeutic activity of the agent that inhibits myeloid-derived suppressor cells is enhanced when it is administered in combination with the inhibitor of cancer-associated phosphatase.

[0104] Myeloid-derived suppressor cells are a heterogeneous population of immature myeloid cells that increase in the setting of cancer, inflammation and infection. In malignant settings, myeloid-derived suppressor cells are induced by growth factors secreted by tumors. Myeloid-derived suppressor cells play a key role in suppressing the host immune response through various mechanisms, such as the production of arginase 1, the release of reactive oxygen species and nitric oxide, and the secretion of immunosuppressive cytokines. This creates a permissive immune environment required for the growth of malignant cells. Myeloid-derived suppressor cells may also contribute to angiogenesis and tumor invasion.

[0105] The agent that inhibits myeloid-derived suppressor cells is known to those skilled in the art and can include small molecule inhibitors of myeloid-derived suppressor cells, antibodies or therapeutic nucleic acids.The agent that inhibits myeloid-derived suppressor cells includes indoleamine 2,3-dioxygenase (IDO) inhibitors, anti-VEGF antibodies, nitric oxide synthetase (NOS) inhibitors, CSF-1R inhibitors, arginase inhibitors, or multikinase inhibitors.Other exemplary agents that inhibit myeloid-derived suppressor cells are provided in Wesolowski et al., J Immunother Cancer. 1: 10 (2013), the entirety of which is incorporated herein by reference.

[0106] Antiproliferative Agents One or more antiproliferative agents (e.g., one or more chemotherapeutic agents) may be administered in combination with the inhibitor of cancer-associated phosphatase. Antiproliferative agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, adrenocorticosteroids, progesterones, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Also included are 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ); acetogenins (especially bullatacin and bullatacinone); camptothecins (including their synthetic analogue topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including their synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatins;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enedi enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma II and calicheamicin omega II; the dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and the enediyne antibiotic chromophores neocarzinostatin chromophore and related chromoproteins), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabimycin, caminomycin, carzinophilin, chromomycin, dactinomycin, dacarbazine ... Unorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, quelamycin, rodorubicin, streptonigrin , streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;Androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal cortices, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elfomithine; elliptic acetate tinium;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidynin;maytansinoids, e.g. maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidamol;nitraelin;pentostatin;phenamet;pirarubicin;rosoxantrone;podophyllic acid;2-ethylhydrazide;procarbazine;razoxane;rhizoxin;schizofuran;spirogermanium;tenuazonic acid;triaziquone;2,2',2''-trichlorothiazide Triethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum derivatives Ligand 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 inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine;As well as pharmaceutically acceptable salts, acids or derivatives of any of the above. Two or more anti-proliferative agents may be used in a cocktail administered in combination with a first therapeutic agent as described herein. Suitable dosing regimens for combination chemotherapy are known in the art.

[0107] Nonpharmacological treatments Another type of agent that can be administered in combination with an inhibitor of a cancer-associated phosphatase is a therapeutic agent that is a non-drug treatment. For example, the second therapeutic agent is radiation therapy, cryotherapy, hyperthermia and / or surgical removal of tumor tissue.

[0108] Phosphonoantigen vaccine Methods of vaccinating a subject (e.g., a human subject having cancer or at risk of developing cancer) with a phosphoneoantigen and, optionally, a suitable adjuvant are also useful in increasing immune recognition of cancer cells. Vaccinating a subject includes administering to the subject a phosphoneoantigen and, optionally, a suitable adjuvant in an amount sufficient to induce an immune response against the phosphoneoantigen. The phosphoneoantigen can be a phosphoneoantigen described herein, e.g., a phosphoneoantigen associated with expression or overexpression of a cancer-associated phosphatase.

[0109] The phosphoneoantigen vaccines described herein can be administered to a subject as a monotherapy or as a combination therapy (e.g., as a combination therapy with an inhibitor of a cancer-associated phosphatase, an immunomodulatory cytokine, an agent that inhibits myeloid-derived suppressor cells, an antiproliferative agent, an immune checkpoint modulator, or any other therapeutic agent described herein). In a preferred embodiment, the phosphoneoantigen vaccine is administered as a combination therapy with an inhibitor of a cancer-associated phosphatase.

[0110] Increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibiting cancer-associated phosphatases) can enhance the immune response to cancer cells upon vaccination with the phosphoneoantigen (e.g., enhancing the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibiting cancer-associated phosphatases) reduces the dose of vaccination with phosphoneoantigens required to achieve a therapeutic effect (e.g., reducing the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more).

[0111] Methods of treating cancer by administering anti-cancer vaccines (e.g., cancer vaccines that include tumor-specific antigens) are known to those of skill in the art; see, e.g., Buonaguro et al. Clin Vaccine Imunol. 18(1): 23-34 (2011); and Tagliamonte et al. Hum Vaccin Immunother. 10(11): 3332-3346 (2014). A phosphoneoantigen vaccine can include all or any portion of a phosphoneoantigen required to elicit an immune response specific to the phosphoneoantigen.

[0112] manipulated T cells Another treatment that can be used either as a monotherapy or in combination with the methods and compositions described herein is chimeric antigen receptor (CAR)-T therapy or therapy using lymphocytes, such as autologous or allogeneic T cells, modified to express a CAR that recognizes a specific cancer antigen (e.g., phosphoneoantigen). In general, CARs comprise a single-chain variable fragment (scFv) region of an antibody or binding domain specific for a tumor-associated antigen (TAA) linked to the cytoplasmic domain of a T cell signaling molecule through a hinge and transmembrane region. The most common lymphocyte activation moiety comprises a T cell costimulatory domain (e.g., CD28 and / or CD137) in series with a T cell effector function-inducing (e.g., CD3ζ) moiety. CARs have the ability to redirect T cell reactivity and become specific for selected targets in a non-MHC class-restricted manner, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC class-restricted antigen recognition endows CAR-T cells with the ability to circumvent major tumor escape mechanisms.

[0113] The invention provides for administration to a subject (e.g., a human subject having or at risk of developing cancer) of a T cell that has been genetically modified to express a T cell receptor (TCR), where the TCR specifically binds to a phosphoneoantigen.

[0114] The genetically modified T cells can be administered to the subject as a monotherapy or as a combination therapy (e.g., as a combination therapy with an inhibitor of cancer-associated phosphatase, an immunomodulatory cytokine, an agent that inhibits myeloid-derived suppressor cells, an antiproliferative agent, an immune checkpoint modulator, or any other therapeutic agent described herein).In a preferred embodiment, the genetically modified T cells are administered as a combination therapy with an inhibitor of cancer-associated phosphatase.

[0115] In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) can enhance the immune response to the cancer cells upon administration of the engineered T cells (e.g., enhance the immune response by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more). In some embodiments, increasing expression of phosphoneoantigens in cancer cells (e.g., by inhibition of cancer-associated phosphatases) reduces the dose of engineered T cells required to achieve a therapeutic effect (e.g., reduce the dose by 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500%, or 1000% or more).

[0116] Immune cell regulation The methods described herein are useful for modulating the antigenicity of cells and / or modulating immune response in a subject or cell by administering a therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) to the subject or cell in a dose (e.g., effective amount) and for a period sufficient to modulate the antigenicity of the cells and / or the immune response of the subject. These methods can be used to treat subjects in need of modulating immune response, such as subjects with cancer. One way to modulate immune response is to modulate the activity of immune cells. This modulation can be performed in vivo (e.g., in a human subject or animal model) or in vitro (e.g., in rapidly isolated or cultured cells, such as human cells from a patient, a depository institution, or cell line, or rodent cells). Cell types that may be modulated include T cells (e.g., peripheral T cells, cytotoxic T cells / CD8+ T cells, T helper cells / CD4+ T cells, memory T cells, regulatory T cells / Tregs, natural killer T cells / NKT, mucosal-associated invariant T cells and gamma delta T cells), B cells (e.g., memory B cells, plasmablasts, plasma cells, follicular B cells / B-2 cells, marginal zone B cells, B-1 cells, regulatory B cells / Bregs), dendritic cells (e.g., myeloid DC / conventional DC, plasmacytoid DC or follicular DC), granulocytes (e.g., eosinophils, mast cells, neutrophils and basophils), monocytes, macrophages (e.g., peripheral macrophages or tissue-resident macrophages or tumor-resident macrophages), myeloid-derived suppressor cells, natural killer (NK) cells, innate lymphoid cells (ILC1, ILC2, ILC3), thymocytes and megakaryocytes.

[0117] Immune cell activities that may be modulated by administering to a subject or contacting a cell with an effective amount of a therapeutic agent described herein (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) can include activation (e.g., activation of macrophages, T cells, NK cells, ILCs, B cells, dendritic cells, neutrophils, eosinophils, or basophils), phagocytosis (e.g., phagocytosis of macrophages, neutrophils, monocytes, mast cells, B cells, eosinophils, or dendritic cells), antibody-dependent cell-mediated phagocytosis (e.g., ADCP by monocytes, macrophages, neutrophils, or dendritic cells), antibody-dependent cell-mediated cytotoxicity (e.g., ADCC by NK cells, ILCs, monocytes, macrophages, neutrophils, eosinophils, dendritic cells, or T cells), polarization (polarization of macrophages or polarization of T cells toward an M1 or M2 phenotype), proliferation (e.g., activation of B cells, T cells, monocytes, macrophages, dendritic cells, NK lymph node homing (e.g., lymph node homing of T cells, B cells, dendritic cells, or macrophages); lymph node egress (e.g., lymph node egress of T cells, B cells, dendritic cells, or macrophages); recruitment (e.g., recruitment of B cells, T cells, monocytes, ILCs, macrophages, dendritic cells, NK cells, mast cells, neutrophils, eosinophils, or basophils); migration (e.g., These include migration of B cells, T cells, monocytes, macrophages, dendritic cells, NK cells, ILCs, mast cells, neutrophils, eosinophils or basophils), differentiation (e.g., differentiation of regulatory T cells), cytokine production of immune cells, antigen presentation (e.g., antigen presentation of dendritic cells, macrophages and B cells), maturation (e.g., maturation of dendritic cells), and degranulation (e.g., mast cells, NK cells, ILCs, cytotoxic T cells, neutrophils, eosinophils or basophils). Innervation of lymph nodes or lymphatic organs, development of high endothelial venules (HEV) and development of ectopic or tertiary lymphatic organs (TLO) can also be modulated using the methods described herein. Modulation can increase or decrease these activities depending on the therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) used to contact the cells or treat the subject.

[0118] In some embodiments, an effective amount of a therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) is an amount sufficient to modulate (e.g., increase or decrease) one or more (e.g., two or more, three or more, four or more) of the following immune cell activities in a subject or cell: T cell polarization; T cell activation, dendritic cell activation; neutrophil activation; eosinophil activation; basophil activation; T cell proliferation; B cell proliferation; T cell proliferation; monocyte proliferation; macrophage proliferation; dendritic cell proliferation; NK cell proliferation; ILC proliferation; mast cell proliferation; neutrophil proliferation; eosinophil proliferation; basophil proliferation; cytotoxic T cell activation; circulating monocytes; peripheral blood hematopoietic stem cells; macrophage polarization; macrophage phagocytosis; macrophage ADCP; neutrophil phagocytosis; monocyte phagocytosis; phagocytosis of mast cells; phagocytosis of B cells; phagocytosis of eosinophils; phagocytosis of dendritic cells; activation of macrophages; antigen presentation (e.g., antigen presentation of dendritic cells, macrophages and B cells); migration of antigen presenting cells (e.g., migration of dendritic cells, macrophages and B cells); lymph node immune cell homing and cell egress (e.g., lymph node homing and egress of T cells, B cells, dendritic cells or macrophages); activation of NK cells; ADCC of NK cells; degranulation of mast cells; degranulation of NK cells; activation of ILCs, ADCC of ILCs; degranulation of ILCs; degranulation of cytotoxic T cells; degranulation of neutrophils; degranulation of eosinophils; degranulation of basophils; neutrophil recruitment; eosinophil recruitment; activation of NKT cells; activation of B cells; differentiation of regulatory T cells; maturation of dendritic cells; development of HEV; development of TLOs; or innervation of lymph nodes or secondary lymphoid organs. In certain embodiments, the immune response (e.g., immune cell activity as enumerated herein) is increased or decreased by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more in the subject or cells compared to before administration. In certain embodiments, the immune response is increased or decreased by 5-20%, 5-50%, 10-50%, 20-80%, 20-70%, 50-200%, 100-500% in the subject or cells.

[0119] After administering a therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally combined with a second therapeutic agent) to treat a patient or contact a cell, the readouts can be used to assess the effect on immune cell activity. Immune cell activity can be assessed by measuring cytokines or markers associated with a particular immune cell type. In certain embodiments, a parameter is increased or decreased by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more in a subject compared to before administration. In certain embodiments, a parameter is increased or decreased by 5-20%, 5-50%, 10-50%, 20-80%, 20-70%, 50-200%, 100-500% in a subject. The therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) may be administered at a dosage (e.g., an effective amount) and for a period of time sufficient to modulate the activity of immune cells as described herein below.

[0120] In another example, after administering a therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) to treat a patient or contact a cell, the readout can be used to evaluate the effect on immune cell migration. Immune cell migration can be evaluated by measuring the number of immune cells in a location of interest (e.g., tumor metastasis site, lymph node, secondary lymphoid organ, tertiary lymphoid organ). Immune cell migration can also be evaluated by measuring chemokines, receptors, or markers associated with immune cell migration. In certain embodiments, a parameter increases or decreases in a subject by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to before administration. In certain embodiments, the parameter is increased or decreased in the subject by 5-20%, 5-50%, 10-50%, 20-80%, 20-70%, 50-200%, 100-500%. Therapeutic agents (e.g., inhibitors of cancer-associated phosphatases, optionally in combination with a second therapeutic agent) may be administered in a dose (e.g., an effective amount) and for a duration sufficient to modulate immune cell migration as described herein below.

[0121] The therapeutic agent described herein (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) can affect the migration of immune cells. The migration of immune cells between peripheral tissues, blood and lymphatic systems, and lymphoid organs is essential for the orchestration of effective innate and adaptive immune responses. The migration of immune cells is regulated in large part by transport molecules, including integrins, immunoglobulin cell adhesion molecules (IgSF CAMs), cadherins, selectins, and a family of small cytokines called chemokines. Cell adhesion molecules and chemokines regulate the migration of immune cells both by inducing their exudation from the circulation into peripheral tissues and by acting as guidance cues within the peripheral tissues themselves. To cause exudation, chemokines must act in concert with multiple trafficking molecules, including C-type lectins (L-, P- and E-selectins), multiple integrins and cell adhesion molecules (ICAM-1, VCAM-1 and MAdCAM-1), to execute a multistep cascade of immune cell capture, rolling, arrest and transmigration through the blood-endothelial barrier. Some trafficking molecules are constitutively expressed and govern immune cell migration during homeostasis, whereas others are differentially upregulated by inflammatory processes, e.g., autoimmunity and cancer.

[0122] In yet another example, the therapeutic agent described herein (e.g., an inhibitor of cancer-associated phosphatase, optionally in combination with a second therapeutic agent) increases one or more of T cell proliferation, T cell activation, T cell differentiation, or T cell cytokine production (e.g., T cell production of pro-inflammatory cytokines, e.g., IFNγ). In some embodiments, the T cell is an effector T cell, a helper T cell, a Th1 cell, a Th2 cell, or a Th17 cell. In some embodiments, the therapeutic agent described herein (e.g., an inhibitor of cancer-associated phosphatase, optionally in combination with a second therapeutic agent) increases inflammation.

[0123] cancer The methods described herein can be used to treat cancer in a subject by administering to the subject an effective amount of a therapeutic agent described herein (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent). The methods can include locally (e.g., intratumorally) administering to the subject a therapeutic agent described herein (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) in a dose (e.g., an effective amount) and for a period sufficient to treat the cancer.

[0124] The methods described herein can also be used to enhance or increase an immune response, e.g., an anti-tumor immune response, in a subject in need thereof. For example, the subject has cancer, e.g., a cancer described herein. The methods described herein can also include a step of selecting a subject in need of an enhanced immune response, e.g., selecting a subject having cancer or at risk of developing cancer.

[0125] The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) can be administered in an amount sufficient to treat cancer. The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) can treat cancer by increasing cancer cell death in a subject (e.g., a human subject or an animal model) or in a cancer cell culture (e.g., a patient tumor sample, a cancer cell line, or a culture obtained from a patient sample depository). The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) can increase cancer cell death by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to before administration to a subject or cancer cell culture. A therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) may increase cancer cell death in a subject or cancer cell culture by 5-20%, 5-50%, 10-50%, 20-80%, or 20-70%.

[0126] A therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) may also act to inhibit the growth, proliferation, metastasis, migration or invasion of cancer cells, e.g., the method may include administering to a subject (e.g., a human subject or animal model) or cancer cell culture (e.g., a patient tumor sample, a cancer cell line, or a culture obtained from a patient sample depository) a therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) in an amount (e.g., an effective amount) and for a period of time sufficient to inhibit the growth, proliferation, metastasis, migration or invasion of cancer cells. Cancer cell growth, proliferation, metastasis, migration or invasion may be reduced by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in a subject or cancer cell culture compared to before administration of a therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent). Cancer cell growth, proliferation, metastasis, migration or invasion may be reduced by 5-20%, 5-50%, 10-50%, 20-80% or 20-70% in a subject or cancer cell culture.

[0127] Cancer Type In the methods described herein, the cancer or neoplasm may be any solid or liquid cancer, including gastrointestinal cancer (e.g., non-metastatic or metastatic colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, oral cancer, lip cancer), genitourinary cancer (e.g., hormone-sensitive or hormone-refractory prostate cancer, renal cell carcinoma, bladder cancer, penile cancer); gynecological cancer (e.g., ovarian cancer, cervical cancer, endometrial cancer); lung cancer (e.g., small cell lung cancer and non-small cell lung cancer); head and neck cancer (e.g., head and neck squamous cell carcinoma); Benign or malignant tumors and hyperplasias, including CNS cancers, including malignant glioma, astrocytoma, retinoblastoma and brain metastases; malignant mesothelioma; non-metastatic or metastatic breast cancer (hormone refractory metastatic breast cancer); skin cancer (e.g., malignant melanoma, basal and squamous cell skin cancer, Merkel cell carcinoma, cutaneous lymphoma, Kaposi's sarcoma); thyroid cancer; bone and soft tissue sarcoma; and hematological neoplasms (e.g., multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, Hodgkin's lymphoma).

[0128] Additional cancers that may be treated according to the methods described herein include breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, kidney cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, head and neck cancer, esophageal cancer, bronchial cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, laryngeal cancer, bile duct cancer, skin cancer, cancer of the central nervous system, cancer of the respiratory system, and cancer of the urinary tract system. Examples of breast cancer include, but are not limited to, triple negative breast cancer, triple positive breast cancer, HER2 negative breast cancer, HER2 positive breast cancer, estrogen receptor positive breast cancer, estrogen receptor negative breast cancer, progesterone receptor positive breast cancer, progesterone receptor negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget's disease of the nipple, and phyllodes tumor.

[0129] Other cancers that may be treated according to the methods described herein include leukemias (e.g., B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), and erythroleukemia), sarcomas (e.g., angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath sarcoma, and sarcomas). sarcoma, malignant fibrous cell carcinoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leiomyosarcoma, and neurofibrosarcoma), carcinoma (e.g., basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung cancer, renal cancer, liver cancer, gastric cancer, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oesophageal) cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, carcinoma), adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma), blastomas (e.g., hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma and glioblastoma multiforme), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma and Burkitt's lymphoma), myelomas (e.g., multiple myeloma, plasmacytoma, localized myeloma and extramedullary myeloma), melanomas (e.g., superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma and amelanotic melanoma), neuromas (e.g., ganglioneuroma, Pacinian neuroma, neuroma and acoustic neuroma), gliomas (e.g., astrocytoma, oligoastrocytoma, ependymoma, brain stem glioma, optic nerve glioma and oligoastrocytoma), pheochromocytoma, meningioma, malignant mesothelioma and virus-induced cancers.

[0130] The cancer may be highly innervated, metastatic, non-metastatic, or benign (e.g., a benign tumor). The cancer may be a primary tumor or a metastatic tumor.

[0131] In some embodiments, the cancer is a cancer that is treated with immunotherapy (e.g., melanoma, non-small cell lung cancer, renal cancer, renal cell carcinoma, bladder cancer, head and neck cancer, Hodgkin's lymphoma, leukemia, urothelial cancer, gastric cancer, microsatellite instability-high cancer, colorectal cancer, or hepatocellular carcinoma). In some embodiments, the cancer is a cancer for which immunotherapy is ineffective (e.g., a cancer that cannot be treated with immunotherapy or that has not responded to treatment with immunotherapy).

[0132] The subjects that can be treated using the methods described herein include those that have one or more tumors removed, those that have undergone chemotherapy or other pharmacological treatments for cancer, those that have undergone radiation therapy, and / or those that have undergone other treatments for cancer.The subjects that can be treated using the methods disclosed herein include those that do not respond to immunotherapy.Subjects that have not undergone cancer treatment before can also be treated using the methods disclosed herein.

[0133] Treatment Administration An effective amount of the therapeutic agent described herein for the treatment of cancer (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) can be administered to a subject by standard methods. For example, the agent can be administered by any of a number of different routes, including, for example, intravenous, intradermal, subcutaneous, percutaneous, injection, oral, transdermal (topical), or mucosal. The therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent) can be administered orally or by injection, for example, intramuscularly or intravenously. The most suitable administration route in any given case will depend on the specific agent being administered, the patient, the specific disease or condition being treated, the method of formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The agent can be encapsulated or injected, for example, in a viscous form, for delivery to a selected site, for example, a tumor site. The drug can be provided in a matrix that can deliver the drug to the selected site. The matrix can provide a slow release of the drug and provide a suitable presentation and suitable environment for cell entry. The matrix can be formed from materials currently used in other implant medical applications. The selection of the matrix material is based on any one or more of biocompatibility, biodegradability, mechanical properties, and cosmetic appearance and interface properties. One example is a collagen matrix.

[0134] The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) can be incorporated into a pharmaceutical composition suitable for administration to a subject, e.g., a human. Such compositions typically include a drug and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., suitable for pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known. Except where a conventional media or agent is incompatible with the active compound, such media can be used in the description herein. Supplementary active compounds can also be incorporated into the composition.

[0135] Pharmaceutical compositions can be formulated to suit their intended route of administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application can contain the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity adjusters, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be contained in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0136] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the example of a dispersion, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0137] Suitable injection solutions can be prepared by adding the required amount of active compound (e.g., therapeutic agent described herein) to a suitable solvent containing one or a combination of the above-listed components, and then optionally performing filtration sterilization.Generally, dispersions are prepared by adding active compound to a sterile vehicle containing a basic dispersion medium and other required components listed above.In the case of sterile powders for preparing sterile injection solutions, the preferred preparation method is vacuum drying and lyophilization, which produces a powder of active ingredient and any additional desired ingredients from its solution that has been previously sterilized and filtered.

[0138] Oral compositions usually contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be combined with an excipient and used in the form of tablets, troches or capsules. Oral compositions can also be prepared with a liquid carrier to be used as a mouthwash, where the compound in the liquid carrier is applied orally, swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.

[0139] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be passed is used in the formulation. Such penetrants are generally known, and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be administered through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels or creams that are generally known in the art.

[0140] The active compound can be prepared with a carrier that can protect the compound from rapid excretion from the body, for example, controlled release formulations, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, can be used.The method of preparing such formulations will be clear to those skilled in the art.Liposomal suspensions (including liposomes that are made to target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in the art.

[0141] The nucleic acid molecule agent (e.g., therapeutic mRNA) described herein can be directly administered or can be inserted into a vector used as a gene therapy vector. Gene therapy vectors can be delivered to a subject, for example, by intravenous injection, local administration (see, for example, U.S. Pat. No. 5,328,470) or by stereotactic injection (see, for example, Chen et al., PNAS 91: 3054 1994). Pharmaceutical preparations of gene therapy vectors can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, in the case where the complete gene delivery vector can be produced intact from recombinant cells, for example, retroviral vectors, pharmaceutical preparations can include one or more cells that produce the gene delivery system.

[0142] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0143] The method of formulating pharmaceutical agents is known in the art, for example, Niazi, Handbook of Pharmaceutical Manufacturing Formulations (Second Edition). CRC Press 2009 describes the development of liquid, sterile, compressed, semi-compressed and OTC form formulations.Transdermal and mucosal delivery, lymphatic delivery, nanoparticles, controlled drug release systems, theranostics, protein and peptide drugs, and biologics delivery are described in Wang et al., Drug Delivery: Principles and Applications (Second Edition), Wiley 2016, and the formulation and delivery of peptide and protein drugs are described in, for example, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems (Third Edition), CRC Press 2015.

[0144] Combination therapy The therapeutic agents described herein may be administered in combination with one or more additional therapeutic agents (e.g., one, two, three or more additional therapeutic agents). The two or more agents may be administered at the same time (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes or less). The agents may also be administered simultaneously through co-formulation. The two or more agents may also be administered sequentially such that the action of the two or more agents overlaps and their combined effect results in a greater reduction in symptoms or other parameters associated with the disorder than would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two or more treatments may be partially additive, totally additive, or more than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, topical, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same or different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection and a second therapeutic agent of the combination may be administered locally via a microcassette having the compound embedded therein. The first therapeutic agent may be administered immediately before or immediately after, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7 days, 1-14 days, 1-21 days, or 1-30 days before or after the second therapeutic agent.

[0145] In other examples, the inhibitor of cancer-associated phosphatase is administered in combination with a second therapeutic agent, which may be an immunomodulatory cytokine, an agent that inhibits myeloid-derived suppressor cells, an immune checkpoint inhibitor and an antiproliferative agent, a phosphoneoantigen vaccine, a CAR-T, or any other therapeutic agent described herein.

[0146] dosage The subject that can be treated as described herein is a subject that has cancer or is at risk of developing cancer.Cancer can be primary cancer or metastatic cancer.In some embodiments, cancer is a cancer associated with expression or overexpression of cancer-associated phosphatase.The subject that can be treated using the methods disclosed herein includes a subject that has one or more tumors removed, a subject that has undergone chemotherapy or other pharmacological treatment for cancer, a subject that has undergone radiation therapy, and / or a subject that has undergone other treatment for cancer.Subjects that have not been previously treated for cancer can also be treated using the methods described herein.

[0147] In some embodiments, the agent is administered in an amount and for a duration effective to result in one (or more, e.g., two or more, three or more, four or more) of the following: (a) a decrease in tumor size; (b) a decrease in tumor growth rate; (c) an increase in tumor cell death; (d) a decrease in tumor progression; (e) a decrease in the number of metastases; (f) a decrease in the rate of metastasis; (g) a decrease in tumor migration; (h) a decrease in tumor invasion; (i) a decrease in tumor volume; (j) a decrease in tumor recurrence; (k) an increase in subject survival; or (l) an increase in subject progression-free survival.

[0148] The methods described herein may include selecting a treatment for a patient. The method includes (a) identifying (e.g., diagnosing) a patient with cancer or at risk of developing cancer, and (b) selecting a therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent) for treating the condition in the patient. In some embodiments, the method includes administering the selected treatment to the patient. In some embodiments, the patient is identified as having cancer based on imaging (e.g., MRI, CT or PET scan), biopsy or blood sample (e.g., blood antigen markers (e.g., by PCR), detection of circulating tumor DNA). In some embodiments, the patient is identified as having cancer after exhibiting one or more symptoms of a paraneoplastic syndrome (e.g., fever, autoantibodies against nervous system proteins, ataxia, dizziness, nystagmus, difficulty swallowing, loss of muscle tone, loss of fine motor coordination, slurred speech, memory loss, loss of vision, sleep disorder, dementia, seizures, loss of taste, cachexia, anemia, itching or loss of sensation in hands and feet). In some embodiments, a patient presents with symptoms of a paraneoplastic syndrome and is subsequently identified as having cancer based on imaging (eg, CT, MRI or PET scan).

[0149] In one example, the method includes (a) identifying (e.g., diagnosing) a patient with cancer, (b) optionally evaluating the subject (e.g., the subject's cancer cells) for expression or overexpression of a cancer-associated phosphatase, and (c) selecting a therapeutic agent (e.g., an inhibitor of the cancer-associated phosphatase, optionally in combination with a second therapeutic agent) to treat the patient if the cancer cells show expression or overexpression of the cancer-associated phosphatase. In some embodiments, the method includes administering the selected treatment to the patient.

[0150] The method may also include evaluating the subject for parameters of cancer progression or remission, such as one or more (e.g., two or more, three or more, four or more) of primary tumor size (e.g., by imaging), number of metastases (e.g., by imaging or biopsy), in situ cell death (e.g., by biopsy), blood antigen markers (e.g., by ELISA), circulating tumor DNA (e.g., by PCR), or affected organ function (e.g., by tests of circulating enzymes for the liver, albuminuria for the kidneys, pulmonary function for the lungs, etc.).

[0151] The methods described herein may also include evaluating the subject for immune response parameters, such as one or more (e.g., two or more, three or more, four or more) of Th2 cells, T cells, circulating monocytes, neutrophils, peripheral blood hematopoietic stem cells, macrophages, degranulation of mast cells, activated B cells, NKT cells, phagocytosis of macrophages, polarization of macrophages, antigen presentation, activation of immune cells, proliferation of immune cells, lymph node homing or egress of immune cells, differentiation of T cells, recruitment of immune cells, migration of immune cells, innervation of lymph nodes, maturation of dendritic cells, occurrence of HEV, occurrence of TLO, or production of cytokines. In an embodiment, the method includes measuring cytokines or markers associated with a particular immune cell type. In some embodiments, the method includes measuring chemokines, receptors, or immune cell trafficking molecules (e.g., performing an assay to measure chemokines, markers, or receptors). The evaluation can be performed after administration, before the first administration and / or during the course of treatment, for example, after the first, second, third, fourth or subsequent administration, or periodically during the course of treatment, for example, once a month or once every three months.In some embodiments, the method includes evaluating the subject before treatment or the first administration and using the evaluation results to select the subject for treatment.In certain embodiments, the method also includes modifying the administration step (for example, stopping administration, increasing or decreasing the period of administration, increasing or decreasing the dose of the therapeutic agent) based on the evaluation results.

[0152] In other examples, the immune effect (e.g., immune cell activity) is modulated in a subject (e.g., a subject with cancer or an inflammatory or autoimmune condition) or in cultured cells by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% compared to before administration of a therapeutic agent (e.g., an inhibitor of a cancer-associated phosphatase, optionally in combination with a second therapeutic agent), e.g., a therapeutic agent described herein, e.g., a dosing regimen. In certain embodiments, the immune effect is modulated by 5-20%, 5-50%, 10-50%, 20-80%, 20-70%, 50-100%, or 100-500% in a subject or in cultured cells. The immune effects described herein can be assessed by standard methods.

[0153] The therapeutic agent described herein (e.g., an inhibitor of cancer-associated phosphatase, optionally in combination with a second therapeutic agent) is administered in an amount (e.g., an effective amount) and for a period sufficient to produce one of the above results. The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally in combination with a second therapeutic agent) can be administered once or more than once. The therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally in combination with a second therapeutic agent) can be administered once a day, twice a day, three times a day, every other day, once a week, twice a week, three times a week, once every two weeks, once a month, once every two months, twice a year, or once a year. Treatment can be individual (e.g., injection) or continuous (e.g., treatment through an implant or infusion pump). The subject can be evaluated for treatment efficacy after 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more after administration of the therapeutic agent (e.g., an inhibitor of cancer-associated phosphatase, optionally combined with a second therapeutic agent), depending on the therapeutic agent and administration route used for treatment. Depending on the evaluation results, treatment can be continued or stopped, treatment frequency or dosage can be changed, or the patient can be treated with a different therapeutic agent. The subject can be treated for a discrete period (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months) or until the disease or condition is alleviated, or treatment can be continuous, depending on the severity and nature of the disease or condition to be treated. EXAMPLES

[0154] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0155] Example 1. Expression of ALPP and ALPP2 in lymphoblastoid cell lines Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects our healthy B cells. This virus is known to transform infected B cells into cancerous ones. However, these transformed B cells are continuously eliminated by T cells, so for most subjects EBV is a lifelong asymptomatic disease. In subjects whose T cells do not function, such as those with primary immunodeficiencies or those under strong immunosuppressive drugs (e.g., transplant patients), T cells are prevented from fighting these cancers. In these patients, EBV-infected B cells grow uncontrolled and develop an aggressive type of lymphoma called post-transplant lymphoproliferative disease (PTLD).

[0156] PTLD-like tumors were generated by infecting B cells from healthy individuals. This infection resulted in lymphoblastoid cell lines (LCLs) that grow indefinitely. When T cells from a healthy donor were added, the T cells killed the LCLs. This system can therefore be used as a model for cancer.

[0157] LCLs express multiple phosphopeptides but not ALPP or ALPP-L2, and culturing LCLs in phosphatase-rich medium removes phosphopeptide surface HLA molecules and reduces recognition of LCLs by autologous T cells by 50%.

[0158] Expression of ALPP and ALPPL2 in LCLs reduces the ability of autologous T cells to recognize phosphatase-expressing LCLs.

[0159] Generation of Epstein-Barr virus (EBV)-lymphoblastoid cell lines (LCL) Peripheral blood mononuclear cells (PBMCs) were counted and cultured at 1 x 10 in supplemented RPMI. 7 EBV stock was thawed and cyclosporine A was added to the EBV stock solution to a final concentration of 800 ng / mL. 1 mL of PBMCs and 1 mL of EBV stock + cyclosporine A were added per well of a 24-well plate. The final number of PBMCs was 1 x 10 per well. 7 The final concentration of LCL cells and cyclosporine A was 400 ng / mL. The plates were incubated at 37° C. and 5% CO2 for 3 weeks. The cells were counted and cultured according to standard LCL cell culture methods known to those skilled in the art.

[0160] Cloning of lentivectors Either ALPP, ALPPL2 or luciferase cDNA sequences were cloned into pLV lenti-backbone (VectorBuilder) using the Gateway method. To confirm gene expression, either GFP or mCherry was used as a marker under the control of the CMV promoter. Details of the vector design are provided in Figure 5.

[0161] Production of lentivirus On day -1, HEK 293FT packaging cells were plated in 15 cm dishes at approximately 40% confluence. On day 0, a cell density of 80-90% was achieved before transfection. HEK293 cells were transfected with the ALPP / ALPPL2 / GL lentivector and packaging vector pSPAX2 / pMD2.G using Mirus-TransIT™ transfection reagent. On day 2, the supernatant was collected and passed through a 0.2 μM filter. The virus was concentrated by ultracentrifugation at 120,000 g for 2 hours at 4°C. The pellet was resuspended in 200 μl PBS. The resuspended virus was aliquoted and stored at -80°C for future use. The virus was titrated on HEK293 cells and tested by flow cytometry.

[0162] Transduction of LCL cells to generate ALPP / ALPPL2 overexpressing lines Viruses were added to LCL cells at MOI=100 in the presence of 4 μg / ml polybrene. After 4 hours, the medium was replaced with LCL cell medium. On day 3, ALPP / ALPPL2 / luciferase overexpressing cells were purified by FACS sorting based on GFP / mCherry expression. GFP / mCherry positive cells were cultured to obtain ALPP / ALPPL2 / luciferase overexpressing lines (LCL-ALPP, LCL-ALPPL2, LCL-GL) (Figure 6).

[0163] Generation of LCL-specific CD8+ T cell lines Autologous PBMCs were seeded together with LCLs in 2 mL RPMI medium at a ratio of PBMC:LCL = 40:1 (2e6:5e4) in 24-well plates. After 10 days, irradiated (40 Gy) LCLs were added at a ratio of 4:1 (1x10 6 :2.5x10 5 Responder cells were restimulated once a week with a responder:stimulator cell ratio of 1000 μg / mL. Beginning on day 14, two weekly doses of rhIL-2 (50-100 IU / mL) were added.

[0164] specific CD8 +Effect of ALPP / ALPPL2 on killing of autologous LCL by T cells Parental LCLs were pre-labeled with violet dye (Thermofisher, CellTrace™ Violet Cell Proliferation Kit, C34557). Violet dye-labeled parental LCLs were seeded in 96-well plates with LCL-ALPP, LCL-ALPPL2 or LCL-GL (at a ratio of 1:1) in the presence (CD8:LCL=10:1) or absence (CD8:LCL=0:1) of autologous LCL-specific CD8 T cells. After 16 hours, cells were harvested and examined by FACS. Both ALPP and ALPPL2 expression negatively regulated the autologous CD8 T cell killing effect. No obvious killing preference was observed between parental LCL and LCL-GL cells (Figure 7A-B).

[0165] Example 2. ALPP / ALPPL2 inhibitor ML095 inhibits specific CD8 expression in tumor cells + Restoring T-cell killing specific CD8 + The influence of ALPP / ALPPL2 on killing of haploidentical targets by T cells The generated tumor-specific CD8 T cell lines were challenged with two haploidentical tumor cell lines and HLA-matched human PBMCs. The tumor cell lines are shown in Table 2.

[0166] [Table 2]

[0167] Transduction of H2009 to generate ALPP / ALPPL2 overexpression strains Viruses were added to H2009 at MOI=100 in the presence of 4 μg / ml polybrene. After 4 hours, the medium was replaced with H2009 medium. On day 3, ALPP / ALPPL2 overexpressing cells were purified by FACS sorting based on GFP / mCherry expression. GFP / mCherry positive cells were cultured to obtain ALPP / ALPPL2 overexpressing lines (H2009ALPP OE, H2009ALPPL2 OE). ALPP or ALPPL2 expression was confirmed by Western blot (Figure 8A) and NBT BCIP reaction assay (Figure 9A).

[0168] Knocking out ALPP / ALPPL2 in A431 using CRISPR-Cas9, transduction of A431 Both Cas9 and gRNA targeting either ALPP, ALPPL2 or an unrelated target (scramble) were introduced into A431 using the lentivector LentiCRISPR-GFP. After 3 days, GFP+ cells were sorted for single cell cloning of ALPP or ALPPL2 knockout strains (A431ALPP KO, A431ALPPL2 KO) and seeded at a concentration of 0.3 cells / well in 96-well plates. Once the cells were grown, knockout efficiency for both ALPP and ALPPL2 was verified by Western blot (Figure 8B) and NBT-BCIP reaction assay (Figure 9B).

[0169] NBT BCIP reaction assay for ALPP / ALPPL2 Cells were fixed in ice-cold acetone-methanol mixture (30:70) for 5 min. The cells were washed twice with PBS and endogenous heat-labile alkaline phosphatase was inactivated by incubation in TMN substrate buffer (0.1 M Tris-HCl, pH 9.5, containing 0.1 M NaCl and 5 mM MgCl2) at 60 °C for 30 min. The substrate buffer was discarded and staining for heat-stable PLAP was performed with fresh TMN buffer containing 0.175 mg / mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma-Aldrich) and 0.45 mg / mL nitrotetrazolium blue chloride (NBT; Sigma-Aldrich). The staining was performed for at least 3 h at room temperature. Samples were washed twice with PBS and observed under an inverted light microscope (Figure 9A-B).

[0170] Haploidentical target-specific CD8 + Generation of T cell lines In a 24-well plate, HLA-matched PBMCs (A * 03:01 / B * 07:02 / C * 07:02) with either H2009 or A431 in 2 ml RPMI medium at a PBMC:tumor cell ratio of 40:1 (2x10 6 :5x10 4 Both H2009 and A431 were irradiated (40 Gy) before use. From day 10, irradiated (40 Gy) H2009 or A431 were seeded at a ratio of 4:1 (1x10 6 :2.5x10 5 Responder cells were restimulated once a week with a responder:stimulator cell ratio of 1000 μg / mL. Beginning on day 14, two weekly doses of rhIL-2 (50-100 IU / mL) were added.

[0171] specific CD8 + Influence of ALPP / ALPPL2 on killing of HLA-matched haploidentical targets by T cells Parental cell lines (H2009 or A431) were pre-labeled with violet dye (Thermofisher, CellTrace™ Violet Cell Proliferation Kit, C34557). Violet dye-labeled parental cell lines were seeded in 96-well plates together with lentivirus-modified cell lines (at a ratio of 1:1) in the presence (CD8:tumor cells=1:1, 10:1) or absence (CD8:tumor cells=0:1) of autologous specific CD8 T cells. After 16 hours, cells were harvested and examined by FACS.

[0172] Results showed that both ALPP / ALPPL2 expression was associated with specific CD8 + Overexpression of either ALPP or ALPPL2 negatively regulated the T cell killing effect of specific CD8 + Knockout of either ALPP or ALPPL2 inhibited the specific CD8 + sensitizing tumor cells to T cell killing (FIG. 10B, A431 results). No obvious killing preference was observed between the parental A431 and the A431-scrambled KO line.

[0173] Specific CD8+ of HLA-matched haploidentical targets + Effect of the ALPP / ALPPL2 inhibitor ML095 on T cell killing Parental cell lines (H2009 or A431) were pre-labeled with violet dye (Thermofisher, CellTrace™ Violet Cell Proliferation Kit, C34557). The violet dye-labeled parental cell lines were cultured in 96-well plates to express autologous specific CD8 + T cells were seeded (at a 1:1 ratio) with lentivirus-modified cell lines in the presence (CD8:tumor cells = 10:1) or absence (CD8:tumor cells = 0:1). ML095 was added at concentrations of 0 uM, 2 uM and 10 uM. After 16 hours, cells were harvested and examined by FACS.

[0174] The results showed that the addition of ML095 increased theALPP OE (Figure 11A-B) and H2009 ALPPL2 OE (Figure 12A-B) + The results show that the addition of ML095 restores T cell killing in A431 ALPP KO (Figure 13A-B) or A431 ALPPL2 KO (Figure 14A-B) compared with A431 specific CD8 + Increases T cell killing, A431 スクランブルKO (Figures 15A-B) show no effect on

[0175] Other Aspects While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and that this application is intended to cover any variation, use, or application of the invention which generally follows the principles of the invention and is made within the scope of known or ordinary skill in the art to which the invention pertains, and which may be applied to the essential features hereinabove, and which includes departures from the invention encompassed by the scope of the claims. Other embodiments are within the scope of the claims.

Claims

1. 1. A method for identifying an inhibitor of placental alkaline phosphatase, comprising: identifying cancer cells containing increased expression of placental alkaline phosphatase selected from alkaline phosphatase placental type (ALPP) or alkaline phosphatase placental-like 2 (ALPP-L2); contacting the cancer cells with a potential therapeutic candidate; and Measuring expression of phosphoneoantigens; wherein increased expression of phosphoneoantigens is associated with the therapeutic candidate being an inhibitor of ALPP or an inhibitor of ALPPL2.

2. 2. The method of claim 1, wherein the placental alkaline phosphatase is ALPP.

3. 2. The method of claim 1, wherein the placental alkaline phosphatase is ALPP-L2.

4. 2. The method of claim 1, wherein the inhibitor of cancer associated phosphatase is ML095, thiophosphate, Lp-bromotetramisole, tetramisole, levamisole, 5804079, L-phenylalanine, 1-paphthyl phosphate, MLS-0315848, MLS0390945, or MLS-0315687.

5. The method of claim 1 , wherein the inhibitor is a nucleic acid molecule, a polypeptide, a nuclease, or a small molecule.

6. The method of claim 5, wherein the polypeptide is an antibody molecule.

7. The method of claim 1, wherein the cancer cells are identified by measuring the enzymatic activity or expression level of a phosphatase.

8. 2. The method of claim 1, wherein the cancer cells have 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000% or more increased phosphatase activity compared to wild type cells of the same type.

9. The method of claim 8, wherein the cancer cells have a 20% increased phosphatase activity compared to wild-type cells of the same type.

10. 10. The method of claim 9, wherein the cancer cells have a 50% increased phosphatase activity compared to wild-type cells of the same type.

11. 2. The method of claim 1, wherein the expression level of a phosphoneoantigen is increased by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 500% or 1000%.

12. The method of claim 11, wherein the expression level of phosphoneoantigens is increased by at least 20% after contact with the therapeutic agent.

13. The method of claim 11, wherein the expression level of phosphoneoantigens is increased by at least 50% after contact with the therapeutic agent.

14. 2. The method of claim 1, wherein the cancer cells are isolated from a patient having gastrointestinal cancer, genitourinary cancer, gynecological cancer, lung cancer, head and neck cancer, cancer of the central nervous system, malignant mesothelioma, breast cancer, skin cancer, thyroid cancer, bone and soft tissue sarcoma, hematological neoplasms, gastric cancer, colon cancer, liver cancer, renal cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, esophageal cancer, bronchial cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, laryngeal cancer, bile duct cancer, respiratory system cancer, or urinary system cancer.

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