Enhancing activity of cell therapy in tumor microenvironments

Abstract

Embodiments of the present disclosure include improving cell therapy by making cells more effective for cancer therapy, including in solid tumor microenvironments. In particular cases, the cells are modified to reduce or inhibit the expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, or CREB1, such as by CRISPR gene editing. In some cases, the cells are modified to reduce or inhibit the expression level of CREM. In some cases, the cells are further modified to express, for example, one or more engineered receptors, one or more cytokines, and / or optionally one or more suicide genes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 352,516, filed on June 15, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in ST26 format and is hereby incorporated by reference in its entirety. The ST26 copy created on June 11, 2023 is named MDAC_P1332WO_Sequence_Listing.xml and is 240,688 bytes in size. Technical Field

[0005] Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine, including cancer medicine.

[0006] background

[0007] Following the 2017 approval by the U.S. Food and Drug Administration (FDA) of chimeric antigen receptor (CAR)-T cell therapy for the treatment of patients with lymphoma and leukemia, adoptive cell therapy has rapidly become a focal point for stakeholders in the field of cancer immunotherapy. While this treatment modality has demonstrated unprecedented patient responses and offers significant curative potential for certain hematological malignancies, success in solid tumors remains elusive, in part due to the unique characteristics of the solid tumor microenvironment (TME) with hypoxia, acidic pH, nutrient depletion, and immunosuppression (see, e.g., Renner et al., 2017). Acidity is a prominent feature of the tumor microenvironment, primarily due to acidic metabolites such as lactate produced by active glycolysis under hypoxic conditions (see, e.g., Huber et al., 2017). Acidity mediates immunosuppression, tumor progression, and poor prognosis. Specifically, tissue acidosis leads to inhibition of immune cell-mediated responses, such as reduced natural killer (NK) and T cell cytotoxicity, cytokine production, and tumor surveillance.

[0008] Provided herein are techniques for overcoming the immunosuppressive phenotype associated with an acidic environment and / or TME.

[0009] The present disclosure provides a solution to a long-standing need in the field of cancer treatment by manipulating the cAMP signaling pathway through engineering mutations in the G protein-coupled receptor 4 (GPR4), G protein-coupled receptor 31 (GPR31), G protein-coupled receptor 68 (GPR68), G protein-coupled receptor 81 (GPR81), G protein-coupled receptor 132 (GPR132), G protein-coupled receptor 151 (GPR151), cAMP response element modulator (CREM), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes to promote immune effector cell activity in the microenvironment of solid tumors.

[0010] Brief Overview

[0011] Embodiments of the present disclosure include methods and compositions related to cell therapy (including adoptive cell therapy). Specific embodiments of the present disclosure include methods and compositions for cancer immunotherapy, antipathogen immunotherapy, or both. Pathogens include at least viruses, bacteria, fungi, and parasites. The present disclosure includes immune effector cell therapy, which has been improved for the explicit purpose of conferring one or more characteristics on cells to improve their efficacy. In specific embodiments, immune effector cells are modified so that they can better kill target cells, such as cancer cells. In specific embodiments, immune effector cells are engineered to reduce the expression of one or more gene products, which makes the engineered cells effective in acidic environments (such as solid tumor microenvironments) compared to those without engineering, although the cells are also effective for cancers lacking solid tumors (such as blood cancers). In specific embodiments, engineered cells are more suitable for effectively killing cancer cells in an environment of hypoxia, acidic pH, nutrient depletion, and / or immunosuppression.

[0012] In certain embodiments, immune effector cells are included in the compositions and used in the methods contemplated herein, and have been engineered to have reduced expression levels of G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), cAMP response element modulator (CREM), inducible cAMP early repressor (ICER), and / or cyclic AMP response element binding protein 1 (CREB1). In some embodiments, immune effector cells completely inhibit the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, e.g., lack the expression of the above genes detectable by conventional methods in the art.

[0013] In a specific embodiment, endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes have been modified by genetic manipulation of the genomic sites of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. The immune effector cells expressed by GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 with reduction or complete inhibition may or may not be modified by artificial in other ways, such as expressing one or more exogenous gene products. In a specific embodiment, the gene product is a receptor, a cytokine, a chemokine, a suicide gene or a combination thereof. In a specific case, the receptor is an antigen receptor, wherein the antigen may or may not be a cancer antigen, including an antigen on a solid tumor cell. In certain instances, the antigen receptor is a chimeric antigen receptor (CAR) or a non-natural T cell receptor.

[0014] In some embodiments, the disclosure knocks out or knocks down genes encoding GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 from immune effector cells used for various cell therapies to make them insensitive to the immunosuppressive effects of acidity, thereby increasing their survival rate, proliferation rate and immune function, including at least in an acidic solid tumor microenvironment. Taking gene editing CRISPR / Cas9 technology as an example, the feasibility of knocking out cAMP sensing and / or signaling pathways using Cas9 preloaded with chemically synthesized crFNA:tracrRNA duplexes targeting CREM has been confirmed. The data disclosed herein show that knocking out CREM from NK cells leads to its cytotoxic effects and the improvement of the anti-tumor activity of cancer cell lines characterized by active glycolysis and significant acidosis of the microenvironment. In some embodiments, the genetic engineering strategy targeting CREM can be combined with different forms of cell therapy, including CAR-T cells, CAR-NK cells, T cell receptor (TCR)-T cells, T cell receptor (TCR)-NK cells, tumor infiltrating lymphocytes (TILs), or a combination thereof, to enhance their therapeutic effects on various types of cancer, including solid tumors.

[0015] The engineered immune effector cells can be of any kind, but in certain embodiments, the immune effector cells are T cells, natural killer (NK) cells, NK T cells, macrophages, B cells, tumor infiltrating lymphocytes, dendritic cells, mesenchymal stem cells (MSC), combinations thereof, etc. In certain instances, the immune effector cells are NK cells, including NK cells derived from umbilical cord blood.

[0016] Any medical condition can be treated by administering a therapeutically effective amount of the engineered immune effector cells of the encompassed disclosure. In certain embodiments, these cells are used in compositions for treating any type of cancer.

[0017] The present disclosure relates to new strategies for knocking out GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes from immune cells using gene editing technology (e.g., CRISPR / Cas9) to empower them and enhance their anti-tumor activity as cell therapy against any type of cancer, including at least solid tumors.

[0018] The embodiments of the present disclosure include compositions and uses thereof for engineered immune effector cells, wherein the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes in the cells is reduced or completely suppressed. In a specific embodiment, the cell is a T cell, NK cell, NK T cell, macrophage, B cell, unchanged NK T cell, γδT cell, MSC, tumor infiltrating lymphocyte, dendritic cell or a mixture thereof. In a specific embodiment, NK cells are derived from cord blood. In some cases, the cell comprises one or more engineered receptors, including engineered antigen receptors, such as CAR, chemokine receptors, homing receptors and / or non-natural T cell receptors. Antigens can be cancer antigens, including solid tumor antigens. Specific examples of antigens include antigens selected from the group consisting of 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, TROP2, HMW-MAA, VEGFR2, and combinations thereof (a receptor can have two or more antigen binding domains that bind different antigens).

[0019] In certain embodiments, the cell comprises expression of one or more exogenous chemokines or one or more cytokines. Examples of cytokines include IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof. Additionally, or alternatively, the cell comprises a suicide gene.

[0020] The expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes can be reduced or inhibited by homologous recombination or non-homologous recombination. In some cases, endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes are knocked out by CRISPR-Cas9. Any cell of the present disclosure includes autologous, allogeneic or xenogeneic cells relative to the recipient individual.

[0021] In specific embodiments, the cells further reduce or inhibit the expression of one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR and CD7.

[0022] Embodiments of the present disclosure include populations of any of the cells contemplated herein. In specific embodiments, the population is contained in a pharmaceutically acceptable excipient.

[0023] Specific embodiments of the present disclosure include methods for engineering NK cells so that their function is improved in any way (including non-transiently) and relative to NK cells that are not so engineered. In specific embodiments, the genetic modification in NK cells causes the cells to have enhanced cytotoxicity to cancer cells and / or have enhanced amplification, persistence and / or proliferative properties compared to NK cells that are not so engineered. The methods of the present disclosure include methods for suppressing immune cell-mediated responses in vivo in individuals receiving any type of adoptive cell therapy (including using T cells and / or NK cells (only as an example)), and in this case, the cells are engineered to reduce or completely inhibit the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1.

[0024] Embodiments of the present disclosure include improving any type of adoptive cell therapy in a tumor microenvironment by utilizing engineered cells as described herein compared to cells without such engineering. In a specific embodiment, the present disclosure includes producing and using immune effector cells, which have an engineered (as opposed to being natural for cells) reduction or fully inhibited endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression in cells and have enhanced cytotoxicity, persistence and amplification compared to cells without engineering to cause reduction or fully inhibited expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 in cells.

[0025] Any type of immune effector cells, such as NK cells, can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue, tumors or commercially available. Any number of immune cell lines known and available to those skilled in the art can be used.

[0026] In addition to immune effector cells being engineered to have endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 reduction or completely suppressed expression, in at least some cases, engineered immune effector cells are engineered in one or more other aspects. In a specific embodiment, cells are also engineered to express one or more engineered receptors (contrary to the receptor endogenous to the cell), one or more cytokines and / or one or more suicide genes. Engineered receptors can be any kind, including at least one or more CAR, one or more T cell receptors, one or more chemokine receptors, their combination, etc. Any engineering of immune effector cells may occur or may not occur after knocking out (or knocking down) GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression. In the case of the engineered immune effector cells of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 with reduction or complete suppression, it is also engineered to express two or more other genes, and the engineering for expressing two or more other genes can occur simultaneously with each other, or can not occur simultaneously with each other.For example, in the case of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 knockout (KO) (or knocking low) cells are engineered to express CAR and cytokines, the engineering of expressing CAR and cytokines may or may not occur substantially simultaneously.Any other transgenic of cell may or may not be expressed from the same vector.In exemplary cases, after transfection or conversion of immune effector cells, CAR and cytokines (only as representatives) may or may not be expressed from the same vector.

[0027] Embodiments of the present disclosure include methods for treating individual cancers, including the step of administering a therapeutically effective amount of a cell population of the present disclosure to an individual. In some cases, the cancer is a solid tumor or is not a solid tumor. The cancer can be lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer or cervical cancer. The individual can be a mammal, such as a human, dog, cat, horse, cattle, sheep, pig or rodent. The individual may or may not receive other cancer treatments, such as surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy or a combination thereof. In a specific embodiment, the method also includes the step of diagnosing the individual's cancer. In some cases, the method also includes the step of producing a cell population. Relative to the individual, the cell can be autologous or allogeneic.

[0028] In certain embodiments, the cell is a NK cell, such as a cord blood NK cell, including a cell expressing one or more engineered antigen receptors. The cell can be a NK cell expressing a CAR or a NK cell expressing a TCR.

[0029] In certain embodiments, disclosed herein are engineered immune effector cells, wherein the cells comprise one or more engineered mutations in endogenous G protein-coupled receptor 4 (GPR4), G protein-coupled receptor 31 (GPR31), G protein-coupled receptor 68 (GPR68), G protein-coupled receptor 81 (GPR81), G protein-coupled receptor 132 (GPR132), G protein-coupled receptor 151 (GPR151), cAMP response element regulator (CREM), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes in the cell. In certain embodiments, the mutation is a partial or complete loss of function and / or a knockout (KO) mutation. In certain embodiments, the mutation reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene. In certain embodiments, the mutation is a new morphological mutation or a gain-of-function mutation. In certain embodiments, the mutation increases the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene. In certain embodiments, the mutation results in a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative mRNA population encoded by a non-mutated locus encoding the same endogenous gene. In certain embodiments, the mutation results in a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative protein population encoded by a non-mutated locus encoding the same endogenous gene.

[0030] In certain embodiments, relative to control non-engineered cells, the mutation causes engineered cells to improve cytotoxicity in acidic microenvironments and / or tumor microenvironments (TME). In certain embodiments, relative to control non-engineered cells, the mutation causes engineered cells to improve cytotoxicity in an acidic microenvironment of pH less than or equal to about 7.0. In certain embodiments, relative to control non-engineered cells, the mutation causes engineered cells to improve cytotoxicity in an acidic microenvironment of pH less than or equal to about 5.9. In certain embodiments, relative to control non-engineered cells, the mutation causes engineered cells to improve cytotoxicity in an acidic microenvironment of pH less than or equal to about 5.9. In certain embodiments, relative to control non-engineered cells, the mutation causes engineered cells to respond to the enhanced multifunctionality of the stimulation of tumor cells. In certain embodiments, the enhanced multifunctionality is demonstrated by the increased cytokine release in response to the stimulation of tumor cells. In certain embodiments, multifunctionality is demonstrated by the increase of cytokine release, and the increase of cytokine release includes interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a) and / or degranulation marker CD107a increased in response to the stimulation of tumor cells. In certain embodiments, multifunctionality is demonstrated by an increase in cytokine release, including increased granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin in response to stimulation of tumor cells.

[0031] In certain embodiments, relative to control non-engineered cells, the mutation provides enhanced activation and / or cytotoxic phenotype for engineered cells. In some embodiments, relative to control non-engineered cells, the mutation causes enhanced activation and / or cytotoxic phenotype of engineered cells, wherein the enhanced activation and / or cytotoxic phenotype is related to one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV reaction and / or estrogen early response. In some embodiments, mutation causes the upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling. In certain embodiments, relative to control non-engineered cells, the mutation provides enhanced proliferation capacity and / or persistence phenotype for engineered cells. In certain embodiments, the enhanced proliferation capacity and / or persistence occur in the absence of exogenous interleukin 2 (IL-2) stimulation. In certain embodiments, the enhanced proliferation capacity and / or persistence do not result in autonomous growth. In certain embodiments, relative to control non-engineered cells, the mutation provides an enhanced metabolic adaptability phenotype for engineered cells. In certain embodiments, the enhanced metabolic adaptability is higher glycolytic capacity and / or improved oxygen consumption rate (OCR).

[0032] In certain embodiments, the mutation of the endogenous gene is located in the gene CREM. In certain embodiments, the CREM mutation results in decreased expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217, and / or CREM-225. In certain embodiments, the CREM mutation results in increased expression of CREM RNA isoform CREM-218. In certain embodiments, the CREM mutation is the result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO: 142. In certain embodiments, CREM mutations result in a decrease of more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein. In certain embodiments, CREM mutations result in a decrease of more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and / or 70%. In certain embodiments, CREM mutations result in a decrease of more than 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%.

[0033] In certain embodiments, by contacting the cell with an acidic stimulus in vitro, the cell is further adapted to an acidic environment. In some embodiments, the acidic stimulus is provided at a concentration greater than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM. In some embodiments, the acidic stimulus is provided at a concentration greater than or equal to about 2 to 3 mM. In some embodiments, the acidic stimulus is provided at a concentration greater than or equal to about 2.5 mM. In some embodiments, the adaptation to an acidic microenvironment is achieved by contacting the acidic stimulus with an increment and / or accumulation. In some embodiments, the cell is adapted in a time period of at least about 10 to 18 days, optionally at least about 14 days. In some embodiments, cell adaptation includes adding an acidic stimulus approximately every 48-72 hours, optionally approximately every 48 hours. In some embodiments, the acidic stimulus comprises lactic acid or is substantially composed of lactic acid. In some embodiments, the cell is adapted to an acidic microenvironment less than or equal to about pH 6.0.

[0034] In certain embodiments, the cell is a T cell, a natural killer (NK) cell, a NK T cell, a macrophage, a B cell, an invariant NK T cell, a γδT cell, a MSC, a tumor infiltrating lymphocyte, or a dendritic cell. In certain embodiments, the cell is a NK cell derived from cord blood (CB), peripheral blood (PB), a NK cell line, a bone marrow, a stem cell, or a mixture thereof. In certain embodiments, the NK cell is derived from cord blood.

[0035] In certain embodiments, the cell comprises one or more engineered receptors. In certain embodiments, the engineered receptor includes an engineered antigen receptor. In certain embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In certain embodiments, the engineered antigen receptor is a CAR. In certain embodiments, the antigen is a cancer antigen. In certain embodiments, the antigen is a solid tumor antigen. In certain embodiments, the antigen is selected from 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TROP2, TEMs, HMW-MAA, VEGFR2 and combinations thereof.

[0036] In certain embodiments, one or more engineered receptors include cytokine receptors, chemokine receptors, homing receptors or combinations thereof. In certain embodiments, cells include expression of one or more exogenous chemokines and / or one or more cytokines. In certain embodiments, cytokines are IL-15, IL-12, IL-21, IL-2, IL-18, IL-7 or combinations thereof. In certain embodiments, cytokines are IL-15. In certain embodiments, cells include suicide genes.

[0037] In certain embodiments, endogenous genes are mutated due to homologous recombination or non-homologous recombination. In certain embodiments, endogenous genes are mutated by endonucleases. In certain embodiments, endonucleases are RNA-guided endonucleases. In certain embodiments, RNA-guided endonucleases are CRISPR-Cas9. In certain embodiments, cells include one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR and CD7.

[0038] Also disclosed herein are populations of any of the cells described herein.In some embodiments, the population of cells is contained in a pharmaceutically acceptable excipient.

[0039] Also disclosed herein is a method for treating cancer in an individual, comprising administering to an individual a therapeutically effective amount of a colony of the claimed cells. In some embodiments, the cells are autologous, allogeneic or xenogeneic for the individual. In some embodiments, the cells are allogeneic for the individual. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer does not comprise a solid tumor. In some embodiments, the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer and / or cervical cancer. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human, dog, cat, horse, cattle, sheep, pig or rodent. In some embodiments, the individual is a human. In some embodiments, additional cancer therapies are administered to the individual. In some embodiments, additional cancer therapies are surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy or a combination thereof. In some embodiments, the individual is diagnosed with cancer.

[0040] Also disclosed herein is a method for engineering immune effector cells. In some embodiments, the method for engineering immune effector cells includes endogenous cAMP response element regulators (CREMs), G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes in mutated cells. In some embodiments, mutations produce partial or complete loss of function and / or knockout (KO) mutations. In some embodiments, mutations reduce or inhibit the transcription or post-transcriptional processing of one or more mRNA isomers encoded by mutated endogenous genes relative to non-mutated loci encoding the same endogenous genes. In some embodiments, mutations produce new morphologies or functional gain mutations. In some embodiments, the mutation increases the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene. In some embodiments, the mutation produces a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative population of mRNAs encoded by a non-mutated locus encoding the same endogenous gene. In some embodiments, the mutation produces a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative population of proteins encoded by a non-mutated locus encoding the same endogenous gene.

[0041] In some embodiments, the mutation produces improved cytotoxicity of engineered cells in an acidic microenvironment and / or tumor microenvironment (TME) relative to control non-engineered cells. In some embodiments, the mutation produces improved cytotoxicity of engineered cells in an acidic microenvironment with a pH less than or equal to about 7.0 relative to control non-engineered cells. In some embodiments, the mutation produces improved cytotoxicity of engineered cells in an acidic microenvironment with a pH less than or equal to about 5.9 relative to control non-engineered cells.

[0042] In some embodiments, mutations cause engineered cells to produce enhanced multifunctionality relative to control non-engineered cells in response to stimulation of tumor cells. In some embodiments, enhanced multifunctionality is demonstrated by an increase in cytokine release in response to stimulation of tumor cells. In some embodiments, the increase in cytokine release includes increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or degranulation marker CD107a in response to stimulation of tumor cells. In some embodiments, the increase in cytokine release includes increased granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin in response to stimulation of tumor cells.

[0043] In some embodiments, relative to control non-engineered cells, mutations are engineered cells that produce enhanced activation and / or cytotoxic phenotypes. In some embodiments, relative to control non-engineered cells, mutations are engineered cells that provide enhanced activation and / or cytotoxic phenotypes, wherein the enhanced activation and / or cytotoxic phenotypes are associated with one or more pathways identified by GSEA: G2M checkpoints, E2F targets, P53 pathways, mitotic spindles, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response and / or early estrogen response. In some embodiments, mutations result in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

[0044] In some embodiments, relative to the control non-engineered cells, the mutation is that the engineered cells produce enhanced proliferation capacity and / or persistence phenotype. In some embodiments, the enhanced proliferation capacity and / or persistence occur in the absence of exogenous interleukin 2 (IL-2) stimulation. In some embodiments, the enhanced proliferation capacity and / or persistence do not lead to autonomous growth. In some embodiments, relative to the control non-engineered cells, the mutation is that the engineered cells produce an enhanced metabolic adaptability phenotype. In some embodiments, the enhanced metabolic adaptability is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0045] In some embodiments, the method of engineering immune effector cells comprises mutating the endogenous cAMP response element regulator (CREM) gene. In some embodiments, the mutation of CREM results in decreased expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225. In some embodiments, the mutation of CREM results in increased expression of CREM RNA isoform CREM-218. In some embodiments, the mutation of CREM comprises exposing the cell to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO: 142. In some embodiments, the mutation of CREM results in a decrease of more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% or any range derivable therein. In some embodiments, the mutation of CREM results in a decrease of more than 80% in one or more CREM protein isoforms.

[0046] In certain embodiments, method also comprises making cell adapt to acidic environment by making cell contact with acidic stimulus in vitro.In some embodiments, making cell adapt to acidic environment comprises providing acidic stimulus with the concentration greater than or equal to about 2 to 3mM, optionally greater than or equal to about 2.5mM.In some embodiments, adaptation to acidic microenvironment is realized by contacting with the increment and / or accumulation of acidic stimulus.In some embodiments, adaptation continues at least about 10 to 18 days, optionally at least about 14 days time period.In some embodiments, adaptation comprises adding acidic stimulus about every 48-72 hours, optionally about every 48 hours.In some embodiments, acidic stimulus comprises lactic acid or is substantially composed of lactic acid.In some embodiments, adaptation is to adapt to the acidic microenvironment less than or equal to about pH6.0.

[0047] In some embodiments, the method of engineering immune effector cells includes mutation T cells, natural killer (NK) cells, NK T cells, macrophages, B cells, constant NK T cells, γδ T cells, MSCs, tumor infiltrating lymphocytes, dendritic cells or endogenous genes in their precursor cells. In some embodiments, cells are NK cells derived from cord blood (CB), peripheral blood (PB), NK cell lines, bone marrow, stem cells or their mixtures. In some embodiments, NK cells are derived from cord blood.

[0048] In some embodiments, the method of engineering immune effector cells comprises mutating an endogenous gene in a cell, wherein the cell further comprises one or more engineered receptors. In some embodiments, the one or more engineered receptors comprise an engineered antigen receptor. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In some embodiments, the engineered antigen receptor is a CAR. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is a solid tumor antigen. In some embodiments, the antigen is selected from 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, F BP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TROP2, TEMs, HMW-MAA, VEGFR2, and combinations thereof. In some embodiments, one or more engineered receptors include cytokine receptors, chemokine receptors, homing receptors, or combinations thereof.

[0049] In some embodiments, the method of engineering immune effector cells includes mutating endogenous genes in cells, wherein the cells also include expression of one or more exogenous chemokines and / or one or more cytokines. In some embodiments, the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof. In some embodiments, the cytokine is IL-15. In some embodiments, wherein the cell comprises a suicide gene. In some embodiments, the mutation of the endogenous gene includes homologous recombination or non-homologous recombination. In some embodiments, the mutation of the endogenous gene is mediated by an endonuclease. In some embodiments, the endonuclease is an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is CRISPR-Cas9. In some embodiments, the cells comprise one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR, and CD7.

[0050] The following aspects describe certain inventions disclosed herein.

[0051] Aspect 1 is an engineered immune effector cell, wherein the cell comprises one or more engineered mutations in the cell's endogenous cAMP response element regulator (CREM), G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes.

[0052] Aspect 2 is the cell of aspect 1, wherein the mutation is a partial or complete loss of function and / or a knockout (KO) mutation.

[0053] Aspect 3 is a cell of aspect 1 or 2, wherein the mutation reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

[0054] Aspect 4 is the cell of aspect 1, wherein the mutation is a neomorphic mutation or a gain-of-function mutation.

[0055] Aspect 5 is a cell of aspect 1 or 4, wherein the mutation increases transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

[0056] Aspect 6 is the cell of any one of aspects 1-5, wherein the mutation results in a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative population of mRNAs encoded by a non-mutated locus encoding the same endogenous gene.

[0057] Aspect 7 is the cell of any one of aspects 1-4, wherein the mutation results in a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative population of proteins encoded by a non-mutated locus encoding the same endogenous gene.

[0058] Aspect 8 is the cell of any one of Aspects 1-7, wherein the mutation comprises a knockout (KO) mutation in an endogenous gene of the cell.

[0059] Aspect 9 is the cell of any one of aspects 1-7, wherein the mutation comprises a homozygous mutation in an endogenous gene of the cell.

[0060] Aspect 10 is the cell of any one of aspects 1-7, wherein the mutation comprises a heterozygous mutation in an endogenous gene of the cell.

[0061] Aspect 11 is the cell of any one of aspects 1-10, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment and / or tumor microenvironment (TME) relative to a control non-engineered cell.

[0062] Aspect 12 is the cell of any one of aspects 1-11, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment of pH less than or equal to about 7.0 relative to a control non-engineered cell.

[0063] Aspect 13 is the cell of any one of aspects 1-12, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment of pH less than or equal to about 5.9 relative to a control non-engineered cell.

[0064] Aspect 14 is the cell of any one of aspects 1-13, wherein the mutation results in improved cytotoxicity of the engineered cell, optionally in an acidic microenvironment characterized by increased lactate levels relative to a non-acidic microenvironment.

[0065] Aspect 15 is the cell of any one of aspects 1-14, wherein the mutation results in enhanced multifunctionality of the engineered cell in response to stimulation by a tumor cell relative to a control non-engineered cell.

[0066] Aspect 16 is the cell of aspect 15, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation of tumor cells.

[0067] Aspect 17 is the cell of aspect 16, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or degranulation marker CD107a in response to stimulation of tumor cells.

[0068] Aspect 18 is a cell of Aspect 15 or 16, wherein the increased cytokine release includes increased granulocyte-macrophage colony stimulating factor (GMCSF) in response to stimulation of tumor cells, soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin.

[0069] Aspect 19 is the cell of any one of aspects 1-18, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxicity phenotype relative to a control non-engineered cell.

[0070] Aspect 20 is a cell of any one of Aspects 1-19, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxic phenotype relative to a control non-engineered cell, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response and / or estrogen early response.

[0071] Aspect 21 is the cell of any one of aspects 1-20, wherein the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

[0072] Aspect 22 is the cell of any one of aspects 1-21, wherein the mutation provides the engineered cell with enhanced proliferation capacity and / or persistence phenotype relative to a control non-engineered cell.

[0073] Aspect 23 is the cell of aspect 22, wherein the enhanced proliferation capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

[0074] Aspect 24 is the cell of aspect 22 or 23, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

[0075] Aspect 25 is the cell of any one of aspects 1-24, wherein the mutation provides the engineered cell with an enhanced metabolic fitness phenotype relative to a control non-engineered cell.

[0076] Aspect 26 is the cell of aspect 25, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0077] Aspect 27 is a cell of any one of aspects 1-26, wherein the mutation provides increased chromatin accessibility across the genome when the cell is contained in an acidic microenvironment.

[0078] Aspect 28 is the cell of any one of aspects 1-27, wherein the endogenous gene is CREM.

[0079] Aspect 29 is a cell according to aspect 28, wherein the CREM mutation results in reduced expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

[0080] Aspect 30 is the cell of aspect 28 or 29, wherein the CREM mutation results in increased expression of the CREM RNA isoform CREM-218.

[0081] Aspect 31 is the cell of any one of aspects 28-30, wherein the CREM mutation is the result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO:140 and / or SEQ ID NO:142.

[0082] Aspect 32 is the cell of any one of aspects 28-31, wherein the CREM mutation results in a reduction of one or more CREM protein isoforms by more than 60%.

[0083] Aspect 33 is the cell of any one of aspects 28-32, wherein the CREM mutation results in a greater than 80% reduction in one or more CREM protein isoforms.

[0084] Aspect 34 is the cell of any one of aspects 1-33, wherein the cell has been further adapted to an acidic microenvironment by contacting the cell with an acidic stimulus ex vivo.

[0085] Aspect 35 is the cell of aspect 34, wherein the acidic stimulator is provided at a concentration greater than or equal to about 2 to 3 mM, optionally greater than or equal to about 2.5 mM.

[0086] Aspect 36 is the cell of any one of aspects 34-35, wherein adaptation to the acidic microenvironment is achieved by incremental and / or cumulative contact with an acidic stimulus.

[0087] Aspect 37 is the cell of any one of aspects 34-36, wherein the cell is adapted over a period of at least about 10 to 18 days, optionally at least about 14 days.

[0088] Aspect 38 is the cell of aspect 36 or 37, wherein cell adaptation comprises adding an acidic stimulus about every 48-72 hours, optionally about every 48 hours.

[0089] Aspect 39 is the cell of any one of aspects 34-37, wherein the acidic stimulus comprises or consists essentially of lactic acid.

[0090] Aspect 40 is the cell of any one of aspects 34-39, wherein the cell is adapted to an acidic microenvironment of less than or equal to about pH 6.0.

[0091] Aspect 41 is the cell of any one of aspects 1-40, wherein the cell is a T cell, a natural killer (NK) cell, a NK T cell, a macrophage, a B cell, an invariant NK T cell, a γδ T cell, a MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

[0092] Aspect 42 is the cell of any one of aspects 1-41, wherein the cell is a NK cell derived from cord blood (CB), peripheral blood (PB), NK cell lineage, bone marrow, stem cells or a mixture thereof.

[0093] Aspect 43 is the cell of Aspect 41 or 42, wherein the NK cell is derived from umbilical cord blood.

[0094] Aspect 44 is the cell of any one of aspects 1-43, wherein the cell comprises one or more engineered receptors.

[0095] Aspect 45 is a cell of aspect 44, wherein the one or more engineered receptors comprise an engineered antigen receptor that specifically targets an antigen.

[0096] Aspect 46 is the cell of Aspect 45, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0097] Aspect 47 is a cell of Aspect 46, wherein the engineered antigen receptor is a CAR.

[0098] Aspect 48 is the cell of any one of aspects 45-47, wherein the antigen is a cancer antigen.

[0099] Aspect 49 is the cell of any one of aspects 45-48, wherein the antigen is a solid tumor antigen.

[0100] Aspect 50 is a cell of any one of Aspects 45-49, wherein the antigen is selected from 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM , EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TROP2, TEM, HMW-MAA, VEGFR2 and combinations thereof.

[0101] Aspect 51 is a cell according to any one of aspects 45-50, wherein the antigen comprises TROP2 and / or CD70.

[0102] Aspect 52 is the cell of any one of aspects 44-51, wherein the one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

[0103] Aspect 53 is the cell of any one of aspects 1-52, wherein the cell comprises expression of one or more exogenous chemokines and / or one or more cytokines.

[0104] Aspect 54 is the cell of Aspect 53, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7 or a combination thereof.

[0105] Aspect 55 is the cell of Aspect 54, wherein the cytokine is IL-15.

[0106] Aspect 56 is the cell of any one of aspects 1-55, wherein the cell comprises a suicide gene.

[0107] Aspect 57 is the cell of any one of aspects 1-56, wherein the endogenous gene is mutated due to homologous recombination or non-homologous recombination.

[0108] Aspect 58 is a cell according to any one of aspects 1-57, wherein the endogenous gene is mutated by an endonuclease.

[0109] Aspect 59 is the cell of aspect 58, wherein the endonuclease is an RNA-guided endonuclease.

[0110] Aspect 60 is the cell of Aspect 59, wherein the RNA-guided endonuclease is CRISPR-Cas9.

[0111] Aspect 61 is a cell of any one of Aspects 1-60, wherein the cell comprises one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR and CD7.

[0112] Aspect 62 is a population of cells, said cells being cells according to any one of aspects 1-61.

[0113] Aspect 63 is the cell population of Aspect 62, wherein the cell population is contained in a pharmaceutically acceptable excipient.

[0114] Aspect 64 is a method of treating cancer in an individual in need thereof, comprising the step of administering to the individual a therapeutically effective amount of the cell population of aspect 62 or 63.

[0115] Aspect 65 is the method of aspect 64, wherein the cells are autologous, allogeneic or xenogeneic to the individual.

[0116] Aspect 66 is the method of aspect 64 or 65, wherein the cells are allogeneic to the individual.

[0117] Aspect 67 is the method of any one of aspects 64-66, wherein the cancer comprises a solid tumor.

[0118] Aspect 68 is the method of any one of aspects 64-66, wherein the cancer does not comprise a solid tumor.

[0119] Aspect 69 is the method of any one of Aspects 64 to 68, wherein the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer and / or cervical cancer.

[0120] Aspect 70 is the method of any one of aspects 64-69, wherein the individual is a mammal.

[0121] Aspect 71 is the method of aspect 70, wherein the subject is a human, dog, cat, horse, cow, sheep, pig or rodent.

[0122] Aspect 72 is the method of aspect 70 or 71, wherein the individual is a human.

[0123] Aspect 73 is the method of any one of aspects 64-72, wherein an additional cancer therapy is administered to the individual.

[0124] Aspect 74 is the method of aspect 73, wherein the additional cancer treatment is surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0125] Aspect 75 is the method of any one of aspects 64-74, further comprising the step of diagnosing cancer in the individual.

[0126] Aspect 76 is a method for engineering immune effector cells, comprising mutating the cells' endogenous cAMP response element modulator (CREM), G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes.

[0127] Aspect 77 is the method of aspect 76, wherein the mutation produces a partial or complete loss of function and / or a knockout (KO) mutation.

[0128] Aspect 78 is the method of aspect 76 or 77, wherein the mutation reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

[0129] Aspect 79 is the method of aspect 76, wherein the mutation produces a neomorphic mutation or a gain-of-function mutation.

[0130] Aspect 80 is the method of aspect 76 or 79, wherein the mutation increases transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

[0131] Aspect 81 is the method of any one of aspects 76-80, wherein the mutation produces a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative population of mRNAs encoded by a non-mutated locus encoding the same endogenous gene.

[0132] Aspect 82 is the method of any one of aspects 76-79, wherein the mutation produces a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative population of proteins encoded by a non-mutated locus encoding the same endogenous gene.

[0133] Aspect 83 is the method of any one of aspects 76-82, wherein the mutation produces a knockout (KO) mutation in an endogenous gene of the cell.

[0134] Aspect 84 is the method of any one of aspects 76-82, wherein the mutation produces a homozygous mutation in an endogenous gene of the cell.

[0135] Aspect 85 is the method of any one of aspects 76-82, wherein the mutation produces a heterozygous mutation in an endogenous gene of the cell.

[0136] Aspect 86 is the method of any one of aspects 76-85, wherein the mutation results in improved cytotoxicity of the engineered cells in an acidic microenvironment and / or a tumor microenvironment (TME) relative to control non-engineered cells.

[0137] Aspect 87 is the method of any one of aspects 76-86, wherein the mutation results in improved cytotoxicity of the engineered cells in an acidic microenvironment at a pH less than or equal to about 7.0 relative to control non-engineered cells.

[0138] Aspect 88 is the method of any one of aspects 76-87, wherein the mutation results in improved cytotoxicity of the engineered cells in an acidic microenvironment at a pH less than or equal to about 5.9 relative to control non-engineered cells.

[0139] Aspect 89 is the method of any one of aspects 76-88, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment characterized by increased lactate levels relative to a non-acidic microenvironment.

[0140] Aspect 90 is the method of any one of aspects 76-89, wherein the mutations result in enhanced pluripotency of the engineered cells in response to stimulation by tumor cells relative to control non-engineered cells.

[0141] Aspect 91 is the method of aspect 90, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation of tumor cells.

[0142] Aspect 92 is the method of aspect 91, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or degranulation marker CD107a in response to stimulation of tumor cells.

[0143] Aspect 93 is the method of aspect 90 or 91, wherein the increased cytokine release comprises increased granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin in response to stimulation of tumor cells.

[0144] Aspect 94 is the method of any one of aspects 76-93, wherein the mutation results in an enhanced activation and / or cytotoxicity phenotype for the engineered cell relative to a control non-engineered cell.

[0145] Aspect 95 is the method of any one of Aspects 76-93, wherein the mutation causes the engineered cells to have an enhanced activation and / or cytotoxic phenotype relative to control non-engineered cells, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response and / or estrogen early response.

[0146] Aspect 96 is the method of any one of aspects 76-95, wherein the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

[0147] Aspect 97 is the method of any one of aspects 76-96, wherein the mutation results in an enhanced proliferation capacity and / or persistence phenotype for the engineered cell relative to a control non-engineered cell.

[0148] Aspect 98 is the method of aspect 97, wherein the enhanced proliferation capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

[0149] Aspect 99 is the method of aspect 97 or 98, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

[0150] Aspect 100 is the method of any one of aspects 76-99, wherein the mutation produces an enhanced metabolic fitness phenotype for the engineered cell relative to a control non-engineered cell.

[0151] Aspect 101 is the method of aspect 100, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0152] Aspect 102 is the method of any one of aspects 76-101, wherein the mutation provides increased chromatin accessibility throughout the genome when the cell is contained in an acidic microenvironment.

[0153] Aspect 103 is the method of any one of aspects 76-102, wherein the endogenous gene is CREM.

[0154] Aspect 104 is the method of aspect 103, wherein the mutation of CREM results in reduced expression of CREM RNA isoform CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

[0155] Aspect 105 is the method of aspect 103 or 104, wherein the mutation in CREM results in increased expression of the CREM RNA isoform CREM-218.

[0156] Aspect 106 is the method of any one of aspects 103-105, wherein the mutation of CREM comprises exposing the cell to a polynucleotide comprising the sequence of SEQ ID NO:140 and / or SEQ ID NO:142.

[0157] Aspect 107 is the method of any one of aspects 103-106, wherein the mutation of CREM results in a greater than 60% reduction in one or more CREM protein isoforms.

[0158] Aspect 108 is the method of any one of aspects 103-107, wherein the mutation of CREM results in a greater than 80% reduction in one or more CREM protein isoforms.

[0159] Aspect 109 is the method of any one of aspects 76-108, further comprising adapting the cells to the acidic microenvironment by contacting the cells with an acidic stimulus ex vivo.

[0160] Aspect 110 is the method of aspect 109, comprising providing the acidic stimulus at a concentration greater than or equal to about 2 to 3 mM, optionally greater than or equal to about 2.5 mM.

[0161] Aspect 111 is the method of aspect 109 or 110, wherein adaptation to the acidic microenvironment is achieved by incremental and / or cumulative contact with an acidic stimulus.

[0162] Aspect 112 is the method of any one of aspects 109-111, wherein the adapting continues for a period of at least about 10 to 18 days, optionally at least about 14 days.

[0163] Aspect 113 is the method of any one of aspects 109-112, wherein the adapting comprises adding the acidic stimulus about every 48-72 hours, optionally about every 48 hours.

[0164] Aspect 114 is the method of any of aspects 109-113, wherein the acidic stimulus comprises or consists essentially of lactic acid.

[0165] Aspect 115 is the method of any one of aspects 109-114, wherein the adapting is to an acidic microenvironment of less than or equal to about pH 6.0.

[0166] Aspect 116 is the method of any one of aspects 76-115, wherein the cell is a T cell, a natural killer (NK) cell, an NK T cell, a macrophage, a B cell, an invariant NK T cell, a γδ T cell, a MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

[0167] Aspect 117 is the method of any one of aspects 76 to 116, wherein the cell is a NK cell derived from cord blood (CB), peripheral blood (PB), NK cell lineage, bone marrow, stem cells or a mixture thereof.

[0168] Aspect 118 is the method of aspect 116 or 117, wherein the NK cells are derived from umbilical cord blood.

[0169] Aspect 119 is the method of any one of aspects 76-118, wherein the cell comprises one or more engineered receptors that specifically bind an antigen.

[0170] Aspect 120 is the method of aspect 119, wherein the one or more engineered receptors comprise an engineered antigen receptor.

[0171] Aspect 121 is the method of Aspect 120, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0172] Aspect 122 is the method of aspect 121, wherein the engineered antigen receptor is a CAR.

[0173] Aspect 123 is the method of any one of aspects 120-122, wherein the antigen is a cancer antigen.

[0174] Aspect 124 is the method of any one of aspects 120-123, wherein the antigen is a solid tumor antigen.

[0175] Aspect 125 is the method of any one of Aspects 120-124, wherein the antigen is selected from 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EP CAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TROP2, TEM, HMW-MAA, VEGFR2 and combinations thereof.

[0176] Aspect 126 is the method of any one of aspects 120-125, wherein the antigen comprises TROP2 and / or CD70.

[0177] Aspect 127 is the method of any one of aspects 119-126, wherein the one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

[0178] Aspect 128 is the method of any one of aspects 76-127, wherein the cells comprise expression of one or more exogenous chemokines and / or one or more cytokines.

[0179] Aspect 129 is the method of aspect 128, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof.

[0180] Aspect 130 is the method of aspect 129, wherein the cytokine is IL-15.

[0181] Aspect 131 is the method of any one of aspects 76-130, wherein the cell comprises a suicide gene.

[0182] Aspect 132 is the method of any one of aspects 76 to 131, wherein the mutation of the endogenous gene comprises homologous recombination or non-homologous recombination.

[0183] Aspect 133 is the method of any one of aspects 76-132, wherein the mutation of the endogenous gene is mediated by an endonuclease.

[0184] Aspect 134 is the method of aspect 133, wherein the endonuclease is an RNA-guided endonuclease.

[0185] Aspect 135 is the method of aspect 134, wherein the RNA-guided endonuclease is CRISPR-Cas9.

[0186] Aspect 136 is the method of any one of Aspects 76 to 135, wherein the cell comprises one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR and CD7.

[0187] Aspect 137 is an engineered natural killer (NK) cell, wherein the cell comprises an engineered partial or complete loss-of-function and / or knockout (KO) mutation in the cell's endogenous cAMP response element modulator (CREM) gene.

[0188] Aspect 138 is the cell of aspect 137, wherein the cell comprises a knockout (KO) mutation in the endogenous CREM gene of the cell.

[0189] Aspect 139 is the cell of aspect 138, wherein the cell comprises a homozygous knockout (KO) mutation in the endogenous CREM gene.

[0190] Aspect 140 is the cell of aspect 138, wherein the cell comprises a heterozygous KO mutation in the endogenous CREM gene of the cell.

[0191] Aspect 141 is the cell of aspect 137, wherein the mutation results in improved cytotoxicity of the engineered cell relative to a reference cell lacking the mutation.

[0192] Aspect 142 is the cell of aspect 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment and / or a tumor microenvironment (TME) relative to a control non-engineered cell.

[0193] Aspect 143 is the cell of aspect 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment of pH less than or equal to about 7.0 relative to a control non-engineered cell.

[0194] Aspect 144 is the cell of aspect 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment of pH less than or equal to about 5.9 relative to a control non-engineered cell.

[0195] Aspect 145 is the cell of aspect 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic environment characterized by increased lactate levels relative to a non-acidic microenvironment.

[0196] Aspect 146 is the cell of aspect 137, wherein the mutation results in enhanced multifunctionality of the engineered cell in response to stimulation by a tumor cell relative to a control non-engineered cell.

[0197] Aspect 147 is the cell of aspect 146, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation of tumor cells.

[0198] Aspect 148 is the cell of aspect 137, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or degranulation marker CD107a in response to stimulation of tumor cells.

[0199] Aspect 149 is a cell of Aspect 148, wherein the increased cytokine release includes increased granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin in response to stimulation of tumor cells.

[0200] Aspect 150 is the cell of aspect 137, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxicity phenotype relative to a control non-engineered cell.

[0201] Aspect 151 is a cell of aspect 150, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxic phenotype relative to a control non-engineered cell, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response and / or estrogen early response.

[0202] Aspect 152 is a cell of aspect 137, wherein the mutation provides upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

[0203] Aspect 153 is the cell of aspect 137, wherein the mutation provides the engineered cell with enhanced proliferation capacity and / or persistence phenotype relative to a control non-engineered cell.

[0204] Aspect 154 is the cell of aspect 153, wherein the enhanced proliferation capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

[0205] Aspect 155 is the cell of aspect 153 or 154, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

[0206] Aspect 156 is the cell of aspect 137, wherein the mutation provides the engineered cell with an enhanced metabolic fitness phenotype relative to a control non-engineered cell.

[0207] Aspect 157 is the cell of aspect 137, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0208] Aspect 158 ​​is a cell of aspect 137, wherein the mutation provides increased chromatin accessibility throughout the genome when the cell is contained in an acidic microenvironment.

[0209] Aspect 159 is a cell according to aspect 137, wherein the CREM mutation results in reduced expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

[0210] Aspect 160 is the cell of aspect 137, wherein the CREM mutation is the result of exposing the cell to a polynucleotide comprising the sequence of SEQ ID NO:140 and / or SEQ ID NO:142.

[0211] Aspect 161 is the cell of aspect 137, wherein the CREM mutation results in a reduction of CREM protein isoforms by more than 60%; or wherein the CREM mutation results in a reduction of CREM protein isoforms by more than 80%.

[0212] Aspect 162 is the cell of aspect 137, wherein the cell is derived from umbilical cord blood (CB).

[0213] Aspect 163 is the cell of aspect 137, wherein the cell is derived from peripheral blood (PB).

[0214] Aspect 164 is the cell of aspect 137, wherein the cell is derived from a stem cell.

[0215] Aspect 165 is the cell of aspect 137, wherein the cell comprises an engineered receptor.

[0216] Aspect 166 is the cell of Aspect 137, wherein the cell comprises a T cell receptor (TCR).

[0217] Aspect 167 is the cell of aspect 137, wherein the cell comprises a chimeric antigen receptor (CAR).

[0218] Aspect 168 is a cell of any one of Aspects 165-167, wherein the engineered receptor specifically binds an antigen selected from the group consisting of 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB 3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TROP2, TEM, HMW-MAA, VEGFR2 and combinations thereof.

[0219] Aspect 169 is the cell of aspect 168, wherein the antigen is TROP2.

[0220] Aspect 170 is the cell of aspect 168, wherein the antigen is CD70.

[0221] Aspect 171 is a population of cells, said cells being cells according to any one of aspects 137-170.

[0222] Aspect 172 is a pharmaceutical composition comprising the cell of any one of aspects 137-170 and optionally a pharmaceutically acceptable excipient.

[0223] Aspect 173 is a method of treating cancer in an individual in need thereof, comprising the step of administering to the individual a therapeutically effective amount of the cell of any one of Aspects 137-170, the cell population of Aspect 171, or the pharmaceutical composition of Aspect 172.

[0224] Aspect 174 is the method of aspect 173, wherein the cancer comprises a solid tumor.

[0225] Aspect 175 is the method of aspect 173, wherein the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer and / or cervical cancer.

[0226] Aspect 176 is the method of aspect 173, wherein the individual is a mammal.

[0227] Aspect 177 is the method of Aspect 173, wherein the individual is a human being.

[0228] Aspect 178 is a method of engineering a cell according to any one of aspects 137-170.

[0229] Aspect 179 is a method of killing cancer cells, comprising contacting the cancer cells with an engineered NK cell according to any one of aspects 137-170.

[0230] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. In addition, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any composition of the invention. Aspects of the embodiments set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in the application, such as in the Brief Summary, Detailed Description, Claims, Abstract, and Brief Description of the Drawings.

[0231] The features and technical advantages of the present disclosure have been outlined quite extensively above so that the following detailed description may be better understood. Additional features and advantages that form the subject matter of the claims herein will be described below. It will be appreciated by those skilled in the art that the disclosed concepts and specific embodiments may be readily used as a basis for modifying or designing other structures to achieve the same purpose of the present design. It will also be appreciated by those skilled in the art that such equivalent structures do not depart from the spirit and scope set forth in the appended claims. The novel features believed to be design features disclosed herein, including with respect to organization and methods of operation and further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. However, it should be expressly understood that each figure is provided for illustration and description purposes only and is not intended as a definition of limitations to the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0232] For a more complete understanding of the present disclosure, reference is now made to the following description in conjunction with the accompanying drawings.

[0233] Figure 1AB. Differential upregulation of genes involved in acidic signaling in tumor-infiltrating NK cells in hyperglycolytic tumors. (1A) High relative expression of genes involved in glycolytic enzymatic reactions leading to the accumulation of lactate and other acids in the tumor microenvironment in primary pancreatic cancer tissues analyzed by single-cell RNA sequencing. (1B) Pancreatic cancer-infiltrating natural killer (NK) cells show upregulation of many proton-sensing G protein-coupled receptors (GPRs) such as GPR68 and GPR132, as well as upregulation of downstream signaling mediators of the cyclic AMP (cAMP) pathway in response to acid such as CAMP response element modulator (CREM) and CAMP response element binding protein 1 (CREB1).

[0234] Figure 2 AC. Upregulation of CREM expression in tumor-infiltrating immune cells of hyperglycolytic tumors. (2A) Analysis of The Cancer Genome Atlas (TCGA) project data revealed high relative expression of genes involved in glycolytic enzymatic reactions that lead to the accumulation of lactate and other acids in the tumor microenvironment of clear cell renal cell carcinoma. (2B) Analysis of single-cell RNA sequencing data available in the Tumor Immunity Single Cell Center (TISCH) database revealed low expression of CREM in peripheral blood mononuclear cells (PBMCs) of healthy donors. (C) Analysis of single-cell RNA sequencing data available in the TISCH database revealed high relative expression of CREM in infiltrating immune cells of renal cell carcinoma.

[0235] Figure 3AB. High knockout (KO) efficiency of CREM using CRISPR-Cas9. (3A) CREM knockout (KO) confirmation was performed by PCR on DNA obtained from CAR-NK cells with CREM wild-type (WT) loci relative to CREM KO loci. 1KbPlusDNALadder (Invitrogen) was used. The DNA band amplified from CREM WT NK cells was located at the 300bp band, while the DNA band amplified from CREM KO NK cells was located near the 200bd band (3B) Selected CREM isoforms were analyzed from bulk RNA sequencing data obtained from CREM WT relative to CREM KO NK cells from 3 umbilical cord blood donors (D1, D2 and D3). The heatmap color scale represents the number of expressing cells from 0 (dark blue) to 400 (red); the FDR column corresponds to the false discovery rate (significance < 0.05); and the logPostFC column represents the logarithm of the fold change in WT relative to KO expression levels, with positive values ​​indicating higher expression in WT and negative values ​​indicating higher expression in KO cells. Isoform expression analysis showed that multiple CREM RNA isoforms were significantly knocked down in NK cells. Examples include: ICER isoform (CREM-228), CREM-207, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, and CREM-217.

[0236] Figure 4 AD. CREM KO significantly enhances NK cell cytotoxicity against a hyperglycolytic tumor model. (4A) Photographs representing NK cell cytotoxicity against a hyperglycolytic UMRC3 cell line expressing green fluorescent protein (GFP) after 0 hours (T=0) and 48 hours (T=48h) of co-culture with CREM WT or CREM KO CAR-NK cells. (4B) Depicts quantification of GFP+UMRC3 tumor growth over time in a long-term cytotoxicity assay using INCUCYTE®. (4C) Photographs representing NK cell cytotoxicity against a hyperglycolytic GFP+UMRC3 cell line grown into spheroids (3D tumor blocks) after T=42 hours (when spheroids form) and 48 hours (when NK cells are added; (T=48h)) and 7 days (T=7d). (4D) Depicts quantification of GFP+UMRC3 spheroid growth over time in a long-term cytotoxicity assay using INCUCYTE®.

[0237] Figure 5AB. CREM KO overcomes cytotoxic inhibition of NK cells under acidic conditions. (5A) depicts cytotoxicity of NK cells to renal cancer cells (e.g., A498 cell line) at typical culture medium pH (pH=7.2) compared to acidic culture medium pH (pH=5.9), as determined by chromium release assay at effector to target (E:T) ratios of 20:1, 10:1, 5:1, 2.5:1, and 1.25:1. Multiple paired t-tests were used to compare cytotoxicity at 2 pHs at each E:T ratio. (5B) Comparison of cytotoxicity of CREM WT NK cells relative to CREM KO NK cells at pH7.2 or pH5.9 was shown. One-way Anova using multiple comparisons performed using the corrected method of Benjamini and Yekutieli was used to compare the overall cytotoxicity of CREM WT NK cells relative to CREM KO NK cells.

[0238] Figure 6 AC. CREM KO CAR-NK cells exhibit enhanced polyfunctionality as demonstrated by increased cytokine release. (6A) shows FACS plots representing cytokine release (INF-gamma (INF-γ or IFN-g) and TNF-alpha (TNF-α or TNF-a) and the degranulation marker CD107a) by NT NK, CREM WT CAR-NK, or CREM KO CAR-NK cells in response to stimulation with glycolytic tumor cells SKOV3 followed by intracellular staining. (6B) shows a summary of the percentage of INF-g, TNF-a, and CD107a release under NK cell conditions: non-transduced (NT) NK cells, CREM WT CAR-NK cells, and CREM KO CAR-NK cells. (6C) shows a heat map of the levels of various cytokines released by NT NK cells, CREM WT CAR-NK cells, or CREM KO CAR-NK cells when assayed by LUMINEX® (multiplex assay for the indicated analytes). Each column shows normalization to the condition with the highest expression level under the tested condition (e.g., typically CREM KO The mean levels of markers for CAR-NK (CAR-NK) are shown on a scale of 0 (blue) to 1 (yellow).

[0239] Figure 7.CREM KO CAR-NK cells have an immunophenotype characterized by activation and cytotoxicity. It is a heat map showing the mass spectrometry flow cytometry proteomics marker levels (by time-of-flight flow cytometry (CyTOF)) of NK cells, CREM WT CAR-NK cells and CREM KO CAR-NK cells that are not transduced (NT). The columns correspond to various NK cell conditions. Each row shows the average level of markers normalized to a range of 0 (blue) to 1 (yellow) on the column.

[0240] Figure 8 . In the absence of IL-2 stimulation, CREM KO enhanced the proliferation capacity and persistence of CAR-NK cells, but did not lead to autonomous growth. Shown is the absence of IL-2 stimulation in non-transduced (NT) NK cells, CREM WT CAR-NK cells, and CREM KO CAR-NK cells (x10 6 ) after culture, in days.

[0241] Fig. 9 AB. CREM KO enhances metabolic fitness of CAR-NK cells. (9A) Shown are extracellular acidification rate (ECAR) measurements determined by the SEAHORSE® Glycolytic Stress Test assay as a representative of aerobic glycolysis in non-transduced (NT) NK cells, CREM WT CAR-NK cells, and CREM KO CAR-NK cells. (9B) Oxygen consumption rate (OCR) measurements determined by the SEAHORSE® Mitochondrial Stress Test assay as a representative of oxidative phosphorylation under various NK cell conditions.

[0242] Fig.10 AH. CREM KO renders CAR-NK cells resistant to the inhibitory effects of lactate at concentrations similar to those observed in the tumor microenvironment (TME). (10A) Depicts the results of a cytotoxicity assay (e.g., 51Cr release assay) of anti-CD70 CAR-NK (CAR.70) cells against renal cell carcinoma (e.g., UMRC3 renal cell carcinoma tumor cells) at different E:T ratios (e.g., 20:1, 10:1, 5:1, 2.5:, and 1.25:1, respectively). NK cells were CREM wild-type (WT) or CREM knockout (KO) and incubated at a normal pH (pH Reg ) or using lactic acid to generate an acidic pH (pH Lac ) (e.g., pH about 6). (10B) shows that the Reg ) or acidic pH (pH LacRepresentative bright field microscopy images of CAR-NK cells and UMRC3 tumor cells (elongated cells) co-cultured under pH 5.5. (10C) depicts the active engagement area of ​​cytotoxic CAR-NK cells with tumor cells during attack against UMRC3 cells. Reg or pH Lac Results of a representative long-term cytotoxicity (e.g., Incucyte®) assay of CAR.70 NK cells and their non-transduced (NT) counterparts in a 2-week culture environment. Over a 2-week culture period, NK cells were gradually adapted (Inc) to more acidic conditions (e.g., by adding approximately 2.5 mM lactate every 2-3 days) (pH Lac20(Inc) resulting in a pH of approximately 6.0), or maintaining a pH of Reg All conditions started with the normal pH of the culture medium (pH Reg After a 2-week adaptation period, NK cells in various conditions were cultured at the specified pH values ​​(pH Reg or pH Lac20 (Inc) ) were attacked by UMRC3 cells at acidic (pH ). The results showed that chronic lactate exposure inhibited the cytotoxicity of CAR-NK cells compared with that under conventional pH conditions, but despite the relatively extreme conditions of low pH in the tumor microenvironment that mimics glycolytic tumors, CREM KO CAR-NK cells were still able to maintain strong anti-tumor killing. (10D) Shown in acidic (pH Lac20 (Inc) Representative Incucyte® images of NT NK cells (NT), CAR.70 CREMWT NK cells (CAR.70), or CAR.70 CREM KO NK cells challenged with GFP+UMRC3 cells at an E:T ratio of 1:1 or 2:1 under 10E . (10E ) shows multiparametric spectral flow cytometry-mediated phenotypic characterization of CAR.70 NK cells that are CREM WT or CREM KO and challenged with GFP+UMRC3 cells at an E:T ratio of 1:1 or 2:1. Reg or pH Lac Co-cultured with UMRC3 tumor cells under different conditions. ViSNE analysis was performed for each condition, followed by FlowSOM clustering. (10F) shows the distribution of four FlowSOM metaclusters (MCs) under different conditions, indicating that the pH Lac MC2 (orange) increased in CREM WT CAR.70 NK cells co-cultured with UMRC3 cells at pH Lac CREM KO CAR.70 NK cells co-cultured with UMRC3 cells showed the same Reg(10G) Shows the expression of various markers in 4 MCs locally normalized to minimum and maximum expression under various conditions on a color scale from blue (minimum) to red (maximum). Percent expression is superimposed by circles of various sizes proportional to % expression. MC2 is generally characterized by relatively low expression of activation markers and relatively high expression of certain checkpoints (e.g., TIGIT and LAG3) and terminal differentiation markers (e.g., CD57). (10H ... Reg or pH Lac , and contour tSNE plots of CREM WT or CREM KO), showing the overall phenotypic clustering differences in NK cell distribution after viSNE analysis.

[0243] Fig.11 AF. CREM KO improves CAR-NK cell activation and cytotoxicity in a long-term assay of multiple tumor re-attacks. (11A) shows the results of a representative re-attack cytotoxicity assay for UMRC3 tumor cells using CREM WT and CREM KO anti-CD70 CAR-NK cells (CAR.70) and their non-transduced (NT) counterparts xCELLigence®. UMRC3 cells were seeded in E-plates and allowed to adhere and grow overnight. Twenty-four hours later, the NK cells were added at an E:T ratio of 2:1. Every 2-4 days, new tumor cells were seeded in a new E-plate. Twenty-four hours later, the NK cells in the first E-plate were collected and counted and transferred to a new tumor plate at the same E:T ratio of 2:1. (11B) Phenotypic characterization of CAR.70 NK cells as CREM WT or CREM KO at the end of the re-attack assay shown in (11A). ViSNE analysis was performed on each condition, followed by FlowSOM clustering into five FlowSOM groups (C1-C5). (11C) Fig.11A-11B describes the distribution of 5 FlowSOM element clusters (MC, C1-C5) of cells, and the results show that compared with CREM WT cells, MC5 in CREM KO CAR.70 NK cells increases and MC4 decreases. (11D) shows the profile tSNE diagram of CREM WT CAR.70 NK cells (top) and CREM KO CAR.70 NK cells (bottom), showing the overall phenotypic clustering differences of their distribution after viSNE analysis. (11E) is shown in the color scale of blue (minimum) to orange (maximum) under various conditions. Local standardization to the expression of various markers in 5 MCs (C1-C5) with minimum and maximum expression. Percent expression is superimposed by circles of various sizes proportional to % expression. MC4 is usually characterized by relatively low activation marker expression levels, while MC5 is characterized by the opposite pattern of overall increase of activation markers. (11F) shows the expression of selected markers (ICOS, DNAM1, CD16, OX40, TIGIT and TIM3) at the single cell level when comparing between CREM WT CAR.70 NK cells and CREM KO CAR.70 NK cells in MC4 and MC5. The results showed that the expression of markers in CREM KO CAR.70 NK cells was significantly different compared with control CREM WT CAR.70 NK cells by Kruskal-Wallis test using Dunn's multiple comparisons.

[0244] Fig.12AC. CREM KO enhances the cytotoxicity of CAR-NK cells in a variety of different tumor models and using a variety of different CAR constructs, while targeting a variety of different tumor antigens in an in vitro 3D tumor spheroid model and in an in vivo mouse model. (12A) By ordinary one-way ANOVA and Tukey's multiple comparison test with a single pooled variance, it is shown how CREM KO significantly enhances the cytotoxicity of TROP2-directed CAR-NK cells (CAR.TROP2) to pancreatic cancer cell 3D spheroids (e.g., GFP+PATC148 cells) relative to CREM WTTROP-2 directed CAR-NK cells. (12B) Depicts representative bioluminescence imaging (BLI) of mouse groups injected intraperitoneally with TROP2+ expressing ovarian cancer cells (luciferase-positive SKOV3 cells), injected with tumor cells and CREM WT CAR.TROP2 CAR-NK cells, or injected with tumor cells and CREM KO CAR.TROP2 CAR-NK cells. The image shows the burden of luciferase-positive tumor cells on day 47 after NK cell injection. Six days after tumor implantation, mice were irradiated and treated intraperitoneally with 4 million NK cells (NK cells transduced with CAR.TROP2 of CREMWT or CREM KO, respectively) the next day. The results showed that CREM KO enhanced the in vivo cytotoxicity of CARNK cells against solid tumors. (12C) shows representative images (in duplicate) of cytotoxicity assays of CREMWT or CREM KO anti-CD70 (CAR.70) CAR-NK cells against CD70-positive breast cancer (e.g., breast cancer cell line BCX010) in spheroid assays using Incucyte®. BCX010 (GFP-positive) cells were inoculated in ultra-low attachment (ULA) plates and allowed to form spheroids within 24 hours. The next day, NK cells were added to the corresponding wells. The results showed that 3 days after the addition of NK cells, CREM KO CAR-NK cells reduced CD70+ tumor cell fluorescence relative to CREM WT CAR-NK cells.

[0245] Fig.13AC. CREM KO enhances the metabolic adaptability of NK cells at baseline and when cultured at acidic pH. (13A) depicts the extracellular acidification rate (ECAR; as a substitute for glycolysis, upper figure) of CREM WT or CREM KO anti-CD70 CAR-NK cells (CAR.70) and their non-transduced (NT) counterparts. The CAR.70 construct is designed to also secrete interleukin 15 (IL15), and control NK cells (IL15NK cells) transduced with constructs expressing IL15 in the absence of CAR are included in the assay. The assay is performed using Seahorse® Sugar Stress Test to determine NK cells cultured in conventional culture medium without stimulation according to the manufacturer's protocol. The oxygen consumption rate (OCR; as a substitute for oxidative phosphorylation (OXPHOS), lower figure) was analyzed using Seahorse® Mitochondrial Stress Test according to the manufacturer's protocol. (13B) shows ECAR (top) and OCR (bottom) of NT, CREM WT CAR-NK cells, and CREM KO CAR-NK cells incubated in lactate. (13C) shows how the pathways of glycolysis and OXPHOS are significantly upregulated in CREM KO NK cells compared to CREM WT NK cells after incubation with lactate (analyzed using unbiased GSEA performed on bulk RNA sequencing of these conditions).

[0246] Fig.14 AB. After incubation with lactate, CREM KO NK cells showed different transcriptomes and epigenetic profiles compared to CREM WT NK cells. (14A) Bar graph showing HALLMARK pathways differentially upregulated or downregulated in CREM KO relative to CREM WT NK cells incubated in lactate for 24 hours. Bulk RNA sequencing of samples was analyzed using gene set enrichment analysis (GSEA). Notably, CREM KO was associated with upregulation of proliferation pathways (G2M, E2F), MYC, MTORC1, oxidative phosphorylation, and TNFa signaling. (14B) Chromatin accessibility of CREM KO relative to CREM WT NK cells incubated in lactate for 24 hours, as assessed by bulk ATAC sequencing of corresponding samples. Each row represents a peak, displayed on a 4kb window. Three cord blood NK cell donors (WT-1, WT-2, WT-3) and their CREM KO counterparts (KO-1, KO-2, KO-3) were used. Notably, CREM KO was associated with a pattern of markedly increased chromatin accessibility across the genome.

[0247] Although various embodiments of the present disclosure have been shown and described herein, it will be appreciated by those skilled in the art that these embodiments are provided by way of example only. Many variations, changes and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0248] Detailed Description

[0249] I. Definition Examples

[0250] In accordance with long-standing patent law convention, the words "a" and "an" when used with the word "included" in this specification, including the claims, mean "one or more". Some embodiments of the present disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0251] Throughout this specification, unless the context requires otherwise, the words "comprise", "include" and "contain" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in the disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0252] Throughout this specification, reference to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0253] As used herein, the terms "or" and "and / or" are used to describe multiple components that are in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment.

[0254] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0255] As used herein, the term "engineered" refers to artificially produced entities, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and include elements that do not occur in nature or are not configured in the manner in which they are used in the present disclosure.

[0256] As used herein, the term "exogenous" refers to a polynucleotide (e.g., a polynucleotide encoding a gene product or a portion of a gene product) that is not endogenous in a mammalian cell, such as an immune cell, or is synthetically produced outside of a mammalian cell (e.g., by recombinant technology).

[0257] The term "expression" as used herein refers to the process in which a polynucleotide is transcribed into mRNA and / or the process in which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing mRNA in a eukaryotic cell. Therefore, "gene product" as used herein refers to transcribed mRNA, transcribed RNA before splicing (for example, RNA still comprising a non-coding region), translated polypeptide (for example, with or without a signal peptide or other regions not present in the mature protein) and protein. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from a sample can be directly compared with the expression level of the gene from a control or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared with the expression level of the gene from the same sample after the administration of a compound.

[0258] The term "isolated" as used herein refers to a molecule or biological product or cell material that is substantially free of other materials. In one aspect, the term "isolated" refers to a nucleic acid, such as DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, which is separated from other DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ (e.g., present in a natural source). The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cell material, viral material, or culture medium when produced by recombinant DNA technology, or a nucleic acid or peptide that is substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist naturally in the form of fragments and are not found in the natural state. The term "isolated" is also used herein to refer to polypeptides separated from other cell proteins, and is intended to include purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues separated from other cells or tissues, and is intended to include cultured and engineered cells or tissues.

[0259] As used herein, "prevention" and similar words such as "prevent", "preventing", etc. refer to methods for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0260] As used herein, "sample" generally refers to a biological sample. The sample can be taken from an individual's tissue or cell. In some instances, the sample may include or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, discarded tissue. The sample may be separated from a source before collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymph, saliva, urine, feces, tears, sweat or mucosal excretions, and other body fluids separated from the main source before collection. In some instances, the sample is separated from its main source (cells, tissues, body fluids such as blood, environmental samples, etc.) during sample preparation. The sample may or may not be purified or otherwise enriched from its main source. In some cases, the main source is homogenized before further processing. The sample may be filtered or centrifuged to remove white film, lipids or particulate matter. The sample may also be purified or enriched for nucleic acid, or may be treated with RNase. The sample may include intact, fragmented or partially degraded tissue or cells.

[0261] The subject can be any organism or animal subject as the object of the method or material, including mammals, such as humans, experimental animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys and chickens), household pets (e.g., dogs, cats and rodents), horses and transgenic non-human animals. The subject can be a patient, such as one who suffers from or is suspected of having a disease (which can be referred to as a medical condition), such as a benign or malignant neoplasm or cancer. The subject may be receiving or has been receiving treatment. The subject may be asymptomatic. The subject may be a healthy individual, but cancer prevention is desired. The term "individual" is used interchangeably with "subject". As used herein, "subject" or "individual" may or may not be placed in a medical institution, and may be considered an outpatient of a medical institution. An individual may be receiving one or more medical compositions via the Internet. The term "individual" can refer to a person or non-human animal of any age, thus including adults and adolescents (i.e., children) and infants, and including individuals in the womb. It is not intended that the term implies a need for medical treatment, and therefore, individuals may be voluntarily or involuntarily part of an experiment, whether a clinical trial or in support of basic scientific research.

[0262] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include a reduction or amelioration of symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms.

[0263] ********

[0264] While CAR-T cell-based therapeutic modalities have demonstrated unprecedented patient responses and offer significant curative potential for certain hematological malignancies, success in other cancers, particularly solid tumors, remains elusive, in part due to the unique characteristics of the tumor microenvironment, which include hypoxia, acidic pH, nutrient depletion, and / or immunosuppression. Acidity is a prominent feature of the tumor microenvironment, primarily due to acidic metabolites such as lactate resulting from active glycolysis. Acidity mediates immunosuppression, tumor progression, and poor prognosis. Specifically, tissue acidosis results in suppression of immune cell-mediated responses, such as reductions in natural killer (NK) and T cell cytotoxicity, cytokine production, and tumor surveillance.

[0265] The cAMP pathway mediates the main immunosuppressive effect on effector cells and is related to the response of immune cells to acidity and hypoxia through certain G protein-coupled receptors (GPRs). For example, proton receptors are transmembrane GPRs that act as sensors of extracellular acidity, leading to adenylate cyclase activity and subsequent cAMP accumulation. This in turn leads to the activation of transcription factors in the cAMP pathway, promoting anti-inflammatory responses and leading to the inhibition of immune cell function. As described herein, in some embodiments, the key elements of immune cell responses to acidity are genetically engineered. In some embodiments, the cAMP signaling pathway in immune effector cells is targeted for engineered mutations. In some embodiments, mutations in the cAMP signaling pathway can make cell therapy insensitive to the immunosuppressive effects of acidity, thereby increasing the survival rate, proliferation and / or immune function of cells in an acidic tumor microenvironment.

[0266] In some embodiments, the technology described herein includes the use of gene editing technology (e.g., CRISPR-Cas technology), wherein the technology is used to mutate (e.g., knock out) genetic elements disclosed herein. In some embodiments, the prominent and key transcription factor cAMP response element regulator (CREM) is mutated (e.g., knocked out). As shown herein, in some embodiments, knocking out CREM from NK cells leads to improvements in NK cell persistence and proliferation, as well as anti-tumor activity for cancer cell lines characterized by active glycolysis and significant acidosis of the microenvironment. In some embodiments, targeted genetic engineering strategies are used to target CREM and / or other genes in the cAMP signaling pathway, and / or genes encoding proton receptors disclosed herein. In some embodiments, genetic engineering strategies are used in different forms of cell therapy, including CAR-T cells, T cells, CAR-NK cells, NK cells, T cell receptors (TCR)-T cells, TCR-NK cells and / or tumor infiltrating lymphocytes (TIL). In some embodiments, this genetic engineering enhances cell therapy for various types of cancers, including for solid tumors.

[0267] In some embodiments, disclosed herein are compositions and methods comprising genetically engineered immune effector cells and / or methods for making them. In some embodiments, genetic engineering includes knocking out genes encoding proteins involved in the acidic induction of immunosuppressive cAMP pathways and downstream signaling, including but not limited to the following genes: CREM, ICER, CREB1, GPR4, GPR31, GPR68, GPR81, GPR151 and / or GPR132. In some embodiments, genetic engineering is carried out by using endonuclease-mediated cleavage (e.g., CRISPR-Cas technology). In some embodiments, compositions disclosed herein are used for cancer treatment methods.

[0268] In certain embodiments, knockout of genes associated with responses to acidity and / or their associated downstream signaling pathway components improves the effectiveness of engineered immune cells against various tumors. For example, FDA-approved CAR-T cells for the treatment of leukemia, lymphoma, and myeloma can be genetically engineered to include mutations in one or more of these genes: CREM, ICER, CREB1, GPR4, GPR31, GPR68, GPR81, GPR151, and / or GPR132 in an attempt to improve their effectiveness in the acidic TME of various cancers, which may lead to the extension of this therapy to drug-resistant tumors, such as solid tumors. In addition, this genetic engineering strategy can be used in various other forms of cell therapy, such as CAR-NK cells, TCR-T cells, TILs, to enhance their activity against various cancer types.

[0269] As described in this paper, single-cell RNA sequencing data were analyzed and showed higher expression of proton-sensing GPR and transcription factor CREM in tumor-infiltrating immune cells compared with their peripheral blood counterparts ( Figure 1A and 1B). CREM is a transcriptional repressor in the cAMP pathway that is upregulated in response to acidic signals through proton-sensing GPRs. In some embodiments, the cAMP signaling pathway is regulated by mutations in the CREM transcription factor. In some embodiments, the effects of CREM mutations are analyzed in cord blood-derived NK and CAR-NK cells. In some embodiments, genetic engineering techniques, such as the CRISPR-Cas9 editing system, are used to genetically engineer the target gene. In some embodiments, one or more guide RNA molecules are designed for the target target gene described herein. In some embodiments, one or more guide RNA molecules are used to generate mutations in the target target gene described herein. In some embodiments, the target target gene is specifically CREM. In some embodiments, CREM mutations (e.g., CREM knockouts) cause immune effector cells (e.g., NK cells) to have a significant proliferation advantage over CREM wild-type (WT) cells (e.g., WT NK cells). In some embodiments, CREM mutations (e.g., CREM knockouts) cause immune effector cells (e.g., NK cells) to have an immune phenotype characterized by activation and cytotoxicity. In some embodiments, CREM mutation (for example, CREM KO) makes immune effector cells (for example, NK cells) have enhanced metabolic adaptability.In some embodiments, enhanced metabolic adaptability is shown by the increase of oxidative phosphorylation and / or glycolytic activity.In some embodiments, CREM KO effector cells (for example, NK cells) with or without additional engineering (for example, with or without chimeric antigen receptor (CAR) etc.) have higher cytotoxicity relative to CREM WT effector cells (for example, NK cells) with or without additional engineering (for example, with or without CAR etc.).In some embodiments, when inspected under acidic pH conditions that usually inhibit the cytotoxicity of effector cells, the cytotoxicity improvement associated with CREM mutation is particularly obvious.In some embodiments, mutations in CREM (for example, knockout of CREM) improve long-term cytotoxicity, whether in conventional 2D culture or in 3D culture (wherein tumor cells grow in spheroids to simulate the characteristics of solid tumors).In some embodiments, mutations in CREM (for example, knockout of CREM) improve long-term cytotoxicity in solid tumor microenvironment in vivo. In some embodiments, the tumor microenvironment is characterized by increased lactate levels relative to a non-tumor microenvironment.

[0270] II. Gene Editing of Cells with Reduced or Inhibited Expression Levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1

[0271] Before the cells of the amplification and genetic modification of the present disclosure, cell sources can be obtained from a subject by a variety of non-limiting methods. Any type of immune cell, such as NK cells, can be obtained from many non-limiting sources, including tissues from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, infection sites, ascites, pleural effusion, spleen tissue, tumors or commercially available. Any number of immune cell lines known and available to those skilled in the art can be used.

[0272] In a specific embodiment, any type of immune effector cell is subjected to gene editing to modify the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 in the cell. In a particular case, the cell is modified to reduce the expression level of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, including completely suppressing the detectable expression (e.g., can be referred to as knockout) of some isomers of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. Such cells may or may not be amplified before production and / or use.

[0273] In certain cases, the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes is disrupted, wherein expression is partially or completely reduced. In certain cases, the method of the present disclosure is used to knock down or knock out GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes. In certain embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are disrupted (e.g., mutated) so that one or more RNA isomers encoded by the above-mentioned one or more genes of mutation are upregulated relative to the non-mutated copy of the same gene.

[0274] Those skilled in the art know how to engineer any cell, including any immune cell, to reduce or completely inhibit the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes. Specific embodiments utilize means including polynucleotide sequences targeting specific genes whose expression is desired to be reduced or completely inhibited.

[0275] In some embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes are destroyed in a heterozygous manner. In some embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are destroyed in a homozygous manner. In some embodiments, the immune effector cell group with the destruction of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes includes immune cells that are homozygous for wild-type genes, heterozygous for wild-type genes and destroyed genes and / or homozygous for destroyed genes. In some embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes are destroyed in most alleles in immune effector cell populations. In some embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes are destroyed in greater than or equal to, approximately or just 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the alleles of the gene in the colony or any range therefrom.

[0276] Examples of G protein coupled receptor 4 ((GPR4), also known as GPR6C.1) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 2828 (GPR4), NC_000019.10 for genomic sequence with reference to GRCh38.p14 primary assembly (range 45589764 to 45602212, complement), NM_005282.3 for mRNA sequence, and NP_005273.1 for protein sequence, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR4 is mutated as a result of exposure of cells to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR4 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 3-25.

[0277] Examples of G protein coupled receptor 31 ((GPR31), also known as HETER; HETER1; and / or 12-HETER) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 2853 (GPR31), reference GRCh38.p14 primary assembly (range 167155247 to 167157980, complement) for genomic sequence NC_000006.12, for mRNA sequence NM_005299.3, for protein sequence NP_005290.2, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR31 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR31 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 26-42.

[0278] Examples of G protein coupled receptor 68 ((GPR68), also known as OGR1; A12A6; and / or GPR12A) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 8111 (GPR68), NC_000014.9 for the genomic sequence with reference to the GRCh38.p14 primary assembly (range 91232532 to 91270790, complement), NM_001177676.2 for the mRNA sequence encoding transcript variant 1, NM_003485.3 for the mRNA sequence encoding transcript variant 2, NM_001348437.1 for the mRNA sequence encoding transcript variant 3, and NP_001171147.1, NP_001335366.1 and NP_003476.3 for the protein sequences, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR68 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR68 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 43-74.

[0279] Examples of G protein coupled receptor 81 ((GPR81) also known as hydroxycarboxylic acid receptor 1 (HCAR1); HCA1; LACR1; FKSG80; GPR104; TAGPCR; and / or TA-GPCR) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 27198 (HCAR1), NC_000012.12 for genomic sequence with reference to GRCh38.p14 primary assembly (range 122726076 to 122730844, complement), NM_032554.4 for mRNA sequence, and NP_115943.1 for protein sequence, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR81 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR81 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 75-94.

[0280] Examples of G protein coupled receptor 132 ((GPR132), also known as G2A) gene sequences are located in the GENBANK® database of the National Center for Biotechnology Information, gene ID 29933 (GPR132), NC_000014.9 for the genomic sequence with reference to the GRCh38.p14 primary assembly (range 105049395 to 105065430, complement), and NM_001278694.2 and NM_01334 for the mRNA sequence encoding protein isoform 1. 5.4, ​​NM_001278695.2 for the mRNA sequence encoding protein isoform 2, NM_001278696.2 for the mRNA sequence encoding protein isoform 3, NP_001265623.1 and NP_037477.1 for protein isoform 1, NP_001265624.1 for protein isoform 2, and NP_001265625.1 for protein isoform 3, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR132 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR132 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 95-119.

[0281] Examples of G protein coupled receptor 151 ((GPR151) also known as GPCR; PGR7; GALR4; GALRL; and / or GPCR-2037) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 134391 (GPR151), NC_000005.10 for genomic sequence with reference to GRCh38.p14 primary assembly (range 146513144 to 146516190, complement), NM_194251.3 for mRNA sequence, and NP_919227.2 for protein sequence, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR151 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, GPR151 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 120-139.

[0282] Examples of cAMP response element modulator ((CREM) also known as ICER; CREM-2; and / or hCREM-2) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, Gene ID 1390 (CREM), for genomic sequences NC_000010.11 reference GRCh38.p14 primary assembly (range 35126846 to 35212958), or NG_029065.1 RefSeqGene (range 5129 to 91081), each of which is incorporated herein by reference in its entirety. The CREM gene encodes a number of RNA and protein isoforms, some of which are inducible cAMP early repressor (ICER) RNA isoforms encoding ICER proteins.Examples of CREM mRNA isoform sequences include, but are not limited to, NM_001267562.2, NM_001267563.2, NM_001267564.2, NM_001267565.2, NM_001267566.2, NM_001267567.2, NM_001267568.2, NM_001267569.2, NM_001267570.2, NM_001267571.2, NM_001267572.2, NM_001267573.2, NM_001267574.2, NM_001267575.3, NM_001267576.4, NM_001267577.5 2466.2, NM_001352467.2, NM_001394595.1, NM_001394598.1, NM_001394600.1, NM_001394602.1, NM_001394603.1, NM_00139 4605.1, NM_001394608.1, NM_001394610.1, NM_001394613.1, NM_001394614.1, NM_001394615.1, NM_001394616.1, NM_001394 617.1, NM_001394618.1, NM_001394619.1, NM_001394620.1, NM_001394621.1, NM_001394622.1, NM_001394623.1, NM_001394 625.1, NM_001394626.1, NM_001394627.1, NM_001394628.1, NM_001394629.1, NM_001394630.1, NM_001394631.1, NM_001881. 4, NM_181571.3, NM_182717.2, NM_182718.2, NM_182719.2, NM_182720.2, NM_182721.2, NM_182723.2, NM_182724.2, NM_182769.3, NM_182770.3, NM_182771.2, NM_182772.2, NM_183011.2, NM_183012.2, NM_183013.3 and NM_183060.3, each of which is incorporated herein by reference in its entirety.Examples of CREM protein isoform sequences include, but are not limited to: NP_001254491.1, NP_001254492.1, NP_001254493.1, NP_001254494.1, NP_001254495.1, NP_001254496.1, NP_001254497.1, NP_001254498.1, NP_001254499.1, NP_001339374.1, NP_001339375.1, NP_001254497.1, NP_001254498.1, NP_001254499.1. 9395.1, NP_001339396.1, NP_001381524.1, NP_001381527.1, NP_001381529.1, NP_001381531.1, NP_001381532.1, NP_00138 1534.1, NP_001381537.1, NP_001381539.1, NP_001381542.1, NP_001381543.1, NP_001381544.1, NP_001381545.1, NP_001381 546.1, NP_001381547.1, NP_001381548.1, NP_001381549.1, NP_001381550.1, NP_001381551.1, NP_001381552.1, NP_001381 554.1, NP_001381555.1, NP_001381556.1, NP_001381557.1, NP_001381558.1, NP_001381559.1, NP_001381560.1, NP_001872. 3. NP_853549.1, NP_874386.1, NP_874387.1, NP_874388.1, NP_874389.1, NP_874390.1, NP_874392.1, NP_874393.1, NP_877570.1, NP_877571.1, NP_877572.1, NP_877573.1, NP_898829.1, NP_898830.1, NP_898831.1 and NP_898883.1, each of which is incorporated herein by reference in its entirety.CREM RNA isoforms encoding ICER proteins include NM_182717.2, NM_182718.2, NM_182719.2, NM_182720.2, NM_182721.2, NM_182723.2, and NM_182724.2, while the ICER protein variant sequences are represented as NP_874386.1 (isoform ICER1), NP_874387.1 (isoform ICER11gamma), NP_874388.1 (isoform ICER11), NP_874389.1 (isoform ICER1gamma), NP_874390.1 (isoform 8 aka h), NP_874392.1 (isoform 10 aka j), and NP_874393.1 (isoform 11 aka k). In some embodiments, CREM is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, CREM is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one or more of SEQ ID NOs: 140-161, and 181-184. In some embodiments, CREM is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 140 and / or 142.

[0283] Examples of cAMP response element binding protein 1 ((CREB1) also known as CREB; and / or CREB-1) gene sequences are located in the National Center for Biotechnology Information GENBANK® database, gene ID 1385 (CREB1), with reference to the GRCh38.p14 primary assembly (range 207529962 to 207605988) for the genomic sequence as NC_000002.12, and mRNA sequences are represented as including, but not limited to: NM_001320793.2, NM_001371426.1, NM_001371427.1, NM_001371428.2, NM_001371429.3, NM_001371429.4, NM_001371429.5, NM_001371429.6, NM_001371429.7, NM_001371429.8, NM_001371429.9, NM_001371429.1, NM_001371429. 28.1, NM_004379.5, NM_134442.5, protein sequences are represented by, but are not limited to: NP_001307722.1, NP_001358355.1, NP_001358356.1, NP_001358357.1, NP_004370.1, and NP_604391.1, each of which is incorporated herein by reference in its entirety. In some embodiments, CREB1 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence provided in Table 2. In some embodiments, CREB1 is mutated as a result of exposure of a cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one or more of SEQ ID NOs: 162-180.

[0284] In some embodiments, the 5'-3' DNA sequences of exemplary guide RNAs for knocking out GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes are shown in Table 2. In some embodiments, these guide RNA sequences are suitable for use with CRISPR / Cas9 technology, which utilizes guide RNAs (complementary to short target DNA sequences on the target gene) for double-stranded DNA cleavage. The guide RNA can be positive or negative stranded, but since the cleavage performed using CRISPR / Cas9 technology affects both strands of the target DNA, the target sequence on the positive DNA strand of the sequence is shown here.

[0285] In some embodiments, after mutation with an endonuclease, oligonucleotide amplification techniques (e.g., PCR) can be used to determine mutation efficiency and / or mutation type. In some embodiments, for the PCR reaction, primers that include (e.g., flank) the edited region are used to amplify the target sequence. In some embodiments, suitable primers for amplifying the target locus can be, but are not limited to, SEQ ID NOs: 1 and 2.

[0286] Table 1 - Exemplary primer sequences

[0287] Embodiments of the present disclosure include methods for knocking out or reducing the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 in cells, including contacting cells with at least Cas9 or functionally equivalent alternatives and appropriate guide RNAs targeting GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. Cas9 and / or guide RNA can be provided to cells by expressing from one or more expression vectors encoding them. Carriers can be viruses (retroviruses, slow viruses, adenoviruses, adeno-associated viruses) or non-viruses (naked plasmid DNA or chemically modified mRNA).

[0288] In certain cases, genes other than GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are knocked down or knocked out, and this may or may not occur in the same step in which GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is knocked down or knocked out. The reduction or complete inhibition of expression may or may not utilize the same gene editing mechanism as GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and the reduction or complete inhibition of expression of other genes may occur before, during, or after gene editing of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. The genes edited in the cells can be of any kind, but in certain embodiments, these genes are genes whose gene products inhibit the activity and / or proliferation of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 KO cells. In certain cases, in addition to GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, the edited genes enable the cells to work more effectively in the tumor microenvironment. In certain cases, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD38, and CD7. In certain embodiments, the TGFBR2 gene is knocked out or knocked down in the cell. In certain embodiments, the CISH gene is knocked out or knocked down in the cell. In certain embodiments, the CD38 gene is knocked out or knocked down in the cell. In a specific embodiment, the glucocorticoid receptor (GR) gene is knocked out or knocked down in the cell.

[0289] In some embodiments, any gene editing in the cell is performed using one or more DNA binding molecules, such as endonucleases, such as but not limited to Cas enzymes (and variants thereof), zinc finger nucleases, TALENs, and meganucleases, etc. In some embodiments, any gene editing in the cell is performed by one or more DNA binding nucleic acids, such as by RNA-guided endonucleases (RGENs). For example, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins can be used for the changes; in some embodiments, Cpf1 is used instead of Cas9. In general, "CRISPR system" refers collectively to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") genes or direct their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (comprising "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0290] CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA (which sequence-specifically binds to DNA) and a Cas protein (e.g., Cas9) having nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, such as from a specific organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0291] In some aspects, Cas nuclease and gRNA (including fusion of crRNA and fixed tracrRNA specific to target sequence) are introduced into cells. Typically, complementary base pairing is used to target Cas nuclease to target site, such as gene, at the target site at the 5' end of gRNA. The target site can be selected based on the 5' position (e.g., generally NGG or NAG) of its adjacent protospacer sequence adjacent to the motif (PAM) sequence. In this regard, gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11 or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Typically, the CRISPR system is characterized by promoting the elements of the CRISPR complex formation at the target sequence site. Typically, "target sequence" generally refers to a sequence designed to have complementarity with the guide sequence, wherein the hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. If there is enough complementarity to cause hybridization and promote the formation of the CRISPR complex, it is not necessarily necessary to be fully complementary.

[0292] The CRISPR system can induce double-strand breaks (DSBs) at the target site, subsequently causing damage or changes as discussed herein. In other embodiments, Cas9 variants considered to be "nickases" are used to nick single strands at the target site. Paired nickases can be used, for example, to improve specificity, each of which is guided by different gRNA targeting sequences, so that when the nicks are introduced at the same time, 5' overhangs are introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0293] The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of the cell, such as in an organelle of the cell. Typically, a sequence or template that can be used to reorganize a target locus comprising a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide can be referred to as an editing template. In some aspects, recombination is homologous recombination.

[0294] Typically, in the case of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). The tracr sequence (which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or greater than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence) can also form part of a CRISPR complex, e.g., by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally matched.

[0295] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into cells so that the expression of the elements of the CRISPR system guides the formation of a CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, Cas enzymes, guide sequences connected to tracr-pairing sequences, and tracr sequences can each be operably connected to a separate regulatory element on a separate vector. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors provide any components of the CRISPR system not included in the first vector. The vector may include one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in the cell.

[0296] The vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme (e.g., a Cas protein). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0297] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae). In some cases, CpF1 can be used as an endonuclease instead of Cas9. The CRISPR enzyme can guide the cutting of one or two chains at the target sequence position, for example, within the target sequence and / or within the complementary sequence of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cut one or two chains of the target polynucleotide containing the target sequence. For example, the aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with one or more guide sequences (e.g., two guide sequences (which target the sense and antisense strands of the DNA target, respectively)). This combination allows both chains to be nicked and used to induce NHEJ or HDR.

[0298] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in a specific cell (e.g., a eukaryotic cell). The eukaryotic cell can be a eukaryotic cell of a specific organism (e.g., a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates) or a eukaryotic cell derived from the specific organism. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Various species exhibit specific biases for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on (among other things) the nature of the codons translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of the selected tRNA in the cell generally reflects the most commonly used codons in peptide synthesis. Therefore, genes can be customized for optimal gene expression in a given organism based on codon optimization.

[0299] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and guide sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0300] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0301] The CRISPR enzyme can be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may comprise any other protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion and herpes simplex virus (HSV) BP16 protein fusion. Other domains that can form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0302] In some embodiments, the gene editing molecule includes a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include at least ZFNs, TALEs, and TALENs.

[0303] In some embodiments, the gene editing molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof, which bind to DNA in a sequence-specific manner. ZFPs or their domains are proteins or domains within a larger protein that bind to DNA in a sequence-specific manner through one or more zinc fingers, and zinc fingers are amino acid sequence regions within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA binding protein is usually abbreviated as zinc finger protein or ZFP. ZFPs include artificial ZFP domains that target specific DNA sequences, are usually 9-18 nucleotides long, and are produced by assembling a single finger. ZFPs include those with a single finger domain length of about 30 amino acids and comprising an alpha helix, the alpha helix comprising two invariant histidine residues, the histidine residues being coordinated by zinc to two cysteines of a single beta turn, and having two, three, four, five or six fingers. Typically, the sequence specificity of ZFPs can be changed by amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, for example, engineered to bind to a selected target site. In some embodiments, the DNA targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from the Type IIS restriction endonuclease FokI. FokI typically catalyzes double-stranded cleavage of DNA, cleaving one strand at 9 nucleotides from its recognition site and the other strand at 13 nucleotides from its recognition site. Many gene-specific engineered zinc fingers are already commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has collaborated with Sigma-Aldrich (St. Louis, MO, USA) to develop a platform (CompoZr) for zinc finger construction that enables researchers to completely bypass zinc finger construction and validation and provide specific targeted zinc fingers for thousands of proteins (Gaj et al. , Trends in Biotechnology , 10 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers or custom designed zinc fingers are used. (See, e.g., Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTil-1KT, and PZD0020).

[0304] In some embodiments, the gene editing molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, see, for example, U.S. Patent Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety. The TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains / units. The repeat domain participates in the binding of TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Each TALE repeat unit includes 1 or 2 DNA binding residues, which constitute a repeat variable diresidue (RVD), typically located at position 12 and / or 13 of the repeat. The natural (canonical) codes for DNA recognition of these TALEs have been determined, so that the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG to T, NI to A, NN to G or A, NO to T, and non-canonical (atypical) RVDs are also known. In some embodiments, TALE can target any gene by designing a TAL array with specificity for a target DNA sequence. The target sequence typically begins with thymidine. In some embodiments, the molecule is a DNA binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, TALEN is a fusion protein comprising a DNA binding domain derived from TALE and a nuclease catalytic domain for cutting a nucleic acid target sequence. In some embodiments, TALEN recognizes and cuts a target sequence in a gene. In some aspects, the cutting of DNA results in a double-strand break. In some aspects, the break stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Typically, NHEJ is an imperfect repair process that typically results in changes in the DNA sequence of the cutting site. In some aspects, the repair mechanism involves reconnecting the remainder of the two DNA ends by direct reconnection or by so-called microhomology-mediated end connection. In some embodiments, repair by NHEJ will result in small insertions or deletions, and can be used to destroy and thereby inhibit genes. In some embodiments, the modification can be the replacement, deletion or addition of at least one nucleotide. In some aspects, cells in which a cutting-induced mutagenic event (i.e., a mutagenic event occurring after an NHEJ event) has occurred can be identified and / or selected by methods well known in the art. In some embodiments, TALE repeats are assembled to specifically target genes. (Gaj et al., 2013). A TALEN library targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013).Custom designed TALE arrays can be purchased on the market by Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA) and Life Technologies (Grand Island, NY, USA). Specifically, TALEN targeting CD38 is commercially available (see Gencopoeia, catalog number HTN222870-1, HTN222870-2 and HTN222870-3). Exemplary molecules are described in, for example, U.S. Patent Publication Nos. US2014 / 0120622 and 2013 / 0315884. In some embodiments, TALEN is introduced as a transgenic encoded by one or more plasmid vectors. In some aspects, the plasmid vector can include a selection marker for identifying and / or selecting cells receiving the vector.

[0305] III. Immune Effector Cells

[0306] The present disclosure relates to genetically engineered immune effector cells, including partial reduction or complete inhibition of expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. In some embodiments, partial reduction or complete inhibition of expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 can occur by any mechanism, including at least by CRISPR / Cas9 technology, to manufacture innovative and effective cell therapies for treating any type of cancer (including solid tumors).

[0307] The present disclosure encompasses any type of immune effector cells that are modified to have reduced or completely suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. In a specific embodiment, the present disclosure encompasses any type of immune effector cells that are modified to have reduced or completely suppressed CREM expression. In a specific embodiment, the reduction or complete suppression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression in the cell is the direct or indirect result of artificial deliberate manipulation of the cell. Immune effector cells can be manipulated by any mechanism to reduce or completely suppress the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, including homologous or non-homologous recombination. In certain embodiments, cells are manipulated, e.g., by CRISPR technology, to reduce or completely inhibit the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1.

[0308] The immune effector cells have GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression, particularly reduced or suppressed by genetic engineering, rather than natural cells with one or more mutations that cause endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression to be reduced. Therefore, in specific embodiments, immune effector cells are genetically engineered to reduce or suppress the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 in the immune effector cell genome. In specific embodiments, immune effector cells are knocked out to express endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1.

[0309] The present disclosure encompasses any type of immune effector cells, including conventional T cells, γ-δ T cells, NK cells, NK T cells, invariant NK T cells, regulatory T cells, macrophages, B cells, dendritic cells, tumor infiltrating lymphocytes, MSCs, or mixtures thereof. The cells may be allogeneic, autologous, or xenogeneic with respect to an individual, including an individual in need of the cells, such as an individual suffering from cancer.

[0310] In a specific embodiment, immune effector cells are artificially modified to express or otherwise produce one or more gene products, except that cells are also modified to have reduced or completely suppressed GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression. Such additional modifications to cells are not naturally present in cells or are exogenous relative to cells. Additional modifications can be any type, such as immune effector cells expressing receptors, cytokines, suicide genes or chemokines or combinations thereof, as examples.

[0311] When the immune effector cells with reduced or completely suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are additionally modified to produce or express a gene product that is not naturally present in the cell or is of exogenous origin, the order in which the immune effector cells are modified can be of any type. For example, immune effector cells with reduced or completely suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 can be modified to have one or more additional modifications, wherein in other cases, after immune effector cells are modified to produce or express a gene product that is not naturally present in the cell or is of exogenous origin, they are modified to have reduced or completely suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1.

[0312] In a specific embodiment, the immune effector cells lacking the full or partial expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are identical to cells modified to express receptors (e.g., antigen receptors). Any immune effector cell expression encompassed by the present disclosure may be any type of antigen receptor, including receptors for antigens, which are cancer antigens, which may also be tumor antigens. In a specific embodiment, the receptor is a chimeric antigen receptor or a T cell receptor, for example. The immune effector cells may be specifically designed to have the full or partial suppression of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, and are specifically designed to have antigen receptors for antigens on cancer cells of a targeted individual. That is, cells may be customized to include one or more antigen receptors, the antigen receptor targeting being known to be present on individual cancer cells.

[0313] In certain embodiments, the cells of the present disclosure are generated for use as ready-made cells. For example, cells with fully or partially suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are present in a repository, for example, they are obtained from a repository and engineered to have further modifications in addition to fully or partially suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. In other cases, cells with modifications other than the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are fully or partially suppressed are obtained from a repository, and these cells are engineered to have the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are fully or partially suppressed. After such modifications are performed on cells after obtaining them from a repository, these cells can be stored, or an effective amount of cells can be provided to an individual in need. GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1KO or knockdown cells can be further engineered to express engineered receptors, such as engineered antigen receptors targeting tumor antigens, which are suitable for treating individuals with specific cancer expression antigens.

[0314] In a specific embodiment, immune effector cell has the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are suppressed in whole or in part, and also expresses one or more engineering antigen targeting receptors and / or expresses at least one transfected (opposite to being endogenous to cells) cytokine and / or expresses at least one suicide gene. In the case where some cells have the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are suppressed in whole or in part, different vectors encode antigen targeting receptors relative encoding suicide gene and / or cytokine of transfection. Immune cell (including NK cell) can be derived from cord blood, peripheral blood, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), bone marrow or their mixture. NK cell can be derived from cell line, such as but not limited to NK-92 cell. NK cell can be cord blood mononuclear cell, such as CD56+NK cell.

[0315] The present disclosure describes and / or demonstrates the successful knockout (KO) of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 genes in natural killer (NK) cells derived from umbilical cord blood stored in a cord blood bank using CRISPR / Cas9. The present disclosure demonstrates that NK cells with disruption of the cAMP signaling pathway have enhanced anti-tumor activity compared to WT cAMP signaling pathway NK cells under acidic conditions or in vivo-like conditions shown in the literature as acidic. This enhanced anti-tumor activity is shown for solid tumor cell lines known to have active glycolysis and a significantly acidic tumor microenvironment. In certain embodiments, the tumor microenvironment is characterized by an increase in lactic acid levels relative to a non-tumor microenvironment.

[0316] In some cases, immune effector cells expressing GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are completely or partially suppressed are expanded in the presence of an effective amount of universal antigen presenting cells (UAPC) or fragments thereof (including any suitable ratio). The cells can be cultured with UAPC in a ratio of 10:1 to 1:10, 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2 or 1:1 (including, for example, a ratio of 1:2). In some cases, NK cells are expanded in the presence of IL-2, for example, at a concentration of 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0317] After using any carrier to carry out genetic modification, the immune effector cells with the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that are partially or completely reduced can be delivered to individuals immediately or can be stored (or some cells are delivered to individuals and the remaining cells are stored). In some aspects, after genetic modification, in about 1, 2, 3, 4, 5 days or longer after gene transfer in cells, cells can be used as a large number of colonies in vitro breeding for several days, weeks or months. On the other hand, clone transfectants, and in vitro amplification shows the existence of single integration or clones of expression cassettes or plasmids maintained by episomes. The clone selected for amplification shows a reduction or disappearance of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. Recombinant immune cells can be expanded by stimulation with IL-2 or other cytokines that bind to a common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). Recombinant immune cells can be expanded by stimulation with artificial antigen presenting cells. On the other hand, genetically modified cells can be cryopreserved.

[0318] In some embodiments, cells are treated with one or more inactivating agents (e.g., kinase inhibitors, e.g., dasatinib, nilotinib, rapamycin, etc.) before cryopreservation. In some embodiments, the technology described herein includes inactivating NK cells, including treating NK cells with an effective amount of one or more inactivating agents under certain conditions to produce inactivated NK cells. In some embodiments, the inactivating agent is a kinase inhibitor. In some embodiments, the inactivating agent is a mechanistic target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the inactivating agent is a tyrosine kinase (TK) inhibitor. In some embodiments, the TK inhibitor is lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pesidatinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gefitinib, Retinib, midostaurin, tucatinib, neratinib, baricitinib, ruxolitinib, feizotinib, tofacitinib, ripretinib, selumetinib, bemetinib, cobimetinib, trametinib, upadatinib, afatinib, selpatinib, cabozantinib, fotantinib, larotrectinib, entrectinib, axitinib, regorafenib, pazopanib, sorafenib, lenvatinib, vandetanib and / or sunitinib. In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is bosutinib, ponatinib, nilotinib, dasatinib and / or imatinib. In some embodiments, the TK inhibitor is dasatinib and / or nilotinib. In some embodiments, the TK inhibitor is dasatinib.

[0319] In some embodiments, the NK cells are treated with an inactivator at any time point during the NK cell culture. In some embodiments, the treatment lasts about 24 to about 96 hours, about 36 to about 84 hours, or about 48 to about 72 hours. In some embodiments, the treatment lasts about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cells are treated with an inactivator at a concentration of about 1 to about 1000 nM. In some embodiments, the NK cells are treated with an inactivator at a concentration of about 5 to about 500 nM. In some embodiments, the NK cells are treated with an inactivator at a concentration of about 20 to about 200 nM. In some embodiments, the NK cells are treated with an inactivator at a concentration of about 30 to about 100 nM. In some embodiments, relative to activated NK cells, inactivated NK cells have increased expression of one or more of C-kit, CCR-5, CD62L and / or CXCR4, and / or decreased expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS and / or CD95.

[0320] In some embodiments, the technology described herein includes a method for maintaining cell population viability exceeding at least 50% after cryopreservation of a cell population, comprising the following steps: subjecting the cell population to an effective amount of one or more inactivating agents (e.g., tyrosine kinase inhibitors) to inactivate cells before cryopreservation, cryopreserving the cells, and thawing the cell population, wherein after thawing, the viability of the cell population exceeds at least 50%. In some cases, after the cell population is cryopreserved, the viability of the cell population after the cells are thawed is at least more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0321] The embodiment of the present disclosure covers the immune effector cells with complete or partial inhibition of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression and one or more engineered receptors (including one or more antigen receptors).One or more engineered antigen receptors are produced by artificial means, for example, using recombinant technology, and are not natural for immune effector cells.Although engineered receptors can be any kind, in a specific embodiment, receptor is a chimeric antigen receptor, a T cell receptor, a homing receptor, a CRISPR / Cas9-mediated gene mutation, a decoy receptor, a cytokine receptor, a chimeric cytokine receptor, etc.

[0322] The embodiment of the present disclosure covers the cells with the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 completely or partially suppressed and one or more suicide genes.Immune effector cells can have the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 completely or partially suppressed, and can include the recombinant nucleic acid encoding any kind of suicide gene.The example of suicide gene includes the tumor necrosis factor (TNF)-α mutant polypeptide (see PCT / US2019 / 062009, which is incorporated herein by reference in its entirety) that cannot be secreted (including membrane binding) of engineering, and they may be affected by the delivery of the antibody combined with TNF-α mutant. Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxynucleoside into the toxic purine 6-methylpurine, can be used. As examples, other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0323] The cells may be obtained directly from the individual, or may be obtained from a bank or other storage facility. The cells used as therapy may be autologous or allogeneic with respect to the individual receiving the cells as therapy.

[0324] The cells can be derived from an individual in need of treatment for a disease, and after the cells are treated to have reduced or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, optionally a suicide gene, optionally a cytokine, and optionally a receptor (e.g., using standard transduction and expansion techniques for adoptive cell therapy), they can be returned to the individual from which they were originally derived. In some cases, the cells are stored for later use by the individual or another individual.

[0325] The immune cells may be comprised in a cell population, and a majority of the cells in the cell population may have reduced or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 and / or one or more suicide genes and / or one or more cytokines. The cell population may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of cells having reduced or inhibited GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression and / or one or more suicide genes and / or one or more cytokines and / or one or more engineered receptors; each of these gene products may or may not be produced as a separate polypeptide.

[0326] Immune cells with reduced or suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 and / or one or more suicide genes and / or one or more cytokines can be generated to be modularized for specific purposes. For example, cells can be generated (including for commercial distribution) with reduced or suppressed expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 and / or one or more suicide genes and / or one or more cytokines (or distributed with nucleic acids encoding suicide genes for subsequent transduction), and can be modified by the user to express one or more other genes of interest (including therapeutic genes) according to their intended use. For example, an individual interested in treating cancer cells can obtain or generate suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to have reduced or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, or vice versa.

[0327] In a specific embodiment, NK cells are utilized, and the genome of NK cells with reduced or suppressed GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression and / or one or more suicide genes and / or one or more cytokines can be modified. The genome can be modified in any way, but in a specific embodiment, the genome is modified, for example, by CRISPR gene editing. The genome of the cell can be modified to enhance the effectiveness of the cell for any purpose.

[0328] In certain embodiments, at least a method for manufacturing immune effector cells, a method for using immune effector cells, and a composition comprising immune effector cells are provided herein, wherein the immune effector cells have been adapted to (e.g., accustomed to, adapted to, etc.) acidic conditions by contacting acidic stimulants. In some embodiments, cells are adapted in vitro by contacting acidic stimulants. In some embodiments, cells gradually adapt over time. In some embodiments, cells gradually adapt over time by contacting acidic stimulants that accumulate gradually. In some embodiments, cells are adapted to at least or equal to, just or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29 or 30 days or a time period greater than 30 days. In some embodiments, cells are adapted to a time period of just or about 10 to 18 days. In some embodiments, cells are adapted to a time period of just 14 days or about 14 days. In some embodiments, the cells are adapted by contacting the acidic stimulus at a concentration greater than or equal to, at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mM. In some embodiments, the cell is adapted to an acidic stimulus greater than or equal to, just or about 2-3 mM by contact concentration. In some embodiments, the cell is adapted to an acidic stimulus greater than or equal to, just or about 2.5 mM by contact concentration. In some embodiments, the cell is adapted to an acidic stimulus comprising lactic acid or consisting essentially of lactic acid. In some embodiments, at least every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, an acidic stimulus is added. In some embodiments, about or just every 2-3 days, an acidic stimulus is added. In some embodiments, the cell is adapted to an acidic stimulus less than or equal to, just or about 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5.0. In some embodiments, the cells are adapted to an acidic stimulus of less than or equal to, about or exactly pH 6.0 or pH 5.9.

[0329] IV. Treatment Methods

[0330] Embodiments of the present disclosure include treatment methods associated with cancer immunotherapy or anti-pathogen immunotherapy, for example, wherein the cancer immunotherapy and anti-pathogen immunotherapy include at least a composition comprising immune effector cells with reduced or suppressed GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression levels. The method includes providing an effective amount of immune effector cells with reduced or suppressed GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression levels to an individual suffering from cancer and / or a pathogen.

[0331] In certain instances, an effective amount of cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 are provided to an individual. In certain instances, immune cells used in various cell therapies are genetically engineered using CRISPR / Cas9 to knock out GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 to improve their effectiveness against solid tumors, and these cell therapies are provided to an individual.

[0332] As an example, chimeric antigen receptor (CAR)-T cells (e.g., cells approved by the FDA for the treatment of leukemia and lymphoma) are genetically engineered to delete GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes with the goal of increasing their effectiveness in the acidic TME of solid tumors, which in certain embodiments results in the extension of the therapy to solid tumors. In addition, this genetic engineering strategy is used in various other forms of cell therapy, such as CAR-NK cells, engineered TCR-T cells, tumor infiltrating lymphocytes (TILs), to enhance their ability to fight various types of solid tumors.

[0333] In certain embodiments, the cells of the present disclosure are provided to individuals for improving medical conditions, such as any kind of cancer and / or any kind of pathogen infection. The use of the cells contemplated herein (including pharmaceutical compositions comprising them) is for preventing, treating or improving cancer diseases, such as tumor diseases or pathogen infection. In specific embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for preventing, improving and / or treating cancer, including, for example, cancers that may or may not be solid tumors.

[0334] In certain embodiments, the present disclosure contemplates the use of cells encompassed herein, which may be administered alone or in any combination with one or more other therapies, and in at least some aspects, with a pharmaceutically acceptable carrier or excipient. In certain embodiments, any nucleic acid molecule or vector may be stably integrated into the genome of the cell prior to delivery to a subject.

[0335] In addition, the present disclosure relates to a method for preventing, treating or ameliorating a tumor disease, comprising the step of administering to a subject in need thereof an effective amount of any cell having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1, as described herein.

[0336] In one embodiment, the isolated cells obtained by any suitable method or from a cell line and engineered as contemplated herein can be used as a medicament. The medicament can be used to treat cancer or infection in an individual in need thereof. In one embodiment, the isolated cells according to the present disclosure can be used to manufacture a medicament for treating cancer or infection in an individual in need thereof.

[0337] In some embodiments, the present disclosure provides a method for treating an individual in need thereof, the method comprising at least one of the following steps: (a) providing immune effector cells; (b) engineering the immune effector cells to reduce or inhibit the expression of at least GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1; (c) engineering the immune effector cells to express one or more engineered receptors (and step (c) can be performed simultaneously with step (b) or before step (b)); (d) engineering the immune effector cells to express one or more cytokines (and step (d) can be performed simultaneously with step (b) or (c) or before step (b) or (c)); (e) administering the engineered cells to an individual in need thereof, including an individual who has been determined to have cancer or to be at risk of developing cancer (e.g., above the average level in the population).

[0338] In specific embodiments, the engineered cells are engineered specifically for enhanced expansion, persistence, and / or cytotoxicity compared to any type of non-engineered cell.

[0339] In a specific embodiment, the engineered cell is engineered to improve cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) relative to non-engineered cells in a microenvironment (e.g., an acidic environment) at a pH of less than or equal to, just or about 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5.0, or less than 5.0. In a specific embodiment, the engineered cell is engineered to improve cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) relative to non-engineered cells in an acidic microenvironment comprising a low pH representing an increase in lactic acid levels relative to a non-acidic microenvironment. In certain embodiments, engineered cells are engineered to improve cell function (e.g., amplification, persistence, cytotoxicity, etc.) relative to non-engineered cells in an acidic microenvironment, and the acidic microenvironment includes a low pH of less than or equal to, about or just 6.0. In certain embodiments, engineered cells are engineered to improve cell function (e.g., amplification, persistence, cytotoxicity, etc.) relative to non-engineered cells in an acidic microenvironment, and the acidic microenvironment includes a low pH of less than or equal to, about or just 5.9. In certain embodiments, the acidic microenvironment includes or is a tumor microenvironment (TME).

[0340] Any treatment method of the present disclosure can be ameliorative, curative or preventive for an individual. It can be part of an autoimmune therapy or part of an allogeneic immunotherapy treatment. In certain cases, the method is used for allogeneic immunotherapy, as long as it can convert NK cells usually obtained from a donor into non-alloreactive cells. This can be done under standard protocols and replicated multiple times as needed. The resulting engineered immune cells can be pooled and administered to one or several patients, providing them as "off-the-shelf" therapeutic products. The cells can be stored, such as cryopreserved.

[0341] In some embodiments, the cell composition is administered for any type of cancer disease, including tumor diseases, such as B cell malignancies, multiple myeloma, lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer, or cervical cancer. Exemplary indications for the administration of the composition of the cell are cancerous diseases, including any malignant tumor that expresses one or more certain antigens associated with an individual's cancer. The administration of the composition of the present disclosure can be used for all stages (I, II, III, and / or IV) and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer.

[0342] The disclosure further encompasses co-administration schemes with other compounds (e.g., bispecific antibody constructs, targeted toxins, or other compounds) that work through immune cells. Clinical schemes for co-administration of the compounds of the invention may include co-administration while, before, or after administration of other components. Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0343] Embodiments relate to kits comprising a construct that produces cells, a nucleic acid sequence defined herein, a vector defined herein, and / or a host cell (e.g., immune effector cell) defined herein. It is also contemplated that the kit of the present disclosure comprises a pharmaceutical composition as described above, alone or in combination with other drugs to be administered to an individual in need of medical treatment or intervention.

[0344] V. Genetically Engineered Receptors

[0345] Immune cells of the present disclosure having reduced or suppressed GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression can be further modified to express one or more non-endogenous gene products. The gene product may or may not be a genetically engineered receptor. The receptor may be of any kind, including, for example, a receptor for an antigen, a chemokine, or a cytokine. In the case where the receptor is an antigen, the antigen may be a cancer antigen, including a solid tumor antigen.

[0346] Immune effector cells with reduced or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 can be genetically engineered to express antigen receptors that target specific antigens, and such cells can be specifically designed to target one or more antigens present on an individual's cancer cells.

[0347] In a specific embodiment, the immune effector cells expressed by GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 with reduction or inhibition may include engineered antigen receptors, such as engineered TCR or CAR. For example, immune cells may be NK cells modified to express one or more CARs and / or TCRs with antigen-specificity for one or more specific antigens. In some aspects, immune cells are engineered to express antigen-specific CAR or antigen-specific TCR by knocking in, for example, CAR or TCR using CRISPR.

[0348] Suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, cells can be transduced to express TCRs with antigen specificity for cancer antigens using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0349] In some embodiments, the cell comprises one or more nucleic acids encoding one or more antigen receptors introduced by genetic engineering, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, that is, it is not usually present in the cell or sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, which is, for example, not usually found in the cell being engineered and / or the organism from which such cells are derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not present in nature (e.g., chimeric).

[0350] Exemplary antigen receptors (including CARs and recombinant TCRs) and methods of engineering and introducing the receptors into cells include, for example, those described in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and European Patent Application No. EP2537416 and / or those described in Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically engineered antigen receptor includes a CAR as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1.

[0351] A. Chimeric Antigen Receptor

[0352] In some embodiments, the CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising one or more antigen binding regions.

[0353] In some embodiments, engineered antigen receptors include CAR, including activation or stimulatory CAR, costimulatory CAR (see WO2014 / 055668) and / or inhibitory CAR (iCAR, see, e.g., Fedorov et al., 2013). CAR generally comprises an extracellular antigen (or ligand) binding domain connected to one or more intracellular signal transduction components (in some aspects by a joint and / or one or more transmembrane domains). Such molecules generally simulate or approximate the signal caused by a natural antigen receptor, the signal caused by such a receptor combined with a costimulatory receptor, and / or the signal caused only by a costimulatory receptor.

[0354] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including CARs (hCARs) that have been humanized to reduce immunogenicity, which include at least one intracellular signal transduction domain, a transmembrane domain, and an extracellular domain comprising one or more signal transduction motifs). In certain embodiments, antigen-specific CARs can recognize epitopes contained in a shared space between one or more antigens. In certain embodiments, the binding region may include a complementary determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0355] It is contemplated that the human antigen targeting CAR components may be encoded by nucleic acids derived from human genes. In some embodiments, such components may be used to enhance human genes for cellular immunotherapy of human patients. In a specific embodiment, the present disclosure includes a full-length antigen-specific CAR cDNA or coding region. The antigen binding region or domain may comprise a V fragment of a single chain variable fragment (scFv) derived from a specific human monoclonal antibody. H and V L The fragments of the antibody chain are fragments of the antigen binding domains of the human antigen-specific antibody, such as those described in U.S. Pat. No. 7,109,304, which is incorporated herein by reference. The fragments can also be any number of different antigen-binding domains of human antigen-specific antibodies. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells.

[0356] The arrangement can be a multimer, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing the variable parts of the light chain and the heavy chain into a diabody. The hinge portion of the construct can have a variety of alternatives, from complete deletion to maintaining the first cysteine, to proline instead of serine replacement, truncated to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerized protein can achieve this purpose. Only one Fc domain can be used, such as the CH2 or CH3 domain of a human immunoglobulin. The hinge region, CH2 region, and CH3 region of a human immunoglobulin modified to improve dimerization can also be used. It is also possible to use only the hinge portion of an immunoglobulin. A portion of CD8α can also be used.

[0357] In some embodiments, CAR nucleic acid comprises a sequence encoding other co-stimulatory receptors (e.g., transmembrane domains and modified CD28 intracellular signal transduction domains).Other co-stimulatory receptors include but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137).In addition to the main signal triggered by CD3ζ, other signals provided by the human co-stimulatory receptors inserted into human CAR are also important for the complete activation of NK cells and can help improve the in vivo persistence and the therapeutic success of adoptive immunotherapy.

[0358] In some embodiments, antigen-specific CAR is constructed in a manner specific to antigens expressed on normal or non-diseased cell types or diseased cell types. Therefore, CAR generally includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or parts, or one or more antibody variable domains, and / or antibody molecules in its extracellular part. In some embodiments, antigen-specific CAR includes one or more antigen binding portions of antibody molecules, such as single-chain antibody fragments (scFv) of variable heavy chains (VH) and variable light chains (VL) derived from monoclonal antibodies (mAb).

[0359] In certain embodiments, antigen-specific CAR can be co-expressed with cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, CAR can be co-expressed with one or more cytokines (e.g., IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof).

[0360] The sequence of the open reading frame encoding the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, or may be synthesized (e.g., via PCR) or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA. In addition, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0361] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA by electroporation are known in the art. See, e.g., U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in a suitable orientation for expression.

[0362] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use according to the methods of the present disclosure are non-replicative in immune cells. Many viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0363] In some aspects, antigen-specific binding or recognition components are connected to one or more transmembrane and intracellular signal transduction domains. In some embodiments, CAR includes a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid replacement to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, so as to minimize the interaction with other members of the receptor complex.

[0364] In some embodiments, the membrane-spanning domain is derived from a natural source or from a synthetic source. If the source is natural, the domain is derived from any membrane-bound protein or a membrane-spanning protein in some aspects. The membrane-spanning region includes those derived from the following (i.e., at least including one or more membrane-spanning regions): α, β or ζ chains of T cell receptors, CD28, CD30, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules. Alternatively, in some embodiments, the membrane-spanning domain is synthetic. In some aspects, the synthetic membrane-spanning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be found at each terminus of the synthetic transmembrane domain.

[0365] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells (e.g., NK cells) includes (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) a CAR that signals through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) a CAR with a variable length of an extracellular domain that connects a CD70 recognition domain to the cell surface, and in some cases (iv) a CAR derived from K562 to enable robust and digital expansion of the CAR +Artificial antigen presenting cells (aAPCs) of immune cells (see, e.g., Singh et al., 2008; Singh et al., 2011).

[0366] In certain embodiments, CAR recognizes TROP2 antigen. In certain embodiments, anti-TROP2 CAR and / or NK cells comprising the CAR are as described in International Patent Application Publication No. WO2023 / 283644A2, which was published on January 12, 2023, and is incorporated herein by reference in its entirety.

[0367] In certain embodiments, CAR recognizes CD70 antigen. In certain embodiments, anti-CD70 CAR comprises a CD27-derived polypeptide. In certain embodiments, anti-CD70 CAR comprises a sequence having at least or equal to, exactly or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one or more of SEQ ID NOs: 185-193, or is encoded by it. In certain embodiments, anti-CD70 CAR and / or NK cells comprising the CAR are as described in International Patent Application Publication No. WO2021 / 142127A1, which was published on July 15, 2021, and is incorporated herein by reference in its entirety. In certain embodiments, the anti-CD70 CAR and / or NK cells comprising the CAR are as described in International Patent Application Publication No. WO2022 / 159791A1, which was published on July 28, 2022, and is incorporated herein by reference in its entirety. In certain embodiments, the anti-CD70 CAR and / or NK cells comprising the CAR are as described in International Patent Application Publication No. WO2023 / 278520A1, which was published on January 5, 2023, and is incorporated herein by reference in its entirety.

[0368] SEQ ID NO: 185—Exemplary truncated CD27 amino acid sequence

[0369] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNG WQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV

[0370] SEQ ID NO: 186 - Exemplary Truncated CD27 Amino Acid Sequence

[0371] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR

[0372] SEQ ID NO: 187 - Exemplary CD28 TMD

[0373] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0374] SEQ ID NO: 188, Exemplary Full - Length WT CD27 Amino Acid Sequence

[0375] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0376] SEQ ID NO: 189, Exemplary Truncated WT CD27 Amino Acid Sequence

[0377] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR

[0378] SEQ ID NO: 190—Exemplary CD27 signal peptide (SP) amino acid sequence

[0379] MARPHPWWLCVLGTLVGLS

[0380] SEQ ID NO: 191 - Exemplary CD3z-derived intracellular signaling domain (ICD) amino acid sequence

[0381] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG

[0382] SEQ ID NO: 192—Exemplary CD28-derived intracellular signaling domain (ICD) amino acid sequence

[0383] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0384] SEQ ID NO: 193—Exemplary DAP10-derived intracellular signaling domain (ICD) amino acid sequence

[0385] LCARPRRSPAQEDGKVYINMPGRG

[0386] T cell receptor (TCR)

[0387] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells."T cell receptor" or "TCR" refers to a molecule comprising variable α and β chains (referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (referred to as TCRγ and TCRδ, respectively), and capable of specifically binding to antigenic peptides bound to major histocompatibility complex (MHC) receptors. In some embodiments, TCR is in the αβ form.

[0388] Generally, TCRs in the form of αβ and γδ are usually similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), and are generally responsible for identifying antigens bound to MHC molecules. In some embodiments, TCRs may also include constant domains, transmembrane domains and / or short cytoplasmic tails (see, e.g., Janeway et al., 1997). For example, in some aspects, each chain of TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with the invariant proteins of the CD3 complexes involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be understood to encompass its functional TCR fragments. The term also encompasses complete or full-length TCRs, including TCRs in the form of αβ or γδ.

[0389] Therefore, for the purposes of this article, the TCR referred to includes any TCR or functional fragment, such as the antigen binding portion of a TCR that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. The "antigen binding portion" or "antigen binding fragment" of a TCR (which can be used interchangeably) refers to a molecule that contains a portion of the structural domain of a TCR but binds to an antigen (e.g., an MHC-peptide complex) bound to a full-length TCR. In some cases, the antigen binding portion comprises a variable domain of a TCR, such as a variable α chain and a variable β chain of a TCR, which is sufficient to form a binding site that binds to a specific MHC-peptide complex, such as typically each chain comprising three complementary determining regions.

[0390] In some embodiments, the variable domains of the TCR chains associate to form a loop or complementary determining region (CDR) similar to an immunoglobulin, which confers antigen recognition and determines peptide specificity by forming the binding site of the TCR molecule and determining peptide specificity. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigens, although it has also been shown that the CDR1 of the α chain interacts with the N-terminal portion of the antigenic peptide, while the CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is considered to recognize MHC molecules. In some embodiments, the variable region of the β chain may include an additional hypervariable (HV4) region.

[0391] In some embodiments, the TCR chain comprises a constant domain. For example, like an immunoglobulin, the extracellular portion of the TCR chain (e.g., an alpha chain, a beta chain) can comprise two immunoglobulin domains, namely a variable domain at the N-terminus (see, e.g., V a or Vp; usually amino acids 1 to 116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.), and the constant domains adjacent to the cell membrane (e.g., the α chain constant domain or C a , typically amino acids 117 to 259 based on Kabat, and a beta chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat). For example, in some cases, the extracellular domain of a TCR formed by two chains comprises two membrane proximal constant domains and two membrane distal variable domains comprising CDRs. The constant domain of the TCR domain comprises a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby forming a connection between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the alpha and beta chains, such that the TCR comprises two disulfide bonds in the constant domain.

[0392] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, the structure allows TCR to associate with other molecules (e.g., CD3). For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunit of the CD3 signal transduction device or complex.

[0393] Generally, CD3 is a multiprotein complex, which can have three different chains (γ, δ and ε) and ζ chain in mammals. For example, in mammals, the complex can contain a homodimer of CD3γ chain, CD3δ chain, two CD3ε chains and CD3ζ chain. CD3γ, CD3δ and CD3ε chains are highly correlated cell surface proteins of the immunoglobulin superfamily comprising a single immunoglobulin domain. The transmembrane region of CD3γ, CD3δ and CD3ε chains is negatively charged, which is a feature that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ and CD3ε chains each contain a conserved motif, referred to as tyrosine-based immunoreceptor activation motifs or ITAMs, and each CD3ζ chain has three. Generally, ITAMs are related to the signal transduction ability of TCR complexes. These auxiliary molecules have negatively charged transmembrane regions and play a role in propagating signals from TCR to cells. CD3- and ζ- chains form so-called T cell receptor complexes together with TCR.

[0394] In some embodiments, TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, TCR is a heterodimer comprising two separate chains (α and β chains or γ and δ chains), which are, for example, connected by one or more disulfide bonds. In some embodiments, the TCR for the target antigen (e.g., cancer antigen) is identified and introduced into the cell. In some embodiments, the nucleic acid encoding TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, TCR is obtained from biological sources, such as obtained from cells, such as obtained from T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, T cells can be obtained from cells separated in vivo. In some embodiments, high-affinity T cell clones can be isolated from patients, and TCR is isolated. In some embodiments, T cells can be cultured T cell hybridomas or clones. In some embodiments, TCR clones directed against a target antigen have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs directed against a target antigen (see, for example, Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, a TCR or antigen binding portion thereof can be synthetically generated based on knowledge of the TCR sequence.

[0395] VI. Cytokines

[0396] One or more cytokines can be used in immune effector cells with reduced or suppressed expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1. In some cases, one or more cytokines are present on the same carrier molecule as the engineered receptor, but in other cases, they are present on separate molecules. In a specific embodiment, one or more cytokines are co-expressed with the engineered receptor from the same carrier. One or more cytokines can be produced as polypeptides separated from antigen-specific receptors. In some embodiments, NK cells do not include one or more engineered receptors, but include one or more heterologous cytokines. As an example, interleukin-15 (IL-15) is used. IL-15 can be used because, for example, it is confined to tissues, and only under pathological conditions will it be observed in serum or any level of the whole body. IL-15 has a variety of characteristics suitable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors by alleviating the functional inhibition of tumor-resident cells, and inhibits activation-induced cell death. In certain embodiments, NK cells expressing IL-15 can continue to support cytokine signaling, which is useful for its survival after infusion. In addition to IL-15, other cytokines are also envisioned. These include but are not limited to cytokines, chemokines and other molecules that contribute to the activation and proliferation of cells for human applications. For example, cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-7 or a combination thereof. In certain embodiments, NK cells expressing IL-21 can continue to support cytokine signaling, which is useful for its survival after infusion. In certain embodiments, cytokines are expressed as part of a polycistronic construct with one or more functional proteins and / or marker proteins. Cells with reduced or suppressed expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 and that may express one or more cytokines may be utilized and are able to continue to support cytokine signaling that is useful for their survival following infusion.

[0397] In a specific embodiment, the NK cell expression level of the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 that has been reduced or suppressed expresses one or more exogenously provided cytokines. The cytokine can be provided to the cell exogenously because it is expressed by an expression vector in the cell. In an alternative case, when manipulating the regulation of the expression of the endogenous cytokine (for example, carrying out genetic recombination at the promoter site of the cytokine), the endogenous cytokine in the cell is upregulated. In the case where the cytokine is provided to the cell on the expression construct, the cytokine can be encoded by the same vector as the vector expressing another gene product (for example, a suicide gene). The cytokine can be expressed as a polypeptide molecule independent of the suicide gene, and can be expressed as a polypeptide independent of the engineered receptor of the cell. In some embodiments, the disclosure relates to the co-utilization of CAR and / or TCR vectors with IL-15, particularly in NK cells with reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1.

[0398] VII. Suicide Gene

[0399] In certain embodiments, suicide genes are used in conjunction with any type of cell therapy to control its use and allow cell therapy to be terminated at a desired event and / or time. Suicide genes are used in transduced cells for the purpose of inducing the death of transduced cells when needed. The immune effector cells of the present disclosure that have been modified to accommodate the vectors covered by the present disclosure may include one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene whose gene product is converted into a compound that kills its host cell after administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that, when needed, is targeted by an agent (e.g., an antibody) that targets the suicide gene product.

[0400] Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase, a so-called suicide gene, can be used to convert the prodrug 6-methylpurine deoxynucleoside into the toxic purine 6-methylpurine. Other examples of suicide genes used with prodrug therapy are the Escherichia coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0401] Exemplary suicide genes also include CD20, CD52, EGFRv3 or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be ablated by cetuximab. Other suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine-α, γ-lyase (MET) and thymidine phosphorylase (TP).

[0402] In a specific embodiment, any vector in the vector encoding antigen-targeted CAR or NK cells covered herein includes one or more suicide genes. The suicide gene may or may not be located on the same vector as the antigen-targeted CAR. In the case where the suicide gene and the antigen-targeted CAR are located on the same vector, the suicide gene and CAR can be separated by, for example, an internal ribosome entry site (IRES) element or a 2A element.

[0403] In a specific embodiment, the suicide gene is a tumor necrosis factor (TNF)-α mutant that cannot be cleaved by a standard enzyme that naturally cleaves TNF, such as TNF-α converting enzyme (also known as TACE). Thus, in a specific embodiment, the TNF-α mutant is membrane-bound and non-secretable. The TNF-α mutant used in the present disclosure can be targeted by one or more agents (including at least one antibody) that bind to the mutant, such that after the agent binds to the TNF-α mutant on the cell surface, the cell dies. Embodiments of the present disclosure allow the use of a TNF-α mutant as a marker for cells that express it.

[0404] Cells expressing non-cleavable TNF-α mutants can be targeted for selective deletion, including, for example, using FDA-approved TNF-α antibodies currently in clinical use, such as etanercept, infliximab, or adalimumab. The mutated TNF-α polypeptide can be co-expressed with one or more therapeutic transgenes in the cell, such as genes encoding TCRs or CARs (including CD70-targeted TCRs and / or CARs). In addition, cells expressing TNF-α mutants have excellent activity against tumor targets, which is mediated by the biological activity of membrane-bound TNF-α proteins.

[0405] Relative to the wild type, TNF-α has a 26 kD transmembrane form and a 17 kD secretory component. Some mutants described by Perez et al. (1990) can be used in the present disclosure. In a specific embodiment, examples of TNF-α mutants of the present disclosure include at least the following relative to 17 kD TNF: (1) Val1 deletion and Pro112 deletion; (2) Val13 deletion; (3) Val1 deletion and Val13 deletion; (4) Val1 to Pro112 (including Pro112) deletion and Val13 deletion (deletion of 13 aa); (5) Ala-3 to Val13 (including Val13) deletion (deletion of 14 aa). In a specific embodiment, the TNF-α mutant includes a deletion of the corresponding amino acid at position -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or a combination thereof. Particular combinations include positions -3 to and including 13; -3 to and including 12; -3 to and including 11; -3 to and including 10; -3 to and including 9; -3 to and including 8; -3 to and including 7; -3 to and including 6; -3 to and including 5; -3 to and including 4; -3 to and including 3; -3 to and including 2; -3 to and including 1; -3 to and including -1; -3 to and including -2; -2 to and including 13; -2 to and including 12; -2 to and including 11; -2 to and including 10; -2 to and including 9; -2 to and including 8; -2 to and including 7; -2 to and including 6; -2 to and including 5; -2 to and including 4; -2 to and including 3 ; -2 to and including 2; -2 to and including 1; -2 to and including -1; -1 to and including 13; -1 to and including 12; -1 to and including 11; -1 to and including 10; -1 to and including 9; -1 to and including 8; -1 to and including 7; -1 to and including 6; -1 to and including 5; -1 to and including 4; -1 to and including 3; -1 to and including 2; -1 to and including 1; 1 to and including 13; 1 to and including 12; 1 to and including 11; 1 to and including 10; 1 to and including 9; 1 to and including 8; 1 to and including 7; 1 to and including 6; 1 to and including 5; 1 to and including 4; 1 to and including 3; 1 to and including 2; and so on.

[0406] TNF-α mutants can be produced by any suitable method, but in certain embodiments, they are produced by site-directed mutagenesis. In some cases, TNF-α mutants may have mutations other than mutations that render the protein non-cleavable. In certain cases, TNF-α mutants may have 1, 2, 3 or more mutations other than deletions of Val1, Pro12 and / or Val13 or regions therebetween. Mutations other than mutations that render the mutant non-secretable may be one or more of amino acid substitutions, deletions, additions, inversions, etc. In the case where the additional mutations are amino acid substitutions, for example, the substitutions may or may not be conservative amino acids. In some cases, 1, 2, 3, 4, 5 or more additional amino acids may be present at the N-terminus and / or C-terminus of the protein. In some cases, TNF-α mutants have (1) one or more mutations that render the mutant non-secretable; (2) one or more mutations that prevent the mutant from signaling from the outside to the inside; and / or (3) one or more mutations that interfere with the binding of the mutant to TNF receptor 1 and / or TNF receptor 2.

[0407] In a specific embodiment, after delivering an effective amount of one or more agents to target cells in combination with antigen CAR expressing TNF-α mutants, most of the cells expressing TNF-α mutants are eliminated. In a specific embodiment, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the cells expressing TNF-α mutants are eliminated in individuals. After recognizing the need to eliminate cells, agents can continue to be delivered to individuals until one or more symptoms no longer occur or until a sufficient number of cells are eliminated. TNF-α mutants can be used as markers to monitor the number of cells in individuals.

[0408] Embodiments of the methods of the present disclosure may include a first step of providing an effective amount of cell therapy to an individual in need, wherein the cells comprise one or more non-secretory TNF-α mutants; and a second step of eliminating the cells using the TNF-α mutants as suicide genes (directly or indirectly by any mechanism of cell death). The second step may be initiated when at least one adverse event occurs in the individual, and the adverse event may be identified in any manner, including routine monitoring, which may or may not continue from the start of the cell therapy. The adverse event may be detected after an examination and / or test. In the case where an individual suffers from cytokine release syndrome (also referred to as a cytokine storm), the individual may have elevated inflammatory cytokines (by way of example only: interferon-γ, granulocyte macrophage colony stimulating factor, IL-10, IL-6, and TNF-α); fever; fatigue; for example, hypotension; hypoxia, tachycardia; nausea; capillary leakage; cardiac / renal / liver dysfunction; or a combination thereof. In the case where an individual has neurotoxicity, the individual may have confusion, delirium, dysplasia, and / or seizures. In some cases, individuals are tested for markers associated with the onset and / or severity of cytokine release syndrome (e.g., C-reactive protein, IL-6, TNF-α, and / or ferritin).

[0409] In another embodiment, for example, during cytokine release syndrome or neurotoxicity, one or more agents that bind to non-secretory TNF-α are administered with the additional benefit of neutralizing high levels of soluble TNF-α that cause therapeutic toxicity. Soluble TNF-α is released at high levels during cytokine release syndrome and is a toxic medium for CART cell therapy. In this case, the administration of the TNF-α antibody described herein has a dual beneficial effect, namely selectively deleting cells expressing TNF-α mutants and neutralizing soluble TNF-α that causes toxicity. Therefore, embodiments of the present disclosure include methods for eliminating or reducing the severity of cytokine release syndrome in individuals who receive or have received adoptive cell therapy, wherein the cells express non-secretory TNF-α mutants, including providing an effective amount of a step of a medicament that binds to non-secretory TNF-α mutants, the medicament causing the individual (a) to eliminate at least some cells of cell therapy; and (b) reducing the level of soluble TNF-α.

[0410] Embodiments of the present disclosure include methods of reducing the effects of cytokine release syndrome in an individual who has received or is receiving cell therapy using cells expressing a non-secretory TNF-α mutant, comprising the step of providing an effective amount of one or more agents that bind to the mutant to cause in the individual (a) elimination of at least some cells of the cell therapy; and (b) a reduction in the level of soluble TNF-α.

[0411] When it is necessary to use the TNF-α suicide gene, an effective amount of one or more inhibitors is provided to the individual, and the inhibitor is capable of inhibiting the TNF-α mutant on the cell surface, for example, by direct binding. In some embodiments, the inhibitor can be provided to the individual systemically and / or locally. The inhibitor can be a polypeptide (e.g., an antibody), a nucleic acid, a small molecule (e.g., a xanthine derivative), a peptide, or a combination thereof. In a specific embodiment, the antibody is approved by the FDA. When the inhibitor is an antibody, in at least some cases, the inhibitor can be a monoclonal antibody. When an antibody mixture is used, one or more antibodies in the mixture can be a monoclonal antibody. Examples of small molecule TNF-α inhibitors include small molecules as described in U.S. Patent No. 5,118,500, which is incorporated herein by reference in its entirety. Examples of polypeptide TNF-α inhibitors include polypeptides, such as the polypeptides described in U.S. Patent No. 6,143,866, which is incorporated herein by reference in its entirety.

[0412] In certain embodiments, at least one antibody is used to target a TNF-α mutant to trigger its activity as a suicide gene. Examples of antibodies include at least, for example, adalimumab, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, golimumab, infliximab, infliximab-dyyb, or a mixture thereof.

[0413] Embodiments of the present disclosure include methods of reducing the risk of toxicity of cell therapy to an individual by modifying the cells of the cell therapy to express a non-secretory TNF-α mutant. In certain embodiments, the cell therapy is for cancer and may include engineered receptors that target antigens, including cancer antigens.

[0414] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, the individual may have received, may receive, and / or may receive additional therapy for the medical condition. Where the medical condition is cancer, the individual may be provided with one or more of surgery, radiation, immunotherapy (in addition to the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, etc.

[0415] Provide an effective level of a cell population with reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 to an individual in need thereof. The cells can be administered to an individual by injection, intravenous, intraarterial, intraperitoneal, intratracheal, intratumoral, intramuscular, intraendoscopic, intralesional, intracranial, transdermal, subcutaneous, regional, by perfusion, in a tumor microenvironment, or a combination thereof.

[0416] In certain embodiments of the method, the cells may be administered to the individual once or more than once. The duration between administrations of cells to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or 1 year or more.

[0417] VIII. Vector

[0418] In the case where the immune effector cells with reduced or suppressed expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 comprise non-endogenous engineered gene products or exogenously provided gene products, the gene products can be delivered to the receptor immune effector cells by any suitable vector, including by viral vectors or by non-viral vectors. Examples of viral vectors include at least retrovirus, slow virus, adenovirus or adeno-associated virus vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles, etc.

[0419] In the case where immune cells are transduced with vectors encoding antigen targeting receptors and another gene or multiple genes (such as suicide genes and / or cytokines and / or optional therapeutic gene products) are also required to be transduced into cells, antigen targeting receptors, suicide genes, cytokines and optional therapeutic genes may or may not be included on the same vector or together with the same vector. In some cases, antigen targeting CAR, suicide genes, cytokines and optional therapeutic genes are expressed from the same vector molecule, such as the same viral vector molecule. In this case, the expression of antigen targeting CAR, suicide genes, cytokines and optional therapeutic genes may or may not be regulated by the same regulatory elements. When antigen targeting CAR, suicide genes, cytokines and optional therapeutic genes are located on the same vector, they may or may not be expressed as separate polypeptides. For example, in the case where they are expressed as separate polypeptides, they can be separated by 2A elements or IRES elements on the vector (or two types can be used once or multiple times on the same vector).

[0420] A. General Implementation Plan

[0421] Those skilled in the art will be able to construct vectors for expression of the antigen receptors of the present disclosure by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference).

[0422] 1. Regulatory elements

[0423] The expression cassette included in the vector of the present disclosure particularly comprises (along 5' to 3' direction) a eukaryotic transcription promoter operably connected to a protein coding sequence, a splicing signal including an intervening sequence and a transcription termination / polyadenylation sequence. The promoter and enhancer controlling the transcription of a protein coding gene in a eukaryotic cell can be composed of a variety of genetic elements. The cell machinery can collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to evolve unique, usually complex transcriptional regulatory patterns. For example, the promoter used in the context of the present disclosure includes constitutive, inducible and tissue-specific promoters. In the case where the vector is used to produce cancer treatment, the promoter may be effective under hypoxic conditions.

[0424] 2. Promoter / enhancer

[0425] The expression construct provided herein comprises a promoter that drives the expression of antigen receptor and other cistron gene products. The promoter generally comprises a sequence for locating the start site of RNA synthesis. The most well-known example is the TATA box, but in some promoters lacking the TATA box, such as, for example, the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, the discrete elements covering the start site themselves help fix the start position. Other promoter elements regulate the frequency of transcription initiation. Usually, these regions located upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. In order to make the coding sequence "under the control of the promoter", the 5' end of the transcription start site of the transcription reading frame is positioned at the "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0426] The spacing between promoter elements is often flexible so that promoter function is retained when the elements are inverted or moved relative to each other. For example, in the tk promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0427] The promoter may be a promoter naturally associated with a nucleic acid sequence, such as one that can be obtained by isolating a 5′ non-coding sequence upstream of a coding segment and / or exon. Such a promoter may be referred to as “endogenous”. Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, prokaryotic or eukaryotic cell, and promoters or enhancers that are not “naturally present”, i.e., different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the promoters most commonly used for recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthetically generating promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can also be used in conjunction with the compositions disclosed herein. <tm>) to generate the sequence. In addition, it is contemplated that control sequences that direct transcription and / or expression of the sequence in non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.) may also be employed.

[0428] Naturally, it is important to use promoters and / or enhancers that effectively direct the expression of the DNA fragment in the organelle, cell type, tissue, organ or organism selected for expression. The use of promoters, enhancers and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology (see, e.g., Sambrook et al., 1989, incorporated herein by reference). The promoter used can be constitutive, tissue-specific, inducible and / or can be used to direct high-level expression of the introduced DNA fragment under appropriate conditions, such as in large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0429] In addition, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB, accessed via the website epd.isb-sib.ch / ) can also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription of certain bacterial promoters if the appropriate bacterial polymerase is provided (either as part of the delivery complex or as an additional gene expression construct).

[0430] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as β-actin promoter, GADPH promoter, metallothionein promoter; and tandem response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter (tre) near the minimal TATA box. Human growth hormone promoter sequences (e.g., human growth hormone minimal promoter described in Genbank® Accession No. X05244, nucleotides 283-341) or mouse mammary tumor promoter (available from ATCC Accession No. ATCC 45007) can also be used. In certain embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I, or MHC class II promoter, but any other promoter that can be used to drive expression of a therapeutic gene is suitable for the practice of the present disclosure.

[0431] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase the activity of a promoter and have the potential to act in cis, regardless of their orientation, even at relatively long distances (up to several kilobases from the target promoter). However, enhancer function is not necessarily limited to such long distances, as they can also act in close proximity to a given promoter.

[0432] 3. Initiation Signal and Linked Expression

[0433] Specific start signals can also be used in the expression constructs provided in the present disclosure to effectively translate the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art will be able to easily determine this and provide the necessary signals. It is well known that the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be improved by including appropriate transcription enhancer elements.

[0434] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to generate polygenic or polycistronic information. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and start translation at the internal site. IRES elements from two members of the picornavirus family (poliomyelitis and encephalomyocarditis) have been described, as well as IRES from mammalian information. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by IRES, thereby generating polycistronic information. With the help of IRES elements, ribosomes can approach each open reading frame for effective translation. Using a single promoter / enhancer to transcribe a single message can effectively express multiple genes.

[0435] As described in detail elsewhere herein, certain 2A sequence elements can be used to produce linked expression or co-expression of genes in constructs provided by the present disclosure. For example, cleavage sequences can be used to co-express genes by connecting open reading frames to form a single cistron. Exemplary cleavage sequences are equine rhinitis A virus (E2A) or F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thoseaasigna virus 2A; T2A) or porcine Teschovirus-1 (P2A). In specific embodiments, in a single vector, multiple 2A sequences are not identical, although in alternative embodiments the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are provided in US2011 / 0065779, which is incorporated herein by reference in its entirety.

[0436] 4. Starting point of replication

[0437] In order to propagate the vector in the host cell, it may contain one or more replication origin sites (commonly referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV as described above or an oriP (which is a specific nucleic acid sequence at the origin of replication) genetically engineered in programming with similar or enhanced functions. Alternatively, the replication origin or autonomous replication sequence (ARS) of other extrachromosomal replication viruses as described above may be used.

[0438] 5. Selective and screenable markers

[0439] In some embodiments, NK cells containing a receptor construct targeting CD70 of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer a recognizable change to the cell, allowing easy identification of cells containing the expression vector. In general, a selection marker is a marker that confers a property that allows selection. A positive selection marker is a marker in which the presence of the marker allows its selection, while a negative selection marker is a marker in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0440] The inclusion of drug selection markers usually helps to clone and identify transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to conferring markers that allow the phenotype of transformants to be distinguished based on conditional implementation, other types of markers are also contemplated, including screenable markers, such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is believed to be unimportant, as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selectivity and screenable markers are well known to those skilled in the art.

[0441] B. Polycistronic vectors

[0442] In a specific embodiment, an antigen targeting receptor, an optional suicide gene, an optional cytokine and / or an optional therapeutic gene are expressed from a polycistronic vector (the term "cistron" as used herein refers to a nucleic acid sequence from which a gene product can be produced). In a specific embodiment, a polycistronic vector encodes an antigen targeting receptor, a suicide gene and at least one cytokine, a suicide gene and / or an engineered receptor, such as a T cell receptor and / or another non-antigen targeting CAR. In some cases, a polycistronic vector encodes at least one antigen targeting CAR, at least one TNF-α mutant and at least one cytokine. Cytokines can be specific types of cytokines, such as humans or mice or any species. In particular cases, cytokines are IL-15, IL-12, IL-2, IL-18 and / or IL-21.

[0443] In certain embodiments, the present disclosure provides a flexible modular system (the term "modularity" as used herein refers to a cistron or a component of a cistron that allows its interchangeability, such as by removing and replacing the entire cistron or a component of a cistron, respectively, such as by using standard recombinant techniques), which utilizes a polycistronic vector with the ability to express multiple cistrons at substantially the same level. The system can be used to allow cell engineering of combined expression (including overexpression) of multiple genes. In a specific embodiment, the one or more genes expressed by the vector include one, two or more antigen receptors. A variety of genes may include, but are not limited to, CAR, TCR, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, etc. The vector may further include: (1) one or more reporter molecules, such as fluorescent or enzymatic reporter molecules, such as for cell assays and animal imaging; (2) one or more cytokines or other signaling molecules; and / or (3) suicide genes.

[0444] In certain cases, the vector may include at least 4 cistrons separated by any kind of cleavage site (e.g., 2A cleavage site). The vector may or may not be based on Moloney murine leukemia virus (MoMLV or MMLV), including 3' and 5' LTR, with psi packaging sequence in the pUC19 backbone. The vector may include 4 or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene exchange. The system allows the combined overexpression of multiple genes (7 or more) flanked by restriction sites for rapid integration by subcloning, and in some embodiments, the system also includes at least three 2A self-cleavage sites. Therefore, the system allows the expression of a variety of CARs, TCRs, signaling molecules, cytokines, cytokine receptors and / or homing receptors. The system may also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV and non-viral plasmids.

[0445] The modular nature of this system also enables efficient subcloning of genes into each of the 4 cistrons in a polycistronic expression vector and exchange of genes, for example for rapid testing. Restriction sites strategically located in the polycistronic expression vector allow for efficient exchange of genes.

[0446] Embodiments of the present disclosure encompass systems utilizing multicistronic vectors, wherein at least a portion of the vector is modular, such as by allowing removal and replacement of one or more cistrons (or components of one or more cistrons), such as by utilizing one or more restriction enzyme sites, the nature and location of which are specifically selected to facilitate modular use of the vector. The vector also has embodiments in which multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby conferring the advantage of the vector to express separate gene products in substantially equimolar concentrations.

[0447] The vectors of the present disclosure are configured for modularity to be able to change one or more cistrons of the vector and / or change one or more components of one or more specific cistrons. The vectors can be designed to utilize unique restriction enzyme sites flanking the ends of one or more cistrons and / or flanking the ends of one or more components of a specific cistron.

[0448] Embodiments of the present disclosure include polycistronic vectors, which include at least two, at least three or at least four cistrons, each cistron flanked by one or more restriction enzyme sites, wherein at least one cistron encodes at least one antigen receptor. In some cases, two, three, four or more cistrons are translated into a single polypeptide and cut into separate polypeptides, while in other cases multiple cistrons are translated into a single polypeptide and cut into separate polypeptides. Adjacent cistrons on the vector can be separated by self-cleavage sites (e.g., 2A self-cleavage sites). In some cases, each cistron expresses a separate polypeptide from the vector. In particular cases, adjacent cistrons on the vector are separated by IRES elements.

[0449] In certain embodiments, the disclosure provides a system for cell engineering, which allows, for example, the combined expression of multiple cistrons that can include one, two or more antigen receptors, including overexpression. In a specific embodiment, the use of a polycistronic vector as described herein allows the vector to produce multiple gene products at equimolar levels from the same mRNA. Multiple genes may include, but are not limited to, CAR, TCR, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, etc. The vector may further include one or more fluorescent or enzymatic reporters, such as for cell determination and animal imaging. The vector may also include a suicide gene product for terminating the cells containing the vector when the cells containing the vector are no longer needed or the cells containing the vector are harmful to the host to which they have been provided.

[0450] In a specific embodiment of the present disclosure, at least one cistron on the vector comprises two or more modular components, wherein each modular component within the cistron is flanked by one or more restriction enzyme sites. For example, a cistron may include three, four, or five modular components. In at least some cases, a cistron encodes an antigen receptor having different portions of a receptor encoded by the corresponding modular components. The first modular component of a cistron may encode an antigen binding domain of a receptor. In addition, the second modular component of a cistron may encode a hinge region of a receptor. In addition, the third modular component of a cistron may encode a transmembrane domain of a receptor. In addition, the fourth modular component of a cistron may encode a first costimulatory domain. In addition, the fifth modular component of a cistron may encode a second costimulatory domain. In addition, the sixth modular component of a cistron may encode a signaling domain.

[0451] In a particular aspect of the present disclosure, two different cistrons on the vector each encode different antigen receptors. Both antigen receptors can be encoded by a cistron comprising two or more modular components, including a separate cistron comprising two or more modular components. For example, the antigen receptor can be a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0452] In a specific embodiment, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may comprise a Moloney murine leukemia virus (MMLV) 5'LTR, a 3'LTR, and / or a psi packaging element. In certain cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not comprise a pUC19 sequence. In some aspects of the vector, at least one cistron encodes a cytokine (e.g., interleukin 15 (IL-15), IL-7, IL-21, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0453] When a 2A cleavage site is used in a vector, the 2A cleavage site may comprise a P2A, T2A, E2A and / or F2A site.

[0454] In addition to a cistron encoding a CAR targeting CD70, any cistron of the vector may contain a suicide gene. Any cistron of the vector may encode a reporter gene. In a specific embodiment, the first cistron encodes a suicide gene, the second cistron encodes a CAR targeting CD70, the third cistron encodes a reporter gene, and the fourth cistron encodes a cytokine. In certain embodiments, the first cistron encodes a suicide gene, the second cistron encodes a CAR targeting CD70, the third cistron encodes a second CAR or another antigen receptor, and the fourth cistron encodes a cytokine. In a specific embodiment, the different parts of the CAR targeting CD70 and / or another receptor are encoded by corresponding modular components, and the first component of the second cistron encodes an antigen binding domain, the second component encodes a hinge and / or a transmembrane domain, the third component encodes a costimulatory domain, and the fourth component encodes a signaling domain.

[0455] In a specific embodiment, at least one of the cistrons encodes a suicide gene. In some embodiments, at least one of the cistrons encodes a cytokine. In certain embodiments, at least one of the cistrons encodes an antigen targeting CAR. A cistron may or may not encode a reporter gene. In certain embodiments, at least two cistrons encode two different antigen receptors (e.g., CAR and / or TCR). A cistron may or may not encode a reporter gene.

[0456] In a specific configuration of a genetic cargo of interest, a single vector may include a cistron encoding a CAR targeting an antigen and a cistron encoding a second antigen receptor different from an antigen targeting receptor. In a specific embodiment, the first antigen receptor encodes an antigen targeting CAR, and the second antigen receptor encodes a TCR, or vice versa. In a specific embodiment, the vector comprising a separate cistron encoding an antigen targeting CAR and a second antigen receptor, respectively, further comprises a third cistron encoding a cytokine or chemokine and a fourth cistron encoding a suicide gene. However, suicide genes and / or cytokines (or chemokines) may not be present on the vector.

[0457] In a particular embodiment, at least one cistron itself includes modular multiple components. For example, a cistron can encode a multi-component gene product, such as an antigen receptor with multiple parts; in particular cases, the antigen receptor is encoded by a single cistron, thereby ultimately producing a single polypeptide. The cistron encoding multiple components can have multiple components separated by 1, 2, 3, 4, 5 or more restriction enzyme digestion sites (including 1, 2, 3, 4, 5 or more restriction enzyme digestion sites unique to the vector containing the cistron). In a specific embodiment, the cistron with multiple components encodes an antigen receptor with multiple corresponding parts (each giving a unique function to the receptor). In a specific embodiment, each or most of the components of the multi-component cistron are separated by one or more restriction endonuclease digestion sites unique to the vector, thereby allowing the interchangeability of the separated components when necessary.

[0458] In a specific embodiment, each component of the multi-component cistron corresponds to a different part of the encoded antigen receptor, such as an antigen-targeted CAR. In an illustrative embodiment, component 1 can encode the antigen binding domain of the receptor; component 2 can encode the hinge domain of the receptor; component 3 can encode the transmembrane domain of the receptor; component 4 can encode the costimulatory domain of the receptor, and component 5 can encode the signaling domain of the receptor. In a specific embodiment, the antigen-targeted CAR can include one or more costimulatory domains, each separated by a unique restriction enzyme digestion site for interchangeability of the costimulatory domains within the receptor.

[0459] In a specific embodiment, there is a polycistronic vector with four separate cistrons, wherein adjacent cistrons are separated by a 2A cleavage site, although in a specific embodiment, there is an element that directly or indirectly causes the production of separate polypeptides from the cistron (e.g., an IRES sequence) in place of the 2A cleavage site. For example, the four separate cistrons may be separated by three 2A peptide cleavage sites, and each cistron has restriction sites (X1, X2, etc.) flanking each end of the cistron to allow for interchangeability of a particular cistron, such as interchangeability with another cistron or other type of sequence, and after using standard recombination techniques. In a specific embodiment, the restriction enzyme sites flanking each cistron are unique to the vector to allow for easy recombination, although in alternative embodiments the restriction enzyme sites are not unique to the vector.

[0460] In certain embodiments, the vector provides a unique second level of modularity by allowing interchangeability within a particular cistron, including within multiple components of a particular cistron. Multiple components of a particular cistron can be separated by one or more restriction enzyme sites, including those unique to the vector, to allow interchangeability of one or more components within the cistron. As an example, cistron 2 can include five separate components, although each cistron can have 2, 3, 4, 5, 6 or more components. For example, a vector can include cistron 2, which has five components, each of which is separated by unique restriction enzyme sites X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X52, X53, X54, X55, X56, X57, X58, X59, X60, X61, X62, X71, X72, X73, X74, X75 10 , X 11 , X 12 , X 13 and X 14 Spaced to allow standard recombination to exchange different components 1, 2, 3, 4 and / or 5. In some cases, there may be multiple restriction sites between different components (which are unique, although alternatively one or more are not unique) and there may be sequences between multiple restriction sites (although alternatively there may be none). In certain embodiments, all components encoded by a cistron are designed for interchangeable purposes. In certain cases, one or more components of a cistron are designed to be interchangeable, while one or more other components of a cistron may not be designed to be interchangeable.

[0461] In a specific embodiment, the cistron encodes an antigen-targeted CAR molecule with multiple components. For example, cistron 2 can be composed of a sequence encoding an antigen-targeted CAR molecule having a separate component represented by component 1, component 2, component 3, etc. The CAR molecule may include 2, 3, 4, 5, 6, 7, 8 or more interchangeable components. In a specific example, component 1 encodes scFv; component 2 encodes hinge; component 3 encodes transmembrane domain; component 4 encodes costimulatory domain (although there may also be a component 4′ encoding a second or more costimulatory domain with restriction sites on both sides for exchange); component 5 encodes signaling domain. In a specific example, component 1 encodes scFv; component 2 encodes IgG1 hinge and / or transmembrane domain; component 3 encodes CD28; and component 4 encodes CD3ζ.

[0462] Those skilled in the art recognize that in the design of the vector, the various cistrons and components must be configured so that they are held in frame where necessary.

[0463] In a specific example, cistron 1 encodes a suicide gene; cistron 2 encodes an antigen-targeting CAR; cistron 3 encodes a reporter gene; cistron 4 encodes a cytokine; component 1 of cistron 2 encodes a scFv; component 2 of cistron 2 encodes an IgG1 hinge; component 3 of cistron 2 encodes CD28; and component 4 encodes CD3ζ.

[0464] Restriction enzyme sites can be of any kind and can include any number of bases in their recognition sites, such as 4-8 bases; the number of bases in the recognition site can be at least 4, 5, 6, 7, 8 or more. The site may produce a blunt cut or sticky end when cut. For example, the restriction enzyme can be type I, type II, type III or type IV. Restriction enzyme sites can be obtained from available databases, such as the Integrated Relational Enzyme Database (IntEnz) or BRENDA (Integrated Enzyme Information System).

[0465] Exemplary vectors may be circular, and by convention, with position 1 (12 o'clock at the top of the circle, with the rest of the sequence going clockwise) being placed at the beginning of the 5' LTR.

[0466] In embodiments using a self-cleaving 2A peptide, the 2A peptide can be a viral oligopeptide 18-22 amino acids (aa) long that mediates "cleavage" of polypeptides during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viral 2As are usually named after the viruses from which they originated. The first 2A discovered was F2A (foot-and-mouth disease virus), followed by E2A (equine rhinitis A virus), P2A (porcine Teschovirus-1 2A), and T2A (thosea asigna virus 2A). The mechanism of 2A-mediated "self-cleavage" was found to be that the ribosome skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0467] In certain cases, the vector can be a γ-retroviral transfer vector. The retroviral transfer vector can include a plasmid-based backbone, such as a pUC19 plasmid (a large fragment (2.63 kb) between HindIII and EcoRI restriction enzyme sites). The backbone may carry viral components from Moloney murine leukemia virus (MoMLV), including 5'LTR, psi packaging sequence, and 3'LTR. LTR is a long terminal repeat found on either side of the retroviral provirus, and in the case of a transfer vector, the genetic cargo of interest (e.g., antigen-targeted CAR and related components) is encapsulated. The psi packaging sequence (which is a target site for packaging by nucleocapsid) is also incorporated in cis, sandwiched between the 5'LTR and CAR coding sequences. Therefore, the basic structure of an example of a transfer vector can be configured as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence. The system can also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0468] A. Pharmaceutical Composition

[0469] Also provided are pharmaceutical compositions and formulations comprising transduced NK cells and a pharmaceutically acceptable carrier.The transduced cells can be contained in a culture medium suitable for transfer to an individual and / or a culture medium suitable for storage (e.g., cryopreservation, including prior to transfer to an individual).

[0470] The pharmaceutical compositions and formulations described herein can be prepared in the form of lyophilized formulations or aqueous solutions by mixing the active ingredients (e.g., cells) having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) (e.g., hydroxyethyl) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX ® , Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglucanases (e.g., chondroitinases).

[0471] B. Combination therapy

[0472] In certain embodiments, the compositions and methods of the present embodiment relate to immune cell populations (including NK cell populations) combined with at least one additional therapy. Additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination of the foregoing. Additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0473] In some embodiments, additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, additional therapy is the administration of side effect limiters (e.g., agents intended to reduce the occurrence and / or severity of the side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation. In some embodiments, additional therapy is targeted PBK / AKT / mTOR pathway therapy, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors and / or chemopreventive agents. Additional therapy can be one or more chemotherapeutic agents known in the art.

[0474] Immune cell therapy can be administered before, during, after or in various combinations relative to other cancer therapies (such as immune checkpoint therapy).The administration interval can be from simultaneously to several minutes to several days to several weeks.In the embodiment in which immune cell therapy is provided to the patient separately from other therapeutic agents, it is generally ensured that there is no significant time period between the time of each delivery, so that the two compounds will still be able to play a combined effect that is beneficial to the patient.In such a case, it is considered that antibody therapy and anticancer therapy can be provided to patients within about 12 to 24 or 72 hours each other, more specifically within about 6-12 hours each other.In some cases, it may be desirable to significantly extend the treatment time, wherein a few days (2,3,4,5,6 or 7) to several weeks (1,2,3,4,5,6,7 or 8) pass between each administration.

[0475] Various combinations can be used. For the following example, immune cell therapy is "A", and anti-cancer therapy is "B":

[0476] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0477] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0478] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0479] Administration of any compound or cell therapy of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agents. Thus, in some embodiments, there is a step of monitoring toxicity due to the combination therapy.

[0480] 1. Chemotherapy

[0481] According to embodiments of the present invention, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to represent a compound or composition used in the treatment of cancer. These agents or drugs are classified according to their intracellular activity (e.g., whether they affect the cell cycle and at what stage the cell cycle is affected). Alternatively, agents can be characterized based on their ability to directly crosslink DNA, insert DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0482] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, and melamine. , trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adolesin derivatives); zelesin, carzelesin and bizelesin synthetic analogs); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthalene mustard, and chloramphenicol. aphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarcinogen chromophores and related chromoprotein enediyne antibiotics. Chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as leucovorin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine;Pyrimidine analogs such as cyclocytidine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; adrenal antistagnation drugs such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; androgens such as oxadiazine, dapoxetine, dapoxetine, and dapoxetine. glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazines; methylbenzylhydrazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, mucin A, baculosporin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman;gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. ;

[0483] 2. Radiation therapy

[0484] Other factors that cause DNA damage and have been widely used include the directional delivery of commonly known gamma rays, X-rays and / or radioisotopes to tumor cells. Other forms of DNA damaging factors may also be considered, such as microwaves, proton beam irradiation (USPs 5,760,395 and 4,870,287) and UV irradiation. Most likely, all of these factors affect extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dosage range of X-rays ranges from daily doses of 50 to 200 roentgens for a long time (3-4 weeks) to single doses of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the irradiation emitted, and the absorption of tumor cells.

[0485] 3. Immunotherapy

[0486] Those skilled in the art will appreciate that other immunotherapies may be combined or used in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN ® ) is such an example. Immune effectors can be, for example, antibodies that are specific for certain markers on the surface of tumor cells. A single antibody can serve as an effector of therapy, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used as targeting agents. Alternatively, effectors can be lymphocytes that carry surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0487] Antibody-drug conjugates have become a breakthrough approach to developing cancer therapies. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cytocidal drug. This approach combines the high specificity of MAbs for their antigenic targets with highly effective cytotoxic drugs, resulting in "armed" MAbs that deliver payloads (drugs) to tumor cells with abundant antigen levels. Targeted delivery of drugs also minimizes their exposure to normal tissues, thereby reducing toxicity and improving the therapeutic index. Two ADC drugs approved by the FDA (ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (Trastuzumab Emtansine or T-DM1) in 2013) validate this approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (see, e.g., Leal et al., 2014). As antibody engineering and linker-payload optimization become more sophisticated, the discovery and development of new ADCs increasingly relies on the identification and validation of novel targets amenable to this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high-level expression in tumor cells and robust internalization.

[0488] In one aspect of immunotherapy, tumor cells must carry some markers that are easy to target, i.e., they are not present on most other cells. There are many tumor markers, and in the context of embodiments of the present invention, any of these tumor markers may be suitable for targeting. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigens, MucA, MucB, PLAP, laminin receptor, erbB and p155. An optional aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand.

[0489] Examples of immunotherapies currently under investigation or in use are immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998); , 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

[0490] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints turn up signals (e.g., co-stimulatory molecules) or turn down signals. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0491] Immune checkpoint inhibitors can be drugs, such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Patent Publication No. WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, in particular chimeric, humanized or human forms of antibodies may be used. As will be known to the skilled artisan, alternative and / or equivalent names may be used for certain antibodies mentioned in the present disclosure. In the context of the present disclosure, such alternative and / or equivalent names are interchangeable. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0492] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509 and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, for example, as described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0493] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO ® ) is an anti-PD-1 antibody described in WO2006 / 121168, pembrolizumab (also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA ® and SCH-900475) is an anti-PD-1 antibody described in WO2009 / 114335. CT-011 (also known as hBAT or hBAT-1) is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 (also known as B7-DCIg) is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0494] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also referred to as CD152. The Genbank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is found on the surface of T cells, and when combined with CD80 or CD86 on the surface of antigen presenting cells, it acts as a "close" switch. CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to T cell co-stimulatory protein CD28, and both molecules are combined with CD80 and CD86 (also referred to as B7-1 and B7-2, respectively) on antigen presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells, and may be important for their function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4 (inhibitory receptors for B7 molecules).

[0495] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0496] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, disclosed in US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly known as tesimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No.2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 of the anti-CTLA-4 antibodies can be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; all are incorporated herein by reference.

[0497] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101 and YERVOY®) or an antigen-binding fragment and variant thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the above-mentioned antibodies and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95% or 99% variable region identity with ipilimumab).

[0498] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as described in U.S. Pat. Nos. 5,844,905, UA5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins, such as described in U.S. Pat. No. 8,329,867, incorporated herein by reference.

[0499] 4. Surgery

[0500] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection (in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed) and may be used in conjunction with other therapies (e.g., treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy). Tumor resection refers to the removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0501] After removing part or all of the cancer cells, tissues or tumors, a cavity may be formed in the body. Treatment may be accomplished by perfusing, directly injecting or topically applying other anti-cancer therapies to the area. For example, this treatment may be repeated every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also have different dosages.

[0502] 5. Other medicines

[0503] It is contemplated that other agents may be used in combination with certain aspects of this embodiment to improve the efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducing agents, or other biological agents. Increasing intercellular signal transduction by increasing the number of GAP connections will increase the anti-overproliferation effect on neighboring overproliferation cell groups. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-overproliferation efficacy of treatment. It is contemplated that inhibitors of cell adhesion can improve the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of overproliferation cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of the present embodiment of the invention to improve the efficacy of treatment.

[0504] IX. Kits of the Disclosure

[0505] Any composition described herein may be included in a kit. In a non-limiting example, cells containing GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 expression levels with reduction or inhibition, reagents for producing cells, vectors and reagents for producing vectors and / or its components may be included in a kit. In certain embodiments, NK cells may be included in a kit, and they may or may not have been modified in any way. Such a kit may or may not have one or more reagents for operating cells. For example, such reagents include small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts and / or combinations thereof. Nucleotides encoding CRISPR reagents, suicide gene products, receptors and / or cytokines for KOGPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER and / or CREB1 may be included in the kit. Proteins, such as cytokines or antibodies, including monoclonal antibodies, may be included in the kit. The kits may include nucleotides encoding components of an engineered CAR receptor or TCR receptor, including reagents for producing them.

[0506] In certain aspects, the kit includes the NK cell therapy of the present disclosure and another cancer therapy. In some cases, in addition to the cell therapy embodiment, the kit also includes a second cancer therapy, such as chemotherapy, hormone therapy and / or immunotherapy. The kit can be customized for an individual's specific cancer and includes a corresponding second cancer therapy for the individual.

[0507] The kit may include a composition of the present disclosure in appropriate equal parts. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. The container means of the kit will typically include at least one vial, test tube, flask, bottle, syringe or other container means, in which the components may be placed, and preferably appropriately aliquoted. In the case where there is more than one component in the kit, the kit may also typically include a second, third or other additional container, in which the additional components may be placed individually. However, a combination of various components may be included in a vial. The kit of the present invention typically also includes a device for containing the composition and any other reagent containers within a closed range for commercial sale. Such a container may include an injection or blow-molded plastic container, in which the required vial is maintained. X. Examples

[0508] The following examples are included to demonstrate certain non-limiting aspects of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the subsequent examples represent techniques that the inventors have found to work well in the practice of the disclosed subject matter. However, it will be appreciated by those skilled in the art based on the present disclosure that many changes can be made in the disclosed specific embodiments and still similar or similar results can be obtained without departing from the spirit and scope of the disclosed subject matter.

[0509] Example 1 - Proton-sensing GPRs and CREMs are highly expressed in tumor-infiltrating immune cells of glycolytic tumors

[0510] Single-cell RNA sequencing (scRNAseq) data from tumor samples from pancreatic cancer patients were interrogated for expression of genes encoding enzymes involved in glycolysis. The inventors determined that tumor cells had significant upregulation of glycolytic genes ( Figure 1 A). This reflects active aerobic and anaerobic glycolysis leading to accumulation of organic and inorganic acids in the tumor microenvironment (TME), as previously shown in the literature for multiple tumor types. Next, the expression of proton-sensing G protein-coupled receptors (GPRs) (e.g., GPR68 and GPR132) of the corresponding tumor-infiltrating NK cells, as well as downstream signaling mediators in the cAMP pathway in response to acidity (e.g., CREM and CREB1), was determined. Many of these genes were found to be significantly upregulated in NK cells within the TME compared to NK cells in peripheral blood ( Figure 1 B) Since CREM is a key transcription factor in the cAMP pathway, exhibits significant upregulation in NK cells in the TME, and is implicated in common downstream signaling mediating these proton-sensing GPRs, the CREM gene was identified as a potential target for gene modulation to potentially counteract acidity-mediated immunosuppression.

[0511] Expression of CREM on tumor-infiltrating immune cells in clear cell renal cell carcinoma (CC-RCC) was investigated due to the well-established "Warburg effect" and hyperglycolytic state of clear cell renal cell carcinoma tumors (see, e.g., Courtney K.D. et al., 2018; and because it is associated with von Hippel-Lindau (VHL) gene loss, which mechanistically drives the development of most CC-RCCs), and was confirmed by high expression of glycolytic enzymes ( Figure 2 A, from TCGA data). Public scRNAseq data from the TISCH database were used to compare the expression of CREM in renal cancer infiltrating immune cells relative to peripheral blood immune cells of healthy donors (see, e.g., Sun D. et al., 2020). Figure 2 As shown in B, CREM is significantly upregulated in various immune cells in the TME compared with its rare expression in immune cells in the peripheral blood of healthy donors.

[0512] Example 2 - Endonuclease-mediated knockout of endogenous genes

[0513] Guide RNAs (gRNAs) were designed that induce double-strand breaks in GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, or CREB1 with both high on-target activity scores and low off-target activity scores. Table 2 provides exemplary crispr RNA (crRNA) sequences suitable for incorporation into gRNAs.

[0515] Table 2 - Exemplary crRNA sequences

[0516] For each gene candidate for genetic targeting in immune effector cells, a set of crisprRNAs (crRNAs) (see Table 2) were designed, which were complexed with tracrRNA (a binding scaffold for the Cas nuclease) to form a guide RNA (gRNA).

[0517] Any one or more gene candidates for genetic targeting in immune effector cells are knocked out and / or otherwise mutated by one or more crRNAs that are complexed with tracrRNA to form guide RNAs.

[0518] Example 3 - CREM KO significantly improved the acidic environment and NK cell cytotoxicity in the TME

[0519] As described above, CREM was found to be consistently upregulated in TME-infiltrating immune cells in various glycolytic tumor models. Since CREM protein is a key transcription factor in the cAMP pathway, the inventors created a knockout (KO) CREM locus in NK cells. CREM locus KO was used as a proof of principle for targeting cAMP pathway activation in response to acidic conditions in an attempt to eliminate acid-mediated immunosuppression. Using the CRISPR-Cas9 system with SEQ ID NOs: 140 and 142, the CREM KO locus can be effectively and stably created in NK cells ( Figure 3 A and 3B). Figure 3 B shows selected CREM isoforms from bulk RNA sequencing data of CREM WT and CREM KO NK cells obtained from three umbilical cord blood donors (D1, D2, and D3). The heat map color scale represents the count of expressing cells from 0 (dark blue) to 400 (red); the FDR column corresponds to the false discovery rate (significance < 0.05); and the logPostFC column represents the logarithm of the fold change in WT versus KO expression levels, with positive values ​​indicating that RNA isoform expression is higher in CREM WT NK cells and negative values ​​indicating that RNA isoform expression is higher in CREM KO NK cells. RNA isoform expression analysis showed that multiple CREM isoforms were effectively knocked out and / or significantly knocked down in mutant NK cells. Examples of knocked out and / or significantly knocked down RNA isoforms include: ICER isoform (CREM-228), CREM-207, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, and CREM-217. RNA isoform CREM-218 was found to be upregulated in CREM KO NK cells relative to CREM WT NK cells.

[0520] To test the killing potential of CREM WT vs CREM KO CAR-NK cells, live cell imaging of tumor cell growth and NK cell killing was performed using the INCUCYTE® device, and a long-term NK cell killing assay was performed on the glycolytic UMRC3 renal cell carcinoma cell line. The results showed that the activity of CAR-NK cells after CREM KO was enhanced compared with wild-type CAR-NK cells targeting UMRC3 ( Figure 4 A and 4B). To investigate the effect of CREM KO on the anti-tumor function of NK cells under conditions that mimic in vivo solid tumor models, NK cell killing assays were performed on a 3D tumor spheroid culture model of the UMRC3 cell line. Tumor spheroids mimic solid tumor masses and have been shown in the literature to have an acidic pH (e.g., see Nunes et al., 2019). To perform these assays, GFP+UMRC3 cells were seeded in ultra-low attachment plates and 3D tumor spheroids were allowed to form in an INCUCYTE® machine for 48 hours before NK cells were added. Tumor growth and cell death were monitored in real time. The data showed that CREM KO CAR-NK cells had enhanced cytotoxicity against 3D tumor spheroids compared to CREMWT CAR-NK cells targeting UMRC3 ( Figure 4 C and 4D).

[0521] To test the specific effect of CREM KO on abrogating acid-mediated immunosuppression, chromium release killing assays were performed against the A498 cell line by CREM WT versus CREM KO NK cells at different effector:target (E:T) ratios under 2 pH conditions, typical culture medium pH (pH=7.2) and acidic pH (pH=5.9). For CREM WT NK cells, the data showed the expected attenuation of NK cell cytotoxicity at acidic pH ( Figure 5 A), but when killing assays were performed using CREM KO NK cells, cytotoxicity was restored ( Figure 5 B).

[0522] like Fig.10 As shown in AH, CREM KO renders CAR-NK cells resistant to the inhibitory effects of lactate at concentrations similar to those observed in the tumor microenvironment (TME). Fig.10 As shown in A, the results of 51Cr release assays show that in an acidic environment, the cytotoxicity of anti-CD70 CAR-NK (CAR.70) cells against renal cell carcinoma (e.g., UMRC3 renal cell carcinoma tumor cells) at different E:T ratios (e.g., 20:1, 10:1, 5:1, 2.5: and 1.25:1, respectively) is enhanced in CREM KO CAR-NK cells relative to CREM WT CAR-NK cells and is comparable to CREM WT CAR-NK cells at a more neutral pH level. NK cells were CREM wild-type (WT) or CREM knockout (KO) and were maintained at a normal pH (pH Reg ) or using lactic acid to generate an acidic pH (pH Lac ) and incubated in . Fig.10 B shows the pH Reg ) or acidic pH (pH Lac Representative bright field microscopy images of CAR-NK cells and UMRC3 tumor cells (elongated cells) co-cultured under pH 7. Cell clumps indicate areas of active contact between cytotoxic CAR-NK cells and tumor cells. The results showed that CREM KOCAR.70 NK cells showed higher levels of UMRC3 cell clearance compared to CREM WT CAR.70 NK cells. Reg or pH Lac Representative long-term cytotoxicity (e.g., Incucyte®) assays of CAR.70 NK cells and their non-transduced (NT) counterparts in the setting of CREM WT or CREM KO CAR.70 NK cells were analyzed for long-term cytotoxicity ( Fig.10 C) Acidic conditions (pH 5.4) were achieved over 2 weeks by a stepwise increase in cumulative lactate concentration (Inc). Lac20(Inc) ) (e.g., adding about 2.5 mM lactate every 2-3 days during a 2-week incubation period to gradually adapt NK cells to more acidic conditions; pH Lac20 (Inc) resulting in a pH of approximately 6.0; all conditions were set from the normal pH of the culture medium (pH Reg The results showed that, although chronic lactate exposure suppressed the cytotoxicity of CAR-NK cells compared with conditions at conventional pH, CREM KO CAR-NK cells were able to maintain robust anti-tumor killing despite this relatively extreme condition that mimics the low pH of the tumor microenvironment of glycolytic tumors. Fig.10 D indicates acidity (pH Lac20(Inc) Representative Incucyte® images of NT NK cells (NT), CAR.70 CREM WT NK cells (CAR.70), or CAR.70 CREM KO NK cells challenged with GFP+UMRC3 cells at an E:T ratio of 1:1 or 2:1 under 2 conditions.

[0523] After confirming the enhanced cytotoxicity of CREM KO CAR.70 NK cells relative to CREM WT CAR.70 NK cells when analyzed at acidic pH, phenotype analysis of NK cells was performed. Fig.10 E-10H shows the use of multiparameter spectral flow cytometry to characterize the pH Reg or pH Lac NK cells co-cultured with UMRC3 tumor cells under different conditions (e.g., CREM WT or CREM KO). ViSNE analysis was performed for each condition followed by FlowSOM clustering. Fig.10 E shows a colored FlowSOM cluster map showing composite metaclusters 1-4 for all 4 conditions tested. Fig.10 F shows the distribution of four FlowSOM metaclusters (MCs) under different test conditions. Lac MC2 (orange) increased in CREM WT CAR.70 NK cells co-cultured with UMRC3 cells at pH Lac CREM KO CAR.70 NK cells co-cultured with UMRC3 cells showed the same Reg The potential phenotypes associated with the meta-clusters were then analyzed. Fig.10 G shows the expression of various markers in 4 MCs, which are locally normalized to minimum and maximum expression under various conditions, with colors ranging from blue (minimum) to red (maximum). Percent expression is covered by circles of various sizes proportional to % expression. The general characteristics of MC2 are relatively low expression of activation markers and relatively high expression of certain checkpoints (such as TIGIT and LAG3) and terminal differentiation markers (such as CD57). Fig.10 H shows various test conditions (pH Reg or pH Lac and CREM WT or CREM KO), where the overall phenotypic clustering differences in NK cell distribution after viSNE analysis can be observed.

[0524] Example 4 - CREM KO enhances NK cell activation and multifunctionality

[0525] To further elucidate the effect of CREM KO on NK cell function, a co-culture assay of NK cells with the glycolytic ovarian cancer cell line SKOV3 was performed, and the expression of cytokines in NK cells, such as INF-γ and TNF-α, and the degranulation marker CD107a were measured. The results showed that CREM KO further enhanced the ability of CAR-NK cells targeting SKOV3 to produce effective cytokines and express NK cell activation markers ( Figure 6 A and 6B). In addition, in a larger scale multifunctional assay, a LUMINEX® assay was used to measure multiple cytokines as markers of NK cell activation. Again, the results showed an overall increase in cytokine secretion levels by CREM KO CAR-NK cells compared to CREM WT CAR-NK cells ( Figure 6 C). The expression of activation, inhibition, and cytotoxic markers of various NK cell genotypes was analyzed by mass cytometry. The data showed that CREM KO CAR-NK cells had an immunophenotype consistent with activated and cytotoxic cells ( Figure 7 ).

[0526] Example 5 - CREM KO confers a proliferation advantage to NK cells but does not result in autonomous growth

[0527] In the above studies involving CREM KO NK cells, an increase in the proliferation rate of CREM KO NK cells relative to their CREM WT counterparts was observed. To assess the effect of CREM KO on NK cell proliferation while ensuring that it did not result in autonomous NK cell growth, the growth of NK cells with or without CREM KO was measured in the absence of IL-2 stimulation, which is typically an essential cytokine for NK cell survival in vitro. While enhanced proliferation of CREMWT CAR-NK cells compared to non-transduced (NT) NK cells was observed, which could be explained by the CAR construct also secreting IL-15, the results indicate that CREMKO CAR-NK cells proliferated more and survived longer in the absence of additional IL-2. However, no autonomous growth over time was observed ( Figure 8 ).

[0528] Example 6 - CREM KO confers improved metabolic function to NK cells

[0529] Since the above results show that the activity of NK cells in TME and acidic environment is enhanced after CREM KO, the inventors speculate that CREM KO enhances NK cells through metabolic reprogramming, enabling them to compete for metabolites in TME and resist immunosuppression produced by highly metabolically active tumors. To verify this hypothesis, the glycolytic capacity of CREM KO cells was determined by measuring the extracellular acidification rate (ECAR) and the oxidative phosphorylation rate through the oxygen consumption rate (OCR). The results showed that compared with CREM WT CAR-NK cells, CREM KO CAR-NK cells have higher glycolytic capacity ( Fig. 9 A) and better OCR ( Fig. 9 B) These results suggest that CREM KO can reprogram NK cells’ metabolic program, making them better suited to resist the negative effects of acidity in the TME.

[0530] The above results were confirmed by Fig.13 AC, where CREM KO was found to enhance the metabolic fitness of NK cells at baseline as well as when cultured at acidic pH. Fig.13 A depicts the extracellular acidification rate (ECAR; as a substitute for glycolysis, upper figure) of CREM WT or CREM KO anti-CD70 CAR-NK cells (CAR.70) and their non-transduced (NT) counterparts. The CAR.70 construct is also designed to secrete interleukin 15 (IL15), and control NK cells transduced with constructs expressing IL15 in the absence of CAR are included in the assay (IL15NK cells). According to the manufacturer's protocol, the Seahorse® sugar stress test assay was used to assay NK cells cultured in conventional culture medium and not stimulated. According to the manufacturer's protocol, the Seahorse® mitochondrial stress test assay was used to analyze oxygen consumption rate (OCR; as a substitute for oxidative phosphorylation (OXPHOS), lower figure). Fig.13 B provides the ECAR (top) and OCR (bottom) results of NT, CREM WT CAR-NK cells, and CREM KO CAR-NK cells incubated in lactate. Fig.13 C shows that both glycolysis and OXPHOS pathways are significantly upregulated in CREM KO NK cells compared to CREM WT NK cells following incubation with lactate (unbiased GSEA analysis using bulk RNA sequencing of these conditions).

[0531] Together, these results demonstrate that CREM KO can rewire the metabolic program of NK cells and render them better suited to resist the negative effects caused by acidity in the TME.

[0532] Example 7 - CREM KO improves CAR-NK cell activation and cytotoxicity in vitro and / or in vivo

[0533] like Fig.11 As shown in AF, the inventors demonstrated that CREM KO improved CAR-NK cell activation phenotype and cytotoxicity, including in long-term assays involving multiple tumor re-challenges. Fig.11 A shows the results of a representative re-challenge cytotoxicity assay of xCELLigence® against UMRC3 tumor cells using CREM WT and CREM KO anti-CD70 CAR-NK cells (CAR.70) and their non-transduced (NT) counterparts. UMRC3 cells were seeded into E-plates and allowed to adhere and grow overnight. Twenty-four hours later, the above NK cells were added at an E:T ratio of 2:1. Every 2-4 days, new tumor cells were seeded into new E-plates. Twenty-four hours later, the NK cells in the first E-plate were collected and counted and transferred to a new tumor plate at the same E:T ratio of 2:1. The results showed that CAR.70 NK cells had a strong cytolytic effect on UMRC3 cells during the first attack, and CREM KO CAR.70 NK cells showed increased cytolysis of UMRC3 cells during the first and second tumor cell re-challenge compared to CREM WT CAR.70 NK cells. CREM WT and CREM KO CAR.70 NK cells were then phenotypically characterized at the end of the re-challenge assay as shown in (11A). ViSNE analysis was performed for each condition and then clustered by FlowSOM into 5 FlowSOM groups (C1-C5), as shown in Fig.11 B. Then analyze Fig.11 The distribution of the five FlowSOM metaclusters (MC, C1-C5) of the cells described in A-11B is shown in Fig.11 C. The results showed that MC5 was increased, while MC4 was decreased in CREM KO CAR.70 NK cells compared with CREM WT cells. Fig.11 D provides the underlying profile tSNE plots of CREM WT CAR.70 NK cells (top) and CREM KO CAR.70 NK cells (bottom) and shows the overall phenotypic clustering differences in their distribution after viSNE analysis. Fig.11 The relative abundance of some underlying cell markers contributing to the observed phenotypic clustering is determined and provided in E, which shows the expression of various markers in 5 MCs (C1-C5) locally normalized to the minimum and maximum expression under various conditions on a color scale of blue (minimum) to orange (maximum). Percent expression is covered by circles of various sizes proportional to % expression. The results show that MC4 (which is enriched in CREM WT cells relative to CREM KO cells) is generally characterized by relatively low expression levels of activation markers, while MC5 (which is enriched in CREM KO cells relative to CREM WT cells) is characterized by the opposite pattern of overall increase of activation markers. The selected markers of interest are then quantified at the single cell level. Fig.11 F shows the expression of selected markers (ICOS, DNAM1, CD16, OX40, TIGIT, and TIM3) at the single cell level when comparing between CREM WT CAR.70 NK cells and CREM KO CAR.70 NK cells in MC4 and MC5. The results show that there are significant differences in the expression of markers in CREM KO CAR.70 NK cells compared to control CREM WT CAR.70 NK cells. These results confirm that when cultured with target tumor cells, CREM KO NK cells display different phenotypic characteristics compared to CREM WT NK cells, representing a higher level of activation.

[0534] like Fig.12 As shown in Figures 5A and 5C, the inventors subsequently demonstrated that CREM KO enhanced the cytotoxicity of CAR-NK cells in multiple different tumor models and using multiple different CAR constructs, while targeting multiple different tumor antigens in an in vitro 3D tumor spher...

Claims

1. An engineered immune effector cell, wherein the cell comprises one or more engineered mutations in the cell's endogenous cAMP response element modulator (CREM), G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes.

2. The cell of claim 1, wherein the mutation is a partial or complete loss of function and / or a knockout (KO) mutation.

3. The cell of claim 1 or 2, wherein the mutation reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

4. The cell of claim 1, wherein the mutation is a neomorphic mutation or a gain-of-function mutation.

5. The cell of claim 1 or 4, wherein the mutation increases transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

6. The cell of any one of claims 1-5, wherein the mutation results in a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative population of mRNAs encoded by a non-mutated locus encoding the same endogenous gene.

7. The cell of any one of claims 1-4, wherein the mutation results in a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative population of proteins encoded by a non-mutated locus encoding the same endogenous gene.

8. The cell of any one of claims 1-7, wherein the mutation comprises a knockout (KO) mutation in an endogenous gene of the cell.

9. The cell of any one of claims 1-7, wherein the mutation comprises a homozygous mutation in an endogenous gene of the cell.

10. The cell of any one of claims 1-7, wherein the mutation comprises a heterozygous mutation in an endogenous gene of the cell.

11. The cell of any one of claims 1-10, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment and / or a tumor microenvironment (TME) relative to a control non-engineered cell.

12. The cell of any one of claims 1-11, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment at a pH of less than or equal to about 7.0 relative to a control non-engineered cell.

13. The cell of any one of claims 1-12, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment of pH less than or equal to about 5.9 relative to a control non-engineered cell.

14. The cell of any one of claims 1-13, wherein the mutation results in improved cytotoxicity of the engineered cell, optionally in an acidic microenvironment characterized by increased lactate levels relative to a non-acidic microenvironment.

15. The cell of any one of claims 1-14, wherein the mutation results in enhanced multifunctionality of the engineered cell in response to stimulation by a tumor cell relative to a control non-engineered cell.

16. The cell of claim 15, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation by tumor cells.

17. The cell of claim 16, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or the degranulation marker CD107a in response to stimulation of tumor cells.

18. The cell of claim 15 or 16, wherein the increased cytokine release comprises granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin increased in response to stimulation of tumor cells.

19. The cell of any one of claims 1-18, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxicity phenotype relative to a control non-engineered cell.

20. The cell of any one of claims 1-19, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxic phenotype relative to a control non-engineered cell, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or estrogen early response.

21. The cell of any one of claims 1-20, wherein the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

22. The cell of any one of claims 1-21, wherein the mutation provides the engineered cell with enhanced proliferation capacity and / or persistence phenotype relative to a control non-engineered cell.

23. The cell of claim 22, wherein the enhanced proliferation capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

24. The cell of claim 22 or 23, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

25. The cell of any one of claims 1-24, wherein the mutation provides the engineered cell with an enhanced metabolic fitness phenotype relative to a control non-engineered cell.

26. The cell of claim 25, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

27. The cell of any one of claims 1-26, wherein the mutation provides increased chromatin accessibility throughout the genome when the cell is contained in an acidic microenvironment.

28. The cell of any one of claims 1-27, wherein the endogenous gene is CREM.

29. The cell of claim 28, wherein the CREM mutation results in reduced expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

30. The cell of claim 28 or 29, wherein the CREM mutation results in increased expression of the CREM RNA isoform CREM-218.

31. The cell of any one of claims 28-30, wherein the CREM mutation is the result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO:

142.

32. The cell of any one of claims 28-31, wherein the CREM mutation results in a greater than 60% reduction in one or more CREM protein isoforms.

33. The cell of any one of claims 28-32, wherein the CREM mutation results in a greater than 80% reduction in one or more CREM protein isoforms.

34. The cell of any one of claims 1-33, wherein the cell has been further adapted to an acidic microenvironment by contacting the cell with an acidic stimulus ex vivo.

35. The cell of claim 34, wherein the acidic stimulator is provided at a concentration greater than or equal to about 2 to 3 mM, optionally greater than or equal to about 2.5 mM.

36. The cell of any one of claims 34-35, wherein adaptation to an acidic microenvironment is achieved by incremental and / or cumulative contact with an acidic stimulus.

37. The cell of any one of claims 34-36, wherein the cell is adapted over a period of at least about 10 to 18 days, optionally at least about 14 days.

38. The cell of claim 36 or 37, wherein cell adaptation comprises adding an acidic stimulus about every 48-72 hours, optionally about every 48 hours.

39. The cell of any one of claims 34-37, wherein the acidic stimulus comprises or consists essentially of lactic acid.

40. The cell of any one of claims 34-39, wherein the cell is adapted to an acidic microenvironment of less than or equal to about pH 6.

0.

41. The cell of any one of claims 1-40, wherein the cell is a T cell, a natural killer (NK) cell, an NK T cell, a macrophage, a B cell, an invariant NKT cell, a γδ T cell, a MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

42. The cell of any one of claims 1-41, wherein the cell is a NK cell derived from cord blood (CB), peripheral blood (PB), NK cell line, bone marrow, stem cells, or a mixture thereof.

43. The cell of claim 41 or 42, wherein the NK cell is derived from umbilical cord blood.

44. The cell of any one of claims 1-43, wherein the cell comprises one or more engineered receptors.

45. The cell of claim 44, wherein the one or more engineered receptors comprise an engineered antigen receptor that specifically targets an antigen.

46. ​​The cell of claim 45, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

47. The cell of claim 46, wherein the engineered antigen receptor is a CAR.

48. The cell of any one of claims 45-47, wherein the antigen is a cancer antigen.

49. The cell of any one of claims 45-48, wherein the antigen is a solid tumor antigen.

50. The cell of any one of claims 45-49, wherein the antigen is selected from 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, F BP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TROP2, TEM, HMW-MAA, VEGFR2 and combinations thereof.

51. The cell of any one of claims 45-50, wherein the antigen comprises TROP2 and / or CD70.

52. The cell of any one of claims 44-51, wherein the one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

53. The cell of any one of claims 1-52, wherein the cell comprises expression of one or more exogenous chemokines and / or one or more cytokines.

54. The cell of claim 53, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof.

55. The cell of claim 54, wherein the cytokine is IL-15.

56. The cell of any one of claims 1-55, wherein the cell comprises a suicide gene.

57. The cell of any one of claims 1-56, wherein the endogenous gene is mutated due to homologous recombination or non-homologous recombination.

58. The cell of any one of claims 1-57, wherein the endogenous gene is mutated by an endonuclease.

59. The cell of claim 58, wherein the endonuclease is an RNA-guided endonuclease.

60. The cell of claim 59, wherein the RNA-guided endonuclease is CRISPR-Cas9.

61. The cell of any one of claims 1-60, wherein the cell comprises one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR and CD7.

62. A cell population, wherein the cells are the cells according to any one of claims 1-61.

63. The cell population of claim 62, wherein the cell population is contained in a pharmaceutically acceptable excipient.

64. A method of treating cancer in an individual in need thereof, comprising the step of administering to the individual a therapeutically effective amount of the cell population of claim 62 or 63.

65. The method of claim 64, wherein the cells are autologous, allogeneic, or xenogeneic to the individual.

66. The method of claim 64 or 65, wherein the cells are allogeneic to the individual.

67. The method of any one of claims 64-66, wherein the cancer comprises a solid tumor.

68. The method of any one of claims 64-66, wherein the cancer does not comprise a solid tumor.

69. The method of any one of claims 64-68, wherein the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer and / or cervical cancer.

70. The method of any one of claims 64-69, wherein the individual is a mammal.

71. The method of claim 70, wherein the subject is a human, dog, cat, horse, cow, sheep, pig, or rodent.

72. The method of claim 70 or 71, wherein the individual is a human.

73. The method of any one of claims 64-72, wherein the individual is administered an additional cancer therapy.

74. The method of claim 73, wherein the additional cancer therapy is surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

75. The method of any one of claims 64-74, further comprising the step of diagnosing cancer in the individual.

76. A method for engineering immune effector cells, comprising mutating the cell's endogenous cAMP response element modulator (CREM), G protein coupled receptor 4 (GPR4), G protein coupled receptor 31 (GPR31), G protein coupled receptor 68 (GPR68), G protein coupled receptor 81 (GPR81), G protein coupled receptor 132 (GPR132), G protein coupled receptor 151 (GPR151), inducible cAMP early repressor (ICER) and / or cyclic AMP response element binding protein 1 (CREB1) genes.

77. The method of claim 76, wherein the mutation produces a partial or complete loss of function and / or a knockout (KO) mutation.

78. The method of claim 76 or 77, wherein the mutation reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

79. The method of claim 76, wherein the mutation produces a neomorphic mutation or a gain-of-function mutation.

80. The method of claim 76 or 79, wherein the mutation increases transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene relative to a non-mutated locus encoding the same endogenous gene.

81. The method of any one of claims 76-80, wherein the mutation produces a population of modified mRNA isoforms encoded by the mutated endogenous gene relative to a representative population of mRNAs encoded by a non-mutated locus encoding the same endogenous gene.

82. The method of any one of claims 76-79, wherein the mutation produces a population of modified protein isoforms encoded by the mutated endogenous gene relative to a representative population of proteins encoded by a non-mutated locus encoding the same endogenous gene.

83. The method of any one of claims 76-82, wherein the mutation produces a knockout (KO) mutation in an endogenous gene of the cell.

84. The method of any one of claims 76-82, wherein the mutation produces a homozygous mutation in an endogenous gene of the cell.

85. The method of any one of claims 76-82, wherein the mutation produces a heterozygous mutation in an endogenous gene of the cell.

86. The method of any one of claims 76-85, wherein the mutation results in improved cytotoxicity of the engineered cells in an acidic microenvironment and / or a tumor microenvironment (TME) relative to control non-engineered cells.

87. The method of any one of claims 76-86, wherein the mutation results in improved cytotoxicity of the engineered cells in an acidic microenvironment at a pH of less than or equal to about 7.0 relative to a control non-engineered cell.

88. The method of any one of claims 76-87, wherein the mutation results in improved cytotoxicity of the engineered cells relative to control non-engineered cells in an acidic microenvironment at a pH of less than or equal to about 5.

9.

89. The method of any one of claims 76-88, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment characterized by increased lactate levels relative to a non-acidic microenvironment.

90. The method of any one of claims 76-89, wherein the mutations result in enhanced pluripotency of the engineered cells in response to stimulation by tumor cells relative to control non-engineered cells.

91. The method of claim 90, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation of the tumor cells.

92. The method of claim 91, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or the degranulation marker CD107a in response to stimulation of tumor cells.

93. The method of claim 90 or 91, wherein the increased cytokine release comprises granulocyte-macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin increased in response to stimulation of tumor cells.

94. The method of any one of claims 76-93, wherein the mutations produce an enhanced activation and / or cytotoxicity phenotype in the engineered cells relative to control non-engineered cells.

95. The method of any one of claims 76-93, wherein the mutation causes the engineered cells to have an enhanced activation and / or cytotoxic phenotype relative to control non-engineered cells, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or estrogen early response.

96. The method of any one of claims 76-95, wherein the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

97. The method of any one of claims 76-96, wherein the mutations produce an enhanced proliferation capacity and / or persistence phenotype for the engineered cells relative to control non-engineered cells.

98. The method of claim 97, wherein the enhanced proliferative capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

99. The method of claim 97 or 98, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

100. The method of any one of claims 76-99, wherein the mutation produces an enhanced metabolic fitness phenotype for the engineered cell relative to a control non-engineered cell.

101. The method of claim 100, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

102. The method of any one of claims 76-101, wherein the mutation provides increased chromatin accessibility throughout the genome when the cell is contained in an acidic microenvironment.

103. The method of any one of claims 76-102, wherein the endogenous gene is CREM.

104. The method of claim 103, wherein the mutation of CREM results in reduced expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

105. The method of claim 103 or 104, wherein the mutation in CREM results in increased expression of the CREM RNA isoform CREM-218.

106. The method of any one of claims 103-105, wherein the mutation of CREM comprises exposing the cell to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO:

142.

107. The method of any one of claims 103-106, wherein the mutation of CREM results in a greater than 60% reduction in one or more CREM protein isoforms.

108. The method of any one of claims 103-107, wherein the mutation of CREM results in a greater than 80% reduction in one or more CREM protein isoforms.

109. The method of any one of claims 76-108, further comprising adapting the cells to an acidic microenvironment by contacting the cells with an acidic stimulus ex vivo.

110. The method of claim 109, comprising providing the acidic stimulus at a concentration greater than or equal to about 2 to 3 mM, optionally greater than or equal to about 2.5 mM.

111. The method of claim 109 or 110, wherein adaptation to an acidic microenvironment is achieved by incremental and / or cumulative contact with an acidic stimulus.

112. The method of any one of claims 109-111, wherein the adapting continues for a period of at least about 10 to 18 days, optionally at least about 14 days.

113. The method of any one of claims 109-112, wherein the adapting comprises adding an acidic stimulus about every 48-72 hours, optionally about every 48 hours.

114. The method of any one of claims 109-113, wherein the acidic stimulus comprises or consists essentially of lactic acid.

115. The method of any one of claims 109-114, wherein the adaptation is to an acidic microenvironment of less than or equal to about pH 6.

0.

116. The method of any one of claims 76-115, wherein the cell is a T cell, a natural killer (NK) cell, a NKT cell, a macrophage, a B cell, an invariant NK T cell, a γδ T cell, a MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

117. The method of any one of claims 76-116, wherein the cell is a NK cell derived from cord blood (CB), peripheral blood (PB), NK cell line, bone marrow, stem cells, or a mixture thereof.

118. The method of claim 116 or 117, wherein the NK cells are derived from umbilical cord blood.

119. The method of any one of claims 76-118, wherein the cells comprise one or more engineered receptors that specifically bind an antigen.

120. The method of claim 119, wherein the one or more engineered receptors comprise an engineered antigen receptor.

121. The method of claim 120, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

122. The method of claim 121, wherein the engineered antigen receptor is a CAR.

123. The method of any one of claims 120-122, wherein the antigen is a cancer antigen.

124. The method of any one of claims 120-123, wherein the antigen is a solid tumor antigen.

125. The method of any one of claims 120-124, wherein the antigen is selected from 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, F BP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TROP2, TEM, HMW-MAA, VEGFR2 and combinations thereof.

126. The method of any one of claims 120-125, wherein the antigen comprises TROP2 and / or CD70.

127. The method of any one of claims 119-126, wherein the one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

128. The method of any one of claims 76-127, wherein the cells comprise expression of one or more exogenous chemokines and / or one or more cytokines.

129. The method of claim 128, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof.

130. The method of claim 129, wherein the cytokine is IL-15.

131. The method of any one of claims 76-130, wherein the cell comprises a suicide gene.

132. The method of any one of claims 76-131, wherein the mutation of the endogenous gene comprises homologous recombination or non-homologous recombination.

133. The method of any one of claims 76-132, wherein the mutation of the endogenous gene is mediated by an endonuclease.

134. The method of claim 133, wherein the endonuclease is an RNA-guided endonuclease.

135. The method of claim 134, wherein the RNA-guided endonuclease is CRISPR-Cas9.

136. The method of any one of claims 76-135, wherein the cells comprise one or more additional mutations in one or more genes, wherein the genes are selected from NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR, and CD7.

137. An engineered natural killer (NK) cell, wherein the cell comprises an engineered partial or complete loss-of-function and / or knockout (KO) mutation in the cell's endogenous cAMP response element modulator (CREM) gene.

138. The cell of claim 137, wherein the cell comprises a knockout (KO) mutation in the cell's endogenous CREM gene.

139. The cell of claim 138, wherein the cell comprises a homozygous knockout (KO) mutation in an endogenous CREM gene.

140. The cell of claim 138, wherein the cell comprises a heterozygous KO mutation in the cell's endogenous CREM gene.

141. The cell of claim 137, wherein the mutation results in improved cytotoxicity of the engineered cell relative to a reference cell lacking the mutation.

142. The cell of claim 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment and / or a tumor microenvironment (TME) relative to a control non-engineered cell.

143. The cell of claim 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment having a pH of less than or equal to about 7.0 relative to a control non-engineered cell.

144. The cell of claim 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment having a pH of less than or equal to about 5.9 relative to a control non-engineered cell.

145. The cell of claim 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic environment characterized by increased lactate levels relative to a non-acidic microenvironment.

146. The cell of claim 137, wherein the mutation results in enhanced multifunctionality of the engineered cell in response to stimulation by a tumor cell relative to a control non-engineered cell.

147. The cell of claim 146, wherein the enhanced multifunctionality is demonstrated by increased cytokine release in response to stimulation of tumor cells.

148. The cell of claim 137, wherein the increased cytokine release comprises increased interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or the degranulation marker CD107a in response to stimulation of tumor cells.

149. The cell of claim 148, wherein the increased cytokine release comprises granulocyte macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, granzyme A, interleukin 13 (IL-13), granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein-1α (MIP-1a), macrophage inflammatory protein-1β (MIP-1b), TNF-a and / or perforin increased in response to stimulation of tumor cells.

150. The cell of claim 137, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxicity phenotype relative to a control non-engineered cell.

151. The cell of claim 150, wherein the mutation provides the engineered cell with an enhanced activation and / or cytotoxic phenotype relative to a control non-engineered cell, wherein the enhanced activation and / or cytotoxic phenotype is associated with one or more pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNFα signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or estrogen early response.

152. The cell of claim 137, wherein the mutation provides upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation and / or TNFa signaling.

153. The cell of claim 137, wherein the mutation provides the engineered cell with enhanced proliferation capacity and / or persistence phenotype relative to a control non-engineered cell.

154. The cell of claim 153, wherein the enhanced proliferation capacity and / or persistence occurs in the absence of exogenous interleukin 2 (IL-2) stimulation.

155. The cell of claim 153 or 154, wherein the enhanced proliferation capacity and / or persistence does not result in autonomous growth.

156. The cell of claim 137, wherein the mutation provides the engineered cell with an enhanced metabolic fitness phenotype relative to a control non-engineered cell.

157. The cell of claim 137, wherein the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

158. The cell of claim 137, wherein the mutation provides increased chromatin accessibility throughout the genome when the cell is contained in an acidic microenvironment.

159. The cell of claim 137, wherein the CREM mutation results in reduced expression of CREM RNA isoforms CREM-228 (ICER), CREM-207, CREM-230, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, CREM-217 and / or CREM-225.

160. The cell of claim 137, wherein the CREM mutation is the result of exposing the cell to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO:

142.

161. The cell of claim 137, wherein the CREM mutation results in a greater than 60% reduction in CREM protein isoforms; or wherein the CREM mutation results in a greater than 80% reduction in CREM protein isoforms.

162. The cell of claim 137, wherein the cell is derived from umbilical cord blood (CB).

163. The cell of claim 137, wherein the cell is derived from peripheral blood (PB).

164. The cell of claim 137, wherein the cell is derived from a stem cell.

165. The cell of claim 137, wherein the cell comprises an engineered receptor.

166. The cell of claim 137, wherein the cell comprises a T cell receptor (TCR).

167. The cell of claim 137, wherein the cell comprises a chimeric antigen receptor (CAR).

168. The cell of claim 165, wherein the engineered receptor specifically binds an antigen selected from the group consisting of 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, F BP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TROP2, TEMs, HMW-MAA, VEGFR2 and combinations thereof.

169. The cell of claim 168, wherein the engineered receptor specifically binds TROP2.

170. The cell of claim 168, wherein the engineered receptor specifically binds CD70.

171. A cell population, wherein the cells are cells according to any one of claims 137-170.

172. A pharmaceutical composition comprising the cell of any one of claims 137-170 and optionally a pharmaceutically acceptable excipient.

173. A method of treating cancer in an individual in need thereof, comprising the step of administering to the individual a therapeutically effective amount of a cell of any one of claims 137-170, a cell population of claim 171, or a pharmaceutical composition of claim 172.

174. The method of claim 173, wherein the cancer comprises a solid tumor.

175. The method of claim 173, wherein the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer and / or cervical cancer.

176. The method of claim 173, wherein the individual is a mammal.

177. The method of claim 173, wherein the individual is a human.

178. A method of engineering a cell according to any one of claims 137-170.

179. A method of killing cancer cells, comprising contacting the cancer cells with an engineered NK cell according to any one of claims 137-170.

Images