GENETICALLY ENGINEERED DRUG-RESISTANT T CELLS AND METHODS OF USE THEREOF

Abstract

Novel cell compositions genetically engineered to express at least a chimeric antigen receptor (CAR) and a survival factor, and methods of using such cell compositions, are provided for treating a subject suffering from cancer by administering to the subject a cell composition comprising γδ T cells that express at least a chimeric antigen receptor (CAR) directed against a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that enables the immune system cells expressing the CAR to survive in the treatment environment resulting from additional therapeutic treatment.

Description

[Background technology]

[0001] background Although traditional therapeutic strategies for cancer treatment are promising in many ways, they still require improvement. For example, despite significant efforts, treatments for aggressive primary brain tumors such as glioblastoma multiforme (GBM) have not been able to significantly and consistently extend median survival beyond two years.

[0002] One area of ​​research being explored is immunotherapy. Such treatments harness the power of a patient's immune system to fight diseases such as cancer. In particular, adoptive cell transfer is being explored. In many forms of adoptive cell transfer, a patient's immune cells are harvested, expanded, and modified for a more efficient response. In this context, chimeric antigen receptors (CARs) have been used. CARs are genetically engineered receptors that graft arbitrary and defined specificities onto immune effector cells. Typically, CARs are used to graft the specificity of monoclonal antibodies onto T cells. In this approach, T cells are harvested from the patient or a suitable donor and modified to express receptors specific for a particular form of cancer. The T cells, capable of recognizing and killing cancer cells, are then reintroduced into the patient. Early clinical studies of this approach have shown efficacy.

[0003] However, there are still some difficulties in using CAR in adoptive cell transfer. The present disclosure provides a new immune system cell composition that is genetically engineered to express a CAR specific for a tumor antigen and a survival factor that confers resistance to additional therapeutic treatments (e.g., chemotherapy treatment regimens) that may be administered to patients. The cell composition may further include suicide genes and additional elements. Methods of using the cell composition for the treatment of disease are also provided. Thus, the present disclosure provides a new and necessary solution to the problems of treatment using immunotherapy. In a particular embodiment, the cell composition provided is a γδ T cell. Summary of the Invention

[0004] [The present invention 1001] a. administering to a subject a cell composition comprising γδ T cells expressing at least a chimeric antigen receptor (CAR) directed against a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that allows immune system cells expressing the CAR to survive in a treatment environment resulting from an additional therapeutic treatment; and b. administering an additional therapeutic treatment to the subject prior to administration of the cellular composition to the subject, after administration of the cellular composition to the subject, simultaneously with administration of the cellular composition to the subject, or any combination thereof. 23. A method of treating a subject suffering from cancer with an immunotherapy treatment and an additional therapeutic treatment comprising: [The present invention 1002] The method of claim 1001, further comprising at least one additional administration of the cell composition to the subject. [The present invention 1003] The method of the present invention 1001, wherein an additional therapeutic treatment is optionally administered prior to administering at least one additional administration of the cellular composition to the subject, after administering at least one additional administration of the cellular composition to the subject, simultaneously with administering at least one additional administration of the cellular composition to the subject, or any combination thereof. [The present invention 1004] The method of claim 1001, wherein the cell composition is administered to the subject on day X, and the additional therapeutic treatment is administered to the subject 12-72 hours before day X, 12-72 hours after day X, or both 12-72 hours before and after day X. [The present invention 1005] The method of the present invention 1001, comprising administering a cellular composition to a subject on day X and administering an additional therapeutic treatment to the subject 12-72 hours before day X, 12-72 hours after day X, or both 12-72 hours before and after day X, followed by additional administration of a cellular composition to the subject on day Y and, optionally, administering an additional therapeutic treatment to the subject 12-72 hours before day Y, 12-72 hours after day Y, or both 12-72 hours before and after day Y. [The present invention 1006] The method of the present invention 1001, wherein the cancer is selected from the group consisting of brain cancer, breast cancer, prostate cancer, lung cancer, colon cancer, epithelial cancer, head and neck cancer, skin cancer, cancer of the genitourinary tract, ovarian cancer, uterine cancer, cervical cancer, kidney cancer, stomach cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gallbladder cancer, cancer of the bile duct, esophageal cancer, cancer of the salivary glands, thyroid cancer, and hematological malignancies, leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome. [The present invention 1007] The method of the present invention 1001, wherein the cancer is selected from the group consisting of pineal tumor, pituitary tumor, PNET, Schwannoma, lymphoma, medulloblastoma, meningioma, metastatic brain tumor, neurofibroma, neuronal and mixed neuronal-glial tumors, oligoastrocytoma, oligodendroglioma, astrocytoma, atypical teratoid rhabdoid tumor (ATRT), chondrosarcoma, tumor of the choroid plexus, craniopharyngioma, ependymoma, germ cell tumor, neuroblastoma, glioblastoma and glioma. [The present invention 1008] The method of claim 1001, wherein the cancer is susceptible to additional therapeutic treatment. [The present invention 1009] The method of claim 1001, wherein the cancer is resistant to additional therapeutic treatment. [The present invention 1010] Tumor antigens include EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, ROR1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA-125, glypican-3, epithelial tumor antigen, melanoma-associated antigen, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, folate receptor alpha, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV- 1 envelope glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD23, CD33, CD30, CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL-11Rα, IL-13Rα, CSPG4, Lewis-Y, MCSP, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Sp17, SURVIVIN, TAG72, TEM1, TEM8, EGFRvIII, and VEGFR2. [The present invention 1011] 1001. The method of claim 1001, wherein the cancer is glioblastoma and the tumor antigen is selected from the group consisting of NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, EGFRvIII and IL13Rα. [The present invention 1012] The method of claim 1001, wherein the cancer is glioblastoma and the tumor antigen is EGFRvIII or IL13Rα. [The present invention 1013] 1001. The method of claim 1001, wherein the cancer is neuroblastoma and the tumor antigen is selected from the group consisting of NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B and GD2. [The present invention 1014] The method of claim 1001, wherein the cancer is neuroblastoma and the tumor antigen is GD2. [The present invention 1015] The method of claim 1001, wherein the cell composition comprises γδ T cells and at least one of αβ T cells and NK cells. [The present invention 1016] The method of claim 1001, wherein the cell composition comprises γδ T cells, and optionally αβ T cells and NK cells. [The present invention 1017] 1001. The method of claim 1001, wherein the cell composition comprises 60% or more γδ T cells, 5% or less αβ T cells, and 25% or less NK cells. [The present invention 1018] The method of claim 1001, wherein the receptor for a stress-induced antigen is an NKG2D receptor. [The present invention 1019] The method of claim 1001, wherein the additional therapeutic treatment is treatment with a nucleoside analog, an alkylating agent, an antimetabolite, an antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a differentiation inducer, or a hormone therapy agent, and the survival factor confers resistance to the additional therapeutic treatment. [The present invention 1020] Survival factor O 6 1001. The method of claim 1001, wherein the enzyme is -methylguanine-DNA methyltransferase, multidrug resistance protein 1, or 5'-nucleotidase II. [The present invention 1021] The additional therapeutic treatment is treatment with an alkylating agent, and the survival factor is O 6 1001. The method of claim 1001, wherein the enzyme is -methylguanine-DNA methyltransferase. [The present invention 1022] The additional therapeutic treatment was temozolomide, and the survival factor was O 6 1001. The method of claim 1001, wherein the enzyme is -methylguanine-DNA methyltransferase. [The present invention 1023] The method of claim 1001, wherein the γδ T cells are genetically engineered to express a CAR directed against a tumor antigen and a survival factor, and a receptor for a stress-induced antigen is naturally present on an immune system cell. [The present invention 1024] The method of claim 1001, wherein the γδ T cells are genetically engineered to express a CAR directed against a tumor antigen, a survival factor, and a receptor for a stress-induced antigen. [The present invention 1025] The method of claim 1001, wherein the γδ T cells further express a suicide gene. [The present invention 1026] The additional therapeutic procedure has the following advantages: a. increasing the efficacy of the cellular composition by induction of stress-induced antigens; b. Increased homeostatic reconstitution of cellular composition; c. increasing the in vivo expansion of the cellular composition; and d. Increased persistence of cellular composition The method of the present invention 1001, further comprising providing at least one of the following: [The present invention 1027] The method of claim 1001, wherein the additional therapeutic treatment increases expression of a stress-induced antigen selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A and MIC-B. [The present invention 1028] The method of claim 1001, wherein γδ T cells are obtained from a subject and optionally expanded ex vivo. [The present invention 1029] A cell composition comprising a γδ T cell that expresses at least a chimeric antigen receptor (CAR) directed against a tumor antigen and a survival factor that enables the immune system cell to survive in the treatment environment provided by an additional therapeutic treatment, and further expresses a receptor for a stress-inducing antigen. [The present invention 1030] The cell composition of the present invention 1029, wherein the cancer is selected from the group consisting of brain cancer, breast cancer, prostate cancer, lung cancer, colon cancer, epithelial cancer, head and neck cancer, skin cancer, cancer of the genitourinary tract, ovarian cancer, cancer of the uterine corpus, cancer of the cervix, kidney cancer, stomach cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gallbladder cancer, cancer of the bile duct, cancer of the esophagus, cancer of the salivary glands, thyroid cancer, and hematological malignancies, leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome. [The present invention 1031] The cell composition of the present invention 1029, wherein the cancer is selected from the group consisting of pineal tumor, pituitary tumor, PNET, Schwannoma, lymphoma, medulloblastoma, meningioma, metastatic brain tumor, neurofibroma, neuronal and mixed neuronal-glial tumors, oligoastrocytoma, oligodendroglioma, astrocytoma, atypical teratoid rhabdoid tumor (ATRT), chondrosarcoma, choroid plexus tumor, craniopharyngioma, ependymoma, germ cell tumor, neuroblastoma, glioblastoma and glioma. [The present invention 1032] The cell composition of the present invention 1029, wherein the cancer is susceptible to additional therapeutic treatment. [The present invention 1033] Tumor antigens include EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, ROR1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA-125, glypican-3, epithelial tumor antigen, melanoma-associated antigen, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, folate receptor alpha, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 E. The cell composition of the present invention 1029, wherein the cell is selected from the group consisting of envelope glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD23, CD33, CD30, CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL-11Rα, IL-13Rα, CSPG4, Lewis-Y, MCSP, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Sp17, SURVIVIN, TAG72, TEM1, TEM8, EGFRvIII, and VEGFR2. [The present invention 1034] The cell composition of the present invention 1029, wherein the cancer is glioblastoma and the tumor antigen is selected from the group consisting of NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, EGFRvIII and IL13Rα. [The present invention 1035] The cell composition of the present invention 1029, wherein the cancer is glioblastoma and the tumor antigen is EGFRvIII or IL13Rα. [The present invention 1036] The cell composition of the present invention 1029, wherein the cancer is neuroblastoma and the tumor antigen is selected from the group consisting of NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B and GD2. [The present invention 1037] The cell composition of the present invention 1029, wherein the cancer is neuroblastoma and the tumor antigen is GD2. [The present invention 1038] The cell composition of the present invention 1029, comprising γδ T cells and at least one of αβ T cells and NK cells. [The present invention 1039] A cell composition of the present invention 1029 comprising γδ T cells, and optionally αβ T cells and NK cells. [The present invention 1040] The cell composition of the present invention 1029, comprising 60% or more of γδ T cells, 5% or less of αβ T cells, and 25% or less of NK cells. [The present invention 1041] The cell composition of the present invention 1029, wherein the γδ T cells are genetically engineered to express a CAR and a survival factor, and optionally further to express a receptor for a stress-induced antigen. [The present invention 1042] The cell composition of the present invention 1041, wherein the receptor for a stress-induced antigen is an NKG2D receptor. [The present invention 1043] The cell composition of the present invention 1029, wherein the γδ T cells naturally express a receptor for a stress-induced antigen. [The present invention 1044] The cell composition of the present invention 1043, wherein the receptor for a stress-induced antigen is an NKG2D receptor. [The present invention 1045] The cell composition of the present invention 1029, wherein the additional therapeutic treatment is treatment with a nucleoside analog, an alkylating agent, an antimetabolite, an antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a differentiation inducer, or a hormone therapy agent, and the survival factor confers resistance to the additional therapeutic treatment. [The present invention 1046] Survival factor O 6 -methylguanine-DNA methyltransferase, multidrug resistance protein 1, or 5'-nucleotidase II. [The present invention 1047] The additional therapeutic treatment is treatment with an alkylating agent, and the survival factor is O 6 The cell composition of the present invention, wherein the enzyme is -methylguanine-DNA methyltransferase. [The present invention 1048] The additional therapeutic treatment was temozolomide, and the survival factor was O 6 The cell composition of the present invention, wherein the enzyme is -methylguanine-DNA methyltransferase. [The present invention 1049] The cell composition of the present invention 1029, wherein the γδ T cells are genetically engineered to further express a suicide gene. [The present invention 1050] The cell composition of the present invention 1029, wherein the additional therapeutic treatment increases the expression of a stress-induced antigen. [The present invention 1051] The cell composition of the present invention 1029, wherein the additional therapeutic treatment increases the expression of a stress-induced antigen selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A and MIC-B. [Brief description of the drawings]

[0005] [Figure 1A] 1 shows an EGFRvIII CAR control vector co-expressing eGFP, which can be used to easily track cell transduction by flow cytometry. [Figure 1B]FIG. 1 shows an EGFRvIII CAR control vector co-expressing firefly luciferase that can be used to track cell migration / homing in vivo. [Figure 1C] The control vector shown is an EGFRvIII CAR-encoding vector that lacks the sequence encoding the survival factor and cannot confer drug resistance. This control construct can be used as a positive control for CAR expression and a negative control for drug resistance. [Figure 1D] Shown is an EGFRvIII CAR that co-expresses a sequence encoding a survival factor (here MGMT) that confers resistance to temozolomide (TMZ). This construct is expected to provide the greatest efficacy for the in vivo treatment of glioblastoma. [Diagram 2] 1 shows an exemplary experimental design for treatment. [Diagram 3] Figure 3A shows a SPECT / CT fusion image (axial view) showing the distribution of 111[In]-labeled γδ T cells in a 4T1 mammary fat pad tumor 48 hours after injection performed with untreated γδ T cells, indicating that untreated γδ T cells bound to the tumor; the dotted circle delineates the tumor area. Figure 3B shows the same imaging as described in Figure 3A performed with γδ T cells pretreated with anti-γδ TCR monoclonal antibody prior to injection, indicating that treated γδ T cells did not bind to the tumor; the dotted circle delineates the tumor area. [Figure 4A] Expression of stress-related NKG2DL on X12T and X22T glioma xenografts. Both cell types showed autofluorescence. Black = isotype control, blue = tumor, red = tumor exposed to 400 μM TMZ for 4 hours. [Figure 4B] We show that ex vivo expanded / activated γδ T cells engineered to be resistant to TMZ were cytotoxic against cells derived from X12T and X22T glioma xenografts cultured in vitro with increasing effector:target (E:T) ratios of X12T and X22T; % lysis is expressed as live / dead cell ratio by flow cytometry. [Diagram 5]Figure 5A shows the survival rates of X12P glioma-bearing mice and untreated control mice after treatment with TMZ alone (TMZ), with a composition comprising γδ T cells and NK cells modified to express survival factors for TMZ resistance in combination with TMZ (DRI), with unmodified γδ T cells and NK cells (CT). Figure 5B shows the survival rates of X12T glioma-bearing mice and untreated control mice after treatment with TMZ alone (TMZ), with a composition comprising γδ T cells and NK cells modified to express survival factors for TMZ resistance in combination with TMZ (DRI), with unmodified γδ T cells and NK cells (CT). [Figure 6] 1 shows that expression of stress-induced antigens is not increased in normal brain tissue adjacent to metastatic brain tumor tissue. [Figure 7] 1 shows that expression of stress-induced antigens is not increased in normal brain tissue following radiation treatment. [Figure 8A] 1 shows one embodiment of a prior art method. [Figure 8B] 1 illustrates one embodiment of the method of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description Conventional therapeutic strategies for cancer need improvement. This disclosure provides novel solutions to problems encountered in the art and describes novel cell compositions and methods of using the cell compositions to treat various cancers, including but not limited to GBM. This disclosure provides examples of cell compositions using γδ T cells and provides specific examples of CARs and survival factors. However, other immune system cells, other CARs and other survival factors can also be utilized with this disclosure. The selection of immune system cells, CARs and survival factors can be influenced, at least in part, by the type of cancer to be treated and the additional therapies utilized to treat the patient.

[0007] The present disclosure provides novel cellular compositions, as well as the use of such cellular compositions in therapeutic methods.

[0008] In a first aspect, the cell composition comprises immune system cells genetically engineered to express a CAR directed against a tumor antigen and a survival factor that enables the immune system cells to survive in the treatment environment provided by the additional therapeutic treatment (e.g., a polypeptide that confers resistance to the chemotherapeutic agent(s) used in combination with the described cell composition).

[0009] In a second aspect, the cell composition comprises a γδ T-cell genetically engineered to express a CAR directed against a tumor antigen and a survival factor that enables the γδ T-cell to survive in the treatment environment provided by the additional therapeutic treatment (e.g., a polypeptide that confers resistance to the chemotherapeutic agent(s) used in combination with the described cell composition).

[0010] In a third aspect, the cell composition comprises γδ T cells genetically engineered to express a CAR directed against a tumor associated antigen (TAA) specific for glioma and a survival factor for resistance to, for example, temozolomide (TMZ) chemotherapy, enabling the γδ T cells to survive in the treatment environment of TMZ chemotherapy.

[0011] In a fourth aspect, the cell composition comprises a CAR directed against EGFRvIII of a glioma-specific TAA and an O-antigen that enables γδ T cells to survive in a treatment environment, e.g., TMZ chemotherapy, e.g., for resistance to TMZ chemotherapy. 6 -methylguanine-DNA methyltransferase (MGMT), and γδ T cells genetically engineered to express .

[0012] In a fifth aspect, the cell composition comprises γδ T cells genetically engineered to express a CAR directed against IL13Rα of a glioma-specific TAA and MGMT, e.g., for resistance to TMZ chemotherapy, enabling the γδ T cells to survive in a treatment environment, e.g., of TMZ chemotherapy.

[0013] In a sixth aspect, the present disclosure provides a method of treating cancer comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0014] In a seventh aspect, the present disclosure provides a method of treating glioma comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0015] In an eighth aspect, the present disclosure provides a method of treating GBM comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0016] In a ninth aspect, the present disclosure provides a method of treating GBM comprising administering to a subject a cellular composition of the fourth or fifth aspect, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the disclosure.

[0017] In a tenth aspect, the present disclosure provides a method of treating GBM comprising administering to the subject, e.g., TMZ chemotherapy, administering to the subject a cell composition of the fourth or fifth aspect, and optionally administering an additional course of, e.g., TMZ chemotherapy and / or an additional cell composition of the disclosure.

[0018] In an eleventh aspect, the present disclosure provides a method of treating a subject suffering from cancer with an immunotherapy treatment and an additional therapeutic treatment, the method comprising the steps of: (i) administering to the subject a cell composition comprising immune system cells expressing a CAR directed to a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that enables the immune system cells expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment; and (ii) administering to the subject the additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0019] In a twelfth aspect, the present disclosure provides a method of treating a subject with an immunotherapeutic treatment and an additional therapeutic treatment, the method comprising: (i) administering to the subject a cell composition comprising a γδ T-cell expressing a CAR directed to a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that enables the γδ T-cell expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment; and (ii) administering the additional therapeutic treatment to the subject prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0020] In a thirteenth aspect, the present disclosure provides a method of treating a subject with an immunotherapy treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising: (i) administering to the subject a cell composition comprising a γδ T-cell expressing a CAR directed to a glioma-specific TAA, a receptor for a stress-inducing antigen, and a survival factor, e.g., for resistance to TMZ chemotherapy, that enables the γδ T-cell expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment; and (ii) administering the additional therapeutic treatment to the subject prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0021] In a fourteenth aspect, the present disclosure provides a method of treating a subject with an immunotherapy treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising the steps of: (i) administering to the subject a CAR directed against EGFRvIII of a glioma-specific TAA and an NKGD2 receptor, and enabling γδ T cells expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment, e.g., conferring resistance to TMZ chemotherapy; 6 -methylguanine-DNA methyltransferase; and (ii) administering to the subject an additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0022] In a fifteenth aspect, the present disclosure provides a method of treating a subject with an immunotherapeutic treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising the steps of: (i) administering to the subject a CAR directed to IL13Rα of a glioma-specific TAA and an NKGD2 receptor, and a γδ T cell expressing the CAR that is capable of surviving in a treatment environment resulting from the additional therapeutic treatment, e.g., conferring resistance to TMZ chemotherapy; 6 -methylguanine-DNA methyltransferase; and (ii) administering to the subject an additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0023] cell composition In general, the present disclosure utilizes survival factors to provide drug resistant cellular immunotherapy (DRI). Through this mechanism, the cell compositions of the present disclosure are provided with the ability to survive, proliferate, and exert anti-tumor activity in a treatment environment following administration to a subject. Survival factors are selected based on additional therapeutic treatments that may be used in conjunction with the cell compositions and methods of the present disclosure.

[0024] As used herein, the phrase "survive in a treatment environment provided by an additional therapeutic treatment" can be used interchangeably with the phrase "survive in the presence of an additional therapeutic treatment", each of which refers to the ability of cells to survive direct contact with the agent used in the additional therapeutic treatment, or to survive in the presence of cytotoxicity in the environment of the cell composition of the present invention resulting from the use of the additional therapeutic agent. Additional therapeutic treatment includes, but is not limited to, treatment with the following agents: anticancer agents, antimetabolites, DNA demethylating agents, plant-derived antitumor agents, nucleoside / nucleotide analog chemotherapeutic agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation inducers, hormonal therapy agents, and combinations thereof. As known in the art and discussed herein, a variety of specific agents can be used in additional therapeutic treatment.

[0025] In one embodiment, the cell composition comprises immune system cells genetically engineered to express at least a CAR directed against a tumor antigen and a survival factor that allows the immune system cells to survive in the treatment environment provided by the additional therapeutic treatment, such as a polypeptide that confers resistance to the chemotherapeutic agent(s) that are combined with the described cell composition. Such polypeptides that confer drug resistance to certain chemotherapeutic agents include, but are not limited to, MGMT, 5' nucleotidase II (NT5C2), drug-resistant mutants of dihydrofolate reductase (L22Y-DHFR), thymidylate synthase, and multidrug resistance protein 1 (MDR1).

[0026] In another embodiment, the cell composition comprises at least a γδ T-cell genetically engineered to express a CAR directed against a tumor antigen and a survival factor that enables the γδ T-cell to survive in the treatment environment provided by the additional therapeutic treatment (e.g., a polypeptide that confers resistance to the chemotherapeutic agent(s) used in combination with the described cell composition).

[0027] In another embodiment, the cell composition comprises a γδ T-cell genetically engineered to express at least a CAR directed against a glioma-specific TAA and a survival factor that enables the γδ T-cell to survive in the treatment environment provided by the additional therapeutic treatment (e.g., a polypeptide that confers resistance to the chemotherapeutic agent(s) used in combination with the described cell composition).

[0028] In another embodiment, the cell composition comprises at least a γδ T-cell genetically engineered to express a CAR directed against a TAA and a survival factor that enables the γδ T-cell to survive in a treatment environment, e.g., for resistance to TMZ chemotherapy.

[0029] In another embodiment, the cell composition comprises at least a γδ T-cell genetically engineered to express a CAR directed against EGFRvIII of a glioma-specific TAA and MGMT, e.g., for resistance to TMZ chemotherapy, enabling the γδ T-cell to survive in a treatment environment of, e.g., TMZ chemotherapy.

[0030] In another embodiment, the cell composition comprises at least a γδ T cell genetically engineered to express a CAR directed to IL13Rα of a glioma-specific TAA and MGMT, e.g., for resistance to TMZ chemotherapy, enabling the γδ T cell to survive in a treatment environment of, e.g., TMZ chemotherapy.

[0031] In another embodiment, the cell composition comprises a γδ T-cell genetically engineered to express at least a CAR directed against a neuroblastoma-specific TAA and a survival factor that enables the γδ T-cell to survive in the treatment environment provided by the additional therapeutic treatment (e.g., a polypeptide that confers resistance to the chemotherapeutic agent(s) used in combination with the described cell composition).

[0032] In another embodiment, the cell composition comprises at least a γδ T-cell genetically engineered to express a CAR directed against a neuroblastoma-specific TAA and a survival factor that enables the γδ T-cell to survive in the treatment environment, e.g., for resistance to TMZ chemotherapy.

[0033] In another embodiment, the cell composition comprises at least a CAR directed to GD2 of a neuroblastoma-specific TAA and an O-CAR that enables γδ T cells to survive in a treatment environment, e.g., TMZ chemotherapy, e.g., for resistance to TMZ chemotherapy. 6 -methylguanine-DNA methyltransferase (MGMT) gene, and γδ T cells genetically engineered to express the gene.

[0034] In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen or glioma-specific TAA and a survival factor, such as, for example, a survival factor for resistance to TMZ. In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen, glioma-specific TAA or neuroblastoma-specific TAA, a suicide gene, and a survival factor, such as, for example, a survival factor for resistance to TMZ. In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen, glioma-specific TAA or neuroblastoma-specific TAA, a receptor for a stress-inducing antigen, and a survival factor, such as, for example, a survival factor for resistance to TMZ. In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen, a glioma-specific TAA or a neuroblastoma-specific TAA, a receptor for a stress-inducing antigen, a suicide gene, and a survival factor, such as a survival factor for resistance to TMZ. In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen, a glioma-specific TAA or a neuroblastoma-specific TAA, an NKG2D receptor, and a survival factor, such as a survival factor for resistance to TMZ. In certain embodiments, the cell composition comprises immune system cells or γδ T cells engineered to express only a CAR directed to a tumor antigen, a glioma-specific TAA or a neuroblastoma-specific TAA, an NKG2D receptor, a suicide gene, and a survival factor, such as a survival factor for resistance to TMZ.

[0035] In certain embodiments, the immune system cells or γδ T cells further comprise a receptor for a stress-induced antigen. In certain embodiments, the immune system cells or γδ T cells further comprise an NKGD2 receptor. In certain embodiments, the receptor for a stress-induced antigen or the NKGD2 receptor is naturally present on the immune system cells or γδ T cells. In certain embodiments, the receptor for a stress-induced antigen or the NKGD2 receptor is induced at an increased level on the immune system cells or γδ T cells. Thus, in embodiments where the cell composition comprises immune system cells or γδ T cells genetically engineered to express only a CAR directed against a tumor antigen, a glioma-specific TAA or a neuroblastoma-specific TAA, a survival factor, and an optional suicide gene, the immune system cells or γδ T cells may further express a stress-induced antigen or an NKGD2 receptor.

[0036] In certain embodiments, the immune system cell is any immune system cell useful for immunotherapy. In another aspect above, the immune system cell is any cell expressing a receptor for a stress-induced antigen. In certain embodiments, the immune system cell is any cell expressing an NKG2D receptor. In certain embodiments, the immune system cell is an NK cell or a γδ T cell. In certain embodiments, the immune system cell is a γδ T cell. In certain embodiments, the immune system cell is a combination of an NK cell and a γδ T cell. In certain embodiments, the immune system cell is a combination of an NK cell and a γδ T cell, where at least a portion of the NK cell and / or the γδ T cell expresses a receptor for a stress-induced antigen. In certain embodiments, the immune system cell is a combination of an NK cell and a γδ T cell, where at least a portion of the NK cell and / or the γδ T cell expresses an NKGD2 receptor.

[0037] In certain embodiments, the cell composition comprises γδ T cells and additional immune system cells. For example, the cell composition may comprise γδ T cells and NK cells, or may comprise γδ T cells, αβ T cells and NK cells. In certain embodiments, the cell composition comprises γδ T cells and additional immune system cells, where the γδ T cells are present at 60% or more of the total cell population. In certain embodiments, the cell composition comprises γδ T cells and NK cells, where the γδ T cells are present at 60% or more of the total cell population. In certain embodiments, the cell composition comprises γδ T cells and NK cells, where the γδ T cells are present at 60% or more of the total cell population, and the NK cells are present at 25% or less. In certain embodiments, the cell composition comprises γδ T cells and αβ T cells, where the γδ T cells are present at 60% or more of the total cell population. In certain embodiments, the cell composition comprises γδ T cells and αβ T cells, wherein γδ T cells are present at 60% or more of the total cell population, and αβ T cells are present at 5% or less. In certain embodiments, the cell composition comprises γδ T cells, αβ T cells, and NK cells, wherein γδ T cells are present at 60% or more of the total cell population. In certain embodiments, the cell composition comprises γδ T cells, αβ T cells, and NK cells, wherein γδ T cells are present at 60% or more of the total cell population, αβ T cells are present at 5% or less, and NK cells are present at 25% or less. The percentage of the different cell types present is, in one embodiment, measured by flow cytometry as described in Example 3.

[0038] In certain embodiments, at least some of the cells (e.g., immune system cells or γδ T cells) that make up the cell composition further comprise a receptor for a stress-induced antigen. In certain embodiments, at least some of the cells (e.g., immune system cells or γδ T cells) that make up the cell composition further comprise an NKGD2 receptor. In certain embodiments, the receptor for the stress-induced antigen or the NKGD2 receptor is naturally present on at least some of the cells (e.g., immune system cells or γδ T cells) that make up the cell composition. In certain embodiments, the receptor for the stress-induced antigen or the NKGD2 receptor is induced at increased levels on at least some of the cells (e.g., immune system cells or γδ T cells) that make up the cell composition. In certain embodiments, at least some of the cells (e.g., immune system cells or γδ T cells) that make up the cell composition are genetically engineered to express a receptor for a stress-induced antigen or an NKGD2 receptor. In certain embodiments, the cell composition comprises γδ T cells, and at least some of the γδ T cells express a receptor for a stress-induced antigen or an NKGD2 receptor. In certain embodiments, the cell composition comprises γδ T cells, αβ T cells and NK cells, at least a portion of the γδ T cells express a receptor for a stress-induced antigen or an NKGD2 receptor. In certain embodiments, the cell composition comprises γδ T cells, αβ T cells and NK cells, at least a portion of the γδ T cells express a receptor for a stress-induced antigen or an NKGD2 receptor, where the γδ T cells are present at 60% or more of the total cell population. In certain embodiments, the cell composition comprises γδ T cells, αβ T cells and NK cells, at least a portion of the γδ T cells express a receptor for a stress-induced antigen or an NKGD2 receptor, where the γδ T cells are present at 60% or more of the total cell population, the αβ T cells are present at 5% or less and the NK cells are present at 25% or less. In certain embodiments, the receptor for a stress-induced antigen or the NKGD2 receptor is naturally present on at least a portion of the γδ T cells, the αβ T cells and / or the NK cells. In certain embodiments, a receptor for a stress-induced antigen or the NKGD2 receptor is induced at increased levels in at least a portion of γδ T cells, αβ T cells and / or NK cells.In a particular embodiment, at least a portion of the γδ T cells, αβ T cells and / or NK cells are genetically engineered to express a receptor for a stress-induced antigen or the NKGD2 receptor. The proportion of different cell types present is, in one embodiment, measured by flow cytometry as described in Example 3.

[0039] In one aspect above, tumor antigen is any tumor antigen known in the art.In certain embodiments, tumor antigen is selected so that it has at least one of the following characteristics: it is expressed in as many stages of cancer as possible; it is expressed on the surface of tumor; it is important for the viability of tumor cells; and it is not expressed on non-tumor tissue or is expressed at such a level that off-target effects are clinically acceptable. In certain embodiments, the tumor antigen is selected from the group consisting of EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, ROR1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA-125, glypican-3, epithelial tumor antigen, melanoma associated antigen, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, folate receptor alpha, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV- 1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD23, CD33, CD30, CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL-11Rα, IL-13Rα, CSPG4, Lewis-Y, MCSP, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Sp17, SURVIVIN, TAG72, TEM1, TEM8, EGFRvIII, and VEGFR2.

[0040] In one aspect above, the glioma-specific TAA is any glioma-specific TAA known in the art. In certain embodiments, the glioma-specific TAA is selected so that at least one of the following characteristics is present: the glioma-specific TAA is expressed in as many stages of cancer as possible; the glioma-specific TAA is expressed on the surface of the tumor; the glioma-specific TAA is important for tumor cell viability; and the glioma-specific TAA is not expressed on non-tumor tissue or is expressed at a level such that off-target effects are clinically acceptable. In certain embodiments, the glioma-specific TAA is selected from the group consisting of: NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, EGFRvIII and IL13Rα. In certain embodiments, the glioma-specific TAA is EGFRvIII. In certain embodiments, the glioma-specific TAA is IL13Rα.

[0041] In one aspect above, the neuroblastoma-specific TAA is any glioma-specific TAA known in the art.In certain embodiments, the neuroblastoma-specific TAA is selected so that it has at least one of the following characteristics: the neuroblastoma-specific TAA is expressed in as many stages of cancer as possible; the neuroblastoma-specific TAA is expressed on the surface of tumor; the neuroblastoma-specific TAA is important for tumor cell viability; and the neuroblastoma-specific TAA is not expressed on non-tumor tissue or is expressed at a level such that off-target effects are clinically acceptable.In certain embodiments, the neuroblastoma-specific TAA is selected from the group consisting of: NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, and GD2.In certain embodiments, the neuroblastoma-specific TAA is GD2.

[0042] The survival factor can be any factor known in the art that confers resistance to additional therapeutic treatment and / or allows the cells that make up the cell composition to survive in a treatment environment (e.g., a chemotherapy treatment environment). In certain embodiments, the additional therapeutic treatment is a treatment with an anticancer drug, an antimetabolite, a DNA demethylating agent, a plant-derived antitumor agent, a nucleoside / nucleotide analog chemotherapeutic agent, an alkylating agent, an antimetabolite, an anticancer antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a differentiation inducer, a hormonal therapy agent, and combinations thereof, and the survival factor confers resistance to the additional therapeutic treatment. As known in the art and discussed herein, various specific agents can be used in the additional therapeutic treatment. Representative alkylating agents include, but are not limited to, cyclophosphamide, ifosfamide, and melphalan. Representative antimetabolites include, but are not limited to, methotrexate (MTX), 5-fluorouracil or its derivatives. Representative DNA demethylating agents (also known as antimetabolites) include, but are not limited to, azacitidine. Representative nucleoside / nucleotide analog chemotherapeutic agents include, but are not limited to, substituted nucleotides and substituted nucleosides. Representative antitumor antibiotics include, but are not limited to, mitomycin, adriamycin, and doxorubicin. Representative plant-derived antitumor agents include, but are not limited to, vincristine, vindesine, TAXOL®, paclitaxel, abraxane; cisplatin; carboplatin; etoposide, etc. Representative anti-cancer drugs include, but are not limited to, trimethotrexate (TMTX), temozolomide (TMZ), raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), nitrosoureas [e.g., bis-chloronitrosourea (BCNU; carmustine), lomustine (CCNU) + / - procarbazine and vincristine (PCV regimen), doxorubicin, fotemustine, cytarabine, camptothecin, and therapeutic derivatives of any of the above.Preferably, the agents used in the additional therapeutic treatment include TMZ, doxorubicin, melphalan, nitrosoureas, and any combination thereof.

[0043] In certain embodiments, the survival factor is MGMT, multidrug resistance protein 1 (MDR1), or 5' nucleotidase II (NT5C2). Other survival factors include, for example, drug-resistant mutants of dihydrofolate reductase (L22Y-DHFR) and thymidylate synthase. Preferably, the survival factor is MGMT. However, other survival factors may be used depending on the nature of the treatment environment (i.e., what other additional therapeutic treatments are administered to the patient in combination with the cell composition of the present disclosure).

[0044] In one aspect above, the cells of the composition further comprise a suicide gene.

[0045] In one aspect above, the cell composition comprises 60%, 70%, 80%, 90%, 95% or more of a single type of immune system cell. In one aspect above, the cell composition comprises 60%, 70%, 80%, 90%, 95% or more of γδ T cells. In one aspect above, the cell composition comprises 60% or more of γδ T cells, 5% or less of αβ T cells, and 25% or less of NK cells. The proportion of the various cell types present is, in one embodiment, measured by flow cytometry as described in Example 3.

[0046] In another aspect above, γδ T cells are obtained from a patient and expanded / activated ex vivo; the cells may then be reintroduced into the patient.

[0047] The described cell compositions are also referred to as DRI-CAR T cells.

[0048] The cell compositions described herein can be made by incorporating a nucleic acid construct capable of encoding and expressing a CAR and a survival factor, as well as other elements (e.g., a suicide gene and / or a receptor for a stress-induced antigen). In certain embodiments, a single nucleic acid construct encodes a CAR and a survival factor, as well as other elements (e.g., a suicide gene and / or a receptor for a stress-induced antigen). In certain embodiments, separate nucleic acid constructs encode a CAR and a survival factor, as well as other elements (e.g., a suicide gene and / or a receptor for a stress-induced antigen). Methods for making such nucleic acid constructs are known in the art. Representative nucleic acid constructs encoding a CAR and / or a survival factor are shown in Figures 1A-1D.

[0049] In certain embodiments, the nucleic acid construct encodes a CAR and a survival factor.

[0050] In the above embodiments, the CAR may comprise the domains and / or sequences described herein. In certain embodiments, the nucleic acid construct encodes a CAR comprising an ectodomain comprising an scFv against the EGFRvIII antigen, a hinge region, a transmembrane region, and an endodomain comprising at least one signaling domain. In certain embodiments, the nucleic acid construct encodes a CAR comprising an ectodomain comprising an scFv against the IL13Rα antigen, a hinge region, a transmembrane region, and an endodomain comprising at least one signaling domain. In certain embodiments, the nucleic acid construct encodes a CAR comprising an ectodomain comprising an scFv against the EGFRvIII antigen, a hinge region from IgG, a transmembrane region from CD3ζ, and an endodomain comprising CD28, Ox40, and CD32 signaling domains. In certain embodiments, the nucleic acid construct encodes a CAR comprising an ectodomain comprising an scFv against the IL13Rα antigen, a hinge region from IgG, a transmembrane region from CD3ζ, and an endodomain comprising CD28, Ox40, and CD32 signaling domains.

[0051] The nucleic acid construct can be incorporated into an expression vector before being introduced into a host cell. Such methods are known in the art. As mentioned above, a single expression vector or multiple expression vectors can be used depending on the nature of the nucleic acid construct. Various expression vectors can be used. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a viral vector. In certain embodiments, the expression vector is a retroviral vector. In certain embodiments, the expression vector is a lentiviral vector. The expression vector can be replicated inside an immune system cell (also called a host cell) or the expression vector can be fully or partially integrated into the genome of an immune system cell, allowing the nucleic acid construct to replicate along with the genome of the host cell.

[0052] An expression vector contains a nucleic acid construct in a form suitable for the expression of the nucleic acid construct in a host cell. Thus, an expression vector contains elements required for the expression of the nucleic acid construct in a host cell. The required elements may vary depending on the nature of the host cell and the vector, as well as other factors (e.g., the desired expression level). For example, the vector may contain one or more regulatory sequences (e.g., promoter sequences, enhancer sequences, other such sequences) operably linked to the nucleic acid construct that allow the expression of the nucleic acid construct. The regulatory sequences may be cell-specific, such that the nucleic acid construct is expressed only in a particular cell type, or the regulatory sequences may be constitutive, such that the nucleic acid construct is expressed in any cell type.

[0053] Chimeric Antigen Receptor The cells that make up the cell composition of the present disclosure incorporate one or more CARs. A specific example of a CAR is provided for use in treating GBM, but other CARs designed for treating other cancers can also be used. The present disclosure can utilize any CAR known in the art. Methods for making CARs with desired specificity for tumor-associated antigens are known in the art. CARs allow immune system cells of the present disclosure to recognize tumor antigens in a non-MHC-restricted manner. In addition, CARs can also be engineered to recognize antigens that are not derived from proteins.

[0054] CARs generally have the following structure: an ectodomain containing an antigen recognition domain, a transmembrane domain, and an endodomain. In certain embodiments, a peptide linker of 1-15 amino acids may be present in the CAR to separate the various domains of the CAR. For example, a peptide linker can be present between the antigen recognition domain and the transmembrane domain, or between the transmembrane domain and the endodomain. The peptide linker may be present between all domains, or only between some of the domains. Furthermore, when an endodomain contains two or more elements, a linker peptide may be present between some or all of the individual elements in the endodomain.

[0055] CARs can be made by first incorporating a signal sequence (which is generally subsequently removed during processing of the CAR). The signal peptide directs the expressed nascent protein to the endoplasmic reticulum and allows the CAR to be modified (e.g., glycosylated) and inserted into the cell membrane. Any eukaryotic signal peptide sequence can be used. Generally, the signal peptide naturally linked to the most amino-terminal component is used (e.g., in an scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0056] The antigen recognition domain can be any domain that recognizes a tumor antigen. In certain embodiments, the antigen recognition domain is directed to a glioma-specific TAA. In certain embodiments, the antigen recognition domain is directed to a tumor antigen. In certain embodiments, the antigen recognition domain is directed to a neuroblastoma-specific TAA. Specific antigens for recognition by the antigen recognition domain are shown above. There are many options for the antigen recognition domain. The antigen recognition domains from natural T cell receptor (TCR) α and β single chains have been described as having simple ectodomains (e.g., CD4 ectodomains that recognize HIV-infected cells), and more specialized ectodomains that contain recognition components such as linked cytokines (leading to recognition of cells with cytokine receptors). In fact, any component that binds with high affinity to a given target can be used as the antigen recognition region. In a preferred embodiment, the antigen recognition region is an scFv. In certain embodiments, a linker peptide (i.e., a spacer region or hinge region) links the antigen binding domain to the transmembrane domain.

[0057] In certain embodiments, the antigen recognition domain is an scFv. H -Linker-V L The heavy chains of immunoglobulins (V) are linked by a short linker peptide (usually about 10 to about 25 amino acids) to form a structure (also called an antigen-binding domain). H ) and light chain (V L ) variable region fusion protein. The linker is usually rich in glycine for flexibility, but also rich in serine or threonine for solubility, and H N-terminus of V L or vice versa. This scFv retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0058] In certain embodiments, the antigen recognition domain is a multivalent scFv. A multivalent scFv comprises two immunoglobulin-derived antigen binding domains (e.g., V H-Linker-V L In certain aspects, one of the antigen binding domains recognizes an activation molecule on the target cell. In certain aspects, one of the antigen binding domains recognizes an antigen whose expression on the target cell is increased as a result of an additional therapeutic treatment, such as, but not limited to, a stress-induced antigen.

[0059] In certain embodiments, the antigen recognition domain is a bivalent scFv. Such a bivalent scFv can, for example, be H1 -Linker a -V L1 -V H -Linker b -V L -Linker c -V H -Linker d -V L The structure is (V H and V L may be arranged in different orientations as described above).

[0060] The linker peptide must be sufficiently flexible to allow the antigen-binding domain to adopt a conformation suitable for antigen recognition and binding. The simplest form is the hinge region from IgG1. Various linker peptides can be used in combination with the CAR described herein.

[0061] The transmembrane domain is a hydrophobic region (such as an alpha helix) that spans the membrane of the cell in which the CAR is integrated. A transmembrane domain from any membrane-embedded protein can be used. Typically, a transmembrane domain from the most membrane-proximal component of the endodomain is used to facilitate the transmission of signals from, for example, the antigen recognition domain to the endodomain. Interestingly, the use of the transmembrane domain of CD3ζ can result in the integration of an artificial TCR into the native TCR a factor, which depends on the presence of the transmembrane charged aspartic acid residue of native CD3ζ.

[0062] The endodomain allows the transmission of a signal after antigen binding. After antigen recognition, the receptors cluster and a signal is transmitted to the immune system cell, including the CAR. The endodomain comprises at least one signaling domain. In certain embodiments, the endodomain comprises two or more signaling domains. The most commonly used signaling domain is CD3ζ, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs). Thus, in certain embodiments, the signaling domain comprises at least one ITAM. In certain embodiments, the endodomain comprises CD3ζ. In certain embodiments, the endodomain comprises CD3ζ and at least one additional signaling domain. The endodomain transmits an activation signal to the cell after antigen is bound. "First generation" CARs typically have an endodomain derived from CD3ζ, the main transmitter of signals from endogenous TCRs. In "second generation" CARs, intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, DAP10, OX40, or ICOS) are added to the cytoplasmic tail of the endodomain to provide additional signals to T cells. More recent "third generation" CARs have endodomains that combine multiple signaling domains, such as CD3ζ-CD28-41BB or CD3ζ-CD28-OX40, to further enhance efficacy. In certain embodiments, the endodomain comprises CD3ζ-CD28-OX40.

[0063] How to use Methods of using the cell compositions of the present disclosure are also described.

[0064] In one aspect, the present disclosure provides a method of treating cancer comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0065] In another aspect, the present disclosure provides a method of treating glioma comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0066] In another embodiment, the present disclosure provides a method of treating GBM comprising administering a cellular composition of the present disclosure to a subject, and administering an additional therapeutic treatment before, after, or both before and after administration of the cellular composition of the present disclosure.

[0067] In another embodiment, the disclosure provides a method of treating GBM comprising administering to a subject a cell composition comprising a γδ T-cell genetically engineered to express a CAR directed to glioma-specific TAA EGFRvIII or IL13Rα and an MGMT gene, e.g., for resistance to TMZ chemotherapy, and administering an additional therapeutic treatment before, after, or both before and after administration of the cell composition of the disclosure.

[0068] In another embodiment, the disclosure provides a method of treating GBM comprising administering to a subject e.g., TMZ chemotherapy, administering a cell composition comprising γδ T-cells genetically engineered to express a CAR directed to glioma-specific TAA EGFRvIII or IL13Rα and e.g., an MGMT gene for resistance to TMZ chemotherapy, and optionally administering an additional course of e.g., TMZ chemotherapy and / or an additional cell composition of the disclosure.

[0069] In another aspect, the disclosure provides a method of treating a subject suffering from cancer with an immunotherapy treatment and an additional therapeutic treatment, the method comprising the steps of: (i) administering to the subject a cell composition comprising immune system cells expressing a CAR directed to a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that enables the immune system cells expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment; and (ii) administering to the subject the additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0070] In another embodiment, the disclosure provides a method of treating a subject suffering from cancer with an immunotherapy treatment and an additional therapeutic treatment, the method comprising: (i) administering to the subject a cell composition comprising a γδ T-cell expressing a CAR directed to a tumor antigen, a receptor for a stress-inducing antigen, and a survival factor that enables the γδ T-cell expressing the CAR to survive in a treatment environment resulting from the additional therapeutic treatment; and (ii) administering to the subject the additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0071] In another embodiment, the present disclosure provides a method of treating a subject suffering from glioma with an immunotherapy treatment and an additional therapeutic treatment utilizing TMZ or other suitable chemotherapy, comprising the steps of: (i) administering to the subject a cell composition comprising γδ T-cells expressing a CAR directed to a glioma-specific TAA, a receptor for a stress-inducing antigen, and a survival factor for resistance, e.g., to TMZ chemotherapy, that enables the γδ T-cells expressing the CAR to survive in the treatment environment resulting from the additional therapeutic treatment; and (ii) administering the additional therapeutic treatment to the subject prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0072] In another embodiment, the disclosure provides a method of treating a subject suffering from neuroblastoma with an immunotherapy treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising: (i) administering to the subject a cell composition comprising a γδ T-cell expressing a CAR directed to a neuroblastoma-specific TAA, a receptor for a stress-inducing antigen, and a survival factor for resistance, e.g., to TMZ chemotherapy, that enables the γδ T-cell expressing the CAR to survive in the treatment environment resulting from the additional therapeutic treatment; and (ii) administering the additional therapeutic treatment to the subject prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0073] In another embodiment, the present disclosure provides a method of treating a subject suffering from glioma with an immunotherapy treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising the steps of: (i) administering to the subject a CAR directed against EGFRvIII of a glioma-specific TAA, an NKGD2 receptor, and an O2-mediated CAR-directed γδ T cell expressing the CAR in a treatment environment resulting from the additional therapeutic treatment, e.g., TMZ chemotherapy, that confers resistance to the O2-mediated CAR-directed EGFRvIII of a glioma-specific TAA, e.g., TMZ chemotherapy, that allows survival of γδ T cells expressing the CAR in the treatment environment resulting from the additional therapeutic treatment. 6 -methylguanine-DNA methyltransferase; and (ii) administering to the subject an additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0074] In another embodiment, the present disclosure provides a method of treating a subject suffering from cancer with an immunotherapy treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising the steps of: (i) administering to the subject a CAR directed against IL13Rα of a glioma-specific TAA, an NKGD2 receptor, and an O2-mediated CAR-directed γδ T cell expressing the CAR in a treatment environment resulting from the additional therapeutic treatment, e.g., TMZ chemotherapy, that confers resistance to the O2-mediated CAR-directed γδ T cell expressing the CAR in the treatment environment resulting from the additional therapeutic treatment. 6-methylguanine-DNA methyltransferase; and (ii) administering to the subject an additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0075] In another embodiment, the disclosure provides a method of treating a subject suffering from neuroblastoma with an immunotherapeutic treatment and an additional therapeutic treatment, e.g., utilizing TMZ chemotherapy, comprising the steps of: (i) administering to the subject a CAR directed against GD2 of a neuroblastoma-specific TAA, an NKGD2 receptor, and an O2-mediated CAR-directed γδ T cell expressing the CAR in a treatment environment resulting from the additional therapeutic treatment, e.g., TMZ chemotherapy, that confers resistance to the O2-mediated CAR-directed γδ T cell expressing the CAR in the treatment environment resulting from the additional therapeutic treatment. 6 -methylguanine-DNA methyltransferase; and (ii) administering to the subject an additional therapeutic treatment prior to administration of the cell composition to the subject, after administration of the cell composition to the subject, simultaneously with administration of the cell composition to the subject, or any combination thereof.

[0076] In certain embodiments of any of the aforementioned methods, the cell composition is any of the cell compositions described herein. In certain embodiments of any of the aforementioned methods, the cell composition is administered to the subject more than once, and the additional therapeutic treatment is administered before, after, or both before and after each administration of the cell composition. In certain embodiments of any of the aforementioned methods, the cell composition is administered to the subject more than once, and the additional therapeutic treatment is administered before, after, or both before and after the first administration of the cell composition, and optionally before, after, or both before and after each additional administration of the cell composition. In certain embodiments of any of the aforementioned methods, the cell composition can be administered to the subject once a week for 2, 3, or 4 weeks or more.

[0077] In certain embodiments of any of the aforementioned methods, the additional therapeutic treatment is administered to the subject 24-48 hours before, 24-48 hours after, or both before and after administration of the cellular composition. In certain embodiments of any of the aforementioned methods, the cellular composition is administered to the subject on day X and the additional therapeutic treatment is administered to the subject 12-72 hours before, 12-72 hours after, or both before and after day X. In certain embodiments of any of the aforementioned methods, the cellular composition is administered to the subject on day X and the additional therapeutic treatment is administered to the subject 12-72 hours before, 12-72 hours after, or both before and after day X, followed by additional administration of the cellular composition to the subject on day Y and, optionally, additional therapeutic treatment is administered to the subject 12-72 hours before, 12-72 hours after, or both before and after day Y.

[0078] In one aspect of any of the above methods, the CAR of the cell composition is specific to the cancer being treated. In one aspect of any of the above methods, the survival factor of the cell composition confers resistance to the therapeutic treatment / additional therapeutic treatment.

[0079] The cell composition of the present disclosure can be administered by any method known in the art. In one aspect of any of the aforementioned methods, the cell composition is administered intravenously. In one aspect of any of the aforementioned methods, the cell composition is administered intracranially. In one aspect of any of the aforementioned methods, the cell composition is administered intra-arterially. In one aspect of any of the aforementioned methods, the cell composition is administered directly into the tumor bed. In one aspect of any of the aforementioned methods, the cell composition is administered near or adjacent to the tumor. In one aspect of any of the aforementioned methods, the cell composition is administered by a combination of the above administration methods.

[0080] In one aspect of any of the foregoing methods, the additional therapeutic treatment (e.g., TMZ chemotherapy) provides at least one of the following advantages: increased efficacy of the cellular composition due to induction of stress-induced antigens, increased homeostatic reconstitution of the cellular composition, increased in vivo proliferation, and increased persistence of the cellular composition (compared to the same treatment but omitting the additional therapeutic treatment).

[0081] In certain embodiments, the cancer is susceptible to an additional therapeutic treatment (the cells are genetically engineered to be resistant to this therapeutic treatment). Such additional therapeutic treatments to which the cancer cells may be susceptible include, but are not limited to, anticancer drugs, antimetabolites, DNA demethylating agents, plant-derived antitumor agents, nucleoside / nucleotide analog chemotherapeutic agents, alkylating agents, antimetabolites, anticancer antibiotics, topoisomerase inhibitors, mitotic inhibitors, differentiation inducers, hormonal therapy agents, and combinations thereof. As known in the art and discussed herein, various specific agents may be used in the additional therapeutic treatment. Representative alkylating agents include, but are not limited to, cyclophosphamide, ifosfamide, and melphalan. Representative antimetabolites include, but are not limited to, methotrexate (MTX), 5-fluorouracil or derivatives thereof. Representative DNA demethylating agents (also known as antimetabolites) include, but are not limited to, azacitidine. Representative nucleoside / nucleotide analog chemotherapeutic agents include, but are not limited to, substituted nucleotides and substituted nucleosides.Representative antitumor antibiotics include, but are not limited to, mitomycin, adriamycin and doxorubicin.Representative plant-derived antitumor agents include, but are not limited to, vincristine, vindesine, TAXOL®, paclitaxel, abraxane; cisplatin; carboplatin; etoposide, etc. Representative anti-cancer drugs include, but are not limited to, trimethotrexate (TMTX), temozolomide (TMZ), raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), nitrosoureas [e.g., bis-chloronitrosourea (BCNU; carmustine), lomustine (CCNU) + / - procarbazine and vincristine (PCV regimen), doxorubicin, fotemustine, cytarabine, camptothecin, and therapeutic derivatives of any of the above.Preferably, the agents used in the additional therapeutic treatment to which the cancer cells may be sensitive include TMZ, doxorubicin, melphalan, nitrosoureas, and any combination thereof.

[0082] In certain embodiments, the cancer is resistant to an additional therapeutic treatment, the cells being genetically engineered to be resistant to the therapeutic treatment. In such embodiments, the additional therapeutic treatment provides at least one of the above advantages. In one embodiment, the additional therapeutic treatment increases the expression of a stress-induced antigen that is a ligand of the NKG2D receptor. Specific stress-induced antigens that are upregulated include, but are not limited to, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A and MIC-B.

[0083] In certain embodiments, the cancer is selected from the group consisting of brain cancer, breast cancer, prostate cancer, lung cancer, colon cancer, epithelial cancer, head and neck cancer, skin cancer, cancer of the genitourinary tract, ovarian cancer, uterine cancer, cervical cancer, kidney cancer, stomach cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gallbladder cancer, cancer of the bile duct, esophageal cancer, cancer of the salivary glands, thyroid cancer, as well as hematological malignancies, leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.

[0084] In certain embodiments, the cancer is brain tumor.In certain embodiments, the cancer is pineal tumor, pituitary tumor, PNET, Schwannoma, lymphoma, medulloblastoma, meningioma, metastatic brain tumor, neurofibroma, neuronal and mixed neuroglial tumor, oligoastrocytoma, oligodendroglioma, astrocytoma, atypical teratoid rhabdoid tumor (ATRT), chondrosarcoma, choroid plexus tumor, craniopharyngioma, ependymoma, germ cell tumor, neuroblastoma, glioblastoma (GBM) and glioma.In certain embodiments, the cancer is glioblastoma.In certain embodiments, the cancer is neuroblastoma.

[0085] In certain embodiments, tumor antigen is any tumor antigen known in the art.In certain embodiments, tumor antigen is selected so that it has at least one of the following characteristics: it is expressed in as many stages of cancer as possible; it is expressed on the surface of tumor; it is important for tumor cell viability; and it is not expressed on non-tumor tissue or is expressed at such a level that off-target effect is clinically acceptable. In certain embodiments, the tumor antigen is selected from the group consisting of EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, ROR1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA-125, glypican-3, epithelial tumor antigen, melanoma associated antigen, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, folate receptor alpha, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV- 1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD23, CD33, CD30, CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL-11Rα, IL-13Rα, CSPG4, Lewis-Y, MCSP, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Sp17, SURVIVIN, TAG72, TEM1, TEM8, EGFRvIII, and VEGFR2.

[0086] In certain embodiments, the glioma-specific TAA is any glioma-specific TAA known in the art. In certain embodiments, the glioma-specific TAA is selected so that at least one of the following characteristics is present: the glioma-specific TAA is expressed in as many stages of cancer as possible; the glioma-specific TAA is expressed on the surface of the tumor; the glioma-specific TAA is important for tumor cell viability; and the glioma-specific TAA is not expressed on non-tumor tissue or is expressed at a level such that off-target effects are clinically acceptable. In certain embodiments, the glioma-specific TAA is selected from the group consisting of: NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, GD2, EGFRvIII and IL13Rα. In certain embodiments, the glioma-specific TAA is EGFRvIII. In certain embodiments, the glioma-specific TAA is IL13Rα.

[0087] In certain embodiments, the neuroblastoma-specific TAA is any glioma-specific TAA known in the art.In certain embodiments, the neuroblastoma-specific TAA is selected so that at least one of the following characteristics is present: the neuroblastoma-specific TAA is expressed in as many stages of cancer as possible; the neuroblastoma-specific TAA is expressed on the surface of tumor; the neuroblastoma-specific TAA is important for tumor cell viability; and the neuroblastoma-specific TAA is not expressed on non-tumor tissue or is expressed at such a level that off-target effects are clinically acceptable.In certain embodiments, the neuroblastoma-specific TAA is selected from the group consisting of: NKG2D ligand, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, and GD2.In certain embodiments, the neuroblastoma-specific TAA is GD2.

[0088] DRI genetic engineering, for example, the use of the MGMT gene, allows the cell composition of the present disclosure to function in a chemotherapy-rich environment at a time when the tumor will be maximally stressed. The stress effect on the tumor, in certain embodiments, increases the expression of stress antigens, which are recognized by receptors on gamma delta T cells, such as the NKG2D receptor. The dual effect of induction of stress antigens by chemotherapy and reduction of regulatory T cells greatly improves tumor reduction compared to either individual regimen. The genetic modification protects the cell composition of the present disclosure from the lymphodepleting effect of chemotherapy regimens, such as TMZ, and allows the cell composition of the present disclosure to specifically access the tumor via TAA, in combination with the cytotoxic function of intact T cells, when malignant cells are maximally stressed by chemotherapy. Use of the cell compositions of the present disclosure (referred to as DRI-CAR T cell therapy) is believed to significantly prolong survival and reduce tumor burden when compared to chemotherapy (e.g., TMZ) treatment alone or γδ T cell infusion alone, and to do so without significant adverse systemic or neurological effects.

[0089] The rationale for the disclosed cell compositions and methods is based on previous studies showing that: (a) human GBM cells are highly susceptible to attack by ex vivo expanded / activated γδ T cells (1, 2); (b) local intracranial injection of expanded / activated γδ T cells prolongs survival of tumor-bearing mice by slowing progression of GBM xenografts (3) and GL261-derived tumors in an immunocompetent C56BL / 6 mouse model; (c) TMZ-resistant clones of human SNB-19 and U373 GBM cell lines are killed by MGMT-modified γδ T cells in the presence of TMZ with significantly higher potency than either TMZ or γδ T cells alone (4); and, most importantly, (d) mice bearing either unmodified or TMZ-resistant clones of primary human GBM xenografts show improved survival when treated with intracranial injection of genetically modified γδ T cells compared to either treatment alone. Thus, a cell composition of the disclosure comprising γδ T-cells engineered to confer resistance to an additional therapeutic treatment (e.g., TMZ) is expected to significantly prolong survival and reduce tumor burden compared to either treatment separately, and would do so without significant adverse systemic or neurological effects.

[0090] Several recent studies have demonstrated that strategic timing of chemotherapy and immunotherapy for extracranial solid neoplasms takes advantage of the innate response to chemotherapy-induced expression of stress-associated antigens on tumor cells (8-10) and depletion of regulatory T cells. The dual effect of inducing stress antigens and depleting regulatory T cells can result in greatly improved tumor reduction over either regimen individually (11-17). The successful realization of this strategy for cancers, including but not limited to GBM, represents a significant departure from classical approaches that have relied on adaptive recognition of tumor-associated antigens (18, 19), glioma cell MHC class I expression (20, 21), or lymphokine-activated killer (LAK) cell therapy (22-26).

[0091] Applicant has previously developed a robust system for generating expanded / activated γδ T cells that reliably target high-grade gliomas via stress-induced antigens (i.e., NKG2D ligands) expressed by the tumor (3, 27, 28). Both in vitro cytotoxicity and increased survival / progression-free time in specific in vivo models designed to recapitulate therapeutic conditions have been shown (1, 2). Applicant has shown that TMZ transiently upregulates expression of NKG2D ligands on the surface of glioma cells, rendering tumors more susceptible to recognition by γδ T cells (4, 8), and killing of TMZ-resistant glioma lines is enhanced by MGMT gene-modified γδ T cells in the presence of therapeutic concentrations of TMZ. Through the use of the cell compositions and methods of the present disclosure, the timing of cellular immunotherapy and chemotherapy can be tailored to maximize the effects of both. Thus, the present disclosure not only provides novel cell compositions as described, but also provides novel anti-glioblastoma therapeutic strategies, providing previously unexplored avenues for treating high-grade gliomas.

[0092] Although the cell compositions of the present disclosure can be administered directly to the tumor bed via a surgically placed catheter (e.g., a ventriculostomy catheter), the time interval between resection and increased TMZ dosage can be up to 8 weeks, exposing the patient to potential risks of infection or additional procedures to place the catheter at the time of administering the cell therapy. The CARs incorporated into the described cell compositions are designed to target tumor-associated antigens (e.g., glioma-specific tumor-associated antigens) to concentrate DRI-CAR T cells in the tumor when administered to the patient via various administration methods, allowing various administration methods to be used. For example, when the cell compositions are administered to the patient via intravenous or intra-arterial routes, the CARs incorporated into the described cell compositions help to increase the infiltration of DRI-CAR T cells into the tumor bed, allowing for an increased concentration of DRI-CAR T cells in the tumor bed. Similarly, when administered by other means (e.g., directly to the tumor bed via a Rickham catheter or similar device, or by intraventricular administration), the CARs incorporated into the described cell compositions allow the same results to be achieved. Activation and cytotoxicity are mediated through CAR and / or by γδ T cell recognition of NKG2D ligands, a strategy that is particularly important in heterogeneous tumors that do not express TAAs at sufficient density to activate T cells through CAR. Importantly, this therapy is administered during high-dose chemotherapy, when tumor cells are highly stressed, regulatory T cells are depleted, other tumor-associated immune defenses are compromised, and vascular beds are more permeable. TMZ-mediated lymphodepletion also favors homeostatic reconstitution, in vivo expansion, and persistence of DRI-CAR T cells.

[0093] The conventional method of immunotherapy and the improved method of the present disclosure are outlined in Figures 8A and 8B, respectively. In the conventional method, immunocompetent cells were generated by cytokine treatment and other methods. The immunocompetent cells themselves showed tumoricidal activity. When additional treatments, such as chemotherapy regimens, were administered, the immunocompetent cells were depleted, limiting the effectiveness of the treatment due to the death of the therapeutic cells. The method of the present disclosure overcomes these limitations by providing a cell composition, such as, but not limited to, γδ T cells, that includes a tumor-specific CAR and survival factor (DRI-CAR). When combined with additional therapeutic treatments, such as chemotherapy regimens, the DRI-CAR cells can survive the treatment environment while retaining the ability to attack tumor cells through the recognition of stress-inducing ligands by the present CAR. Furthermore, the reduction of regulatory T cells provides an additional therapeutic advantage. Furthermore, lymphodepletion via chemotherapy regimens favors homeostatic reconstitution, in vivo expansion and persistence of DRI-CAR T cells, further improving the treatment outcome. EXAMPLES

[0094] Example 1 – Development and in vitro testing of genetically modified γδ T cells: To determine the vector configuration that results in optimal transgene expression and to determine the optimal transduction timing for generating genetically modified γδ T cells, lentiviral vectors are generated that co-express the cDNA sequence of a CAR and a cDNA sequence of a survival factor that confers drug resistance, harboring either EGFRvIII or IL13Rα (DRI-CAR) to quantify optimal delivery and persistence. 111 Engineered γδ T cell immunotherapy using [In]-labeled γδ T cells will be evaluated by comparing the potency, durability, and efficacy of intracranial versus intravenous routes of administration against patient-derived glioma xenolines during TMZ chemotherapy in immunodeficient mice.

[0095] A vector is generated that encodes IL13Rα and EGFRvIII CAR, which are known to recognize glioma cells and target glioblastoma (GBM). In addition, a cDNA encoding MGMT, which confers resistance to TMZ, is co-expressed with the CAR. Also, a control vector is generated that encodes a flow or imaging marker for optimal analysis of gene transfer and tracking of genetically modified cells. An exemplary construct encoding EGFRvIII CAR is shown in Figure 1.

[0096] Our previous studies have validated that the optimal transduction window for inserting the simple SIV-encoded p140K-MGMT vector is 3-7 days after initiation of zoledronic acid / IL2-based γδ T cell expansion cultures. Under these conditions, sufficient MGMT copy numbers to confer resistance to TMZ concentrations above 400 μM occurred at an MOI as low as 5. These conditions will be validated and optimized for DRI-CAR transduction using transduction windows and MOIs spanning days 2-7 and a matrix of MOIs from 1-25.

[0097] Table 1: Experimental design TIFF0007662588000001.tif53136

[0098] Intracranial glioma xenografts consisted of parental clones (denoted "P") and TMZ-resistant clones (denoted "T") of GBM-X12 (classical), GBM-X22 (mesenchymal), GBM-X1066 (neural), and GBM-XD456 (proneural) explants from primary GBMs. Mice were inoculated with 1.5 × 10 6 cells / kg (intracranial) or 5 × 10 8 Cells / kg (intravenous) 111 [In]labeled DRI-CAR γδ T cells are injected and various treatments are evaluated based on the experimental design shown in Table 1 (IC = intracranial; IP = intraperitoneal; IV = intravenous) and Figure 2. Imaging is performed at 24-h intervals for a minimum of 3 days after injection by X-SPECT as modeled for breast cancer in Figure 3. Biodistribution studies are also performed using 5 × 108 Cells / kg 111 [In]-labeled γδ T cells / kg were injected and performed separately 24 hours later, with counts of major tissues and tumors and results reported as % 111 [In] is expressed as injected dose (ID) / g. Tumor localization of γδ T cells is quantified by the accumulated radioactivity in the tumor, derived from a color scale associated with the images and expressed (by convention) as a percentage of the injected radiation dose seen per gram of tumor. Intravenous as well as intracranial administration is expected to be effective.

[0099] The potency of each DRI-CAR product is determined using an in vitro cytotoxicity assay against four selected xenogeneic lineages. Efficacy is assessed by comparing the survival rate of tumor-bearing mice treated based on the experimental design shown in Table 1 and Figure 2 with untreated controls. Tumor and normal tissues are harvested for histological and functional evaluations including tumor histopathology, tumor infiltration, lymphocyte infiltration, and lymphocyte immunophenotype. Statistical analysis is performed utilizing non-parametric t-tests and / or ANOVA for migration and distribution studies, and non-parametric log-rank analysis of survival data. Projecting a difference of 0.15 between treated and control groups, 20 replicates / group provides 80% power to detect differences at p≦0.05.

[0100] Example 2 - Evaluation of the safety of DRI-CAR therapy under conditions likely to be prevalent during treatment (e.g., prior radiation therapy, TMZ) The disclosed cell compositions and methods will be tested for potential localized off-target cytotoxicity using an in vitro toxicity assay of DRI-CAR against cultured human astrocytes. In addition, it will be determined whether γδ T cells expressing a suicide gene confer a safety benefit that is deemed favorable. Analysis of the integration site of the vector will also be performed. Since no comparable animal model exists for Vγ9Vδ2 T cells, an in vitro toxicity analysis will be performed. DRI-CAR T cells (20 replicates) will be tested against cultured human astrocytes exposed to either 250 cGy of ionizing radiation or 4 h incubation in 200 μM TMZ and compared to untreated astrocytes by evaluation of NKG2D ligand expression and DRI-CAR cell-mediated cytotoxicity. Initial studies show only slight upregulation of MIC-A and ULBP2, and no significant γδ T cell cytotoxicity against cultured astrocytes.

[0101] To protect against cascading inflammatory responses or off-target cytotoxicity, suicide genes are evaluated for incorporation into the CAR gene product. The gamma-retrovirus, SFG.iCaspase9.2A.DeltaCD19, consists of iC9 linked via a 2A-like sequence to a truncated human CD19 that serves as a selection marker. AP1903-induced activation of the caspase 9 suicide gene is achieved by expressing a chimeric protein (iC9) fused to a drug-binding domain derived from human FK506-binding protein (FKBP). iC9 is quiescent in cells until exposed to AP1903, which crosslinks the FKBP domain and initiates iCasp9 signaling to induce apoptosis of genetically modified cells. The gene and AP1903 are supplied by Bellicum Pharmaceuticals (Houston, TX), which also supports functional studies. The use of this construct has been previously described ( 6 , 7 ) and will be used to perform functional studies in NSG mice aiming to optimize dosing to induce rapid apoptosis of systemic and intracranial DRI-CAR T cells.

[0102] Lentiviral site integration analysis will be initially performed in our laboratory using the Lenti-X™ system (Clontech) using Genome Walker technology.

[0103] Example 3 – Standardization of cell manufacturing and shipping standards for use of DRI-CAR in human therapy Cytotoxicity (potency and toxicity) assays, flow cytometric graft composition analysis, and infectious disease testing for cGMP cell manufacturing will enable transition to clinical trials. Cellular and gene therapy products require release criteria for sterility, purity, identity, and potency as set forth by the FDA in 21 CFR 211.165 and 610. FDA mandates sterility testing procedures. Identity testing is accomplished by clinical HLA typing. Purity / composition and potency testing of cellular products will require standardization in our laboratories. FDA regulations 21 CFR 600.3 require that the specific ability of a product to function as labeled should be established by appropriate testing, and 21 CFR 610.10 clarifies that evaluation of potency should consist of in vitro and / or in vivo tests specifically designed for the product. The FDA also recommends that cytotoxicity correlate with target phenotype; therefore, cytotoxicity will be validated using defined target glioma cell lines of consistent passage number as a standard for evaluating DRI-CAR graft function against clinical requirements set forth in the Clinical Laboratory Improvement Act of 1988 (CLIA) for Laboratory Developed Tests. Optimal function is tentatively defined as 50% killing at an E:T (effector:target) ratio of 20:1. Cultured astrocytes serve as negative targets and K562 erythroleukemia cell targets serve as positive targets. Final purity and composition are assessed by flow cytometry for total T cells (CD3), γδ T cells, αβ T cells (CD3+CD4+γδ- and CD3+CD8+γδ-)%, NK cells (CD16 / 56)%, B cells (CD19) and monocytes (CD14). Target release criteria are γδ T cells ≥ 60%, αβ T cells ≤ 5% and NK cells ≤ 25%. Statistical analysis consisted of establishing the mean, SD and 95% CI for each individual cell phenotype in the purity / composition analysis and for each killing % for each E:T ratio in the functional assays, over a minimum of 20 assays.ANOVA is used to compare differences in individual E:T ratios between groups. In vitro cytotoxicity functions, as well as migration studies and survival outcomes in animal models are incorporated.

[0104] Example 4 – CAR-DRI as post-resection cell therapy in three patients with primary glioblastoma in a phase 0 clinical trial 111 Assessment of safety, toxicity, trafficking and migration of [In]-labeled γδ T cells To confirm TMZ resistance under clinical conditions and migration of residual microscopic disease to occult areas. 111 A small number of initial experiments with [In]labeled DRI-CAR T cells will be performed. The study will be conducted as a phase 0 clinical trial with toxicity being the primary item of interest. Eligible patients will be admitted sequentially and will receive one infusion. The frequency of side effects will be summarized by body tissue, grade and causality by using frequency and percentages. A list of individual subjects will be provided, including the above information plus the test date of symptom onset and the length of the adverse event. No statistical comparisons will be performed. Secondary objectives include monitoring the time to progression, the presence of early and late systemic hematopoietic chimerism, systemic immune function and overall survival for the purposes of hypothesis generation and design of phase I and II clinical trials. Up to three evaluable patients consisting of adult males and non-pregnant females aged 18 years or older undergoing resection of histologically confirmed primary GBM will be enrolled. Patients will receive one leukapheresis therapy after resection and before the initiation of RT (radiotherapy) + TMZ therapy for DRI-CAR production. 1 × 10 6 A single dose of DRI-CAR T cells at 100 / kg will be administered during the high-dose TMZ therapy following the rest period at the end of the RT+TMZ combination therapy. A portion of the cell preparation will be used to track cell migration. 111 Single-photon emission computed tomography (SPECT) was performed 12, 24, and 48 hours after injection. All attenuation-corrected 111[In]oxine-labeled leukocytes planar and SPECT images will be reviewed. Individual patients will be followed for at least 30 days for toxicity before enrolling the next patient. Using the Cancer Therapy Evaluation Program Common Toxicity Criteria for grading adverse events, grade 3 or 4 toxicity involving the liver, lungs, and heart, or other grade 4 toxicity will be defined as dose-limiting toxicity (DLT) if the toxicity is considered possibly related to the DRI-CAR T cells. Important additional events considered DLTs (if they are possibly related) include death, stroke, hematoma requiring surgery, untreatable neurological regression, refractory systemic infection, and graft-versus-host disease. Putative off-target toxicity, or intracranial events such as inflammation or leukoencephalopathy, will trigger AP1903 administration to activate apoptosis of DRI-CAR T cells via the iC9 suicide gene.

[0105] Example 5 –Expression of stress-induced antigens Selected stress-inducible ligands (NKG2DLs) in glioma xenografts are upregulated upon exposure to TMZ (Figure 4A). Figure 4 shows the expression of NKG2DLs MIC-A, MIC-B, ULBP-1, ULBP-2, ULBP-3 and ULBP-4 in X12T and X22T glioma xenografts. X12T glioma xenografts show expression of MIC-A, ULBP-1 and ULBP-4 with minimal upregulation after treatment with 400 μM TMZ for 4 hours. X22T glioma xenografts show expression of MIC-A, MIC-B, ULBP-1 and ULBP-4 with upregulation of MIC-A and ULBP-4 after treatment with 400 μM TMZ for 4 hours. Ex vivo expanded / activated γδ T cells were effective in killing cells derived from X12T and X22T glioma xenografts, as shown in Figure 4B. γδ T cells engineered to express survival factors for TMZ resistance were cultured with cells derived from X12T and X22T glioma xenografts at increasing effector:target (E:T) ratios, and percent lysis (determined by the ratio of live to dead cells measured by flow cytometry) was expressed as a function of E:T ratio. At E:T ratios greater than 10:1, γδ T cells were cytotoxic to cells derived from X12T and X22T glioma xenografts. γδ T cells did not show any evidence of toxicity to cultured human astrocytes at an E:T ratio of 20:1.

[0106] Example 6 – Efficacy of γδ T cell therapy is increased by expression of stress-induced antigens Expression of NKG2DL on tumor cells enhances the overall efficacy of γδ T cell therapy. Figures 5A and 5B show the survival rates of X12P and X12T (TMZ-resistant) glioma-bearing mice, respectively, in response to various treatments. Figure 5A shows the survival rates of X12P glioma-bearing mice treated with TMZ alone (TMZ), X12P glioma-bearing mice treated with a composition comprising γδ T cells and NK cells modified to express survival factors for TMZ resistance in combination with TMZ (DRI), mice receiving unmodified γδ T cells and NK cells (CT), and control mice receiving no treatment. The TMZ treatment regimen (blue) and the DRI treatment regimen (red) increased the survival rate of X12P glioma-bearing mice compared to the CT treatment regimen (green) and untreated mice (black) (p<0.001). The DRI treatment regimen increased the median survival time from 57 days to 75 days relative to the TMZ treatment regimen. FIG. 5B shows the survival rate of X12T glioma-bearing mice treated as described in FIG. 5A. The DRI treatment regimen (red) increased the survival rate of X12T glioma-bearing mice compared to untreated mice (black) (p=0.0147). The TMZ treatment regimen did not improve the survival rate of X12T glioma-bearing mice compared to untreated mice, as expected. The DRI treatment regimen increased the median survival time from 22 to 27 days compared to the TMZ treatment regimen. The effect of the DRI treatment regimen could be enhanced by increasing the administration of γδ T cells and NK cells from once a week (shown in FIG. 2) to twice a week (data not shown). With this modified administration, the DRI treatment regimen increased the survival rate of X12T glioma-bearing mice compared to untreated mice (p=0.0004) and increased the median survival time from 22 to 38 days compared to the TMZ treatment regimen.

[0107] Example 7 – Stress-induced antigens are not upregulated in normal brain tissue Observation of irradiated metastatic brain tumor tissues and adjacent normal brain tissues reveals that stress-induced antigens (NKG2D ligands) are upregulated in tumor tissues, whereas they are essentially not expressed in surrounding normal (i.e., non-tumor) brain tissues (Figure 6). Figure 6 shows the expression of stress-induced antigens from irradiated metastatic brain tumor tissues and adjacent normal brain tissues. Metastatic brain tumor tissues showed strong expression of ULBP-1 and weak expression of ULBP-2 and ULBP-3. No stress-induced antigens were observed in adjacent normal brain tissues. Therefore, the possibility of off-target effects and resulting damage to normal brain tissues by the cell compositions described herein is low.

[0108] Example 8 – Stress-induced antigens are not upregulated by radiation treatment Similarly, stress-induced antigens (NKG2D ligands) are not upregulated in irradiated mouse brains, nor are other signs of inflammation (Figure 7). Figure 7 shows whole brains of WT C56BL / 6 mice irradiated with a set of 160 kV X-ray beams in the anterior-posterior and posteroanterior directions at a dose of 17 Gy (panels b, d) and non-irradiated control mice (panels a, c). Mice were sacrificed on day 70 and brains were removed. Panels a and b compare stress antigen MULT-1 staining between control and irradiated mice. Panels c and d compare staining with anti-CD3 between control and irradiated mice. The irradiated mice were negative for both MULT-1 and anti-CD3, indicating that stress-induced antigens are not upregulated in normal brain tissue in response to radiation treatment, and that lymphocytic infiltration is not induced in normal brain tissue in response to radiation treatment.

[0109] References TIFF0007662588000002.tif195146TIFF0007662588000003.tif226144TIFF0007662588000004.tif221148TIFF0007662588000005.tif242147

Claims

1. A pharmaceutical for treating a tumor by administering to a subject suffering from a tumor, the pharmaceutical comprising a cell composition comprising γδ T cells expressing at least an NKG2D receptor, a polypeptide that confers resistance to a chemotherapeutic agent, and a chimeric antigen receptor (CAR) directed against a tumor antigen, the pharmaceutical comprising: γδ T cells are present at 60% or more of the total cell population of the cell composition as measured by flow cytometry; the endodomain of the CAR comprises only one signaling domain, the one signaling domain being a CD3ζ signaling domain or a costimulatory signaling domain; Chemotherapeutic agents increase the expression of NKG2D ligands on tumors, the polypeptide that confers resistance to a chemotherapeutic agent is O 6 -methylguanine-DNA methyltransferase, multidrug resistance protein 1, 5'-nucleotidase II, a drug-resistant mutant of dihydrofolate reductase (L22Y-DHFR), or thymidylate synthase; A pharmaceutical agent characterized in that it is used in combination with a chemotherapeutic agent administered to a subject before administration of a cell composition to a subject, after administration of a cell composition to a subject, simultaneously with administration of a cell composition to a subject, or any combination thereof.

2. The pharmaceutical composition according to claim 1, characterized in that the cell composition is administered to a subject at least once additionally.

3. The pharmaceutical of claim 1 or 2, wherein the chemotherapeutic agent is optionally administered prior to administering at least one additional dose of the cell composition to the subject, after administering at least one additional dose of the cell composition to the subject, simultaneously with administering at least one additional dose of the cell composition to the subject, or any combination thereof.

4. The pharmaceutical agent of any one of claims 1 to 3, wherein the cell composition is administered to the subject on day X, and the chemotherapeutic agent is administered to the subject 12 to 72 hours before day X, 12 to 72 hours after day X, or both 12 to 72 hours before and after day X.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the cell composition is administered to the subject on day X, and the chemotherapeutic agent is administered to the subject 12 to 72 hours before day X, 12 to 72 hours after day X, or both 12 to 72 hours before and after day X, followed by an additional administration of the cell composition to the subject on day Y, and optionally, the chemotherapeutic agent is administered to the subject 12 to 72 hours before day Y, 12 to 72 hours after day Y, or both 12 to 72 hours before and after day Y.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the tumor is selected from the group consisting of brain cancer, breast cancer, prostate cancer, lung cancer, colon cancer, epithelial cancer, head and neck cancer, skin cancer, cancer of the genitourinary tract, ovarian cancer, uterine cancer, cervical cancer, kidney cancer, stomach cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gallbladder cancer, cancer of the bile duct, esophageal cancer, cancer of the salivary glands, thyroid cancer, and hematological malignancies.

7. The pharmaceutical agent according to any one of claims 1 to 5, wherein the tumor is selected from the group consisting of pineal tumor, pituitary tumor, PNET, Schwannoma, lymphoma, medulloblastoma, meningioma, metastatic brain tumor, neurofibroma, neuronal and mixed neuronal-glial tumors, oligoastrocytoma, oligodendroglioma, astrocytoma, atypical teratoid rhabdoid tumor (ATRT), chondrosarcoma, choroid plexus tumor, craniopharyngioma, ependymoma, germ cell tumor, neuroblastoma, glioblastoma and glioma.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the tumor is sensitive to a chemotherapeutic agent.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the tumor is resistant to a chemotherapeutic agent.

10. Tumor antigens include EphA2, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD22, CD19, CD20, ROR1, kappa or light chain, carcinoembryonic antigen, alpha-fetoprotein, CA-125, glypican-3, epithelial tumor antigen, melanoma-associated antigen, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, PAP, FAR, FBP, fetal AchR, folate receptor alpha, mutated p53, mutated ras, HER2, ERBB2, HER3, folate binding protein, HIV-1 envelope glycoprotein gp120 ... The pharmaceutical agent of any one of claims 1 to 9, wherein the antibody is selected from the group consisting of peptide glycoprotein gp41, 5T4, 8H9, GD2, CD123, CD23, CD33, CD30, CD38, CD56, c-Met, fap, mesothelin, GD3, HERV-K, IL-11Rα, IL-13Rα, CSPG4, Lewis-Y, MCSP, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, EGFR, Sp17, SURVIVIN, TAG72, TEM1, TEM8, EGFRvIII, and VEGFR2.

11. The pharmaceutical agent according to any one of claims 7 to 10, wherein the tumor is glioblastoma and the tumor antigen is selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B, EGFRvIII and IL13Rα.

12. The pharmaceutical composition according to claim 11, wherein the tumor is glioblastoma and the tumor antigen is EGFRvIII or IL13Rα.

13. The pharmaceutical agent according to any one of claims 7 to 10, wherein the tumor is neuroblastoma and the tumor antigen is selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A, MIC-B and GD2.

14. The pharmaceutical composition of claim 13, wherein the tumor is neuroblastoma and the tumor antigen is GD2.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the cell composition further comprises at least one of αβT cells and NK cells.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the cell composition further comprises αβ T cells and NK cells.

17. The pharmaceutical composition of claim 16, wherein the cell composition comprises 5% or less of αβ T cells and 25% or less of NK cells.

18. 18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the chemotherapeutic agent is a nucleoside analogue, an alkylating agent, an antimetabolite, an antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a differentiation inducer, or a hormone therapy.

19. The chemotherapeutic agent is an alkylating agent, and the polypeptide is O 6 The pharmaceutical agent according to claim 1 or 18, which is -methylguanine-DNA methyltransferase.

20. The chemotherapeutic agent is temozolomide and the polypeptide is O 6 The pharmaceutical agent according to claim 1 or 18, which is -methylguanine-DNA methyltransferase.

21. The pharmaceutical of any one of claims 1 to 20, wherein the γδ T cells are genetically engineered to express a CAR directed against a tumor antigen and a polypeptide that confers resistance to the chemotherapeutic agent, and the NKG2D receptor is naturally present on the γδ T cells.

22. The pharmaceutical agent of any one of claims 1 to 20, wherein the γδ T cells are genetically engineered to express a CAR directed against a tumor antigen, a polypeptide that confers resistance to the chemotherapeutic agent, and an NKG2D receptor.

23. The pharmaceutical agent according to any one of claims 1 to 22, wherein the γδ T cells further express a suicide gene.

24. The pharmaceutical agent according to any one of claims 1 to 23, wherein the NKG2D ligand is selected from the group consisting of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, MIC-A and MIC-B.

25. The pharmaceutical agent of any one of claims 1 to 24, wherein the γδ T cells are obtained from a subject and expanded ex vivo before being genetically engineered to express the CAR and the polypeptide that confers resistance to the chemotherapeutic agent.

26. The pharmaceutical of any one of claims 1 to 25, wherein said one signaling domain is a CD3ζ signaling domain.

27. 26. The pharmaceutical of any one of claims 1 to 25, wherein said one signaling domain is a costimulatory signaling domain.

28. The pharmaceutical agent of claim 27, wherein the costimulatory signaling domain is selected from the group consisting of CD28, 41BB, DAP10, OX40 and ICOS.

29. 7. The pharmaceutical of claim 6, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.

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