Compositions and methods for treating cancer

Abstract

The present invention provides combination therapies for treating cancer, including compositions and methods for γδ T cell immunotherapy in combination with DDR inhibitors, including but not limited to PARP inhibitors. Preferably, the combination of γδ T cell immunotherapy and PARP inhibitors for treating cancer further includes a combination with other immunotherapy, such as immune checkpoint (ICP) blockade therapy, and / or a DNA-damaging agent, such as a cytotoxic chemotherapeutic agent. Preferably, when the combination of γδ T cell immunotherapy and DDR inhibitor therapy further includes a chemotherapeutic agent, the γδ T cells are genetically modified to confer resistance to the chemotherapeutic agent.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 757,383, filed November 8, 2018. The entire teachings of the prior application are incorporated herein by reference. [Background technology]

[0002] Gamma delta (γδ) T cells are an important subset of T lymphocytes because they can recognize a wide range of antigens without antigen priming and the presence of major histocompatibility complex (MHC) molecules. γδ T cells can attack target cells directly through their cytotoxic activity or indirectly through the activation of other immune cells. The functional response of γδ T cells is induced by several factors, including the recognition of stress antigens, which promote cytokine production in response to stress and regulate pathogen clearance, inflammation, and tissue homeostasis.

[0003] Both human γδ T-cell subsets exhibit cytotoxic capabilities that are induced, for example, via the expression of cell surface receptors (i.e., γδ TCR (T cell receptor) and NKG2D (natural killer group 2D) and are mediated in part by the release of soluble mediators (i.e., perforin and granzymes). γδ T-cells can produce the potent antimicrobial protein granulysin and express ligands such as CD95L and tumor necrosis factor-related apoptosis-inducing ligand that engage several death receptors on target cells. In addition, γδ T-cells can indirectly kill their cellular targets via antibody-dependent cellular cytotoxicity (ADCC) in a CD16-dependent mechanism. Other molecules such as DNAM-1 (DNAX accessory molecule 1), leukocyte function-associated antigen 1, and the costimulatory receptor CD27 are also involved in γδ T-cell activation and cytotoxicity.

[0004] Human γδ T cells can also display antigen-presenting capabilities. Similar to dendritic cells (DCs), blood Vγ9Vδ2 T cells respond to signals from microorganisms and tumors and express CD4 + T cells and CD8 + γδT-APCs can prime T cells. + It is believed that γδ T cells directly cross-present tumor antigens to T cells. Intracellular protein degradation and endosomal acidification are significantly delayed in γδ T cells compared to monocyte-derived DCs. Antigens are transported across IRAP (insulin-regulated aminopeptidase)-positive early and late endosomes, and antigen processing consists of export to the cytosol for degradation by the proteasome before import into MHC-I-loaded compartments. Activated γδ T cells phagocytose tumor antigens and apoptotic or live cancer cells, possibly via the scavenger receptor CD36 in a C / EBPα (CCAAT / enhancer binding protein α)-dependent mechanism, and express tumor antigen-specific CD8 + γδ T cells can initiate T cell responses. γδ T cells can also induce DC maturation via TNF-α production. Overall, γδ T cells can process a wide range of antigens for presentation and stimulate other immune cells. Therefore, the intimate involvement of γδ T cells in the response to infection or cancer can be exploited to design new strategies to improve the clinical response of human γδ T cell-based immunotherapy.

[0005] Poly(ADP-ribose) polymerase 1 (PARP-1) has been closely implicated in multiple cellular processes such as DNA damage repair (DDR), apoptosis, and genomic stability. Inhibitors of the DDR, including but not limited to PARP inhibitors, result in genotoxic stress, local antigen release, and other immune mechanisms that result in systemic antitumor responses, including recruitment of natural killer (NK) cells and CD8+ cells and γδT cells. The genotoxic stress and stalling of DNA replication forks resulting from DDR repair inhibition using agonists such as PARP inhibitors is believed to induce expression of ligands for the NKG2D receptor. It is also believed that the genomic instability resulting from DDR repair inhibition results in increased tumor mutation burden, neoantigens, and / or activation of the stimulation of interferon genes (STING) pathway, as well as an overall increase in tumor immunogenicity.

[0006] The increased tumor immunogenicity resulting from DDR inhibition (e.g., increased upregulation of ligands for the NKG2D receptor) uniquely aids in γδ T cell-mediated tumor immunosurveillance and, ultimately, tumor cell killing by γδ T cells. The combined action of DDR inhibition with human γδ T cell-based immunotherapy offers many other clinical benefits. For example, PARP inhibitors delivered by oral or intravenous administration to patients are associated with symptoms such as nausea and fatigue, which can be particularly troublesome and affect quality of life. Also, possible interruptions of PARP inhibitors may be indicated. Human γδ T cell-based immunotherapy in combination with PARP inhibitors can avoid the toxicity associated with PARP inhibitors by lowering the dose required for PARP inhibitors while enhancing tumor killing. Summary of the Invention

[0007] The present invention provides combination therapy for treating cancer, including compositions and methods for γδ T cell immunotherapy in combination with DDR inhibitors, including but not limited to PARP inhibitors (PARPi). Preferably, the combination of γδ T cell immunotherapy and PARP inhibitors for treating cancer further includes combination with other immunotherapy, such as immune checkpoint (ICP) blockade therapy and / or therapy with DNA damaging drugs, such as cytotoxic chemotherapy drugs. Preferably, when the combination of γδ T cell immunotherapy and DDR inhibitor therapy further includes one or more chemotherapy drugs, the γδ T cells are genetically modified to confer resistance to one or more chemotherapy drugs used in the therapy.

[0008] Preferably, the present invention provides a method for the treatment of cancer in a patient in need thereof, comprising: i) administering to the patient a composition comprising a therapeutically effective amount of γδ T cells genetically engineered to be resistant to at least one chemotherapeutic agent; ii) administering to the patient a therapeutically effective amount of the chemotherapeutic agent; and iii) administering to the patient a therapeutically effective amount of a DDR inhibitor. Preferably, the DDR inhibitor is a PARP inhibitor. Preferably, the PARP inhibitor is administered prior to administration of the chemotherapeutic agent and prior to administration of the composition comprising the genetically engineered γδ T cells. Preferably, the PARP inhibitor is administered about 1 to about 21 days prior to administration of the chemotherapeutic agent. Preferably, the genetically engineered γδ T cells are administered about 8 to about 72 hours after administration of the chemotherapeutic agent. Preferably, the genetically engineered γδ T cells are administered about 12 to about 36 hours after administration of the chemotherapeutic agent. Preferably, the genetically engineered γδ T cells are administered about 24 hours after administration of the chemotherapeutic agent. Preferably, the chemotherapeutic agent is an alkylating agent, antimetabolite, a DNA demethylating agent, a substituted nucleotide, a substituted nucleoside, an antitumor antibiotic, a plant-derived antitumor agent, or a nitrosourea. Preferably, the chemotherapeutic agent is selected from cisplatin, carboplatin, etoposide, methotrexate (MTX), trimethotrexate (TMTX), temozolomide, dacarbazine (DTIC), raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (alanose), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)), cytarabine, camptothecin, and therapeutic derivatives of any of these. Preferably, the γδ T cells express alkylguanine transferase (AGT), P140KMGMT, O 6The γδ T-cells are genetically modified to encode methylguanine DNA methyltransferase (MGMT), L22Y-DHFR, thymidylate synthase, dihydrofolate reductase, or multidrug resistance type 1 protein (MDR1). Preferably, the γδ T-cells are genetically modified to be resistant to at least two chemotherapeutic agents selected from alkylating agents, antimetabolites, DNA demethylating agents, substituted nucleotides, substituted nucleosides, antitumor antibiotics, plant-derived antitumor agents, and nitrosoureas. Preferably, the γδ T cells are treated with cisplatin, carboplatin, etoposide, methotrexate (MTX), trimethotrexate (TMTX), temozolomide, dacarbazine (DTIC), raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), nitrosourea (rabinopyranosyl-N-methyl-N-nitrosourea (alanose), carmustine (BCNU), The cancer cells are genetically modified to be resistant to at least two chemotherapeutic agents selected from the group consisting of , BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin), cytarabine, camptothecin, and therapeutic derivatives of any of these. Preferably, the chemotherapeutic agent is TMZ, methotrexate, DTIC, BCNU, CCNU, MCNU, NMU, or ENU. [Brief description of the drawings]

[0009] [Figure 1] Four line graphs are shown comparing expression of natural killer group 2D ligand (NKG2DL) at selected time points on LN229 glioblastoma cell lines exposed for 4 hours to olaparib (5 mM or 10 mM) alone, olaparib (an exemplary PARP inhibitor) + 200 mg / mL TMZ, or media alone (RPMI + 10% FBS media) with viability measured at the start and at 8, 16, 24, and 36 hours after treatment. [Diagram 2]Two line graphs are shown comparing expression of natural killer group 2D ligand (NKG2DL) at selected time points on U138 glioblastoma cell lines exposed to olaparib (5 mM or 10 mM) alone, olaparib (an exemplary PARP inhibitor) + 200 mg / mL TMZ, or media alone (RPMI + 10% FBS media) for 4 hours, with viability measured at the start and at 8, 16, 24, and 36 hours after treatment. [Diagram 3] Three line graphs are shown comparing expression of natural killer group 2D ligand (NKG2DL) at selected time points on U251 glioblastoma cell lines exposed for 4 hours to olaparib (5 mM or 10 mM) alone, olaparib (an exemplary PARP inhibitor) + 200 mg / mL TMZ, or media alone (RPMI + 10% FBS media) with viability measured at the start and at 8, 16, 24, and 36 hours after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0011] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "support" includes a plurality of supports. In this specification and the following claims, reference will be made to certain terms that shall be defined to have the following meanings, unless a contrary intention is apparent.

[0012] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed in a range format herein. Such range formats are used for convenience and brevity, and therefore should be understood to be flexibly interpreted to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. By way of example, a concentration range of "about 0.1% to about 5%" is to be interpreted to include not only the explicitly recited concentration of about 0.1% to about 5% by weight, but also the individual concentrations (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more, of the numerical value(s) being modified. In addition, "about 'x' to 'y'" includes "about 'x' to about 'y'."

[0013] "Administering" means introducing the compound of the present invention, biological material including cell population, or combinations thereof into a human or animal subject.One preferred route of administration of the compound is intravenous.Other preferred routes of administration of the compound can be intraperitoneal or intrapleural, or into the brain via catheter.However, any route of administration can be used, such as oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into cerebrospinal fluid, or instillation into a body compartment.Direct injection into target tissue site, such as solid tumor, is also contemplated.

[0014] The term "cancer", as used herein, shall be given its ordinary meaning as a general term for diseases in which abnormal cells divide without control. Cancer cells can invade nearby tissues and spread to other parts of the body via the bloodstream and lymphatic system. Tumors form when normal cells lose their ability to behave as a specified, controlled, and coordinated unit. In general, solid tumors are abnormal tissue masses that do not usually contain cysts or liquid areas (some brain tumors have central necrotic areas filled with cysts and fluid). A single tumor may have different cell populations within it due to a deranged catabolic process. Solid tumors may be benign (not cancerous) or malignant (cancerous). Various types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

[0015] The term "reducing a tumor" as used herein refers to a decrease in the size or amount of a tumor mass, a decrease in the number of metastatic tumors in a subject, a decrease in the proliferative state of cancer cells (the extent to which cancer cells are proliferating), and the like.

[0016] The term "chemotherapeutic agent" as used herein refers to a compound or its derivatives that can interact with cancer cells, thereby reducing the proliferation state of the cells and / or killing the cells, for example by impairing cell division or DNA synthesis, or by damaging DNA, effectively targeting rapidly dividing cells. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX), 5-fluorouracil or its derivatives), substituted nucleotides, substituted nucleosides, DNA demethylating agents (also known as antimetabolites, e.g., azacytidine), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, TAXOL®, paclitaxel, abraxane), cisplatin, carboplatin, etoposide, etc. Such agents can further include, but are not limited to, the anticancer drugs trimethotrexate (TMTX), temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (alanose), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)), cytarabine, and camptothecin, or a therapeutic derivative of any of these.

[0017] The phrase "therapeutically effective amount" or "effective amount" refers to the administration of an agent to a subject in an amount that, when administered to a subject, alone or as part of a pharmaceutical composition, and in a single dose or as part of a series of doses, can have some detectable positive effect on some symptom, aspect, or feature of a disease, disorder, or condition. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect, and can be adjusted in conjunction with dosing regimens and diagnostic analysis of the subject's condition, and the like. As an example, measuring the amount of inflammatory cytokines produced after administration can indicate whether a therapeutically effective amount has been used. In the context of a disease condition involving cancer or unregulated cell division, a therapeutically effective amount refers to an amount that has the effect of (1) reducing tumor size (i.e., tumor regression), (2) inhibiting (i.e., slowing to some extent, preferably stopping) abnormal cell division, e.g., cancer cell division, (3) preventing or reducing metastasis of cancer cells, and / or (4) alleviating to some extent (or preferably eliminating) one or more symptoms associated with a disease condition involving or partially caused by unregulated or abnormal cell division, including, for example, cancer. An "effective amount" is also an amount that results in desired pharmacokinetic and pharmacodynamic properties and desired immune cell profiling upon administration of a therapeutically active composition of the invention.

[0018] The term "treating" or "treatment" of a disease (or condition or disorder) as used herein refers to preventing the occurrence of a disease in a human or animal subject who may be susceptible to the disease but has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), inhibiting the disease (delaying or stopping its onset), providing relief from symptoms or side effects of the disease (including palliative treatment), and causing regression of the disease. In relation to cancer, these terms also mean that the life expectancy of an individual suffering from cancer is extended or one or more of the symptoms of the disease are reduced. In relation to cancer, "treating" may also include enhancing or prolonging the anti-tumor response in a subject.

[0019] As used herein, any dosage form of a "combination," "combination therapy," and / or "combination treatment regimen" refers to at least two therapeutically active drugs or compositions that can be administered either in separate or combined formulations, simultaneously or sequentially at different times separated by minutes, hours, or days, but that can act together in some way to provide a desired therapeutic response.

[0020] The term "enhancing" as used herein refers to allowing a subject or tumor cells to improve their ability to respond to a treatment disclosed herein. For example, an enhanced response can include an increase in responsiveness of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more. As used herein, "enhancing" can also refer to increasing the number of subjects that respond to treatments such as chemotherapy, drug-resistant immune-competent cells, and combination therapies that include immune checkpoint inhibitors. For example, an enhanced response can refer to a total percentage of subjects that respond to a treatment that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

[0021] The terms "subject" and "patient" as used herein include humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and other organisms. An organism may be as simple as, for example, a single eukaryotic cell, or as complex as a mammal. A typical patient is a mammal, particularly a primate, and especially a human. For veterinary applications, a wide variety of subjects are suitable, such as livestock such as cattle, sheep, goats, dairy cows, pigs, poultry such as chickens, ducks, geese, turkeys, and domestic animals, particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs. Preferably, the system includes a sample and a subject. The term "living host" refers to a host or organism as described above that is alive and not dead. The term "living host" refers to the entire host or organism, not just a portion (e.g., liver or other organ) that has been removed from a living host.

[0022] The term "γδ T cells (gamma delta T cells)" as used herein refers to a small subset of T cells that express a distinct T cell receptor (TCR) on the surface of the cell. The majority of T cells have a TCR composed of two glycoprotein chains called α-TCR chain and β-TCR chain. In contrast, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This T cell population is usually much less common than αβ T cells. γδ T cells are unique among T cell types in that they do not require antigen processing and MHC presentation of peptide epitopes. In addition, γδ T cells are believed to have a prominent role in the recognition of lipid antigens and respond to stress-related antigens such as MIC-A and MIC-B and other ligands of the NKG2D receptor.

[0023] The term "drug resistance immunotherapy" or DRI is a genetic-based strategy for treating cancer whereby anti-cancer immune cells, preferably γδ T cells, are genetically engineered to resist the toxic effects of chemotherapeutic drugs, allowing for the combined administration of chemotherapy and immunotherapy.

[0024] The term "enriched", as used herein, refers to increasing the total percentage of one or more cytotoxic immune cell types (e.g., γδ T cells and / or NK cells) present in a sample, as disclosed herein, relative to the total percentage of the same cell types or types prior to enrichment. For example, a sample "enriched" for one or more types of cytotoxic immune cells may contain about 10%-100% of one or more cytotoxic immune cell types in the sample, whereas the total percentage of one or more cytotoxic immune cell types in the sample prior to enrichment was, for example, 0%-10%. Preferably, an enriched sample contains at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of one or more types of cytotoxic immune cells. A sample can be enriched for one or more cell types using standard techniques, e.g., flow cytometry techniques.

[0025] The term "highly enriched," as used herein, refers to increasing the total percentage of one or more cytotoxic immune cell types in a sample such that one or more cytotoxic immune cell types can comprise at least about 70% to about 100% of the cytotoxic immune cell types in the sample, but the total percentage of that same cytotoxic immune cell type prior to enrichment was, for example, 0% to 10%. Preferably, a highly enriched sample contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of one or more types of cytotoxic immune cells. A sample can be highly enriched for one or more cell types using standard techniques, for example, flow cytometry techniques.

[0026] The terms "expanded" and "expansion" as used herein with respect to the expansion of one or more cytotoxic immune cells in a sample means increasing the number of one or more cytotoxic immune cells in a sample, for example, by at least about 2-fold, preferably about 5-fold, preferably at least 10-fold, preferably at least about 50-fold or more. The expansion of the cytotoxic immune cell population can be achieved by any number of methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin 2 (IL-2) or interleukin 15 (IL-15), preferably IL-2. Non-specific T cell receptor stimulation can include about 30 ng / ml of OKT3 (available from ORTHO-MCNEL®, Raritan, NJ), a murine monoclonal anti-CD3 antibody. Alternatively, T cells can be rapidly proliferated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more antigens of the cancer (including antigenic portions thereof, such as epitope(s), or cells), optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gp100:209-217(210M), in the presence of a T cell growth factor, such as 300 IU / ml IL-2 or IL-15, with IL-2 being preferred.

[0027] As used herein, the terms "isolated" and "isolated cell population" refer to one or more cells removed from a tissue or condition found in a subject. The terms can further include cells separated by parameters such as, but not limited to, cell surface markers, reporter markers such as dyes or labels.

[0028] The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule that is at least partially complementary to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation from the RNA nucleic acid molecule to give a protein, polypeptide, or a portion or fragment thereof.

[0029] The term "recombinant cell" refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A wide range of nucleic acid manipulation techniques available to those skilled in the art can be used to introduce new combinations of nucleic acid segments into an organism. A recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. A recombinant cell can have a vector that is extragenomic. An extragenomic nucleic acid vector is not inserted into the genome of the cell. A recombinant cell can also have an intragenomic vector or a portion thereof. The term "intragenomic" defines a nucleic acid construct that is integrated into the genome of a recombinant cell.

[0030] The terms "recombinant nucleic acid" and "recombinant DNA" as used herein refer to a combination of at least two nucleic acid sequences not naturally found in eukaryotic or prokaryotic cells. The nucleic acid sequences include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, appropriate gene sequences that confer antibiotic resistance upon expression, protein coding sequences, etc. The term "recombinant polypeptide" is meant to include polypeptides produced by recombinant DNA technology such that they differ from naturally occurring polypeptides in either their location, purity or structure. Generally, such recombinant polypeptides are present in cells in amounts that differ from those normally observed in nature.

[0031] The term "targeted therapy," as used herein, refers to any therapeutic molecule that targets some aspect of the immune system.

[0032] The terms "transformation," "transduction," and "transduction" all refer to the introduction of a polynucleotide into one or more recipient cells.

[0033] "Immune checkpoint proteins" regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. Checkpoint proteins interact with specific ligands that send signals into T cells and essentially turn off or inhibit T cell function. Cancer cells exploit this system by driving high levels of expression of checkpoint proteins on the cancer cell surface, thereby controlling T cells that express checkpoint proteins on their surface that enter the tumor microenvironment, thus suppressing anti-cancer immune responses. In this way, inhibition of checkpoint proteins by agents referred to herein as "immune checkpoint protein (ICP) inhibitors" will result in restoration of T cell function and immune responses against cancer cells. Examples of checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof.Preferably, the immune checkpoint inhibitor interacts with a ligand of a checkpoint protein, which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, OX40, A2aR, B-7 family ligands, or combinations thereof.Preferably, the checkpoint inhibitor is a biological therapeutic agent or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. There are several PD-1 inhibitors and CTLA-4 inhibitors currently being tested in clinical trials. CT-011 is a humanized IgG1 monoclonal antibody against PD-1. BMS936558 is a fully human IgG4 monoclonal antibody that targets PD-1 agonists. BMS936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1.MK3475 is a humanized IgG4 anti-PD-1 monoclonal antibody in Phase I development in a five-part study evaluating drug dosing, safety, and tolerability in subjects with advanced, locally extensive, or metastatic carcinoma, melanoma, or NSCLC. MPDL3280A is a monoclonal antibody. AMP224 is a fusion protein consisting of the extracellular domain of PD-L2, a second PD-1 ligand, and IgG1 with the potential to block PD-L2 / PD-1 interaction. Medi4736 is an anti-PD-L1 antibody in Phase I clinical trials in subjects with advanced malignant melanoma, renal cell carcinoma, NSCLC, and colorectal cancer. Ipilimumab (YERVOY®), a first-in-class immunotherapy, a monoclonal antibody that targets CTLA-4 on the surface of T cells, was indicated for the treatment of melanoma.

[0034] γδ T cell immunotherapy combinations The present invention provides combination therapies for reducing tumors and treating cancer in patients, including compositions and methods for γδ T cell immunotherapy in combination DDR inhibitors, including but not limited to PARP inhibitors, and in further combination with chemotherapeutic drugs, including but not limited to temozolomide (TMZ). Preferably, the combination of γδ T cell immunotherapy and PARP inhibitors for the treatment of cancer further includes combination with other immunotherapies, such as immune checkpoint (ICP) blockade therapy and / or other immune stimulants.

[0035] The present invention provides a method for treating cancer in a patient comprising: i) administering a composition comprising an optionally enriched and / or optionally expanded population of γδ T cells, ii) administering the composition of (i) to a patient in need thereof; administering at least one therapeutically effective amount of a DDR inhibitor to the patient, and iii) optionally administering a chemotherapeutic agent, thereby treating cancer in the patient.

[0036] Preferably, the γδ T cells are derived from human induced pluripotent stem cells (hiPSCs). Preferably, the pluripotent stem cells can be isolated from a patient suffering from cancer. Preferably, the pluripotent stem cells may be isolated from a source other than a patient suffering from cancer. Preferably, the optionally enriched and / or optionally expanded composition comprising γδ T cells also comprises natural killer (NK) cells, and optionally further comprises other immunocompetent cells, including but not limited to monocytes, macrophages and dendritic cells.

[0037] Preferably, the composition comprising the optionally enriched and / or optionally expanded population of γδ T cells comprises at least about 50%, at least about 60%, at least about 70% or more γδ T cells. Preferably, the composition comprising the optionally enriched and / or optionally expanded population of γδ T cells comprises less than about 35% natural killer (NK) cells. Preferably, the composition comprising the optionally enriched and / or optionally expanded population of γδ T cells comprises less than about 10%, less than about 5% αβ T cells.

[0038] Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded γδ T cell population, comprises approximately 5×10 8 Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded γδ T cell population, comprises no more than about 5×10 γδ T cells per kg of patient body weight. 7 Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded γδ T cell population, comprises no more than about 5×10 γδ T cells per kg of patient body weight. 6 γδ T cells or less.

[0039] Methods for isolating γδ T cells, either from the patient to be treated or from another source, are described, for example, by Lamb LS in US Pat. No. 7,078,034, which is incorporated herein by reference in its entirety.

[0040] A DDR inhibitor suitable for use in the methods described herein may be any compound or entity, such as a small organic molecule, peptide or nucleic acid, that inhibits, reduces or eliminates the activity of one or more components of DNA damage repair pathways. These pathways include base excision repair (BER), homologous recombination (HR)-dependent DNA double-strand break (DSB) repair, non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR).

[0041] Preferably, DDR inhibitor reduces or eliminates the activity of enzyme poly(ADP-ribose) polymerase (PARP).Preferred PARP inhibitors include, but are not limited to, niraparib, olaparib, and rucaparib, all of which are approved by FDA.Olaparib and rucaparib are approved by FDA for the treatment of recurrent BRCA-related ovarian cancer.More recently, these two and the third PARP inhibitor, niraparib, have been approved by FDA as maintenance therapy following platinum-based chemotherapy for recurrent ovarian cancer.

[0042] Other preferred PARP inhibitors include, but are not limited to, talazoparib (Pfizer), veliparib (Abbvie), E7016 (Eisai), CEP-9722 (Teva), and BGB-290 (pamiparib, BeiGene).

[0043] Other examples of compounds that are known PARP inhibitors and can be used in accordance with the present invention include the following: 1. Nicotinamides such as 5-methylnicotinamide and O-(2-hydroxy-3-piperidino-propyl)-3-carboxylic acid amidoxime, and analogues and derivatives thereof; 2. Benzamides, including 3-substituted benzamides such as 3-aminobenzamide, 3-hydroxybenzamide, 3-nitrosobenzamide, 3-methoxybenzamide and 3-chloroprocainamide, as well as 4-aminobenzamide, 1,5-di[(3-carbamoylphenyl)aminocarbonyloxy]pentane, and analogs and derivatives thereof; 3. 5-substituted dihydroisoquinolinones such as 2H-isoquinolin-1-one, 3H-quinazolin-4-one, 5-hydroxydihydroisoquinolinone, 5-methyldihydroisoquinolinone, and 5-hydroxyisoquinolinone; 3,4-dihydroisoquinolin-1(2H)-ones such as 5-aminoisoquinolin-1-one, 5-dihydroxyisoquinolinone, 3,4-dihydro-5-methoxy-isoquinolin-1(2H)-one, and 3,4-dihydro-5-methyl-1(2H)isoquinolinone; isoquinolin-1(2H)-one; 4,5-dihydro-imidazo[4,5,1-ij] tetracyclic lactams including quinolin-6-ones, 1,6-naphthyridin-5(6H)-ones, 1,8-naphthalimides such as 4-amino-1,8-naphthalimide, isoquinolinones, benzpyranoisoquinolinones such as 3,4-dihydro-5-[4-1(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, 2,3-dihydrobenzo[de]isoquinolin-1-one, 1-11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one, and benzopyrano[4,3,2-de]isoquinolinone, and analogs and derivatives thereof; 4. Benzimidazoles and indoles, including benzoxazole-4-carboxamides, 2-substituted benzimidazole 4-carboxamides such as benzimidazole-4-carboxamides and 2-arylbenzimidazole 4-carboxamides, as well as 2-cycloalkylbenzimidazole-4-carboxamides, including 2-(4-hydroxyphenylbenzimidazole 4-carboxamide, quinoxaline carboxamides, imidazopyridine carboxamides, 2-phenylindoles, 2-substituted benzoxazoles, such as 2-phenylbenzoxazole and 2-(3-methoxyphenyl)benzoxazole, 2-phenylbenzimidazole and 2-(3-methoxyphenyl)benzimidazo azepinoindoles and azepinoidolones such as 1,3,4,5 tetrahydro-azepino[5,4,3-cd]indol-6-one, 1,5 dihydro-azepino[4,5,6-cd]indolin-6-one and dihydrodiazapinoindolinones, 3-substituted dihydrodiazapinoindolinones such as 3-(4-trifluoromethylphenyl)-dihydrodiazapinoindolinone, tetrahydrodiazapinoindolinone and 5,6,-dihydroimidazo[4,5,1-j,k][1,4]benzodiazopin-7(4H)-one, 2-phenyl-5,6-dihydro-imidazo[4,5,1-jk][1,4]benzodiazepin-7(4H)-one and 2,3,-dihydro-isoindol-1-one and analogues and derivatives thereof, 5. Phthalazin-1(2H)-ones and quinazolinones such as 4-hydroxyquinazoline, phthalazinone, 5-methoxy-4-methyl-1(2)phthalazinone, 4-substituted phthalazinones, 4-(1-piperazinyl)-1(2H)-phthalazinone, tetracyclic benzopyrano[4,3,2-de]phthalazinone and tetracyclic indeno[1,2,3-de]phthalazinone, and 2-substituted quinazolines, tricyclic phthalazinones and 2-aminophthalhydrazides such as 8-hydroxy-2-methylquinazolin-4-(3H)one, and analogues and derivatives thereof; 6. Isoindolinone and its analogues and derivatives, 7. Phenanthridines and phenanthridinones such as 5[H]phenanthridin-6-one, substituted 5[H]phenanthridin-6-one, in particular 2-substituted 5[H]phenanthridin-6-one, 3-substituted 5[H]phenanthridin-6-one, and sulfonamide / carbamide derivatives of 6(5H)phenanthridinone, thieno[2,3-c]isoquinolones such as 9-aminothieno[2,3-c]isoquinolone and 9-hydroxythieno[2,3-c]isoquinolone, 9-methoxythieno[2,3-c]isoquinolones, and N-(6-oxo-5,6-dihydrophenanthridin-2-yl]-2-(N,N-dimethylamino}acetamide, substituted 4,9-dihydrocyclopenta[lmn]phenanthridin-5-one, and analogues and derivatives thereof, 8. 1,2-Benzopyrones Benzopyrones such as 6-nitrosobenzopyrone, 6-nitroso-1,2-benzopyrone, and 5-iodo-6-aminobenzopyrone, and their analogues and derivatives; 9. Unsaturated hydroximic acid derivatives such as O-(3-piperidino-2-hydroxy-1-propyl)nicotinamide oxime, and analogs and derivatives thereof; 10. Pyridazines, including fused pyridazines, and their analogs and derivatives, and 11. Other compounds such as caffeine, theophylline, and thymidine, as well as their analogues and derivatives.

[0044] Combination γδ T cell DRI and DDR inhibitor therapy Preferably, the combination of γδ T cell immunotherapy and DDR inhibitor therapy as a treatment regimen further comprises a combination with a chemotherapeutic drug, where the γδ T cells are genetically modified to confer resistance to the chemotherapeutic drug, known as drug resistance immunotherapy (DRI). Preferably, the genetic modification of the γδ T cells to render them resistant to the action of the chemotherapeutic drug does not affect the ability of the γδ T cells to kill target cancer cells in the presence or absence of the chemotherapeutic drug. The γδ T cells are genetically modified using standard recombinant techniques, such as those described in the examples and in WO 2011 / 053750 and Lamb et al., PloS ONE 8(1):e51805.doi:10.1371 / journal.pone.0051805).

[0045] Preferably, the composition comprising the optionally enriched and / or optionally expanded population of engineered γδ T cells comprises at least about 50%, at least about 60%, at least about 70% or more γδ T cells. Preferably, the composition comprising the optionally enriched and / or optionally expanded population of engineered γδ T cells comprises less than about 35% Natural Killer (NK) cells. Preferably, the composition comprising the optionally enriched and / or optionally expanded population of engineered γδ T cells comprises less than about 10%, less than about 5% αβ T cells.

[0046] Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded genetically engineered γδ T cell population, comprises approximately 5×10 8 Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded genetically engineered γδ T cell population, comprises no more than about 5×10 γδ T cells per kg of patient body weight. 7Preferably, the therapeutic composition for administration to a patient, comprising an optionally enriched and / or optionally expanded genetically engineered γδ T cell population, comprises no more than about 5×10 γδ T cells per kg of patient body weight. 6 γδ T cells or less.

[0047] The main limitation to chemotherapy treatment for cancer is drug-induced immunotoxicity, which causes the death of immune-competent cells and the loss of an effective immune system that would otherwise prevent unwanted infection or provide protection against cancer cells. One strategy to combat the severe toxic effects of chemotherapy is to selectively genetically modify cytotoxic immune cells by the introduction of retroviral vectors designed to express cDNA sequences that confer drug resistance, thereby actively targeting cancer cells that can resist the co-administration of chemotherapy drugs. Such drug-resistant immunotherapy using engineered γδ T cell immunotherapy has been described, for example, by Spencer HT et al. in US Patent No. 2015 / 0017137 (see also International Publication No. 2011 / 053750 and Lamb et al., PloS ONE 8(1):e51805.doi:10.1371 / journal.pone.0051805).

[0048] The present inventors have previously discovered that administration of chemotherapeutic drugs such as TMZ transiently increases stress-related antigens (e.g., receptors for the NKG2D family of ligands such as MIC A / B and UL16 binding protein (ULBP)) in TMZ-resistant cell lines. The present invention strengthens this previous discovery by combining chemotherapeutic drugs with DDR inhibitors. Without being limited to any theory, DDR inhibitors such as PARP inhibitors act to enhance and prolong the upregulation of stress-related antigens on cancer cells caused by exposure to chemotherapeutic drugs. Therefore, the combination of long-term chemotherapy and DDR inhibitor therapy enhances the forced expression of NKG2D ligands on cancer cells, making the tumor cells highly vulnerable to killing by γδ T cells. The modification of γδ T cells to resist destruction by chemotherapeutic drugs allows the cells to persist in the presence of chemotherapeutic drugs during the optimal and prolonged window of transient NKG2D upregulation by combined chemotherapeutic drugs and DDR inhibitors.

[0049] Preferably, the DDR inhibitor, such as a PARP inhibitor or any other DDR inhibitor previously described, is administered to the patient about 1 to about 21 days prior to administration of the chemotherapeutic agent. Preferably, the PARP inhibitor is administered about 1 to about 14 days prior to administration of the chemotherapeutic agent. Preferably, the PARP inhibitor is administered about 1 to about 7 days prior to administration of the chemotherapeutic agent.

[0050] Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 8 to about 72 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 8 to about 36 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 8 to about 24 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 8 to about 12 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 12 to about 36 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 12 to about 24 hours after administration of the chemotherapy drug. Preferably, the DRI using genetically engineered chemotherapy-resistant γδ T cells is administered about 24 hours after administration of the chemotherapy drug. Preferably, the DRI using engineered chemotherapy-resistant γδ T cells is administered about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours after administration of the chemotherapy agent.

[0051] Combination therapy of DRI and DDR inhibitor using checkpoint inhibitor (ICP) Preferably, the combination therapy of γδ T cell DRI and DDR inhibitor as a treatment regimen further includes a combination with an ICP inhibitor. Preferably, the checkpoint inhibitor is a PD-1 inhibitor or a CTLA-4 inhibitor. Preferably, the immune checkpoint inhibitor may be a biological therapeutic or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, an antigen-binding fragment, or a combination thereof.

[0052] Preferably, immune stimulants, T cell growth factors and interleukins can be used in various combination therapies of the present invention.Immunostimulants are substances (drugs and nutrients) that stimulate the immune system by inducing the activation or increasing the activity of any of its components.Immunostimulants include, but are not limited to, bacterial vaccines, colony stimulating factors, interferons, interleukins, other immune stimulants, therapeutic vaccines, vaccine combinations and viral vaccines.T cell growth factors are proteins that stimulate the proliferation of T cells.Examples of T cell growth factors include Il-2, IL-7, IL-15, IL-17, IL21 and IL-33.

[0053] Combination therapies including drug-resistant γδ T cell immunotherapy, chemotherapeutic drugs, ICP inhibitors, and optionally immune stimulants are described in WIPO patent application WO2018 / 035413. Clinical trials combining PARP inhibitors with immune checkpoint inhibitors are ongoing and have been reported in the literature.

[0054] Preferably, the combination therapy comprising drug-resistant γδ T cell immunotherapy and PARP inhibitor for the treatment of cancer further includes immune checkpoint (ICP) blockade therapy and / or chemotherapy and / or immunostimulatory therapy. These can be administered simultaneously, either in separate or combined formulations, or sequentially at different times separated by minutes, hours or days, but act together in some way to provide the desired therapeutic response. The optimal timing of γδ T cell immunotherapy in combination with DDR inhibition, and optionally further in combination with chemotherapy and ICP inhibitor, will vary depending on the disease context as known in the art, but generally PARP inhibition and / or chemotherapy will be administered before administration of cell therapy to induce DNA damage and increased immunogenicity.

[0055] Preferably, an effective amount of a checkpoint inhibitor is an amount effective to cause tumor regression in combination with a DRI and DDR combination therapy. Preferably, the checkpoint inhibitor is administered at a dose of less than 0.0001 mg / kg, 0.0001-0.001 mg / kg, 0.001-0.01 mg / kg, 0.01-0.05 mg / kg, 0.05-0.1 mg / kg, 0.1-0.2 mg / kg, 0.2-0.3 mg / kg, 0.3-0.5 mg / kg, 0.5-0.7 mg / kg, 0.7-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 4-5 mg / kg, 5-6 mg / kg, 6-7 mg / kg, 7-8 mg / kg, 8-9 mg / kg, 9-10 mg / kg, at least 10 mg / kg, or any combination thereof (per kg of patient body weight). Preferably, the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or multiple months. Preferably, the checkpoint inhibitor is administered as a single dose, two doses, three doses, four doses, five doses, or six doses or more.

[0056] Pharmaceutical Compositions and Methods of Administration Preferably, the combination therapies disclosed herein may be administered to a patient by a variety of routes including, for example, orally or parenterally, including but not limited to, intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsular, intraperitoneally, intrarectally, intracisternally, intratumorally, intravascularly, intradermally, intravaginally (e.g., vaginal suppositories), or topically (e.g., powders, ointments transdermal patches), or by passive or facilitated absorption through the skin, for example, using a skin patch or transdermal iontophoresis, respectively.

[0057] The therapeutic checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition can also be administered to the site of pathological condition, for example, intravenously or intraarterially into the blood vessels that supply the tumor.For example, the composition comprising γδ T cells, PARP inhibitor or any combination thereof can be delivered directly to the target tumor, such as, but not limited to, direct injection into the tumor mass, delivery to the blood vessels that enter the tumor mass, or a combination of both.For example, it is contemplated that the composition can be delivered to the glioblastoma mass in the brain of a patient by direct implantation through an intubation needle or catheter inserted into the tumor mass, the cavity after resection, the peritoneal cavity or the intraventricular cavity.

[0058] Preferably, the total amount of the agent administered in carrying out the method of the present invention can be administered to the subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a divided treatment protocol in which multiple doses are administered over a long period of time.Those skilled in the art will know that the amount of the composition for treating a pathological condition of a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments administered.In view of these factors, those skilled in the art will adjust the specific dose as necessary.

[0059] The pharmaceutical compositions of the present invention can be formulated to be compatible with the intended method or route of administration, exemplary routes of administration being set forth herein. Additionally, the pharmaceutical compositions can be used in combination with other therapeutically active agents or compounds as described herein to treat or prevent diseases, disorders and conditions as contemplated by the present disclosure.

[0060] A pharmaceutical composition typically comprises a therapeutically effective amount of one or more active agents used in the combination therapy of the present invention and one or more pharma- ceutical and physiologically acceptable preparations. Suitable pharma-ceutical or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl propyl or n-propyl, p-hydroxybenzoate), emulsifiers, suspending agents, dispersing agents, solvents, fillers, extenders, detergents, buffers, vehicles, diluents, and / or adjuvants. For example, suitable vehicles may be physiological saline or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Those skilled in the art will readily recognize the various buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffers include, but are not limited to, pharma- ceutically acceptable weak acids, weak bases, or mixtures thereof. By way of example, buffer components can be water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0061] After the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form, a lyophilized form that requires reconstitution before use, a liquid form that requires dilution before use, or in other acceptable forms. Preferably, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, an ampoule, a syringe, or an auto-injector (e.g., similar to EpiPen®)), but in other embodiments, in a multi-use container (e.g., a multi-use vial). Any drug delivery device can be used to deliver IL-10, including implants (e.g., implantable pumps) and catheter systems, slow infusion pumps and devices, all of which are well known to those skilled in the art. Depot injections, which are generally administered subcutaneously or intramuscularly, can also be utilized to release the polypeptides disclosed herein over a defined period of time. Depot injections are usually based on either solids or oils, and generally include at least one of the formulation components disclosed herein. Those of skill in the art are familiar with the possible formulation and uses of depot injectables.

[0062] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated by known techniques using suitable dispersing or wetting agents and suspending agents as described herein. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media that can be employed include water, Ringer's solution, isotonic sodium chloride solution, CREMOPHOR EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any non-irritating fixed oils can be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including agents which delay absorption, for example, aluminum monostearate or gelatin.

[0063] The pharmaceutical composition may be in a form suitable for oral use, for example, as a tablet, capsule, troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, or syrup, liquid, microbeads or elixir.The pharmaceutical composition intended for oral use can be prepared by any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more active agents, such as, for example, sweeteners, flavorings, colorants and preservatives, to provide a medicament-elegant and palatable formulation.The tablet, capsule, etc. contain the active ingredient mixed with non-toxic pharmacopoeiatable excipients suitable for the manufacture of tablets. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricants, for example, magnesium stearate, stearic acid or talc.

[0064] The tablets, capsules, etc. suitable for oral administration can be uncoated or coated by known techniques to delay disintegration and absorption in the digestive tract, thereby providing a sustained action.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be employed.The tablets, etc. can also be coated by known techniques in the art to form osmotic therapeutic tablets for controlled release.Additional agents include biodegradable or biocompatible particles or polymeric materials such as polyesters, polyamic acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolide copolymers, polylactide / glycolide copolymers, or ethylene-vinyl acetate copolymers, to control the delivery of the administered composition. For example, oral agents can be encapsulated in microcapsules prepared by coacervation techniques or by interfacial polymerization, using hydroxymethylcellulose or gelatin microcapsules or poly(methyl methchlorate) microcapsules, respectively, or in colloidal drug delivery systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for the preparation of the above formulations will be clear to those skilled in the art.

[0065] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0066] Aqueous suspensions contain the active substance mixed with excipients suitable for their manufacture. Such excipients can be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, such as natural phosphatides (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearates), or condensation products of ethylene oxide with long-chain fatty alcohols (e.g., in the case of heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyethylene sorbitan monooleate). Aqueous suspensions can also contain one or more preservatives.

[0067] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin. Oily suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, as set forth above, and flavoring agents can be added to provide a palatable oral preparation.

[0068] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0069] Pharmaceutical compositions may also be in the form of oil-in-water emulsion.Oily phase may be vegetable oil, such as olive oil or peanut oil, or mineral oil, such as liquid paraffin, or mixtures thereof.Suitable emulsifiers may be naturally occurring gums, such as gum acacia or gum tragacanth, naturally occurring phosphatides, such as soybean, lecithin, and esters or partial esters derived from fatty acids, hexitol anhydrides, such as sorbitan monooleate, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0070] The formulation can also include carriers to protect the composition against rapid degradation or elimination from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or with a wax can be employed.

[0071] Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

[0072] Pharmaceutical compositions suitable for use in accordance with the present invention may be in any form now known or later developed (eg, a spray for nasal or inhalation use).

[0073] Treatment indications The combined treatment method described herein is particularly suitable for the treatment of cancer. Cancer cells can invade nearby tissues and spread to other parts of the body via the bloodstream and lymphatic system. There are several main types of cancer, for example, carcinoma is cancer that develops in the skin or tissues that line or cover internal organs. Sarcoma is cancer that develops in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is cancer that develops in blood-forming tissues, such as bone marrow, and produces a large number of abnormal blood cells that enter the bloodstream. Lymphoma is cancer that develops in the cells of the immune system.

[0074] Tumors form when normal cells lose the ability to behave as a specified, controlled, and coordinated unit. In general, solid tumors are abnormal tissue masses that do not usually contain cysts or liquid areas (some brain tumors have cysts and fluid-filled central necrotic areas). A single tumor may have different cell populations within it due to a disrupted catabolic process. Solid tumors can be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.

[0075] Tumors can be classified as malignant or benign. In both cases, there is an abnormal aggregation and proliferation of cells. In the case of malignant tumors, these cells behave more aggressively and acquire the properties of increased invasiveness. Eventually, tumor cells can even acquire the ability to leave the microscopic environment in which they originated and spread to another area of ​​the body (with a very different environment that is not usually conducive to their growth) and continue their rapid growth and division in this new place. This is called metastasis. Once malignant cells have metastasized, it is more difficult to achieve a cure. Benign tumors are less prone to invade and are less likely to metastasize.

[0076] Examples of solid tumor cancers that can be treated using the combination treatment regimen described herein include, but are not limited to, pancreatic cancer, colon cancer, non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, bladder cancer, prostate, cervix, stomach, endometrium, brain, liver, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, esophagus, breast, muscle, connective tissue, lung (including small cell lung cancer and non-small cell carcinoma), adrenal gland, thyroid, kidney, or bone cancer, glioblastoma, mesothelioma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, and seminoma of the testis. In a preferred embodiment, the cancer is cervical cancer, non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, bladder cancer, pancreatic cancer, melanoma, lymphoma, or gastric cancer. In a more preferred embodiment, the cancer is melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, bladder cancer, renal cell carcinoma, or gastric cancer. The treatment regimen of the present invention is particularly suitable for treating solid tumors, including, but not limited to, lymphoma, melanoma, renal cell carcinoma (RCC), advanced solid tumors, tumors that have been previously treated with therapeutic therapies but remain refractory to conventional therapies.

[0077] Cancers that may also be treated according to the present invention include, inter alia, adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; adrenal cortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; childhood cerebellar astrocytoma; childhood cerebral astrocytoma; extrahepatic bile duct cancer; bladder cancer; childhood bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; childhood glioblastoma; adult glioblastoma; childhood brain stem glioma; adult brain tumor; childhood brain tumor, childhood brain stem glioma; childhood brain tumor, childhood cerebellar astrocytoma; childhood brain tumor, childhood cerebral astrocytoma / childhood malignant glioma; childhood brain tumor, childhood ependymoma; childhood brain tumor, childhood medulloblastoma; childhood brain tumor, childhood supratentorial hara Primary neuroectodermal tumors;Pediatric brain tumors, pediatric visual pathway and hypothalamic gliomas;Pediatric brain tumors (other);Breast cancer;Breast cancer in pregnancy;Pediatric breast cancer;Male breast cancer;Pediatric bronchial adenoma / pediatric carcinoid tumors:Pediatric carcinoid tumors;Gastrointestinal carcinoid tumors;Adrenal cortical carcinoma;Islet cell carcinoma;Unknown primary cancer;Primary central nervous system lymphoma;Pediatric cerebellar astrocytoma;Pediatric cerebral astrocytoma / pediatric malignant glioma;Cervical cancer;Pediatric cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon cancer;Pediatric colorectal cancer;Cutaneous T-cell lymphoma;Endometrial cancer;Pediatric ependymoma;Ovarian epithelial carcinoma;Esophageal cancer;Pediatric esophageal cancer;Ewing's Family of tumors Tumors);Pediatric Extracranial Germ Cell Tumors;Extragonadal Germ Cell Tumors;Extrahepatic Bile Duct Carcinoma;Eye Cancer, Intraocular Melanoma;Eye Cancer, Retinoblastoma;Gallbladder Cancer;Gastric (Stomach) Cancer;Pediatric Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumors;Pediatric Extracranial Germ Cell Tumors;Extragonadal Germ Cell Tumors;Ovarian Germ Cell Tumors;Gestational Trophoblastic Tumors;Pediatric Brain Stem Glioma;Pediatric Visual and Hypothalamic Glioma;Hairy Cell Leukemia;Head and Neck Cancer;Adult (Primary) Hepatocellular (Liver) Carcinoma;Pediatric (Primary) Hepatocellular (liver) carcinoma;Adult Hodgkin's lymphoma;Childhood Hodgkin's lymphoma;Hodgkin's lymphoma of pregnancy;Hypopharyngeal carcinoma;Childhood hypothalamic and optic tract glioma;Intraocular melanoma;Islet cell carcinoma (pancreatic endocrine);Kaposi's sarcoma;Ringeal carcinoma;Laryngeal cancer;Childhood laryngeal cancer;Adult acute lymphoblastic leukemia;Childhood acute lymphoblastic leukemia;Adult acute myeloid leukemia;Childhood acute myeloid leukemia;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Hairy cell leukemia;Lip and oral cavity cancer;Adult liver cancer (primary);Childhood liver cancer (primary);Non-small cell lung cancer;Small cell lung cancer;Adult acute lymphoblastic leukemia;Childhood acute lymphoblastic leukemia;Chronic lymphocytic leukemia;AIDS-related lymphoma;Central nervous system (primary) lymphoma;Cutaneous T-cell lymphoma;Adult Hodgkin lymphoma;Childhood Hodgkin lymphoma;Hodgkin lymphoma of pregnancy;Adult non-Hodgkin lymphoma;Childhood non-Hodgkin lymphoma;Non-Hodgkin lymphoma of pregnancy;Primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;Male breast cancer;Adult malignant mesothelioma;Childhood malignant mesothelioma;Malignant thymoma;Childhood medulloblastoma;Melanoma;Intraocular melanoma;Merkel cell carcinoma;Malignant mesothelioma;Metastatic squamous neck cancer with occult primary Primary,Metastatic);Childhood multiple endocrine neoplasia;Multiple myeloma / plasma cell neoplasms;Mycosis fungoides;Myelodysplastic syndromes;Chronic myeloid leukemia;Childhood acute myeloid leukemia;Multiple myeloma;Chronic myeloproliferative disorders;Nasal cavity and paranasal sinus cancer;Nasopharyngeal carcinoma;Childhood nasopharyngeal carcinoma;Neuroblastoma;Neurofibroma;Adult non-Hodgkin's lymphoma;Childhood non-Hodgkin's lymphoma;Non-Hodgkin's lymphoma of pregnancy;Non-small cell lung cancer;Small Childhood oral cancer;cancer of the mouth and lips;oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;childhood ovarian cancer;epithelial ovarian cancer;ovarian germ cell tumors;ovarian low malignant potential tumors;pancreatic cancer;childhood pancreatic cancer, islet cell carcinoma;cancer of the paranasal sinuses and nasal cavity;parathyroid cancer;penile cancer;pheochromocytoma;childhood pineal and supratentorial primary neuroectodermal tumors;pituitary tumors;plasma cell tumors / multiple myeloma;pleuropulmonary blastoma;breast cancer in pregnancy;Hodgkin's lymphoma in pregnancy;pregnancy Non-Hodgkin's lymphoma of pregnancy;Primary central nervous system lymphoma;Adult primary liver cancer;Primary liver cancer in children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Childrenal cell carcinoma;Transitional cell carcinoma of the renal pelvis and ureter;Retinoblastoma;Childhood rhabdomyosarcoma;Salivary gland cancer;Childhood salivary gland cancer;Ewing's sarcoma family;Kaposi's sarcoma;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Childhood rhabdomyosarcoma;Adult soft tissue sarcoma;Childhood soft tissue sarcoma;Sezary syndrome;Skin Skin Cancer;Pediatric Skin Cancer;Skin Cancer (Melanoma);Merkel Cell Carcinoma;Small Cell Lung Cancer;Small Intestine Cancer;Adult Soft Tissue Sarcoma;Pediatric Soft Tissue Sarcoma;Metastatic Squamous Neck Cancer with Occult Primary;Stomach (Gastric) Cancer;Pediatric Stomach (Gastric) Cancer;Pediatric Supratentorial Primitive Neuroectodermal Tumor;Cutaneous T-Cell Lymphoma;Testicular Cancer;Pediatric Thymoma;Malignant Thyroid Cancer;Pediatric Thyroid Cancer;These include, but are not limited to, transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic neoplasm; childhood cancer of unknown primary site; atypical childhood cancers; transitional cell carcinoma of the ureter and renal pelvis; urethral carcinoma; uterine sarcoma; vaginal carcinoma; childhood visual tract and hypothalamic glioma; vulvar carcinoma; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0078] Preferably, the cancers treated according to the present invention are CNS tumors, including, but not limited to, intracranial and spinal ependymomas (excluding subependymomas), poorly differentiated infiltrating supratentorial astrocytomas / oligodendrogliomas, medulloblastomas, anaplastic gliomas, glioblastomas, metastatic lesions of the CNS, and primary CNS lymphomas.

[0079] Preferably, the cancer to be treated is melanoma. Preferably, the cancer to be treated is uveal melanoma.

[0080] Preferably, the cancer to be treated is a neuroendocrine or adrenal tumor, examples of which include, but are not limited to, bronchopulmonary disease, gastrointestinal tract, lung or thymus, pancreas, paraganglioma or pheochromocytoma.

[0081] Preferably, the cancer to be treated is non-Hodgkin's lymphoma, including but not limited to mycosis fungoides and Sezary syndrome.

[0082] Preferably, the cancer to be treated is a soft tissue sarcoma, such as angiosarcoma, unresectable or progressive retroperitoneal / intraperitoneal soft tissue sarcoma, rhabdomyosarcoma, extremity / superficial trunk and / or head and neck cancer, or solitary fibrous tumor / hemangiopericytoma.

[0083] Preferably, the cancer to be treated is bone cancer. Examples include Ewing's sarcoma and mesenchymal chondrosarcoma.

[0084] Preferably, the cancer being treated is uterine sarcoma, small cell lung cancer (SCLC) or Zollinger-Ellison syndrome.

[0085] Preferably, the cancer treated according to the present invention is a gynecological cancer (e.g., a cancer of the female reproductive system), including, but not limited to, ovarian cancer, fallopian tube cancer, peritoneal cancer, and breast cancer. Preferably, the cancer treated according to the present invention is ovarian cancer.

[0086] Preferably, the cancer treated according to the present invention is glioblastoma.

[0087] Brain tumors spread widely in the brain, but usually do not metastasize outside the brain. Gliomas are very aggressive in the brain and can even cross hemispheres. However, they divide in an uncontrolled manner. Depending on the location of gliomas, they can be just as life-threatening as malignant lesions. An example of this would be a benign tumor in the brain, which can grow and occupy space in the skull, leading to increased pressure on the brain.

[0088] kit Kits are also provided that contain pharmaceutical compositions that typically include a therapeutically effective amount of one or more of the agents used in the combination therapies of the invention described herein. The kits typically include a label and instructions indicating the intended use of the contents of the kit.

[0089] The kit is generally in the form of a physical structure that contains various components, as described below, and can be utilized, for example, in carrying out the methods described above. The kit can include, for example, a composition that includes one or more of the therapeutic agents (e.g., genetically engineered γδ cells or DRI inhibitors or ICP inhibitors) used in the combination therapy of the present invention, provided in one or more sterile containers, and the composition can be in the form of a pharmaceutical composition suitable for administration to a subject. The pharmaceutical composition can be provided in a form that is ready for use, or in a form that requires, for example, reconstitution or dilution before administration. When they are in a form that requires the composition to be reconstituted by the user, the kit can also include a buffer, a pharma- ceutical acceptable excipient, etc., packaged together with or separately from the therapeutic agent. When a combination therapy is intended, the kit can contain some of the agents separately, or the agents can already be combined in the kit.

[0090] The kits of the invention can be designed for the conditions necessary to properly maintain the components contained within the kit (e.g., refrigeration or freezing). The kits can contain labels or package inserts that contain identifying information for the components in the kit and instructions for use of the components (e.g., clinical pharmacological properties of the active ingredients, including administration parameters, mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.).

[0091] Each component of the kit can be enclosed within an individual container, and all of the various containers can be in a single package. The label or package insert can include manufacturer information such as lot number and expiration date. The label or package insert can, for example, be integrated into the physical structure that houses the component, can be separately contained within the physical structure, or can be affixed to the component of the kit (e.g., an ampoule, syringe, or vial).

[0092] The label or insert may additionally include or be incorporated into a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), an optical disk such as a CD-ROM or DVD-ROM / RAM, a DVD, MP3, magnetic tape, or an electronic storage medium such as RAM and ROM, or a combination thereof such as a magnetic / optical storage medium, FLASH® media, or a memory type card, etc. In some embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via an internet site.

[0093] example The following examples are offered by way of illustration and should not be construed in any way as limiting the claimed invention.

[0094] Example 1 – Cancer Treatment Paradigm Combination therapy including chemotherapy drugs, drug-resistant γδ-T cells, PARP-1 inhibitors, and immune checkpoint inhibitors (multiple inhibitors) is used to treat cancer patients. For the treatment of ovarian cancer, niraparib, olaparib, or rucaparib is used as PARP inhibitors. For the treatment of glioblastoma, olaparib or niraparib is used as PARP inhibitors. The chemotherapy drugs are selected from alkylating drugs (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX), 5-fluorouracil or its derivatives), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor drugs (e.g., vincristine, vindesine, TAXOL®, paclitaxel, abraxane), cisplatin, carboplatin, etoposide, etc. Such agents may further include, but are not limited to, the anticancer drugs trimethotrexate (TMTX), temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), bis-chloronitrosourea (BCNU), cytarabine, and camptothecin, or therapeutic derivatives of any of these. Drug-resistant γδ-T cells are resistant to chemotherapeutic drugs. Immune checkpoint inhibitors, used alone or in combination with other immune checkpoint inhibitors, are inhibitors of immune checkpoint proteins CTLA-4, PDL1 (B7-H1, CD274), PDL2 (B7-DC, CD273), PD1, B7-H3 (CD276), B7-H4 (B7-S1, B7x, VCTN1), BTLA (CD272), HVEM, TIM3 (HAVcr2), GAL9, LAG3 (CD223), VISTA, KIR, 2B4 (CD244, belonging to the CD2 molecule family) and are involved in the regulation of NK cells, γδ cells, and memory CD8 cells. + (expressed in all αβ)T cells), CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, OX40, A2aR and various B-7 family ligands.

[0095] Assessment of immunotherapy activity in combination with chemotherapy: Response evaluation criteria include Response Evaluation Criteria in Solid Tumors (WHO; World Health Organization Offset Publication No. 48, 1979), RECIST (Response Evaluation Criteria in Solid Tumors, J Natl Cancer Inst 2000;92:205-16, Eisenhauer EA et al., Eur J Cancer 2009;45:228-47), immune-related response criteria (Jedd WE et al., Clin Cancer Res 2009;15(23),7412-7420), and iRANO (Response Evaluation of Immunotherapy in Neuro-Oncology) (Okada H et al., Lancet Oncol 2015 Nov;16(15):e534-42).

[0096] result: Chemotherapy as a combination therapy additionally including drug-resistant γδ-T cells (and / or drug-resistant natural killer cells), a PARP-1 inhibitor, and an inhibitor(s) of immune checkpoint proteins results in enhanced or prolonged antitumor responses; medically beneficial responses, additive or synergistic antitumor activity when compared to chemotherapy alone or chemotherapy in combination with drug-resistant γδ-T cells (and / or drug-resistant natural killer cells), reduction in baseline lesions without new lesions; tumor regression, durable stable disease with the possibility of a steady decrease in total tumor burden, responses after an increase in total tumor burden, and responses in the presence of new lesions. Other clinical findings may include an increase in tumor-infiltrating lymphocytes as measured by an increase in tumor size, and enhanced long-term survival.

[0097] For example, temozolomide (TMZ)-treated tumors result in the production of N-methylpurine and O 6 This results in the formation of a significant number of DNA adducts, including N-methylguanine. Such adducts result in the activation of the DNA damage response (DDR) and mismatch repair (MMR) mechanisms. The resistance mechanisms to these DNA adducts are via the base excision repair (BER) pathway, which involves PARP-1 to remove the N-methylpurine adducts, or via the O 6This occurs through the presence of the methylguanine DNA methyltransferase (MGMT) protein. 6 -Methylguanine is the primary mechanism for TMZ resistance, and when MGMT is absent or inactive, activation of the MMR process leads to cross-linking, blocking of replication forks and formation of double-stranded DNA breaks (DSBs), which result in the formation of MSH-2 / MSH-6 dimers and activation of ataxia telangiectasia Rad3-related (ATR) and ataxia telangiectasia mutated (ATM) kinases.

[0098] Activation of this cascade results in the expression of an increased number of stress ligands on the surface of tumor cells, such as MICA, MICB, ULBP1-6, and MSH-2. Tumor cell death is promoted by drug-resistant γδ-T cells and / or drug-resistant natural killer cells, whose receptors (such as NKG2D) recognize tumor-specific stress ligands. However, tumor cells can enzymatically cleave MICA and MICB to release soluble MICA and MICB. When bound by effector cell receptors in plasma, this can lead to downregulation of receptor expression resulting in immune evasion. Chemotherapy such as TMZ has been found to increase cell surface expression of stress ligands on tumor tissues without significantly increasing the concentration of soluble ligands in plasma. PARP-1 inhibitors such as niraparib, olaparib, or rucaparib have been approved for use in ovarian cancer.

[0099] method Ex vivo expansion of γδ T cells Collection of human isolated exchange products from healthy donors. For static culture: all operations were performed in an ISO-5 laminar flow hood surrounded by an ISO-7 classified clean room environment. The product was diluted volume / volume with HBSS (Hank's Balanced Salt Solution) and mononuclear cells were isolated by density gradient separation on Ficoll (Sigma-Aldrich, St. Louis, MO). Interphases were collected and washed twice in HBSS. Cell pellets were resuspended in CTS™ OpTmizer™ T-cell growth serum-free medium (ThermoFisher Scientific, Waltham, MA) supplemented with 2 mM zoledronic acid (Novartis, Basel HV) and 100 u / mL IL2 (Miltenyi Biotec Ltd, Bergisch Gladbach, Germany). Cells were cultured in standard T150 or T75 flasks. For closed culture: All operations, including preparation of media, stock solutions and buffers, were performed in an ISO7 clean room under biosafety cabinet ion and ISO-t clean room environment using GMP-grade reagents. Automated Ficoll separation and cultivation of the products were performed in a CLINIMACS PRODIGY® (Miltenyi Biotec Ltd, Bergisch Gladbach, Germany) bioreactor using an individualized cell processing program and a TS520 capillary set. Cells were then expanded in a CLINIMACS PRODIGY® CentriCult-Unit using a combination of zoledronate (Zometa; Novartis, Basel) and interleukin-2 (Miltenyi Biotec Ltd; Bergisch Gladbach, Germany) (ZOL / IL-2) in OpTmizer serum-free culture (Thermo Fisher Scientific, Waltham, MA). Cultures were grown at 1–2 × 10 6The cells were maintained at a density of 10 cells / ml and supplemented with IL-2 100u / mL every other day until the cells were harvested on day 13. The process was continuously monitored to determine the proliferation and phenotypic kinetics in comparison to small-scale culture in flasks at regular intervals. After 9-14 days of culture, the expanded γδ T cells were phenotyped and cytotoxicity was determined against standard leukemia cell lines and glioma tumor cells.

[0100] Example 2 - Expression of stress ligands on glioma cell lines upon exposure to a combination of PARP inhibitors (PARPi) and temozolomide (TMZ). material and method Cell collection method / processing / culture conditions: The overall strategy for generating the treatment regimen is outlined below: The goal was to determine the total number of γδ T cells required to treat a patient with the initial dose administered as a freshly prepared cell population.

[0101] Following patient enrollment, one 2-volume separated exchange collection was obtained following resection and immediately prior to the start of induction chemotherapy / radiotherapy.

[0102] MNC expansion cultures were performed in a Prodigy® Bioreactor at 2.0 × 10 6 The cells were incubated at 37°C, 5% CO in an OpTimizer (Life Technologies) for 12 h at 4°C for 24 h. 2 + 5 μgM zoledronic acid (Novartis Oncology; East Hanover, NJ) + 50 u / ml IL-2 (Miltenyi, Duarte, CA). Cultures are maintained for 7 days with the addition of 100 u / mL IL-2 on days 2, 5, and 7 of culture, increasing to 400 u / mL on days + 9, + 11, and + 13. Addition of complete medium is determined by pH and cell density.

[0103] All cells were cultured in the PRODIGY® located in an ISO-7 clean room with a minimum of 60 air changes per hour. Operations performed outside the bioreactor are performed in the clean room under an ISO5 biosafety cabinet.

[0104] To assess cell numbers and differentials, samples for analysis were obtained before expansion and on days 2, 5, 7, 9, 11 and 14. FACS analysis was performed to determine cell viability and enumerate relevant cell subsets including total CD3 T cells, total γδ T cells (including Vδ2 and Vδ1 subsets), total αβ T cells (including CD4 and CD8 subsets), as well as NK and B cells. All specimens were analyzed on a FACS Canto® or LSR Fortessa SE® flow cytometer using FACS DiVa® software.

[0105] The final product was harvested on day +14, ±3 days. Samples of the final product were counted, analyzed by flow cytometry, and subjected to vector copy number (VCN) and required microbiological testing. The final product is diluted 1:2 and washed in Plasma-Lyte A and 10% HSA to remove all remaining manufacturing reagents. After washing and concentration, the product is resuspended in Plasma-Lyte A, 5% HSA, and 10% DMSO and diluted at 1 x 10 until needed. 7 Store frozen in 2 mL of Cryovial at a concentration of 100 cells / mL.

[0106] Ex vivo modification: Once cultures reached logarithmic growth phase, typically >20% γδ T cells, cells were transduced with MGMT lentivirus (Miltenyi Lentigen, Baltimore, MD). Cultures were transduced by spinoculation at an MOI of 10 × 2.

[0107] The product was thawed from frozen storage and washed in buffered saline and 5% Plasma-Lyte. After thawing, the final product was diluted to 1×10 7Resuspend the cells in the same wash buffer to a concentration of 10 γδ T cells.

[0108] Assay: To measure putative differential sensitivity of GBM plus temozolomide (TMZ) to PARPi, standardized GBM cell lines LN229, U87MG and U251MG cells (6000 cells per well of a 96-well plate, seeded in triplicate) were exposed to olaparib (5 or 10 mM) alone, olaparib (an exemplary PARP inhibitor) + 200 mg / mL TMZ or media alone (RPMI + 10% FBS media) for 4 hours and viability measured at the start and after 8, 16, 24 and 36 hours of treatment using a standardized MTT viability assay and incorporation of 7-AAD in a suspension flow cytometry assay.

[0109] Data were expressed as %MTT uptake relative to no treatment after cell lysis, and %7-AAD negative cells for suspension flow cytometry assays. Expression of NKG2D ligands MIC A / B and ULBP1-6 was also assessed in suspension assays by flow cytometry for each cell line and condition described above. Data were expressed as logarithmic mean fluorescence intensity (MFI) relative to control (medium only) for all time points.

[0110] result: The data in Figures 1-3 show that the combination of a PARP inhibitor with temozolomide (TMZ) acts synergistically to upregulate selected natural killer group 2D (NKG2D) ligands on three highly malignant glioma cell lines.

[0111] In many cases, even during the optimal treatment window for established DRI treatment with TMZ alone as described, for example, in Lamb et al., PloS ONE 8(1):e51805.doi:10.1371 / journal.pone.0051805, the maximum increase in NKG2DL was transient, with expression beginning to downregulate after approximately 24 hours.

[0112] The patents and scientific literature cited herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

[0113] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. It will also be understood that any of the embodiments described herein are not mutually exclusive and may be combined in various manners without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A pharmaceutical composition for the treatment of glioblastoma, comprising: i) γδ T cells genetically engineered to be resistant to chemotherapeutic drugs, used in combination with ii) and iii) below; ii) a chemotherapeutic agent to which γδ T-cells have been engineered to be resistant, said chemotherapeutic agent being temozolomide; and iii) a PARP inhibitor selected from the group consisting of niraparib and olaparib; The pharmaceutical composition comprising:

2. A pharmaceutical composition for the treatment of glioblastoma, comprising: Chemotherapy drugs used in combination with: γδ T cells genetically engineered to be resistant to said chemotherapeutic agent; and comprising a PARP inhibitor selected from the group consisting of niraparib and olaparib; wherein the chemotherapy drug is temozolomide. The above pharmaceutical composition.

3. A pharmaceutical composition for the treatment of glioblastoma, comprising: comprising a PARP inhibitor selected from the group consisting of niraparib and olaparib; The PARP inhibitor is: γδ T cells engineered to be resistant to chemotherapeutic drugs, and a chemotherapy drug, to which the γδ T-cells have been genetically engineered to be resistant, the chemotherapy drug being temozolomide; The above pharmaceutical composition is used in combination with

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the PARP inhibitor is administered prior to administering the chemotherapeutic agent and prior to administering the composition comprising the engineered γδ T-cells.

5. The method of claim 3, wherein the PARP inhibitor is administered 1 to 21 days prior to administration of the chemotherapeutic agent.

6. 5. The pharmaceutical composition of claim 4, wherein the composition comprising the engineered γδ T-cells is administered 8-72 hours after administration of the chemotherapeutic agent.

7. 5. The pharmaceutical composition of claim 4, wherein the composition comprising the engineered γδ T-cells is administered 12 to 36 hours after administration of the chemotherapeutic agent.

8. 5. The pharmaceutical composition of claim 4, wherein the composition comprising the engineered γδ T-cells is administered 24 hours after administration of the chemotherapeutic agent.

9. 5. The pharmaceutical composition of claim 4, wherein the composition comprising the engineered γδ T-cells is administered 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours after administration of the chemotherapeutic agent.

10. The γδ T cells are ６ The pharmaceutical composition according to any one of claims 1 to 3, which has been genetically modified to encode methylguanine DNA methyltransferase (MGMT).

11. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the composition comprising engineered γδ T cells comprises more than 60% γδ T cells, less than 5% αβ T cells, and less than 25% natural killer (NK) cells.

12. The composition comprising the engineered γδ T cells is administered at a concentration of 5×10 per kg of patient body weight. ８ γδ T cells, 1×10 per kg of patient body weight or less ７ γδ T cells or less per kg of patient body weight ６ The pharmaceutical composition according to any one of claims 1 to 3, comprising no more than γδ T cells.

13. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the PARP inhibitor is administered 1 to 21 days prior to administration of the chemotherapeutic agent and the composition comprising the engineered γδ T-cells is administered 8 to 36 hours after administration of the chemotherapeutic agent.

14. The γδ T cells are ６ 14. The pharmaceutical composition of claim 13, which is genetically modified to encode methylguanine DNA methyltransferase (MGMT).

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the γδ T cells in the composition are an expanded γδ T cell population.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the PARP inhibitor is olaparib.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the PARP inhibitor is administered prior to the γδ T cells.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the chemotherapeutic agent is administered prior to the γδ T-cells.

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