Enhancement of Cell Therapy Activity in the Tumor Microenvironment

Abstract

Embodiments of the present disclosure include improvements in cell therapy to make cells more effective in cancer treatment, including the microenvironment of solid tumors. In certain aspects, the cells are modified such that the expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, or CREB1 are reduced or inhibited, such as by CRISPR gene editing. In certain aspects, the cells are modified such that the expression level of CREM is reduced or inhibited. In certain aspects, the cells are further modified to express, for example, one or more artificial receptors, one or more cytokines, and / or optionally one or more suicide genes.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,516, filed on June 15, 2022, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted in ST26 format, which is hereby incorporated by reference in its entirety. The ST26 copy created on June 11, 2023, is named MDAC_P1332WO_Sequence_Listing.xml and has a size of 240,688 bytes.

[0003] Technical Field Embodiments of the present disclosure include fields of medicine, including at least cell biology, molecular biology, immunology, and cancer medicine.

Background Art

[0004] Following the 2017 Food and Drug Administration (FDA) approval of chimeric antigen receptor (CAR) - T cell therapy for the treatment of patients with lymphoma and leukemia, adoptive cell therapy has rapidly come into focus for stakeholders across the field of cancer immunotherapy. This treatment modality has shown unprecedented patient responses and offers significant curative potential for certain hematological malignancies, while success in solid tumors remains challenging, in part due to the unique characteristics of the solid tumor microenvironment (TME) characterized by hypoxia, acidic pH, nutrient depletion, and immunosuppression (see, e.g., Renner et al., 2017). Acidity is a prominent feature of the tumor microenvironment, mainly due to acidic metabolites, such as lactate from active glycolysis under hypoxic conditions (see, e.g., Huber et al., 2017). Acidity mediates immunosuppression, tumor progression, and poor prognosis. Specifically, tissue acidosis results in the suppression of immune cell - mediated responses, such as a decrease in the cytotoxicity, cytokine production, and tumor surveillance functions of natural killer (NK) cells and T cells.

[0005] This specification provides techniques for overcoming immunosuppressive phenotypes associated with acidic environments and / or the TME.

[0006] The present disclosure provides a solution to a long - felt need in the art of cancer therapy by manipulating the cAMP signaling pathway via engineered mutations in the genes 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) to promote the activity of immune effector cells in the microenvironment of solid tumors.

Summary of the Invention

Means for Solving the Problems

[0007] Embodiments of the present disclosure include methods and compositions related to cell therapies, including adoptive cell therapies. Certain embodiments of the present disclosure encompass methods and compositions for cancer immunotherapy, anti-pathogen immunotherapy, or both. Pathogens include at least viruses, bacteria, fungi, and parasites. The present disclosure includes improved immune effector cell therapies for the express purpose of conferring one or more properties to cells that improve efficacy. In specific embodiments, immune effector cells are modified to better kill target cells, such as cancer cells. In specific embodiments, immune effector cells are effective against cancers lacking solid cancers, such as blood cancers, but are engineered to have reduced expression of one or more gene products that enable engineered cells to be effective in acidic environments, such as the solid cancer microenvironment, compared to non-engineered cells. In certain embodiments, the engineered cells are better equipped to kill cancer cells in an environment that is hypoxic, has an acidic pH, is nutrient depleted, and / or experiences immunosuppression.

[0008] In certain embodiments, immune effector cells engineered to have reduced levels of expression 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) are included in the compositions encompassed herein and used in the methods encompassed herein. In some embodiments, the immune effector cells have complete inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, e.g., lack detectable expression of the aforementioned genes by routine methods in the art.

[0009] In certain embodiments, the endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are modified by genetic manipulation of the genomic loci of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. Immune effector cells having reduced or completely inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 may or may not be modified in additional ways by human means such as expressing one or more exogenously provided gene products. In specific embodiments, the gene products are receptors, cytokines, chemokines, suicide genes, or combinations thereof. In certain cases, the receptor is an antigen receptor, and the antigen may or may not be a cancer antigen including antigens on solid tumor cells. In specific cases, the antigen receptor is a chimeric antigen receptor (CAR) or an unnatural T cell receptor.

[0010] In some embodiments, the present disclosure knocks out or knockdowns genes encoding GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 from immune effector cells used in various cell therapies to render them insensitive to the immunosuppressive effects of acidity, and thus increase their survival, proliferation, and immune function, including in at least an acidic solid tumor microenvironment. As an example, the feasibility of knocking out cAMP sensitivity and / or signaling pathways using Cas9 pre-incorporated with a chemically synthesized crFNA:tracrRNA duplex targeting CREM with gene editing CRISPR / Cas9 technology has been confirmed. The data disclosed herein show that knockout of CREM from NK cells results in improved cytotoxic effects of those cells and improved antitumor activity against cancer cell lines characterized by active glycolysis and marked acidosis in their microenvironment. In some embodiments, the genetic manipulation 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 (TIL), or combinations thereof, to enhance the efficacy against various types of cancer, including solid tumors.

[0011] The immune effector cells to be engineered can be of any type, but in specific 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, and the like. In certain cases, the immune effector cells are NK cells including umbilical cord blood-derived NK cells.

[0012] Any medical condition can be treated by administration of a therapeutically effective amount of the engineered immune effector cells of the disclosure. In specific embodiments, the cells are utilized in a composition for treating any type of cancer.

[0013] The present disclosure relates to a novel strategy that uses gene editing technology (e.g., CRISPR / Cas9) to knockout the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes from immune cells, enhances them as cell therapies for any type of cancer including at least solid cancers, and enhances antitumor activity.

[0014] Embodiments of the present disclosure include compositions related to engineered immune effector cells and their use, wherein the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes within the cells is decreased or completely inhibited. In specific embodiments, the cells are T cells, NK cells, NK T cells, macrophages, B cells, invariant NKT cells, gamma delta T cells, MSCs, tumor infiltrating lymphocytes, dendritic cells, or mixtures thereof. In specific embodiments, the NK cells are derived from umbilical cord blood. In some cases, the cell comprises one or more engineered receptors, such as engineered antigen receptors like CAR, chemokine receptors, homing receptors, and / or non-native T cell receptors. The antigen can be a cancer antigen including a solid tumor antigen. Specific examples of antigens include 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, the 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, TEMs, TROP2, HMW-MAA, VEGFR2, and combinations thereof (the receptor can have two or more antigen binding domains that bind to different antigens).

[0015] In certain embodiments, the cells comprise the 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 combinations thereof. Additionally or alternatively, the cells comprise a suicide gene.

[0016] Endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes may have decreased or inhibited expression from homologous or non-homologous recombination. In certain 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 cells that are autologous, allogeneic, or xenogeneic to the recipient individual.

[0017] In specific embodiments, 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 is further decreased or inhibited.

[0018] Embodiments of the present disclosure include any one population of the cells included herein. In specific embodiments, the population is included in a pharmaceutically acceptable excipient.

[0019] Specific embodiments of the present disclosure include methods of engineering NK cells, including in any manner in which their functionality is not transient, to be engineered in a manner that results in improvement with respect to NK cells that have not been so engineered. In specific embodiments, genetic modification in NK cells results in cells having enhanced cytotoxicity against cancer cells and / or enhanced expansion, persistence, and / or proliferation compared to NK cells that have not been so engineered. The methods of the present disclosure include, by way of example only, methods of suppressing an immune cell-mediated response in vivo in an individual receiving any type of adoptive cell therapy, including T cells and / or NK cells, where the cells are engineered such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is decreased or completely inhibited.

[0020] Embodiments of the present disclosure include improvement of any type of adoptive cell therapy in the tumor microenvironment by utilization of the engineered cells encompassed herein, as compared to cells that have not been so engineered. In specific embodiments, the present disclosure includes the generation and use of immune effector cells having enhanced cytotoxicity, persistence, and expansion due to engineering (as opposed to what is natural to the cell) such that the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 within the cell is decreased or completely inhibited as compared to cells that have not been so engineered.

[0021] Any type of immune effector cell, such as NK cells, can be obtained from a number of non-limiting sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, tumor, or commercially available. Any number of immune cell lines that are available and known to those of skill in the art can be used.

[0022] In addition to being engineered such that the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is reduced or completely inhibited, in at least some cases, the engineered immune effector cells are engineered in one or more other manners. In a specific embodiment, the cells are also engineered to express one or more engineered receptors (as contrasted with receptors endogenous to the cells), one or more cytokines, and / or one or more suicide genes. The engineered receptors can be of any kind and include at least one or more CARs, one or more T cell receptors, one or more chemokine receptors, combinations thereof, and the like. Any engineering of the immune effector cells can be performed before or after knocking out (or knocking down) the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. If the engineered immune effector cells having reduced or completely inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are also engineered to express two or more other genes, the engineering for the expression of the two or more other genes can be performed simultaneously with each other or not. For example, if knockout (KO) (or knockdown) cells of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are engineered to express a CAR and a cytokine, the engineering for expressing the CAR and the cytokine can be performed substantially simultaneously with each other or not. Any other transgenes for the cells can be expressed from the same vector or not. In an exemplary case, upon transfection or transformation of the immune effector cells, a CAR and a cytokine (only representative ones) can be expressed from the same vector or not.

[0023] Embodiments of the present disclosure include a method of treating cancer in an individual, the method including administering to the individual a therapeutically effective amount of the cell population of the present disclosure. In some cases, the cancer is a solid tumor or is not a solid tumor. The cancer can be of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, or cervix. The individual can be a mammal such as a human, dog, cat, horse, cow, sheep, pig, or rodent. The individual can or cannot be subjected to additional cancer therapies, such as surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, or combinations thereof. In a specific embodiment, the method further includes diagnosing the cancer of the individual. In some cases, the method further includes generating the cell population. The cells can be autologous or allogeneic to the individual.

[0024] In a specific embodiment, the cells are NK cells, such as umbilical cord blood NK cells, including those expressing one or more engineered antigen receptors. The cells can be NK cells expressing a CAR or NK cells expressing a TCR.

[0025] In certain embodiments, the present specification discloses engineered immune effector cells that comprise one or more engineered mutations in the 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 modulator (CREM), inducible cAMP early repressor (ICER), and / or cyclic AMP response element-binding protein 1 (CREB1) genes. In certain embodiments, the mutations are partial or complete loss-of-function and / or knockout (KO) mutations. In certain embodiments, the mutations decrease or inhibit the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not carry a mutation in the same endogenous gene. In certain embodiments, the mutations are novel functional or gain-of-function mutations. In certain embodiments, the mutations increase the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not carry a mutation in the same endogenous gene. In certain embodiments, the mutations result in a modified mRNA isoform population encoded by the mutated endogenous gene as compared to a representative mRNA population encoded by a locus that does not carry a mutation in the same endogenous gene. In certain embodiments, the mutations result in a modified protein isoform population encoded by the mutated endogenous gene as compared to a representative protein population encoded by a locus that does not carry a mutation in the same endogenous gene.

[0026] In certain embodiments, the mutation results in improved cytotoxicity of the engineered cells in an acidic environment and / or the tumor microenvironment (TME) compared to the unengineered cells of the control. In certain embodiments, the mutation results in improved cytotoxicity of the engineered cells in an acidic environment where the pH is less than or equal to about 7.0 compared to the unengineered cells of the control. In certain embodiments, the mutation results in improved cytotoxicity of the engineered cells in an acidic environment where the pH is less than or equal to about 5.9 compared to the unengineered cells of the control. In certain embodiments, the mutation results in enhanced multifunctionality of the engineered cells in response to stimulation by tumor cells compared to the unengineered cells of the control. In certain embodiments, the enhanced multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells. In certain embodiments, the multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells and includes an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or the degranulation marker CD107a. In certain embodiments, the multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells and includes an increase in granulocyte macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin.

[0027] In certain embodiments, the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cells as compared to control, non-engineered cells. In some embodiments, the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cells as compared to control, non-engineered cells, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F targets, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response. In some embodiments, the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation, and / or TNFa signaling. In certain embodiments, the mutation results in an enhanced proliferation and / or persistence phenotype for the engineered cells as compared to control, non-engineered cells. In certain embodiments, the enhanced proliferation and / or persistence occurs in the absence of stimulation by exogenous interleukin 2 (IL-2). In certain embodiments, the enhanced proliferation and / or persistence does not result in autonomous growth. In certain embodiments, the mutation results in an enhanced metabolic fitness phenotype for the engineered cells as compared to control, non-engineered cells. In certain embodiments, the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0028] In certain embodiments, the mutation in the endogenous gene is in the gene CREM. In certain embodiments, the CREM mutation results in a decrease in the 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 an increase in the expression of CREM RNA isoform CREM-218. In certain embodiments, the CREM mutation is the result of exposure of cells to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO: 142. In certain embodiments, the CREM mutation results in a decrease of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of one or more CREM protein isoforms, or any range derivable therefrom. In certain embodiments, the CREM mutation results 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% of one or more CREM protein isoforms. In certain embodiments, the CREM mutation results 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% of one or more CREM protein isoforms.

[0029] In certain embodiments, the cells are further adapted to an acidic environment by contacting the cells with an acidic stimulus ex vivo. 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 2-3 mM. In some embodiments, the acidic stimulus is provided at a concentration greater than or equal to about 2.5 mM. In some embodiments, adaptation to the acidic environment is by incremental and / or cumulative contact with the acidic stimulus. In some embodiments, the cells are adapted over a period of at least about 10-18 days, optionally at least about 14 days. In some embodiments, cell adaptation includes adding the acidic stimulus every about 48-72 hours, optionally every about 48 hours. In some embodiments, the acidic stimulus comprises or consists essentially of lactic acid. In some embodiments, the adaptation is to an acidic environment having a pH less than or equal to about 6.0.

[0030] In certain embodiments, the cells are T cells, natural killer (NK) cells, NK T cells, macrophages, B cells, invariant NKT cells, gamma delta T cells, MSCs, tumor infiltrating lymphocytes, or dendritic cells. In certain embodiments, the cells are NK cells derived from umbilical cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, stem cells, or mixtures thereof. In certain embodiments, the NK cells are derived from umbilical cord blood.

[0031] In certain embodiments, the cell comprises one or more engineered receptors. In certain embodiments, the engineered receptor comprises 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 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, the 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.

[0032] In certain embodiments, the one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof. In certain embodiments, the cell comprises the expression of one or more exogenous chemokines and / or one or more cytokines. In certain embodiments, the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof. In certain embodiments, the cytokine is IL-15. In certain embodiments, the cell comprises a suicide gene.

[0033] In certain embodiments, the endogenous gene is mutated as a result of homologous or non-homologous recombination. In certain embodiments, the endogenous gene is mutated by an endonuclease. In certain embodiments, the endonuclease is an RNA-guided endonuclease. In certain embodiments, the RNA-guided endonuclease is CRISPR-Cas9. In certain embodiments, the cell comprises one or more additional mutations in one or more genes selected from the group consisting 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.

[0034] Also disclosed herein is any population of the cells described herein. In some embodiments, the cell population is contained in a pharmaceutically acceptable excipient.

[0035] Also disclosed herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of the cell population of the claim. In some embodiments, the cells are autologous, allogeneic or xenogeneic to the individual. In some embodiments, the cells are allogeneic to the individual. In some embodiments, the cancer includes solid tumors. In some embodiments, the cancer does not include solid tumors. In some embodiments, the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, and / or cervix. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human, dog, cat, horse, cow, sheep, pig, or rodent. In some embodiments, the individual is a human. In some embodiments, the individual is administered additional cancer therapy. In some embodiments, the additional cancer therapy is surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, or a combination thereof. In some embodiments, the individual is diagnosed with cancer.

[0036] Also disclosed herein are methods of engineering immune effector cells. In some embodiments, methods of engineering immune effector cells include mutating an 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-responsive element-binding protein 1 (CREB1) gene within the cell. In some embodiments, mutating comprises generating a partial or complete loss of function and / or a knockout (KO) mutation. In some embodiments, mutating decreases or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to an unmutated locus encoding the same endogenous gene. In some embodiments, mutating generates a neo-functional or gain-of-function mutation. In some embodiments, mutating increases transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to an unmutated locus encoding the same endogenous gene. In some embodiments, mutating generates a modified mRNA isoform population encoded by the mutated endogenous gene as compared to a representative mRNA population encoded by an unmutated locus encoding the same endogenous gene. In some embodiments, mutating generates a modified protein isoform population encoded by the mutated endogenous gene as compared to a representative protein population encoded by an unmutated locus encoding the same endogenous gene.

[0037] In some embodiments, mutating generates improved cytotoxicity of the engineered cells in an acidic environment and / or tumor microenvironment (TME) compared to control non-engineered cells. In some embodiments, mutating generates improved cytotoxicity of the engineered cells in an acidic environment where the pH is less than or equal to about 7.0 compared to control non-engineered cells. In some embodiments, mutating generates improved cytotoxicity of the engineered cells in an acidic environment where the pH is less than or equal to about 5.9 compared to control non-engineered cells.

[0038] In some embodiments, mutating generates enhanced multifunctionality of the engineered cells in response to stimulation by tumor cells compared to control non-engineered cells. In some embodiments, the enhanced multifunctionality is evidenced by an increase in cytokine release in response to stimulation by tumor cells. In some embodiments, the increase in cytokine release includes an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells. In some embodiments, the increase in cytokine release includes an increase in granulocyte-macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

[0039] In some embodiments, mutating results in an enhanced activation and / or a cytotoxic phenotype for the engineered cells as compared to control, non-engineered cells. In some embodiments, mutating results in an enhanced activation and / or a cytotoxic phenotype for the engineered cells as compared to control, non-engineered cells, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F targets, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response. In some embodiments, mutating results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation, and / or TNFa signaling.

[0040] In some embodiments, mutating results in an enhanced proliferative capacity and / or a persistence phenotype for the engineered cells as compared to control, non-engineered cells. In some embodiments, the enhanced proliferative capacity and / or persistence occurs in the absence of stimulation by exogenous interleukin 2 (IL-2). In some embodiments, the enhanced proliferative capacity and / or persistence does not result in autonomous growth. In some embodiments, mutating results in an enhanced metabolic fitness phenotype for the engineered cells as compared to control, non-engineered cells. In some embodiments, the enhanced metabolic fitness is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0041] In some embodiments, a method of engineering immune effector cells includes mutating an endogenous cAMP response element modulator (CREM) gene. In some embodiments, mutating CREM results in a decrease in the 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, mutating CREM results in an increase in the expression of CREM RNA isoform CREM-218. In some embodiments, mutating CREM includes exposing the cells to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO: 142. In some embodiments, mutating CREM produces a decrease in one or more CREM protein isoforms that is greater 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 therefrom. In some embodiments, mutating CREM produces a decrease in one or more CREM protein isoforms that is greater than 80%.

[0042] In certain embodiments, the method further comprises acclimating the cells to an acidic environment by contacting the cells ex vivo with an acidic stimulus. In some embodiments, acclimating the cells to an acidic environment comprises providing the acidic stimulus at a concentration greater than or equal to about 2 - 3 mM, optionally greater than or equal to about 2.5 mM. In some embodiments, acclimation to an acidic environment is by incremental and / or cumulative contact with the acidic stimulus. In some embodiments, acclimation is carried out over a period of at least about 10 - 18 days, optionally at least about 14 days. In some embodiments, acclimation comprises adding the acidic stimulus every about 48 - 72 hours, optionally every about 48 hours. In some embodiments, the acidic stimulus comprises or consists essentially of lactic acid. In some embodiments, acclimation is to an acidic environment having a pH less than or equal to about 6.0.

[0043] In some embodiments, a method of engineering immune effector cells comprises mutating an endogenous gene in a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cell, gamma delta T cell, MSC, tumor infiltrating lymphocyte, dendritic cell, or a progenitor cell thereof. In some embodiments, the cells are NK cells derived from cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, stem cells, or a mixture thereof. In some embodiments, the NK cells are cord blood-derived.

[0044] In some embodiments, a method of engineering immune effector cells comprises mutating an endogenous gene in the cell, and the cell also 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 5T4, 8H9, α vSelected from the group consisting of 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, the 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. In some embodiments, one or more engineered receptors include cytokine receptors, chemokine receptors, homing receptors, or combinations thereof.

[0045] In some embodiments, a method of engineering immune effector cells includes mutating an endogenous gene in the cell, and the cell also includes the 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, the cell includes a suicide gene. In some embodiments, mutating the endogenous gene includes homologous recombination or non-homologous recombination. In some embodiments, mutating 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 cell includes one or more additional mutations in one or more genes selected from the group consisting 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.

[0046] The following aspects describe certain inventions disclosed herein.

[0047] Aspect 1 is an engineered immune effector cell, which contains one or more engineered mutations in the 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-responsive element-binding protein 1 (CREB1) gene.

[0048] 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.

[0049] Aspect 3 is the cell of Aspect 1 or 2, wherein the mutation reduces or inhibits the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not carry a mutation in the same endogenous gene.

[0050] Aspect 4 is the cell of Aspect 1, wherein the mutation is a novel functional mutation or a gain-of-function mutation.

[0051] Aspect 5 is the cell of Aspect 1 or 4, wherein the mutation increases the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not carry a mutation in the same endogenous gene.

[0052] Aspect 6 is the cell of any one of Aspects 1-5, wherein the mutation results in a modified mRNA isoform population encoded by the mutated endogenous gene as compared to a representative mRNA population encoded by a locus that does not carry a mutation in the same endogenous gene.

[0053] Aspect 7 is a cell of any one of Aspects 1 - 4, wherein the mutation results in a modified protein isoform population encoded by the mutated endogenous gene, as compared to a representative protein population encoded by a non-mutated locus encoding the same endogenous gene.

[0054] Aspect 8 is a cell of any one of Aspects 1 - 7, wherein the mutation comprises a knockout (KO) mutation of an endogenous gene of the cell.

[0055] Aspect 9 is a cell of any one of Aspects 1 - 7, wherein the mutation comprises a homozygous mutation in an endogenous gene of the cell.

[0056] Aspect 10 is a cell of any one of Aspects 1 - 7, wherein the mutation comprises a heterozygous mutation in an endogenous gene of the cell.

[0057] Aspect 11 is a 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) as compared to a control non-engineered cell.

[0058] Aspect 12 is a cell of any one of Aspects 1 - 11, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment where the pH is less than or equal to about 7.0 as compared to a control non-engineered cell.

[0059] Aspect 13 is a cell of any one of Aspects 1 - 12, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment where the pH is less than or equal to about 5.9 as compared to a control non-engineered cell.

[0060] Aspect 14 is a cell of any one of Aspects 1 - 13, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment, optionally characterized by an increased lactate level, as compared to a non-acidic microenvironment.

[0061] Aspect 15 is a cell of any one of Aspects 1 to 14, in which the mutation results in enhanced multifunctionality of the engineered cells in response to stimulation by tumor cells, as compared to control non-engineered cells.

[0062] Aspect 16 is a cell of Aspect 15, in which the enhanced multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells.

[0063] Aspect 17 is a cell of Aspect 16, in which the increase in cytokine release includes an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

[0064] Aspect 18 is a cell of Aspect 15 or 16, in which the increase in cytokine release includes an increase in granulocyte macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

[0065] Aspect 19 is a cell of any one of Aspects 1 to 18, in which the mutation results in enhanced activation and / or a cytotoxic phenotype for the engineered cells, as compared to control non-engineered cells.

[0066] Aspect 20 is a cell of any one of aspects 1-19, wherein the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cell as compared to the unengineered cells of the control, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

[0067] Aspect 21 is a 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.

[0068] Aspect 22 is a cell of any one of aspects 1-21, wherein the mutation results in an enhanced proliferation ability and / or persistence phenotype for the engineered cell as compared to the unengineered cells of the control.

[0069] Aspect 23 is a cell of aspect 22, wherein the enhanced proliferation ability and / or persistence occurs in the absence of stimulation by exogenous interleukin 2 (IL-2).

[0070] Aspect 24 is a cell of aspect 22 or 23, wherein the enhanced proliferation ability and / or persistence does not result in autonomous proliferation.

[0071] Aspect 25 is a cell of any one of aspects 1-24, wherein the mutation results in an enhanced metabolic adaptation ability phenotype for the engineered cell as compared to the unengineered cells of the control.

[0072] Aspect 26 is a cell of aspect 25, wherein the enhanced metabolic adaptation ability is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0073] Embodiment 27 is any one cell of embodiments 1-26, wherein the mutation provides increased chromosomal proximity across the genome when the cell is contained in an acidic microenvironment.

[0074] Embodiment 28 is any one cell of embodiments 1-27, wherein the endogenous gene is CREM.

[0075] Embodiment 29 is the cell of embodiment 28, wherein 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.

[0076] Embodiment 30 is the cell of embodiment 28 or 29, wherein the CREM mutation results in increased expression of the CREM RNA isoform CREM-218.

[0077] Embodiment 31 is any one cell of embodiments 28-30, 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.

[0078] Embodiment 32 is any one cell of embodiments 28-31, wherein the CREM mutation results in a decrease in one or more CREM protein isoforms that exceeds 60%.

[0079] Embodiment 33 is any one cell of embodiments 28-32, wherein the CREM mutation results in a decrease in one or more CREM protein isoforms that exceeds 80%.

[0080] Embodiment 34 is any one cell of embodiments 1-33, wherein the cell is acclimated to an acidic microenvironment by further contacting the cell with an acidic stimulus ex vivo.

[0081] Aspect 35 is the cell of Aspect 34 in which the acidic stimulus is provided at a concentration greater than or equal to about 2-3 mM, optionally greater than or equal to about 2.5 mM.

[0082] Aspect 36 is the cell of any one of Aspects 34-35 in which the acclimation to the acidic microenvironment is by a gradual and / or cumulative contact with the acidic stimulus.

[0083] Aspect 37 is the cell of any one of Aspects 34-36 in which the cell is acclimated over a period of at least about 10-18 days, optionally at least about 14 days.

[0084] Aspect 38 is the cell of Aspect 36 or 37 in which the acclimation of the cell includes applying an acidic stimulus every about 48-72 hours, optionally every about 48 hours.

[0085] Aspect 39 is the cell of any one of Aspects 34-37 in which the acidic stimulus contains lactic acid or consists essentially of lactic acid.

[0086] Aspect 40 is the cell of any one of Aspects 34-39 in which the cell is acclimated to an acidic microenvironment with a pH less than or equal to about 6.0.

[0087] Aspect 41 is the cell of any one of Aspects 1-40 in which the cell is a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cell, gamma delta T cell, MSC, tumor infiltrating lymphocyte, or dendritic cell.

[0088] Aspect 42 is the cell of any one of Aspects 1-41 in which the cell is an NK cell derived from cord blood (CB), peripheral blood (PB), NK cell line, bone marrow, stem cells, or a mixture thereof.

[0089] Aspect 43 is the cell of Aspect 41 or 42 in which the NK cell is derived from cord blood.

[0090] Aspect 44 is a cell of any one of aspects 1 - 43, wherein the cell comprises one or more engineered receptors.

[0091] 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.

[0092] Aspect 46 is a cell of aspect 45, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0093] Aspect 47 is a cell of aspect 46, wherein the engineered antigen receptor is a CAR.

[0094] Aspect 48 is a cell of any one of aspects 45 - 47, wherein the antigen is a cancer antigen.

[0095] Aspect 49 is a cell of any one of aspects 45 - 48, wherein the antigen is a solid tumor antigen.

[0096] Aspect 50 is that the antigen is 5T4, 8H9, α vA cell according to any one of aspects 45 to 49, selected from the group consisting of 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, the 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.

[0097] Aspect 51 is a cell according to any one of aspects 45 to 50, wherein the antigen comprises TROP2 and / or CD70.

[0098] Aspect 52 is a cell according to any one of aspects 44 to 51, wherein one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

[0099] Aspect 53 is a cell according to any one of aspects 1 to 52, wherein the cell comprises the expression of one or more exogenous chemokines and / or one or more cytokines.

[0100] Aspect 54 is a cell according to aspect 53, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof.

[0101] Aspect 55 is the cell of Aspect 54, wherein the cytokine is IL-15.

[0102] Aspect 56 is the cell of any one of Aspects 1 to 55, wherein the cell contains a suicide gene.

[0103] Aspect 57 is the cell of any one of Aspects 1 to 56, wherein the endogenous gene is mutated as a result of homologous recombination or non-homologous recombination.

[0104] Aspect 58 is the cell of any one of Aspects 1 to 57, wherein the endogenous gene is mutated by an endonuclease.

[0105] Aspect 59 is the cell of Aspect 58, wherein the endonuclease is an RNA-induced endonuclease.

[0106] Aspect 60 is the cell of Aspect 59, wherein the RNA-induced endonuclease is CRISPR-Cas9.

[0107] Aspect 61 is the cell of any one of Aspects 1 to 60, wherein the cell contains one or more additional mutations in one or more genes, and the genes are selected from the group consisting 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.

[0108] Aspect 62 is a cell population of any one of Aspects 1 to 61.

[0109] Aspect 63 is the cell population of Aspect 62, wherein the population is contained in a pharmaceutically acceptable excipient.

[0110] Aspect 64 is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the cell population of Aspect 62 or 63.

[0111] Aspect 65 is the method of Aspect 64, wherein the cells are autologous, allogeneic or xenogeneic to the individual.

[0112] Aspect 66 is the method of Aspect 64 or 65, wherein the cells are allogeneic to the individual.

[0113] Aspect 67 is any one of the methods of Aspects 64 - 66, wherein the cancer comprises a solid tumor.

[0114] Aspect 68 is any one of the methods of Aspects 64 - 66, wherein the cancer does not comprise a solid tumor.

[0115] Aspect 69 is any one of the methods of Aspects 64 - 68, wherein the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, and / or cervix.

[0116] Aspect 70 is any one of the methods of Aspects 64 - 69, wherein the individual is a mammal.

[0117] Aspect 71 is the method of Aspect 70, wherein the individual is a human, dog, cat, horse, cow, sheep, pig, or rodent.

[0118] Aspect 72 is the method of Aspect 70 or 71, wherein the individual is a human.

[0119] Aspect 73 is any one of the methods of Aspects 64 - 72, wherein the individual is subjected to additional cancer treatment.

[0120] Aspect 74 is the method of Aspect 73, wherein the additional cancer treatment is surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0121] Aspect 75 is any one of the methods of Aspects 64 - 74, further including a step of diagnosing cancer in an individual.

[0122] Aspect 76 is a method of manipulating immune effector cells, including mutating the 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 - responsive element - binding protein 1 (CREB1) gene.

[0123] Aspect 77 is the method of Aspect 76, wherein mutating generates a partial or complete loss of function and / or a knockout (KO) mutation.

[0124] Aspect 78 is the method of Aspect 76 or 77, wherein mutating decreases or inhibits the transcription or post - transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene compared to the non - mutated locus encoding the same endogenous gene.

[0125] Aspect 79 is the method of Aspect 76, wherein mutating generates a novel functional mutation or a gain - of - function mutation.

[0126] Aspect 80 is the method of Aspect 76 or 79, wherein mutating increases the transcription or post - transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene compared to the non - mutated locus encoding the same endogenous gene.

[0127] Aspect 81 is one of the methods of aspects 76 - 80, wherein mutating generates a modified mRNA isoform population encoded by a mutated endogenous gene as compared to a representative mRNA population encoded by an unmutated locus encoding the same endogenous gene.

[0128] Aspect 82 is one of the methods of aspects 76 - 79, wherein mutating generates a modified protein isoform population encoded by a mutated endogenous gene as compared to a representative protein population encoded by an unmutated locus encoding the same endogenous gene.

[0129] Aspect 83 is one of the methods of aspects 76 - 82, wherein mutating generates a knockout (KO) mutation in an endogenous gene of a cell.

[0130] Aspect 84 is one of the methods of aspects 76 - 82, wherein mutating generates a homozygous mutation in an endogenous gene of a cell.

[0131] Aspect 85 is one of the methods of aspects 76 - 82, wherein mutating generates a heterozygous mutation in an endogenous gene of a cell.

[0132] Aspect 86 is one of the methods of aspects 76 - 85, wherein mutating generates improved cytotoxicity of an engineered cell in an acidic microenvironment and / or a tumor microenvironment (TME) as compared to a control non - engineered cell.

[0133] Aspect 87 is one of the methods of aspects 76 - 86, wherein mutating generates improved cytotoxicity of an engineered cell in an acidic microenvironment where the pH is less than or equal to about 7.0 as compared to a control non - engineered cell.

[0134] Aspect 88 is any one of the methods of aspects 76 - 87, wherein mutating generates improved cytotoxicity of the engineered cells in an acidic microenvironment where the pH is less than or equal to about 5.9 as compared to control, non-engineered cells.

[0135] Aspect 89 is any one of the methods of aspects 76 - 88, wherein mutating results in improved cytotoxicity of the engineered cells in an acidic microenvironment characterized by an increase in lactate levels as compared to a non-acidic microenvironment.

[0136] Aspect 90 is any one of the methods of aspects 76 - 89, wherein mutating generates enhanced multifunctionality of the engineered cells in response to stimulation by tumor cells as compared to control, non-engineered cells.

[0137] Aspect 91 is the method of aspect 90, wherein the enhanced multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells.

[0138] Aspect 92 is the method of aspect 91, wherein the increase in cytokine release includes an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

[0139] Aspect 93 is the method of aspect 90 or 91, wherein the increase in cytokine release includes an increase in granulocyte macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

[0140] Aspect 94 is a method according to any one of aspects 76 - 93, wherein mutating results in an enhanced activation and / or a cytotoxic phenotype for the engineered cells as compared to control non-engineered cells.

[0141] Aspect 95 is a method according to any one of aspects 76 - 93, wherein mutating results in an enhanced activation and / or a cytotoxic phenotype for the engineered cells as compared to control non-engineered cells, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F targets, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

[0142] Aspect 96 is a method according to any one of aspects 76 - 95, wherein mutating results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation, and / or TNFa signaling.

[0143] Aspect 97 is a method according to any one of aspects 76 - 96, wherein mutating results in an enhanced proliferation and / or persistence phenotype for the engineered cells as compared to control non-engineered cells.

[0144] Aspect 98 is the method of aspect 97, wherein the enhanced proliferation and / or persistence occurs in the absence of stimulation by exogenous interleukin 2 (IL-2).

[0145] Aspect 99 is the method of aspect 97 or 98, wherein the enhanced proliferation and / or persistence does not result in autonomous growth.

[0146] Aspect 100 is any one of the methods of Aspects 76 - 99, wherein mutating generates a phenotype of enhanced metabolic adaptability for the engineered cells as compared to the unengineered cells of the control.

[0147] Aspect 101 is the method of Aspect 100, wherein the enhanced metabolic adaptability is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0148] Aspect 102 is any one of the methods of Aspects 76 - 101, wherein mutating provides increased chromosomal proximity across the genome when the cells are contained in an acidic microenvironment.

[0149] Aspect 103 is any one of the methods of Aspects 76 - 102, wherein the endogenous gene is CREM.

[0150] Aspect 104 is the method of Aspect 103, wherein mutating CREM results in a decrease in the 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.

[0151] Aspect 105 is the method of Aspect 103 or 104, wherein mutating CREM results in an increase in the expression of CREM RNA isoform CREM - 218.

[0152] Aspect 106 is any one of the methods of Aspects 103 - 105, wherein mutating CREM includes exposing the cells to a polynucleotide comprising the sequence of SEQ ID NO: 140 and / or SEQ ID NO: 142.

[0153] Aspect 107 is any one of the methods of Aspects 103 - 106, wherein mutating CREM generates a decrease in more than 60% of one or more CREM protein isoforms.

[0154] Aspect 108 is the method of any one of Aspects 103 - 107, wherein mutating CREM produces a reduction of one or more CREM protein isoforms exceeding 80%.

[0155] Aspect 109 is the method of any one of Aspects 76 - 108, further comprising acclimating the cells to an acidic microenvironment by contacting the cells with an acidic stimulus ex vivo.

[0156] Aspect 110 is the method of Aspect 109, comprising providing the acidic stimulus at a concentration greater than or equal to about 2 - 3 mM, optionally greater than or equal to about 2.5 mM.

[0157] Aspect 111 is the method of Aspect 109 or 110, wherein acclimation to the acidic microenvironment is by a gradual and / or cumulative contact with the acidic stimulus.

[0158] Aspect 112 is the method of any one of Aspects 109 - 111, wherein acclimation is carried out over a period of at least about 10 - 18 days, optionally at least about 14 days.

[0159] Aspect 113 is the method of any one of Aspects 109 - 112, wherein acclimation comprises applying the acidic stimulus every about 48 - 72 hours, optionally every about 48 hours.

[0160] Aspect 114 is the method of any one of Aspects 109 - 113, wherein the acidic stimulus comprises lactic acid or consists essentially of lactic acid.

[0161] Aspect 115 is the method of any one of Aspects 109 - 114, wherein acclimation is to an acidic microenvironment with a pH less than or equal to 6.0.

[0162] Aspect 116 is any one of aspects 76 - 115, where the cell is a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cell, gamma delta T cell, MSC, tumor infiltrating lymphocyte, or dendritic cell.

[0163] Aspect 117 is any one of aspects 76 - 116, where the cell is an NK cell derived from cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, stem cells, or a mixture thereof.

[0164] Aspect 118 is the method of aspect 116 or 117, where the NK cell is derived from cord blood.

[0165] Aspect 119 is any one of aspects 76 - 118, where the cell contains one or more engineered receptors that specifically bind to an antigen.

[0166] Aspect 120 is the method of aspect 119, where the one or more engineered receptors include engineered antigen receptors.

[0167] Aspect 121 is the method of aspect 120, where the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0168] Aspect 122 is the method of aspect 121, where the engineered antigen receptor is a CAR.

[0169] Aspect 123 is any one of aspects 120 - 122, where the antigen is a cancer antigen.

[0170] Aspect 124 is any one of aspects 120 - 123, where the antigen is a solid tumor antigen.

[0171] Aspect 125 is where the antigen is 5T4, 8H9, α vA method according to any one of aspects 120 to 124, selected from the group consisting of 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, the 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.

[0172] Aspect 126 is a method according to any one of aspects 120 to 125, wherein the antigen comprises TROP2 and / or CD70.

[0173] Aspect 127 is a method according to any one of aspects 119 to 126, wherein one or more engineered receptors comprise a cytokine receptor, a chemokine receptor, a homing receptor, or a combination thereof.

[0174] Aspect 128 is a method according to any one of aspects 76 to 127, wherein the cell comprises the expression of one or more exogenous chemokines and / or one or more cytokines.

[0175] 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.

[0176] Aspect 130 is the method of Aspect 129, wherein the cytokine is IL-15.

[0177] Aspect 131 is the method of any one of Aspects 76 to 130, wherein the cell contains a suicide gene.

[0178] Aspect 132 is the method of any one of Aspects 76 to 131, wherein mutating the endogenous gene involves homologous recombination or non-homologous recombination.

[0179] Aspect 133 is the method of any one of Aspects 76 to 132, wherein mutating the endogenous gene is mediated by an endonuclease.

[0180] Aspect 134 is the method of Aspect 133, wherein the endonuclease is an RNA-induced endonuclease.

[0181] Aspect 135 is the method of Aspect 134, wherein the RNA-induced endonuclease is CRISPR-Cas9.

[0182] Aspect 136 is the method of any one of Aspects 76 to 135, wherein the cell contains one or more additional mutations in one or more genes, and the genes are selected from the group consisting 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.

[0183] Aspect 137 is an engineered natural killer (NK) cell, wherein the cell contains an engineered partial or complete loss of function and / or a knockout (KO) mutation of the endogenous cAMP response element modulator (CREM) gene in the cell.

[0184] Embodiment 138 is the cell of Embodiment 137, wherein the cell contains a knockout (KO) mutation of the endogenous CREM gene in the cell.

[0185] Embodiment 139 is the cell of Embodiment 138, wherein the cell contains a homozygous knockout (KO) mutation of the endogenous CREM gene.

[0186] Embodiment 140 is the cell of Embodiment 138, wherein the cell contains a heterozygous KO mutation of the endogenous CREM gene in the cell.

[0187] Embodiment 141 is the cell of Embodiment 137, wherein the mutation results in improved cytotoxicity of the engineered cell as compared to a reference cell lacking the mutation.

[0188] Embodiment 142 is the cell of Embodiment 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment and / or tumor microenvironment (TME) as compared to a control non-engineered cell.

[0189] Embodiment 143 is the cell of Embodiment 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment where the pH is less than or equal to about 7.0 as compared to a control non-engineered cell.

[0190] Embodiment 144 is the cell of Embodiment 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic microenvironment where the pH is less than or equal to about 5.9 as compared to a control non-engineered cell.

[0191] Embodiment 145 is the cell of Embodiment 137, wherein the mutation results in improved cytotoxicity of the engineered cell in an acidic environment characterized by an increased lactate level as compared to a non-acidic microenvironment.

[0192] Aspect 146 is the cell of Aspect 137 in which the mutation results in enhanced multifunctionality of the engineered cells in response to stimulation by tumor cells, as compared to control non-engineered cells.

[0193] Aspect 147 is the cell of Aspect 146 in which the enhanced multifunctionality is demonstrated by an increase in cytokine release in response to stimulation by tumor cells.

[0194] Aspect 148 is the cell of Aspect 137 in which the increase in cytokine release includes an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

[0195] Aspect 149 is the cell of Aspect 148 in which the increase in cytokine release includes an increase in granulocyte macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

[0196] Aspect 150 is the cell of Aspect 137 in which the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cells as compared to control non-engineered cells.

[0197] Aspect 151 is a cell of Aspect 150 in which the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cell as compared to the unengineered control cells, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F targets, P53 pathway, mitotic spindle, MYC, MTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

[0198] Aspect 152 is a cell of Aspect 137 in which the mutation results in upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation, and / or TNFa signaling.

[0199] Aspect 153 is a cell of Aspect 137 in which the mutation results in an enhanced proliferation ability and / or persistence phenotype for the engineered cell as compared to the unengineered control cells.

[0200] Aspect 154 is a cell of Aspect 153 in which the enhanced proliferation ability and / or persistence occurs in the absence of stimulation by exogenous interleukin 2 (IL-2).

[0201] Aspect 155 is a cell of Aspect 153 or 154 in which the enhanced proliferation ability and / or persistence does not result in autonomous growth.

[0202] Aspect 156 is a cell of Aspect 137 in which the mutation results in an enhanced metabolic adaptability phenotype for the engineered cell as compared to the unengineered control cells.

[0203] Aspect 157 is a cell of Aspect 137 in which the enhanced metabolic adaptability is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

[0204] Aspect 158 is a cell of Aspect 137 in which the mutation provides increased chromosomal proximity across the genome when the cell is contained in an acidic environment.

[0205] Aspect 159 is a cell of Aspect 137 in which the CREM mutation results in a decrease in the 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.

[0206] Aspect 160 is a cell of Aspect 137 in which 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.

[0207] Aspect 161 is a cell of Aspect 137 in which the CREM mutation results in a decrease of more than 60% of the CREM protein isoforms; or the CREM mutation results in a decrease of 80% or more of the CREM protein isoforms.

[0208] Aspect 162 is a cell of Aspect 137 in which the cell is derived from cord blood (CB).

[0209] Aspect 163 is a cell of Aspect 137 in which the cell is derived from peripheral blood (PB).

[0210] Aspect 164 is a cell of Aspect 137 in which the cell is derived from stem cells.

[0211] Aspect 165 is a cell of Aspect 137 in which the cell contains an engineered receptor.

[0212] Aspect 166 is a cell of Aspect 137 in which the cell contains a T cell receptor (TCR).

[0213] Aspect 167 is a cell of Aspect 137 in which the cell contains a chimeric antigen receptor (CAR).

[0214] Embodiment 168 is such that the receptor being operated on is 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, the 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 any one cell of Embodiments 165-167 that specifically binds to an antigen selected from the group consisting of combinations thereof.

[0215] Embodiment 169 is the cell of Embodiment 168, wherein the antigen is TROP2.

[0216] Embodiment 170 is the cell of Embodiment 168, wherein the antigen is CD70.

[0217] Embodiment 171 is a cell population of any one of Embodiments 137-170.

[0218] Embodiment 172 is a pharmaceutical composition comprising any one cell of Embodiments 137-170 and optionally a pharmaceutically acceptable excipient.

[0219] Aspect 173 is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the cells of Aspects 137 - 170, the population of Aspect 171, or the pharmaceutical composition of Aspect 172.

[0220] Aspect 174 is the method of Aspect 173, wherein the cancer comprises a solid tumor.

[0221] Aspect 175 is the method of Aspect 173, wherein the cancer is a cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, and / or cervix.

[0222] Aspect 176 is the method of Aspect 173, wherein the individual is a mammal.

[0223] Aspect 177 is the method of Aspect 173, wherein the individual is a human.

[0224] Aspect 178 is a method of manipulating any one of the cells of Aspects 137 - 170.

[0225] Aspect 179 is a method of killing cancer cells, the method comprising contacting the cancer cells with any one of the manipulated NK cells of Aspects 137 - 170.

[0226] It is specifically contemplated that any limitation considered with respect to one embodiment of the present invention may be applicable to any other embodiment of the present invention. Further, any composition of the present invention can be used in any method of the present invention, and any method of the present invention can be used to produce or utilize any composition of the present invention. Aspects of the embodiments described in the examples may also be embodiments that can be made in the context of embodiments considered elsewhere in different examples or elsewhere in this application, for example, in the context of embodiments considered in the summary of the invention, the detailed description of the invention, the claims, the abstract, and the brief description of the drawings.

[0227] The above has outlined rather broadly the features and technical advantages of the present disclosure, which is for the purpose of enabling a better understanding of the following detailed description. Additional features and advantages forming the subject matter of the claims herein will be described below. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments can be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be understood by those skilled in the art that such equivalent structures do not depart from the spirit and scope defined in the appended claims. The novel features believed to be characteristic of the design disclosed herein, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. However, it should be explicitly understood that each drawing is provided for purposes of illustration and description only and is not intended as a definition of the limits of the present disclosure.

[0228] Brief Description of the Drawings To more fully understand the present disclosure, reference is made to the following description taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0243] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be made by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0244] Detailed Description I. Examples of Definitions In accordance with long-standing patent law practice, as used herein, the words "a" and "an", when used in combination with the word "comprising", including in the claims, represent "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 can be practiced with respect to any other method or composition described herein, and that different embodiments can be combined.

[0245] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean including the stated step or element or steps or element groups but not to mean excluding other steps or elements or steps or element groups. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the expression "consisting of" indicates that the listed elements are essential or essential and that there may be no other elements. "Consisting essentially of" means including the elements listed after the phrase and being limited to other elements that do not prevent or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the expression "consisting essentially of" indicates that the listed elements are essential or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0246] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "an embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the foregoing phrases appear in various places throughout this specification, but not necessarily all refer to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0247] As used herein, the terms "or" and "and / or" are used to combine multiple components or to describe them mutually exclusively. 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 may be specifically excluded from an embodiment.

[0248] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning in the fields of cell biology and molecular biology, indicating that a value includes the standard deviation of error for the apparatus or method employed to determine that value.

[0249] As used herein, the term "engineered" refers to an entity generated by human hand, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and includes elements that do not exist in nature or are not constituted in the manner utilized in the present disclosure.

[0250] As used herein, the term "exogenous" refers to a polynucleotide (such as one encoding a gene product or a part of a gene product) that does not exist endogenously in mammalian cells such as immune cells or is synthetically produced outside mammalian cells by recombinant techniques or the like.

[0251] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. When the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. Thus, as used herein, "gene product" refers to the transcribed mRNA, the transcribed RNA prior to splicing (e.g., RNA still containing non-coding regions), the translated polypeptide (e.g., regardless of whether it has a signal peptide or other regions not present in the mature protein), and the 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 to the expression level of that gene from a control sample or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene from the same sample after administration of a compound.

[0252] As used herein, the term "isolated" refers to a molecule or biological or cellular substance that is substantially free from other substances. 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 that has been separated from other DNA or RNA, or protein or polypeptide, or cell or organelle, or tissue or organ, such as that present in a natural source. Also, the term "isolated" refers to a nucleic acid or peptide that substantially does not contain cell material, viral material, or medium when produced by recombinant DNA technology, or a chemical precursor or other chemical substance when chemically synthesized. Further, "isolated nucleic acid" means including nucleic acid fragments that do not exist naturally as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to a polypeptide isolated from other cellular proteins and means encompassing both purified polypeptides and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue isolated from other cells or tissues and means encompassing both cultured and manipulated cells or tissues.

[0253] As used herein, the terms "prevent", "prevented", "preventing" and the like refer to an approach for preventing, suppressing, or reducing the likelihood of occurrence or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or medical condition, or delaying the occurrence or recurrence of the symptoms of a disease or medical condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0254] As used herein, the term "sample" generally refers to a biological sample. A sample can be taken from tissue or cells derived from an individual. In some examples, a sample can include or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. A sample may be separated from its source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, feces, tears, sweat, or mucosal excretions, and other body fluids separated from their primary source prior to collection. In some examples, a sample is isolated from its primary source (such as cells, tissue, body fluids such as blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or otherwise concentrated from its primary source. In some cases, the primary source is homogenized prior to further processing. A sample can be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. A sample can also be purified or concentrated for nucleic acids or treated with RNase. A sample contains intact, fragmented, or partially degraded tissue or cells.

[0255] As used herein, the term "subject" generally refers to an individual having a biological sample undergoing processing or analysis, and in specific cases, an individual having or suspected of having cancer. The subject can be any living organism or animal subject that is the subject or material of a method, including mammals such as humans, experimental animals (e.g., primates, rats, mice, rabbits), domestic animals (e.g., cows, 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, for example, having or suspected of having a disease (sometimes referred to as a medical condition) such as a benign or malignant neoplasm or cancer. The subject can be undergoing treatment or have been treated. The subject may be asymptomatic. The subject may be a healthy individual but may also wish to prevent cancer. The term "individual" is used interchangeably with "subject". As used herein, a "subject" or "individual" can or cannot be housed in a medical facility and can be treated as an outpatient of a medical facility. An individual can receive one or more medical compositions via the Internet. The term "individual" may refer to a human or non-human animal of any age, thus including both adults and juveniles (i.e., children) and infants, and also including individuals in utero. This term is not intended to imply a need for medical treatment, and thus an individual can be part of an experiment, either voluntarily or involuntarily, whether for clinical or basic science research support.

[0256] As used herein, "treatment" or "therapy" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, including even a minimal decrease in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment can optionally include either a reduction or amelioration of one or more symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or of the symptoms associated therewith.

[0257] CAR-T cell-based treatment means show unprecedented patient responses and offer significant curative potential for certain blood malignancies, while success in other cancers, particularly solid tumors, remains challenging, partly due to the intrinsic characteristics of the tumor microenvironment characterized by hypoxia, acidic pH, nutrient depletion, and / or immunosuppression. Acidity is a prominent feature of the tumor microenvironment mainly due to acidic metabolites such as lactic acid caused by active glycolysis. Acidity mediates immunosuppression, tumor progression, and poor prognosis. Specifically, tissue acidosis leads to the suppression of immune cell-mediated responses such as the reduction of the cytotoxicity, cytokine production, and tumor surveillance functions of natural killer (NK) cells and T cells.

[0258] The cAMP pathway mediates major immunosuppressive effects in effector cells and is involved in the response of immune cells to acidity and hypoxia via 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 then results in the activation of transcription factors in the cAMP pathway that promote an anti-inflammatory response and lead to the suppression of immune cell function. As described herein, in some embodiments, the major elements of the immune cell response to acidity were genetically engineered. In some embodiments, the cAMP signaling pathway in immune effector cells was targeted for engineered mutations. In some embodiments, mutations in the cAMP signaling pathway render cell therapy insensitive to acidic immunosuppressive effects and thus can increase cell survival, proliferation, and / or immune function in the acidic tumor microenvironment.

[0259] In some embodiments, the techniques described herein include the use of gene editing techniques (e.g., CRISPR-Cas techniques), which are utilized to mutate (e.g., knockout) gene elements disclosed herein. In some embodiments, the cAMP response element modulator (CREM), a prominent and important transcription factor, is mutated (e.g., knocked out). As shown herein, in some embodiments, knockout of CREM from NK cells results in improved persistence and proliferation of NK cells, as well as antitumor activity against cancer cell lines characterized by active glycolysis and significant acidosis in their microenvironment. In some embodiments, the targeted gene manipulation strategy is utilized to target CREM and / or other genes of the cAMP signaling pathway, and / or genes encoding proton receptors, as disclosed herein. In some embodiments, the gene manipulation strategy is utilized in different forms of cell therapy, including CAR-T cells, T cells, CAR-NK cells, NK cells, T cell receptor (TCR)-T cells, TCR-NK cells, and / or tumor-infiltrating lymphocytes (TIL). In some embodiments, such gene manipulation enhances cell therapy against various types of cancer, including those against solid tumors.

[0260] In some embodiments, disclosed herein are compositions comprising genetically engineered immune effector cells, and / or methods comprising methods of manufacturing the same. In some embodiments, the gene manipulation includes, but is not limited to, knockout of genes encoding proteins involved in downstream signaling by the acidity-sensing and immunosuppressive cAMP pathway, including CREM, ICER, CREB1, GPR4, GPR31, GPR68, GPR81, GPR151, and / or GPR132. In some embodiments, the gene manipulation is performed by use of endonuclease-mediated cleavage (e.g., CRISPR-Cas techniques). In some embodiments, the compositions disclosed herein are utilized in methods of treating cancer.

[0261] In certain embodiments, gene knockout of genes involved in the response to acidity and / or their associated downstream signaling pathway components improves the effectiveness of engineered immune cells against various tumors. For example, CAR-T cells, which are FDA-approved as a treatment for leukemia, lymphoma, and multiple myeloma, may potentially lead to an expansion of this therapy to resistant tumors such as solid tumors. In attempts to increase their effectiveness in the acidic TME of various cancers, these genes: CREM, ICER, CREB1, GPR4, GPR31, GPR68, GPR81, GPR151, and / or GPR132 can be genetically engineered to contain mutations in one or more of them. Furthermore, this genetic engineering strategy can be used in various other forms of cell therapy such as CAR-NK cells, TCR-T cells, TILs, etc. to enhance their activity against various cancer types.

[0262] As described herein, single-cell RNA sequencing data was analyzed. The results showed that in tumor-infiltrating immune cells, there was higher expression of proton-sensitive GPR and the transcription factor CREM compared to their peripheral blood counterparts (Figures 1A and 1B). CREM is a transcriptional repressor of the cAMP pathway that is upregulated in response to acidity signals via proton-sensitive GPR. 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 umbilical cord blood-derived NK cells and CAR-NK cells. In some embodiments, gene manipulation techniques such as the CRISPR-Cas9 editing system are employed to manipulate the target gene. In some embodiments, one or more guide RNA molecules are designed for the target gene of interest described herein. In some embodiments, one or more guide RNA molecules are utilized to generate mutations in the target gene of interest described herein. In some embodiments, the target gene of interest is specifically CREM. In some embodiments, mutations in CREM (e.g., CREM knockout) provide immune effector cells (e.g., NK cells) that have a significant growth advantage over CREM wild-type (WT) cells (e.g., WT NK cells). In some embodiments, mutations in CREM (e.g., CREM knockout) provide immune effector cells (e.g., NK cells) that have an immune phenotype characterized by activation and cytotoxicity. In some embodiments, mutations in CREM (e.g., CREM KO) provide immune effector cells (e.g., NK cells) that have enhanced metabolic adaptability. In some embodiments, the enhanced metabolic adaptability is indicated by an increase in oxidative phosphorylation and / or glycolytic activity. In some embodiments, CREM KO effector cells (e.g., NK cells) with or without additional manipulations (e.g., the presence or absence of a chimeric antigen receptor (CAR)) have increased cytotoxicity compared to CREM WT effector cells (e.g., NK cells) with or without additional manipulations (e.g., the presence or absence of a CAR).In some embodiments, the improvement in cytotoxicity associated with CREM mutations is particularly sensitive when examined under acidic pH conditions that normally suppress the cytotoxicity of effector cells. In some embodiments, mutations in CREM (e.g., knockout of CREM) improve long-term cytotoxicity in either conventional 2D culture and / or 3D culture where tumor cells grow as spheroids to mimic the characteristics of solid tumors. In some embodiments, mutations in CREM (e.g., knockout of CREM) improve long-term cytotoxicity in the in vivo solid tumor microenvironment. In some embodiments, the tumor microenvironment is characterized by an increased level of lactate compared to the non-tumor microenvironment. II. Gene editing of cells having reduced or inhibited levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 expression

[0263] Prior to the expansion and genetic modification of the cells of the present disclosure, the source of the cells 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, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, tumor, or commercially available sources. Any number of immune cell lines that are available and known to those of skill in the art can be used.

[0264] In certain embodiments, any type of immune effector cell is genetically edited to modify the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 within the cell. In specific cases, the cells are modified to have reduced levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 expression, which includes complete inhibition of detectable expression of certain isoforms of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 (e.g., it can be referred to as a knockout). Such cells may or may not be expanded before and / or before use.

[0265] In certain cases, the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are disrupted and their expression is reduced in part or in whole. In specific cases, the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are knocked down or knocked out using the processes of the present disclosure. In certain embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are disrupted (e.g., mutated) such that one or more RNA isoforms encoded by the one or more mutated genes described above are upregulated compared to the non-mutated copies of the same gene.

[0266] Those skilled in the art will also recognize methods of manipulating any cell, including any immune cell, such that the expression of the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes is reduced or completely inhibited. Certain embodiments utilize means that include targeting the polynucleotide sequence of a particular gene whose expression is desired to be reduced or completely inhibited.

[0267] In some embodiments, the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are disrupted in a heterozygous fashion. In some embodiments, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are disrupted in a homozygous fashion. In some embodiments, a population of immune effector cells comprising disruption of the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes comprises immune cells that are homozygous for the wild-type gene, heterozygous for the wild-type gene and the disrupted gene, and / or homozygous for the disrupted gene. In some embodiments, the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are disrupted in the majority of alleles in a population of immune effector cells. In some embodiments, the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are disrupted in alleles that are greater than or equal to about or exactly 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the genes in the population, or any range derivable therefrom.

[0268] The G protein-coupled receptor 4 ((GPR4), also known as GPR6C.1) gene sequence example is found in the genomic sequence of NC_000019.10, GRCh38.p14 primary assembly (range 45589764 - 45602212 complementary) with gene ID 2828 (GPR4) in the National Center for Biotechnology Information GENBANK® database, the mRNA sequence NM_005282.3, and the protein sequence NP_005273.1, each of which is hereby incorporated by reference in its entirety. In some embodiments, GPR4 mutates as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, GPR4 mutates as a result of exposing cells 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.

[0269] G protein-coupled receptor 31 (also known as HETER; HETER1; and / or 12-HETER) Examples of gene sequences are available in the National Center for Biotechnology Information GENBANK® database under Gene ID 2853 (GPR31), see NC_000006.12, GRCh38.p14 primary assembly (range 167155247 - 167157980 complementary) for the genomic sequence, NM_005299.3 for the mRNA sequence, and NP_005290.2 for the protein sequence, each of which is hereby incorporated by reference in its entirety. In some embodiments, GPR31 mutates as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, GPR31 mutates as a result of exposing cells 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.

[0270] G protein-coupled receptor 68 (also known as OGR1; A12A6; and / or GPR12A) Examples of gene sequences are available in the National Center for Biotechnology Information GENBANK® database under gene ID 8111 (GPR668), NC_000014.9 reference GRCh38.p14 primary assembly (range 91232532-91270790 complementary), the genomic sequence, the mRNA sequence NM_001177676.2 encoding transcript variant 1, the mRNA sequence NM_003485.3 encoding transcript variant 2, the mRNA sequence NM_001348437.1 encoding transcript variant 3, the protein sequences NP_001171147.1, NP_001335366.1, and NP_003476.3, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR68 is mutated as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, GPR68 is mutated as a result of exposing cells 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.

[0271] G protein-coupled receptor 81 (GPR81), also known as hydroxycarboxylic acid receptor 1 (HCAR1); HCA1; LACR1; FKSG80; GPR104; TAGPCR; and / or TA-GPCR), examples of the gene sequences of which can be found in the National Center for Biotechnology Information GENBANK® database under gene ID 27198 (HCAR1), are referenced in NC_000012.12 of the GRCh38.p14 primary assembly (range 122726076~1122730844 complementary) for the genomic sequence, NM_032554.4 for the mRNA sequence, and NP_115943.1 for the protein sequence, each of which is hereby incorporated by reference in its entirety. In some embodiments, GPR81 mutates as a result of exposing cells to a polynucleotide comprising the sequences provided in Table 2. In some embodiments, GPR81 mutates as a result of exposing cells 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.

[0272] The G protein-coupled receptor 132 (GPR132), also known as G2A, gene sequences examples are available at gene ID 29933 (GPR132) in the National Center for Biotechnology Information GENBANK® database, referring to the genomic sequence of NC_000014.9 GRCh38.p14 primary assembly (range 105049395 - 105065430 complementary), the mRNA sequences NM_001278694.2 and NM_013345.4 encoding protein isoform 1, the mRNA sequence NM_001278695.2 encoding protein isoform 2, the mRNA sequence NM_001278696.2 encoding protein isoform 3, the protein sequences NP_001265623.1 and NP_037477.1 of protein isoform 1, the protein sequence NP_001265624.1 of protein isoform 2, and the protein sequence NP_001265625.1 of protein isoform 3, each of which is hereby incorporated by reference in its entirety. In some embodiments, GPR132 mutates as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, GPR132 mutates as a result of exposing cells 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.

[0273] G protein-coupled receptor 151 (also known as GPCR; PGR7; GALR4; GALRL; and / or GPCR-2037) Examples of gene sequences are in the genome sequence of NC_000005.10 reference GRCh38.p14 primary assembly (range 146513144~146516190 complementary), mRNA sequence NM_194251.3, and protein sequence NP_919227.2 of gene ID 134391 (GPR151) in the National Center for Biotechnology Information GENBANK® database, each of which is incorporated herein by reference in its entirety. In some embodiments, GPR151 mutates as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, GPR151 mutates as a result of exposing cells 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.

[0274] The cyclic AMP-responsive element modulator ((CREM), also known as ICER; CREM-2; and / or hCREM-2) gene sequences are exemplified by the genomic sequence of NC_000010.11 (range 35126846 - 35212958 complementary) of gene ID 1390 (CREM) in the National Center for Biotechnology Information GENBANK® database, or in NG_029065.1 RefSeqGene (range 5129 - 91081), each of which is hereby incorporated by reference in its entirety. The CREM gene encodes multiple RNA and protein isoforms, some of which are the inducible cAMP early repressor (ICER) RNA isoforms that encode the ICER protein.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_001352445.1, NM_001352446.1, NM_001352465.2, NM_001352466.2, NM_001352467.2, NM_001394595.1, NM_001394598.1, NM_001394600.1, NM_001394602.1, NM_001394603.1, NM_001394605.1, NM_001394608.1, NM_001394610.1, NM_001394613.1, NM_001394614.1, NM_001394615.1, NM_001394616.1, NM_001394617.1, NM_001394618.1, NM_001394619.1, NM_001394620.1, NM_001394621.1, NM_001394622.1, NM_001394623.1, NM_001394625.1, 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 hereby incorporated 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_001339394.1, NP_001339395.1, NP_001339396.1, NP_001381524.1, NP_001381527.1, NP_001381529.1, NP_001381531.1, NP_001381532.1, NP_001381534.1, NP_001381537.1, NP_001381539.1, NP_001381542.1, NP_001381543.1, NP_001381544.1, NP_001381545.1, NP_001381546.1, NP_001381547.1, NP_001381548.1, NP_001381549.1, NP_001381550.1, NP_001381551.1, NP_001381552.1, NP_001381554.1, 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 hereby incorporated by reference in its entirety.CREM RNA isoforms encoding the ICER protein include NM_182717.2, NM_182718.2, NM_182719.2, NM_182720.2, NM_182721.2, NM_182723.2, NM_182724.2, while the ICER protein variant sequences are represented by NP_874386.1 (isoform ICER1), NP_874387.1 (isoform ICER11 gamma), NP_874388.1 (isoform ICER11), NP_874389.1 (isoform ICER1 gamma), NP_874390.1 (isoform 8 alias h), NP_874392.1 (isoform 10 alias j), and NP_874393.1 (isoform 11 alias k). In some embodiments, CREM mutates as a result of exposure of cells to a polynucleotide comprising the sequences provided in Table 2. In some embodiments, CREM mutates as a result of exposing cells 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: 140-161 and 181-184. In some embodiments, CREM mutates as a result of exposing cells 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 SEQ ID NO: 140 and / or 142.

[0275] Examples of the gene sequence of cyclic AMP-responsive element-binding protein 1 (CREB1, also known as CREB and / or CREB-1) are in the genomic sequence of NC_000002.12, GRCh38.p14 primary assembly (range 207529962-207605988 complementary) with gene ID 1385 (CREB1) in the National Center for Biotechnology Information GENBANK® database. On the other hand, the mRNA sequences are represented by, but not limited to, NM_001320793.2, NM_001371426.1, NM_001371427.1, NM_001371428.1, NM_004379.5, NM_134442.5, and the protein sequences are represented by, but 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 hereby incorporated by reference in its entirety. In some embodiments, CREB1 is mutated as a result of exposing cells to a polynucleotide comprising the sequence provided in Table 2. In some embodiments, CREB1 is mutated as a result of exposing cells 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: 162-180.

[0276] In some embodiments, the 5'-3' DNA sequences of exemplary guide RNAs for knocking out the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes are found in Table 2. In some embodiments, these guide RNA sequences are suitable for use with the CRISPR / Cas9 technology that utilizes guide RNAs (complementary to short target DNA sequences on the targeted gene) to effect double-stranded DNA cleavage. The guide RNA can be either the sense or antisense strand, but since the cleavage performed using the CRISPR / Cas9 technology affects both strands of the target DNA, the target sequences on the sense strand of the sequences are shown here.

[0277] In some embodiments, after mutagenesis by an endonuclease, oligonucleotide amplification techniques (e.g., PCR) can be utilized to determine the mutagenesis efficiency and / or the mutant form. In some embodiments, for the PCR reaction, primers that flank (e.g., sandwich) the editing region are utilized to amplify the target sequence. In some embodiments, primers suitable for amplification of the target locus can include, but are not limited to, SEQ ID NOs: 1 and 2.

Table 1

[0278] Embodiments of the present disclosure include methods of knocking out or knocking down the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 in cells, including contacting the cells with at least Cas9, or a functionally equivalent alternative, and an appropriate guide RNA targeting GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. Cas9 and / or the guide RNA can be provided to the cells through expression from one or more expression vectors encoding the same. The vectors can be viral (retrovirus, lentivirus, adenovirus, adeno-associated virus) or non-viral (naked plasmid DNA or chemically modified mRNA).

[0279] In specific cases, other gene(s) other than GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 may or may not be knocked down or knocked out, which may occur in the same process as the knockout or knockdown of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. The decrease or complete inhibition of expression may or may not utilize the same mechanism as the gene editing of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and the decrease or complete inhibition of the expression of other gene(s) may occur before, during, or after the gene editing of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. The gene to be edited intracellularly can be of any kind, but in a specific embodiment, the gene is one whose gene product inhibits the activity and / or proliferation of KO cells of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. In specific cases, the gene edited in addition to GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 enables the cell to function more effectively in the tumor microenvironment. In specific 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 a specific embodiment, the TGFBR2 gene is knocked out or knocked down intracellularly. In a specific embodiment, the CISH gene is knocked out or knocked down intracellularly. In a specific embodiment, the CD38 gene is knocked out or knocked down intracellularly.In a specific embodiment, the glucocorticoid receptor (GR) gene is knocked out or knocked down intracellularly.

[0280] In some embodiments, any gene editing intracellularly is performed using one or more DNA binding molecules such as, but not limited to, endonucleases such as Cas enzymes (and their mutants), zinc finger nucleases, TALENs, and meganucleases. In some embodiments, any gene editing intracellularly is performed by one or more DNA binding nucleic acids such as modification via an RNA-guided endonuclease (RGEN). For example, the modification can be performed using regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins; in some embodiments, Cpf1 is utilized instead of Cas9. Generally, the "CRISPR system" collectively refers to transcripts and other elements involved in the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active part of tracrRNA), tracr corresponding sequences (including "direct repeats" and processed part direct repeats of tracrRNA in the context of endogenous CRISPR systems), guide sequences (also called "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0281] The CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA sequence specifically, 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 specific organism including type I, type II, or type III CRISPR systems, e.g., an endogenous CRISPR system such as Streptococcus pyogenes.

[0282] In some embodiments, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into a cell. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can typically be selected based on the position immediately 5' of a protospacer adjacent motif (PAM) sequence such as NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence designed such that the guide sequence has complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.

[0283] The CRISPR system can induce a double-strand break (DSB) at the target site, subsequently inducing disruption or modification as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each being directed by a pair of different gRNAs that target sequences such that a 5' overhang is introduced when the nicks are introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, affecting gene expression.

[0284] The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence can be located within the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into a targeted locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide can be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0285] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). Also, the tracr sequence can comprise all or part of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence) or can consist of all or part thereof, and can form part of the CRISPR complex, such as by hybridizing with all or part of a tracr corresponding sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr corresponding sequence to hybridize and participate in the formation of the CRISPR complex, e.g., having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr corresponding sequence when optimally aligned.

[0286] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell such that the expression of the elements of the CRISPR system leads to the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cell as proteins and / or RNAs. For example, a Cas enzyme, a guide sequence linked to a tracr complementary sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. 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 can be provided to supply any components of the CRISPR system not included in the first vector. The vector can 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 multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0287] The vector may include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme such as 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, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are known, for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the accession number Q99ZW2 of the SwissProt database.

[0288] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumoniae). In some cases, Cpf1 can be used as an endonuclease instead of Cas9. The CRISPR enzyme can effect a direct cleavage of one or both strands at the position of the target sequence, such as within and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence(s), e.g., two guide sequences that each target the sense and antisense strands of a DNA target, respectively. This combination makes it possible to use it to nick both strands and induce NHEJ or HDR.

[0289] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in certain cells, such as eukaryotic cells. Eukaryotic cells can be of or derived from certain organisms, such as mammals including, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance its expression in a target host cell by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit specific biases for certain codons of a particular amino acid. Codon bias (the difference in codon usage among organisms) is often correlated with the translation efficiency of messenger RNA (mRNA), and as a result, is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of a selected tRNA within a cell generally reflects the codons that are most frequently used in peptide synthesis. Thus, genes can be modified based on codon optimization to enable optimal gene expression in a given organism.

[0290] Generally, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more when optimally aligned using an appropriate alignment algorithm.

[0291] The optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., 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).

[0292] A CRISPR enzyme can be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and 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, and protein domains having one or more of these. Non-limiting examples of epitope tags include a histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-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 a fragment of a protein 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. Additional domains that can form part of a fusion protein containing a CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, which is incorporated herein by reference.

[0293] In some embodiments, the gene editing molecule comprises one or more DNA-binding proteins such as zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs) fused to an effector protein such as an endonuclease. Examples include at least ZFN, TALE, and TALEN.

[0294] In some embodiments, the gene editing molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. The ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner through an amino acid sequence region within a binding domain whose structure is stabilized by coordination of one or more zinc fingers, zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among ZFPs, there are artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides in length, generated by assembling individual fingers. ZFPs contain an alpha helix approximately 30 amino acids in length per finger domain, containing two invariant histidine residues coordinated via two cysteines and zinc of one beta turn, and include those having 2, 3, 4, 5 or 6 fingers. Generally, the sequence specificity of ZFPs can be altered by making amino acid substitutions at four helix positions (-1, 2, 3 and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is not naturally occurring and has been engineered, for example, 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 derived from the type IIS restriction endonuclease Fok I. Fok I generally catalyzes double-strand cleavage of DNA 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. Many gene-specific engineered zinc fingers are commercially available.For example, Sangamo Biosciences (Richmond, CA, USA) developed a platform (CompoZr) for zinc finger construction in collaboration with Sigma-Aldrich (St. Louis, MO, USA), enabling researchers to completely bypass zinc finger construction and validation and providing zinc fingers specifically targeted to thousands of proteins (Gaj et al., Trends in Biotechnology, October 2013, Vol. 31, No. 7, pp. 397-405). In some embodiments, commercially available zinc fingers are used or custom designed (see, e.g., Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTil-lKT, and PZD0020).

[0295] In some embodiments, the gene editing molecule comprises a naturally occurring or engineered (non-naturally occurring) transcriptional activator-like protein (TAL) DNA binding domain, such as in a transcriptional activator-like effector (TALE) protein. See, for example, U.S. Patent Application Publication No. 2011 / 0301073, which is hereby incorporated by reference in its entirety. A TALE DNA binding domain or TALE is a polypeptide that includes one or more TALE repeat domains / units. The repeat domain is involved in the binding of the TALE to its corresponding 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 typically includes one or two DNA binding residues that constitute a repeat variable diresidue (RVD) at positions 12 and / or 13 of the repeat. The native (canonical) code for DNA recognition by these TALEs has been determined such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T, and non-canonical (non-standard) RVDs are also known. In some embodiments, a TALE can be targeted to any gene by design of a TAL array that has specificity for a target DNA sequence. The target sequence generally begins with thymidine. In some embodiments, the molecule is a DNA-binding endonuclease such as a TALE nuclease (TALEN). In some aspects, a TALEN is a fusion protein that includes a DNA binding domain derived from a TALE and a nuclease catalytic domain that cleaves a nucleic acid target sequence. In some embodiments, a TALEN recognizes and cleaves a target sequence in a gene. In some aspects, the cleavage of DNA results in a double-strand break. In some aspects, the cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process and often results in changes in the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves rejoining of the remaining portions of the two DNA ends via direct religation or via so-called microhomology-mediated end joining.In some embodiments, repair via NHEJ results in small insertions or deletions, which can disrupt and thereby suppress a gene. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event subsequent to 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 a gene (Gaj et al., 2013). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Specifically, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0120622 and 2013 / 0315884. In some embodiments, TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selectable marker that provides for the identification and / or selection of cells that have received the vector.

[0296] III. Immune effector cells The present disclosure relates to genetically engineering immune effector cells such that it involves a partial reduction or complete inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. In some embodiments, the partial reduction or complete inhibition of the 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, and creates an innovative and effective cell therapy for the treatment of any type of cancer including solid tumors.

[0297] The present disclosure encompasses any type of immune effector cells modified such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is reduced or completely inhibited. In specific embodiments, the present disclosure encompasses any type of immune effector cells modified such that the expression of CREM is reduced or completely inhibited. In specific embodiments, the reduction or complete inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 within the cell is a direct or indirect result of the intentional manipulation of the cell by a human. Manipulating immune effector cells such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is reduced or completely inhibited can be by any mechanism including homologous recombination or non-homologous recombination. In specific embodiments, the cell is manipulated such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is reduced or completely inhibited as a result of, for example, CRISPR technology.

[0298] In contrast to natural cells having one or more mutations that result in decreased expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, immune effector cells, in particular, have decreased or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 by genetic manipulation. Thus, in specific embodiments, immune effector cells are genetically engineered to decrease or inhibit the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 in the genome of the immune effector cell. In specific embodiments, the expression of endogenous GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is knocked out in the immune effector cells.

[0299] The present disclosure encompasses any type of immune effector cells, including conventional T cells, gamma-delta 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 can be allogeneic, autologous, or xenogeneic to an individual in need of the cells, such as an individual having cancer.

[0300] In certain embodiments, the immune effector cells are modified by human hand to express or otherwise produce one or more gene products other than cells modified such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is decreased or completely inhibited. Such additional modification(s) to the cells are not naturally present in the cells or are exogenous to the cells. The additional modification(s) can be of any kind, for example, immune effector cells that express a receptor, cytokine, suicide gene, chemokine, or a combination thereof.

[0301] Immune effector cells having a reduced or completely inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, when additionally modified to produce or express a gene product that is not naturally present intracellularly or is of exogenous origin, can be of any kind in the order in which the immune effector cells are modified. For example, immune effector cells having a reduced or completely inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 can be modified to have one or more additional modifications, and in other cases, the immune effector cells are modified to produce or express a gene product that is not naturally present intracellularly or is of exogenous origin and then modified such that the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is reduced or completely inhibited.

[0302] In certain embodiments, immune effector cells lacking complete or partial expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are the same cells modified to express a receptor such as an antigen receptor. Any immune effector cell encompassed by the present disclosure can express an antigen receptor of any kind, including a receptor having specificity for an antigen that can also be a tumor antigen, a cancer antigen. In specific embodiments, the receptor is, for example, a chimeric antigen receptor or a T cell receptor. The immune effector cells can be specifically designed to have a complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 and can be specifically designed to have an antigen receptor that targets an antigen on cancer cells in an individual. That is, the cells can be modified to include one or more antigen receptors that target an antigen known to be present on the cancer cells of an individual.

[0303] In certain embodiments, the cells of the present disclosure are generated for the purpose of being used as off-the-shelf cells. For example, cells having complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are present in, for example, a repository, and they are obtained from the repository and engineered to have additional modifications other than complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. In other cases, cells having modifications other than complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 are obtained from a repository and engineered to have complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. After obtaining the cells from the repository and after subjecting the cells to such modifications, the cells can be stored or an effective amount of the cells can be provided to an individual in need thereof. Further engineering of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 KO or knockdown cells can be to engineer them to express engineered receptors, such as engineered antigen receptors, that target tumor antigens suitable for the treatment of individuals having a particular cancer expressing the antigen.

[0304] In certain embodiments, the immune effector cells have complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and express one or more engineered antigen-targeting receptors, and / or express at least one transfected (not endogenous to the cell) cytokine, and / or express at least one suicide gene. In some cases of cells having complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, different vectors encode the antigen-targeting receptor(s), whereas the suicide gene(s) and / or transfected cytokine(s) are encoded by other vectors. Immune cells including NK cells can be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (hematopoietic stem cells), bone marrow, or mixtures thereof. NK cells can be derived from cell lines such as, but not limited to, NK-92 cells. NK cells can be umbilical cord blood mononuclear cells such as CD56+ NK cells.

[0305] This disclosure describes and / or shows successful knockout (KO) of the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes using CRISPR / Cas9 from natural killer (NK) cells derived from umbilical cord blood stored in an umbilical cord blood bank. This disclosure shows how NK cells with disrupted cAMP signaling pathways have enhanced antitumor activity compared to NK cells with WT cAMP signaling pathways under acidic conditions or in in vivo-like conditions shown to be acidic in the literature. This enhanced antitumor activity was shown against solid tumor cell lines known to have active glycolysis and a prominent acidic tumor microenvironment. In certain embodiments, the tumor microenvironment is characterized by increased levels of lactate compared to the non-tumor microenvironment.

[0306] In some cases, immune effector cells having complete or partial inhibition of the expression of GPR4, GPR31, GPR6, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 were expanded in the presence of an effective amount of universal antigen-presenting cells (UAPCs) or fragments thereof, comprising any suitable ratio. The cells can be cultured with UAPCs, for example, at 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 a ratio of 1:2. In some cases, NK cells were expanded in the presence of IL-2, for example, at a concentration of 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400, or 400 to 500 U / mL.

[0307] After gene modification with any vector(s), immune effector cells having a partial or complete decrease in the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 can be delivered immediately to an individual or can be preserved (or a portion of the cells is delivered to the individual and the remainder of the cells is preserved). In certain embodiments, after gene modification, the cells can be expanded ex vivo as a bulk population for several days, weeks, or months within about 1, 2, 3, 4, 5 days or more after gene introduction into the cells. In further embodiments, the transfectants are cloned and clones demonstrating the presence of a single integrated or episomally maintained expression cassette or plasmid are expanded ex vivo. Clones selected for expansion demonstrate a decrease or absence of 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 the 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. In further embodiments, the gene-modified cells can be cryopreserved.

[0308] In some embodiments, the cells are preconditioned with one or more inactivating agents (e.g., kinase inhibitors such as dasatinib, nilotinib, rapamycin, etc.). In some embodiments, the techniques described herein include inactivating NK cells and treating the NK cells with an effective amount of one or more inactivating agents under conditions that 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, pegdastinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gilteritinib, midostaurin, tucatinib, neratinib, baricitinib, luxolutinib, fedratinib, tofacitinib, repotrectinib, selumetinib, binimetinib, cobimetinib, trametinib, upadacitinib, abemaciclib, serpelitinib, cabozantinib, fostamatinib, 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.

[0309] In some embodiments, the treatment with the inactivating agent is at any point during the culture of the NK cells. In some embodiments, the treatment is from about 24 hours to about 96 hours, from about 36 hours to about 84 hours, or from about 48 hours to about 72 hours. In some embodiments, the treatment is about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cells are treated with an inactivating agent at a concentration of about 1 to about 1000 nM. In some embodiments, the NK cells are treated with an inactivating agent at a concentration of about 5 to about 500 nM. In some embodiments, the NK cells are treated with an inactivating agent at a concentration of about 20 to about 200 nM. In some embodiments, the NK cells are treated with an inactivating agent at a concentration of about 30 to about 100 nM. In some embodiments, the inactivated NK cells have an increase in the expression of one or more of C-kit, CCR-5, CD62L, and / or CXCR4, and / or a decrease in the expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95, as compared to the activated NK cells.

[0310] In some embodiments, the techniques described herein include a method of maintaining the viability of a population of cells at at least 50% or more after cryopreservation of the population, the method comprising subjecting the population to an effective amount of one or more inactivating agents (e.g., tyrosine kinase inhibitors) to inactivate the cells prior to cryopreservation, cryopreserving the cells, and thawing the population, wherein the viability of the population at the time of thawing is at least 50% or more. Optionally, upon thawing of the cells, the viability of the population of cells is at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more after cryopreservation of the population.

[0311] Embodiments of the present disclosure include immune effector cells having complete or partial inhibition of the expression of one or more engineered receptors, including GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and one or more antigen receptors. The one or more engineered antigen receptors are generated by human hand, for example using recombinant techniques, and are not native to the immune effector cells. The engineered receptor(s) can be of any kind, but in a specific embodiment, the 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.

[0312] Embodiments of the present disclosure include cells having complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and one or more suicide genes. Immune effector cells can have complete or partial inhibition of the expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and can contain a recombinant nucleic acid encoding any type of suicide gene. Examples of suicide genes include engineered non-secretory (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see International Application No. PCT / US2019 / 062009, which is incorporated herein by reference in its entirety), which may be affected by the delivery of an antibody that binds to the TNF-alpha mutant. Examples of suicide gene / prodrug combinations that can be used include 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 cytarabine. The so-called suicide gene, Escherichia coli purine nucleoside phosphorylase, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be utilized. Other suicide genes include, by way of example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0313] Cells can be obtained directly from an individual or from a depository facility or other storage facility. Cells as a therapy can be autologous or allogeneic to the individual to whom the cells are provided as a therapy.

[0314] The cells can be from an individual in need of therapy for a medical condition and, after being engineered to have reduced or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, and optionally any suicide gene, any cytokine(s), and any receptor(s) (e.g., using standard techniques of transduction and expansion for adoptive cell therapy), can be returned to the individual from whom they were originally sourced. In some cases, the cells are stored for later use in that individual or another individual.

[0315] Immune cells can be included in a cell population that can have a majority having 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 can contain 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% immune cells having reduced or inhibited expression of these genes, 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 can or cannot be produced as a separate polypeptide.

[0316] Immune cells can be generated to 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, with the intention of being modularized for a particular purpose. For example, cells having 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 can be generated (or can be distributed together with a nucleic acid encoding a suicide gene for subsequent transduction), and the user can modify them to express one or more genes for other purposes (including therapeutic genes) according to the intended purpose(s). For example, an individual interested in treating cancer cells can obtain or produce 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.

[0317] In certain embodiments, NK cells are utilized and the genome of NK cells having 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 can be modified. The genome can be modified in any manner, but in a specific embodiment, the genome is modified, for example, by CRISPR gene editing. The genome of the cells can be modified for any purpose to enhance the effectiveness of the cells.

[0318] Also, in certain embodiments, provided herein are methods of producing immune effector cells that are at least acclimated to acidic conditions (e.g., accustomed to, adapted to, etc. acidic conditions) through contact with an acidic stimulus, methods of using such immune effector cells, and compositions comprising such immune effector cells. In some embodiments, the cells are conditioned ex vivo through contact with an acidic stimulus. In some embodiments, the cells are acclimated stepwise over time. In some embodiments, the cells are acclimated stepwise over time by exposure to an acidic stimulus that accumulates gradually. In some embodiments, the cells are acclimated for at least or equal to, exactly or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, or 30 days or more. In some embodiments, the cells are acclimated for a period of exactly or approximately 10 to 18 days. In some embodiments, the cells are acclimated for a period of exactly or approximately 14 days. In some embodiments, the cells are acclimated by contacting with an acidic stimulus at a concentration greater than or equal to, exactly or approximately 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 cells are acclimated by contacting with an acidic stimulus at a concentration greater than or equal to, exactly or approximately greater than 2 - 3 mM. In some embodiments, the cells are acclimated by contacting with an acidic stimulus at a concentration greater than or equal to, exactly or approximately greater than 2.5 mM. In some embodiments, the cells are acclimated with an acidic stimulus comprising or consisting essentially of lactic acid. In some embodiments, the acidic stimulus is added at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the acidic stimulus is added approximately or exactly every 2 - 3 days.In some embodiments, the cells are acclimated to acidic stimuli at a pH that is exactly or approximately pH 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 less than or equal to 5.0. In some embodiments, the cells are acclimated to acidic stimuli at a pH that is less than or equal to approximately or exactly pH 6.0 or pH 5.9.

[0319] IV. Treatment Methods Embodiments of the present disclosure include treatment methods related to cancer immunotherapy or anti - pathogen immunotherapy. For example, cancer immunotherapy and anti - pathogen immunotherapy include at least a composition comprising immune effector cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. The method includes providing an effective amount of immune effector cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 to an individual having cancer and / or a pathogen.

[0320] In certain cases, the individual is provided with an effective amount of cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1. In specific cases, knockout of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 using CRISPR / Cas9 is utilized to genetically engineer immune cells used in various cell therapies to increase their effectiveness against solid tumors, and these cell therapies are provided to the individual.

[0321] As an example, chimeric antigen receptor (CAR)-T cells, such as those approved by the FDA for treating leukemia and lymphoma, are genetically engineered to delete the GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 genes in order to increase their effectiveness in the acidic tumor microenvironment (TME) of solid tumors, and in certain embodiments, this results in the expansion of this therapy to solid tumors. Further, this genetic engineering strategy is used to enhance the efficacy against various types of solid tumors in various other forms of cell therapy, such as CAR-NK cells, engineered TCR-T cells, tumor infiltrating lymphocytes (TIL).

[0322] In certain embodiments, the cells of the present disclosure are provided to an individual for the purpose of ameliorating a medical condition such as any type of cancer and / or any type of pathogen infection. The uses of the cells contemplated herein, including pharmaceutical compositions containing the same, are for the prevention, treatment, or amelioration of cancerous diseases such as neoplastic diseases, or pathogen infections. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for the prevention, amelioration, and / or treatment of cancers, including cancers that may or may not be solid tumors.

[0323] In certain embodiments, the present disclosure contemplates the use of the cells encompassed herein, which may be administered, in part, alone or in any combination with one or more other therapies, and in at least some embodiments, 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 cells prior to delivery to a subject.

[0324] Furthermore, the present disclosure relates to a method for preventing, treating, or ameliorating a neoplastic disease, the method comprising administering to a subject in need thereof an effective amount of any cell having a reduced or inhibited expression level of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, as contemplated herein.

[0325] In one embodiment, an isolated cell obtained and engineered by any suitable method as encompassed herein, or from a cell line, can be used as a medicament. The medicament can be used for the treatment of cancer or an infectious disease in an individual in need thereof. In one embodiment, the isolated cells according to the present disclosure can be used in the manufacture of a medicament for treating cancer or an infectious disease in an individual in need thereof.

[0326] In some embodiments, the present disclosure provides a method for treating an individual in need thereof, the method comprising the following steps: (a) providing immune effector cells; (b) engineering the immune effector cells to have a reduced or inhibited 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 (step (c) can be simultaneous with or prior to step (b)); (d) engineering the immune effector cells to express one or more cytokines (step (d) can be simultaneous with or prior to step (b) or (c)); (e) administering the engineered cells to an individual in need thereof, including an individual determined to have cancer or an individual at risk of having cancer (such as more than the average person in the population). The method includes at least one of the steps.

[0327] In a specific embodiment, the engineered cells are specifically engineered for the purpose of generating enhanced expansion, persistence, and / or cytotoxicity compared to any type of non-engineered cells.

[0328] In certain embodiments, the engineered cells are specifically engineered for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) compared to non-engineered cells in a microenvironment (e.g., acidic environment) that has a pH of exactly or approximately 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, 5.0, or less than 5.0 or equal thereto. In certain embodiments, the engineered cells are specifically engineered for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) compared to non-engineered cells in an acidic microenvironment that has a representative low pH with an increased level of lactic acid compared to a non-acidic microenvironment. In certain embodiments, the engineered cells are specifically engineered for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) compared to non-engineered cells in an acidic microenvironment that has a low pH of less than or equal to approximately or exactly 6.0. In certain embodiments, the engineered cells are specifically engineered for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) compared to non-engineered cells in an acidic microenvironment that has a low pH of less than or equal to approximately or exactly 5.9. In certain embodiments, the acidic microenvironment comprises or is the tumor microenvironment (TME).

[0329] Any of the treatment methods of the present disclosure can be ameliorative, curative, or prophylactic for an individual. It can be part of an autoimmunotherapy or part of an allogeneic immunotherapy treatment. In certain cases, the method is utilized in allogeneic immunotherapy to the extent that it enables the transformation of NK cells typically obtained from a donor into non-reactive cells. This is performed under a standard protocol and can be reproduced as many times as necessary. The resulting engineered immune cells are pooled and administered to one or more patients and can be utilized as a "commercially available" therapeutic product. The cells can be stored, such as by cryopreservation.

[0330] In some embodiments, the administration of the cell composition(s) is for any type of cancerous disease, such as a neoplastic disease, e.g., B-cell malignancies, multiple myeloma, lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, or cervix. Exemplary indications for the administration of the cell composition(s) are cancerous diseases including any malignant tumor that expresses one or more of certain antigens associated with an individual's cancer. The administration of the composition(s) of the present disclosure is useful for all stages (I, II, III, and / or IV) and types of cancer, including, for example, minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0331] The present disclosure further encompasses co-administration protocols with other compounds that act through immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical regimens for the co-administration of the compound(s) of the invention may include co-administering simultaneously with, prior to, or after the administration of other components. Specific combination therapies include chemotherapy, radiation therapy, surgical therapy, hormonal therapy, or other types of immunotherapy.

[0332] Embodiments relate to kits comprising constructs that generate cells, nucleic acid sequences as defined herein, vectors as defined herein, and / or host cells (such as immune effector cells) as defined herein. Also contemplated is that the kits of the present disclosure include the pharmaceutical compositions described herein alone or in combination with additional agents to be administered to an individual in need of a medical treatment or intervention.

[0333] V. Genetically Engineered Receptors The immune cells of the present disclosure having decreased or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 can be further modified to express one or more non-endogenous gene products. The gene product can or cannot be a genetically engineered receptor. The receptor can be of any kind, including, for example, receptors for antigens, chemokines, or cytokines. When the receptor is for an antigen, the antigen can be a cancer antigen including a solid tumor antigen.

[0334] Immune effector cells having decreased or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 can be genetically engineered to express an antigen receptor that targets a specific antigen, and such cells can be specifically designed to target one or more antigens present on the cancer cells of an individual.

[0335] In a specific embodiment, immune effector cells comprising decreased or inhibited expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 can include engineered antigen receptors such as engineered TCRs or CARs. For example, the immune cells can be NK cells modified to express one or more CARs and / or TCRs having antigen specificity for one or more specific antigens. In some aspects, the immune cells are engineered to express an antigen-specific CAR or antigen-specific TCR, for example, by knocking in a CAR or TCR using, for example, CRISPR.

[0336] Suitable methods of modification are known in the art. See, for example, Sambrook and Ausubel supra. For example, using the transduction techniques described in Heemskerk et al., 2008, and Johnson et al., 2009, cells can be transduced to express a TCR having antigen specificity for a cancer antigen.

[0337] In some embodiments, the cell comprises one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as those obtained from another organism or cell that are not normally found in the cell being engineered and / or the organism from which such cells are derived. In some embodiments, the nucleic acids are not naturally occurring, such as non-naturally occurring nucleic acids (e.g., chimeric).

[0338] Exemplary antigen receptors that include CARs and recombinant TCRs, and methods for engineering and introducing the receptors into cells include, for example, those described in International Patent Application Publications WO2000 / 14257, WO2013 / 126726, WO2012 / 129514, WO2014 / 031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publications 2002131960, 2013287748, 20130149337, U.S. Patents 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 EP2537416; and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically engineered antigen receptors include the CARs described in U.S. Patent 7,446,190 and those described in International Patent Application Publication WO2014 / 055668.

[0339] A. Chimeric Antigen Receptor In some embodiments, the antigen-specific CAR comprises an extracellular domain that targets (including specifically binding to) a desired antigen and that comprises a) one or more intracellular signaling domains, b) a transmembrane domain, and c).

[0340] In some embodiments, the engineered antigen receptor comprises a CAR including an activating CAR or stimulatory CAR (see International Publication No. WO2014 / 055668), and / or an inhibitory CAR (iCAR, see, e.g., Fedorov et al., 2013). CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, and in some embodiments, a linker and / or transmembrane domain(s). Such molecules typically mimic or approximate signals through native antigen receptors, signals through receptors combined with co-stimulatory receptors, and / or signals through co-stimulatory receptors alone.

[0341] Certain embodiments of the present disclosure relate to the use of a nucleic acid encoding an antigen-specific CAR polypeptide, comprising a humanized CAR (hCAR) that is humanized to reduce immunogenicity, comprising at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the antigen-specific CAR can recognize an epitope comprising a shared space between one or more antigens. In certain embodiments, the binding region can comprise a complementarity-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.

[0342] The human antigen-targeted CAR component is contemplated to be encoded by nucleic acids derived from human genes. In some embodiments, such a component can be used to enhance cellular immunotherapy for human patients. In specific embodiments, the present disclosure includes full-length antigen-specific CAR cDNA or coding regions. The antigen-binding region or domain can be, for example, from a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference, the V H chain and fragments of the V L chain. The fragments can also be any number of different antigen-binding domains of human antigen-specific antibodies. In more specific embodiments, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0343] The sequence can be a multimer such as a diabody or multimer. The multimer is likely formed by cross-pairing the variable portions of the light and heavy chains to form a diabody. There can be multiple options for the hinge portion of the construct, such as completely deleted, maintaining the first cysteine, substituting proline instead of serine, cleaving up to the first cysteine, etc. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains, for example, either the CH2 or CH3 domain from human immunoglobulin, can be used. Also, the hinge, CH2, and CH3 regions of human immunoglobulin modified to improve dimerization can be used. Also, only the hinge portion of immunoglobulin can be used. A portion of CD8 alpha can also be used.

[0344] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory 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 primary signal initiated by CD3ζ, the additional signal provided by the human costimulatory receptor inserted into the human CAR is important for the full activation of NK cells and may help improve the in vivo persistence and therapeutic success of adoptive immunotherapy.

[0345] In some embodiments, antigen-specific CARs are constructed with specificity for antigens, such as antigens expressed on normal or non-disease cell types, or disease cell types. Thus, CARs typically comprise one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules, in their extracellular portions. In some embodiments, antigen-specific CARs comprise an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0346] In certain embodiments, the antigen-specific CAR can be co-expressed with cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR can be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof.

[0347] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or synthesis (such as PCR), or combinations thereof. Depending on the size of the genomic DNA and the number of introns, since introns have been found to stabilize mRNA, it may be desirable to use cDNA or combinations thereof. Additionally, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0348] It is contemplated that the chimeric construct can be introduced into immune cells either as naked DNA or within a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained within a plasmid expression vector in the appropriate orientation for expression.

[0349] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric construct into immune cells. Vectors suitable for use according to the methods of the present disclosure are non-replicative in immune cells. A number of vectors are known that are based on viruses where the copy number of the virus maintained within the cell is low enough to maintain cell viability, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0350] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane domains and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains of the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid the binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize the interaction with other members of the receptor complex.

[0351] The transmembrane domain in some embodiments is derived from either a natural or synthetic source. When the source is natural, the domain in some embodiments is derived from any membrane-bound or transmembrane protein. The transmembrane region includes those derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD30, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (i.e., including at least their transmembrane region(s)). Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, the synthetic transmembrane domain mainly contains hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0352] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells such as NK cells includes (i) non-viral gene delivery 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 having a variable-length extracellular domain that connects the CD70 recognition domain to the cell surface, and in some cases, (iv) a CAR + including K562-derived artificial antigen-presenting cells (aAPCs) that can robustly and abundantly expand immune cells (see, e.g., Singh et al., 2008; Singh et al., 2011).

[0353] In certain embodiments, the CAR recognizes the TROP2 antigen. In certain embodiments, the anti-TROP2 CAR and / or NK cells comprising the same are as described in International Patent Application Publication No. WO2023 / 283644A2, published on January 12, 2023, which is hereby incorporated by reference in its entirety.

[0354] In certain embodiments, the CAR recognizes the CD70 antigen. In certain embodiments, the anti-CD70 CAR comprises a polypeptide derived from CD27. In certain embodiments, the anti-CD70 CAR comprises or is encoded by a sequence having at least or equal to any one or more of SEQ ID NOs: 185-193 and exactly or approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In certain embodiments, the anti-CD70 CAR and / or the NK cells comprising the same are as described in International Patent Application Publication No. WO2021 / 142127A1, published on July 15, 2021, which is hereby incorporated by reference in its entirety. In certain embodiments, the anti-CD70 CAR and / or the NK cells comprising the same are as described in International Patent Application Publication No. WO2022 / 159791A1, published on July 28, 2022, which is hereby incorporated by reference in its entirety. In certain embodiments, the anti-CD70 CAR and / or the NK cells comprising the same are as described in International Patent Application Publication No. WO2023 / 278520A1, published on January 5, 2023, which is hereby incorporated by reference in its entirety. SEQ ID NO: 185 - Exemplary truncated CD27 amino acid sequence MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 186 - Exemplary truncated CD27 amino acid sequence MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR SEQ ID NO: 187 - Exemplary CD28 TMD FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 188, Exemplary full - length WT CD27 amino acid sequence MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO: 189, Exemplary truncated WT CD27 amino acid sequence ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR SEQ ID NO: 190 - Exemplary CD27 signal peptide (SP) amino acid sequence MARPHPWWLCVLGTLVGLS SEQ ID NO: 191 - Exemplary intracellular signaling domain (ICD) amino acid sequence derived from CD3z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG SEQ ID NO: 192 - Exemplary CD28-derived intracellular signaling domain (ICD) amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 193 - Exemplary intracellular signaling domain (ICD) amino acid sequence derived from DAP10 LCARPRRSPAQEDGKVYINMPGRG

[0355] B. T cell receptor (TCR) In some embodiments, the genetically engineered antigen receptor comprises a recombinant TCR and / or a TCR cloned from naturally occurring T cells. A "T cell receptor" or "TCR" refers to a molecule that includes variable alpha and variable beta chains (also known as TCRα and TCRβ, respectively) or variable gamma and variable delta chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigen peptide bound to a major histocompatibility complex (MHC) receptor. In some embodiments, the TCR is of the αβ type.

[0356] Generally, TCRs present in the αβ type and γδ type are generally structurally similar, but the T cells expressing them may have different anatomical locations and functions. TCRs may be present on the cell surface or in a soluble form. Generally, TCRs are present on the surface of T cells (or T lymphocytes) and generally play a role in recognizing antigens bound to MHC molecules. In some embodiments, TCRs can also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some aspects, each chain of a TCR can have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass its functional TCR fragments. This term also encompasses intact or full-length TCRs, including αβ-type or γδ-type TCRs.

[0357] Accordingly, as used herein, a TCR refers to any TCR that binds to an MHC molecule, i.e., a specific antigen peptide bound in an MHC-peptide complex, or a functional fragment such as the antigen-binding portion of a TCR. The "antigen-binding portion" or "antigen-binding fragment" of a TCR may be used interchangeably and refers to a molecule that includes a part of the structural domain of a TCR but binds to the antigen (e.g., an MHC-peptide complex) to which the full TCR binds. In some cases, the antigen-binding portion includes the variable domains of a TCR, e.g., the variable α chain or variable β chain of a TCR, and is generally sufficient to form a binding site for binding to a specific MHC-peptide complex such that each chain includes three complementarity-determining regions.

[0358] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity determining regions (CDRs) similar to immunoglobulins, conferring antigen recognition by forming the binding site of the TCR molecule, determining peptide specificity, and determining peptide specificity. Typically, as with immunoglobulins, the 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 major CDR that recognizes the processed antigen, although CDR1 of the α-chain has also been shown to interact with the N-terminal portion of the antigen peptide, and CDR1 of the β-chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β-chain can include an additional hypervariable (HV4) region.

[0359] In some embodiments, the TCR chains include constant domains. For example, similar to immunoglobulins, the extracellular portion of the TCR chains (e.g., the α-chain, β-chain) is a variable domain (e.g., V a or Vβ; typically, amino acids 1-116 based on the Kabat numbering of Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and one constant domain adjacent to the cell membrane (e.g., the α-chain constant domain or C a, typically from amino acids 117 to 259 based on Kabat, the beta-chain constant domain or Cp, typically from amino acids 117 to 295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains a short linker sequence where cysteine residues form disulfide bonds, creating a link between the two chains. In some embodiments, the TCR has additional cysteine residues in each of the alpha and beta chains, allowing the TCR to contain two disulfide bonds in the constant domain.

[0360] In some embodiments, the TCR chain can 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, this structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with an invariant subunit of the CD3 signaling apparatus or complex.

[0361] Generally, CD3 is a multi-protein complex and can have three different chains (γ, δ, ε) and the ζ chain in mammals. For example, in mammals, it can include the CD3γ chain, CD3δ chain, two CD3ε chains, and a homodimer of the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a feature that allows these chains to associate with the positively charged T cell receptor chains. Each of the intracellular tails of the CD3γ, CD3δ, and CD3ε chains contains one conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while the CD3ζ chain has three. Generally, ITAM is involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signals from the TCR intracellularly. The CD3- and ζ-chains together with the TCR form what is called the T cell receptor complex.

[0362] In some embodiments, the TCR may be a heterodimer of two chains, an α-chain and a β-chain (or optionally a γ-chain and a δ-chain), or a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (an α-chain and a β-chain, or a γ-chain and a δ-chain) linked by, for example, disulfide bonds. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, the nucleic acid encoding the TCR can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source such as a cell from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from a patient and the TCRs isolated. In some embodiments, the T cells can be cultured T cell hybridomas or clones. In some embodiments, TCR clones against a target antigen are generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, for example, tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or its antigen-binding portion can be generated synthetically from knowledge of the TCR's sequence.

[0363] VI. Cytokine One or more cytokines can be utilized in immune effector cells having reduced or inhibited 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 vector molecule as the engineered receptor, while in other cases, they are present on separate molecules. In certain embodiments, one or more cytokines are co-expressed from the same vector as the engineered receptor. One or more cytokines can be produced as a polypeptide separate from the antigen-specific receptor. In some embodiments, NK cells do not contain one or more engineered receptors but contain one or more heterologous cytokines. As an example, interleukin-15 (IL-15) can be utilized. IL-15 can be employed, for example, because IL-15 is tissue-restricted and is observed at any level in serum or systemically only under pathological conditions. IL-15 has several properties desirable 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 are able to continue supportive cytokine signaling, which is useful for survival after injection. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used for human applications. As an example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or a combination thereof. In certain embodiments, NK cells expressing IL-21 are able to continue supportive cytokine signaling, which is useful for survival after injection. In certain embodiments, the cytokine is expressed as part of a multicistronic construct with one or more functional proteins and / or marker proteins.Cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 and capable of expressing one or more cytokines can be utilized, and it is possible to continue supportive cytokine signaling, which is useful for survival after injection.

[0364] In a specific embodiment, NK cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 express one or more exogenously provided cytokines. Since the cytokines are expressed from an expression vector intracellularly, they may be supplied exogenously to the cells. In an alternative case, endogenous cytokines within the cell are upregulated by manipulation of the expression control of the endogenous cytokines, such as genetic recombination at the promoter site(s) of the cytokine. When the cytokine is supplied to the cell on an expression construct, the cytokine can be encoded from the same vector as a vector expressing another gene product such as a suicide gene. The cytokine can be expressed as a polypeptide molecule separate from the suicide gene and also as a polypeptide separate from the engineered receptor of the cell. In some embodiments, the present disclosure relates particularly to the co-utilization of CAR and / or TCR vectors with IL-15 in NK cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1.

[0365] VII. Suicide gene In certain embodiments, the suicide gene is utilized with any type of cell therapy to control its use and enable the termination of cell therapy at a desired event and / or time. The suicide gene is employed in transduced cells for the purpose of inducing the death of the transduced cells when needed. The immune effector cells of the present disclosure modified to carry the vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that, upon administration of a prodrug or other agent, results in the conversion of the gene product into a compound that kills the host cell. In other embodiments, the suicide gene encodes a gene product that is targeted, optionally, by an agent (such as an antibody) that targets the suicide gene product.

[0366] 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 and thymidylate kinase (Tdk:Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase can use a so - called suicide gene that converts the prodrug 6 - methylpurine deoxyriboside into the toxic purine 6 - methylpurine. Other examples of suicide genes used in prodrug therapy are the Escherichia coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0367] In addition, exemplary suicide genes include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated version of epidermal growth factor receptor variant III (EGFRv3) can be used as a suicide antigen that can be excised by cetuximab. Further suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyl transferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0368] In certain embodiments, a vector encoding an antigen-targeted CAR, or any vector in the NK cells encompassed herein, contains one or more suicide genes. The suicide gene may or may not be on the same vector as the antigen-targeted CAR. When the suicide gene is present on the same vector as the antigen-targeted CAR, the suicide gene and the CAR can be separated, for example, by an internal ribosome entry site (IRES) element or a 2A element.

[0369] In certain embodiments, the suicide gene is a TNF-alpha mutant that cannot be cleaved by a standard enzyme that cleaves TNF in nature, such as tumor necrosis factor (TNF)-alpha converting enzyme (also called TACE). Thus, the TNF-alpha mutant is, in certain embodiments, membrane-bound and non-secretable. The TNF-alpha mutant used in the present disclosure can be targeted by one or more agents that bind to the mutant, including at least an antibody, and after binding of the agent(s) to the TNF-alpha mutant on the cell surface, the cell dies. Embodiments of the present disclosure enable the use of the TNF-alpha mutant as a marker of cells that express it.

[0370] Cells expressing non-cleavable TNF-alpha mutants can be targeted for selective deletion, for example, using FDA-approved TNF-α antibodies such as etanercept, infliximab, or adalimumab currently in clinical use. The mutant TNF-alpha polypeptide can be co-expressed with one or more therapeutic transgenes in cells such as those encoding a TCR or CAR, including a CD70-targeted TCR and / or CAR. Furthermore, cells expressing TNF-alpha mutants have excellent activity against tumor targets, mediated through the biological activity of the membrane-bound TNF-alpha protein.

[0371] With respect to the wild type, TNF-alpha has a 26 kD transmembrane form and a 17 kD secreted component. Some mutants described by Perez et al. (1990) can be utilized in the present disclosure. In a specific embodiment, examples of the TNF-alpha mutants of the present disclosure include, with respect to 17 kD TNF, at least the following: (1) deletion of Val1 and deletion of Pro112; (2) deletion of Val13; (3) deletion of Val1 and deletion of Val13; (4) deletion from Val1 to Pro112 and deletion of Val13 (13 aa deletion); (5) deletion from Ala-3 to Val13 (14 aa deletion). In a specific embodiment, the TNF-alpha mutant includes a deletion of each amino acid at positions -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or combinations thereof. Specific combinations include positions from -3 to 13; positions from -3 to 12; positions from -3 to 11; positions from -3 to 10; positions from -3 to 9; positions from -3 to 8; positions from -3 to 7; positions from -3 to 6; positions from -3 to 5; positions from -3 to 4; positions from -3 to 3; positions from -3 to 2; positions from -3 to 1; positions from -3 to -1; positions from -3 to -2; positions from -2 to 13; positions from -2 to 12; positions from -2 to 11; positions from -2 to 10; positions from -2 to 9; positions from -2 to 8; positions from -2 to 7; positions from -2 to 6; positions from -2 to 5; positions from -2 to 4; positions from -2 to 3; positions from -2 to 2; positions from -2 to 1; positions from -2 to -1; positions from -1 to 13; positions from -1 to 12; positions from -1 to 11; positions from -1 to 10; positions from -1 to 9; positions from -1 to 8; positions from -1 to 7; positions from -1 to 6; positions from -1 to 5; positions from -1 to 4; positions from -1 to 3; positions from -1 to 2; positions from -1 to 1; positions from 1 to 13; positions from 1 to 12; positions from 1 to 11; positions from 1 to 10; positions from 1 to 9; positions from 1 to 8; positions from 1 to 7; positions from 1 to 6; positions from 1 to 5; positions from 1 to 4; positions from 1 to 3; positions from 1 to 2, etc.

[0372] TNF-alpha mutants can be generated by any suitable method, but in a specific embodiment, they are generated by site-directed mutagenesis. In some cases, TNF-alpha mutants can have mutations other than those that render the protein uncleavable. In a specific case, TNF-alpha mutants can have deletions at Val1, Pro12, and / or Val13 or one, two, three, or more mutations outside the intervening region. Mutations other than those that render the mutant non-secretory can be one or more of amino acid substitutions, deletions, additions, inversions, etc. If the additional mutation is an amino acid substitution, the substitution can or cannot be, for example, a substitution to a conservative amino acid. In some cases, one, two, three, four, five, or more additional amino acids can be present at the N-terminus and / or C-terminus of the protein. In some cases, TNF-alpha mutants have (1) one or more mutations that render the mutant non-secreted; (2) one or more mutations that prevent outside-in signaling of the mutant; and / or (3) one or more mutations that prevent binding of the mutant to TNF receptor 1 and / or TNF receptor 2.

[0373] In a particular embodiment, delivery of one or more effective amounts of an agent that binds to antigen CAR-targeted cells expressing a TNF-alpha mutant results in the removal of most of the cells expressing the TNF-alpha mutant. In a specific embodiment, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of the cells expressing the TNF-alpha mutant are removed in an individual. After the need to remove the cells is recognized, delivery of the agent(s) to the individual can be continued until one or more symptoms are no longer present or until a sufficient number of cells are removed. The number of cells in the individual can be monitored using the TNF-alpha mutant as a marker.

[0374] Embodiments of the method of the present disclosure include a first step of providing an effective amount of cell therapy to an individual in need thereof, wherein the cells comprise one or more non-secretable TNF-alpha mutants; and a second step of using the TNF-alpha mutant(s) as a suicide gene to remove the cells (either directly or indirectly, through cell death by any mechanism). The second step can be initiated when at least one adverse event occurs for the individual, and the adverse event can be recognized by any means, including continuous or non-continuous periodic monitoring starting from the initiation of the cell therapy. The adverse event(s) can be detected by tests and / or examinations. When the individual has cytokine release syndrome (also sometimes called cytokine storm), the individual can have, for example, an elevation of inflammatory cytokine(s) (merely by way of example: interferon-gamma, granulocyte macrophage colony-stimulating factor, IL-10, IL-6, and TNF-alpha); fever; fatigue; hypotension; hypoxia; tachycardia; nausea; capillary leakage; heart / kidney / liver dysfunction; or a combination thereof. When the individual has neurotoxicity, the individual may have confusion, delirium, aphasia, and / or seizures. In some cases, the individual is tested for markers associated with the onset and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-alpha, and / or ferritin.

[0375] In a further embodiment, for example, administration of one or more agents that bind to non-secreted TNF-α during cytokine release syndrome or neurotoxicity has the additional advantage of neutralizing high levels of soluble TNF-alpha that contribute to the toxicity of the treatment. Soluble TNF-alpha is released at high levels during cytokine release syndrome and is a mediator of the toxicity of CAR T cell therapy. In such cases, administration of the TNF-alpha antibodies encompassed herein has a dual beneficial effect, namely, selective deletion of TNF-alpha mutant-expressing cells as well as neutralization of soluble TNF-alpha that causes toxicity. Accordingly, embodiments of the present disclosure include a method of eliminating or reducing the severity of cytokine release syndrome in an individual who has received or had received adoptive cell therapy in which the cells express a non-secreted TNF-alpha mutant, the method comprising providing an effective amount of an agent that binds to the non-secreted TNF-alpha mutant, the agent causing, in the individual, (a) elimination of at least a portion of the cells of the cell therapy; and (b) a decrease in the level of soluble TNF-alpha.

[0376] Embodiments of the present disclosure include a method of reducing the effects of cytokine release syndrome in an individual who has received or is receiving cell therapy by cells that express a non-secreted TNF-alpha mutant, the method comprising providing, in the individual, one or more effective amounts of an agent that binds to the mutant to cause (a) elimination of at least a portion of the cells of the cell therapy; and (b) a decrease in the level of soluble TNF-α.

[0377] When the need arises to utilize a TNF-alpha suicide gene, an individual is provided with one or more effective amounts of an inhibitor that can inhibit, such as by directly binding to a TNF-alpha mutant on the cell surface. The inhibitor(s) can, in some embodiments, be provided to the individual systemically and / or locally. The inhibitor can be a polypeptide (such as 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 FDA-approved. When the inhibitor is an antibody, the inhibitor can be a monoclonal antibody in at least some cases. When a mixture of antibodies is employed, one or more of the antibodies in the mixture can be a monoclonal antibody. Examples of small molecule TNF-alpha inhibitors include small molecules such as those described in U.S. Patent No. 5,118,500, which is hereby incorporated by reference in its entirety. Examples of polypeptide TNF-alpha inhibitors include polypeptides such as those described in U.S. Patent No. 6,143,866, which is hereby incorporated by reference in its entirety.

[0378] In certain embodiments, at least one antibody is utilized to target a TNF-alpha mutant in order to induce activity as a suicide gene. Examples of antibodies include at least adalimumab, adalimumab-atto, certolizumab pegol, etanercept, etanercept-szzs, golimumab, infliximab, infliximab-dyyb, or a mixture thereof.

[0379] Embodiments of the present disclosure include a method of reducing the risk of toxicity of a cell therapy to an individual by modifying the cells of the cell therapy to express a non-secretable TNF-alpha mutant. In a specific embodiment, the cell therapy is for cancer and can include an engineered receptor that targets an antigen, including a cancer antigen.

[0380] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, an individual has been, can be, and / or will be provided with additional therapy for a medical condition. If the medical condition is cancer, the individual can be provided with one or more of surgery, radiation therapy, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0381] A cell population having a reduced or inhibited expression level of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 is provided at an effective level to an individual in need thereof. The cells can be administered to the individual by injection, intravenous, intraarterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intracerebral, percutaneous, subcutaneous, topical, perfusion, tumor microenvironment, or a combination thereof.

[0382] In certain embodiments of the method, the cells can be administered to the individual one or more times. The period between administrations of the cells to the individual can be 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or more than 1 year.

[0383] VIII. Vectors If immune effector cells having a reduced or inhibited expression level of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1 contain a non-endogenous engineered gene product or an exogenously provided gene product, the gene product can be delivered to the recipient immune effector cells by any suitable vector including a viral vector or a non-viral vector. Examples of viral vectors include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles, and the like.

[0384] Immune cells are transduced with a vector encoding an antigen-targeting receptor, and if further intracellular transduction of another gene or multiple genes such as a suicide gene and / or a cytokine and / or any therapeutic gene product is required, the antigen-targeting receptor, suicide gene, cytokine, and any therapeutic gene may or may not be included on the same vector or with the same vector. In some cases, the antigen-targeting CAR, suicide gene, cytokine, and any therapeutic gene are expressed from the same vector molecule, for example, the same viral vector molecule. In such cases, the expression of the antigen-targeting CAR, suicide gene, cytokine, and any therapeutic gene may or may not be controlled by the same control element(s). When the antigen-targeting CAR, suicide gene, cytokine, and any therapeutic gene are on the same vector, they may or may not be expressed as separate polypeptides. When expressed as separate polypeptides, they can be separated on the vector, for example, by a 2A element or an IRES element (or both types can be used once or more than once on the same vector).

[0385] A. General embodiments One of ordinary skill in the art is sufficiently capable of constructing vectors by standard recombinant techniques for expressing the antigen receptors of the present disclosure (see, for example, Sambrook et al., 2001, and Ausubel et al., 1996; both incorporated herein by reference).

[0386] 1. Control elements The expression cassettes contained in the vectors useful in the present disclosure contain, in particular, a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal containing intervening sequences, and a transcription termination / polyadenylation sequence (in the 5' to 3' direction). Promoters and enhancers that control the transcription of genes encoding proteins in eukaryotic cells can be composed of multiple genetic elements. Cellular machinery can collect and integrate the control information transmitted by each element, allowing different genes to evolve different, often complex patterns of transcriptional control. Promoters used in the context of the present disclosure include, for example, constitutive, inducible, and tissue-specific promoters. When the vector is utilized in the production of cancer therapy, the promoter can be effective under hypoxic conditions.

[0387] 2. Promoter / Enhancer The expression constructs provided herein contain a promoter for driving the expression of antigen receptors and other cistron gene products. A promoter generally contains a sequence that functions to position the start site of RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, discrete elements overlapping the start site itself assist in fixing the starting location. Additional promoter elements control the frequency of transcription initiation. Typically, these are located in the upstream region of the start site, but many promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcription reading frame is placed "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0388] The spacing between promoter elements is often flexible so that promoter function is maintained 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 up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription. A promoter may or may not be used in combination with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0389] A promoter can be naturally associated with a nucleic acid sequence such that it is obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter is called "endogenous". Similarly, an enhancer can be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with the nucleic acid sequence in its natural environment. Also, a recombinant or heterologous enhancer typically refers to an enhancer not associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, and any other viral, or prokaryotic or eukaryotic cell isolated promoters or enhancers, as well as "non-naturally occurring" promoters or enhancers, i.e., promoters or enhancers containing different elements of different transcriptional control regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA constructs include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, the sequences can be generated using recombinant cloning and / or nucleic acid amplification techniques including PCR™ in connection with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. can similarly be employed.

[0390] Of course, it is important to employ promoters and / or enhancers that effectively direct the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those of ordinary skill in the art of molecular biology generally know the use of combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoter employed is useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as being constitutive, tissue-specific, inducible, and / or advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0391] Furthermore, any combination of promoters / enhancers (e.g., by the World Wide Web of the Eukaryotic Promoter Data Base EPDB, 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 from certain bacterial promoters when the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0392] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, the Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, such as the beta-actin promoter, the GAPDH promoter, the metallothionein promoter; and linked response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA) and the response element promoter near the minimal TATA box (tre). Also, the human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in GenBank®, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC, catalog number ATCC 45007) can be used. In certain embodiments, the promoter is the CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, but any other promoter useful for driving the expression of a therapeutic gene is applicable to the practice of the present disclosure.

[0393] 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 can act in cis, regardless of their orientation, even at relatively long distances (up to several kilobases away from the target promoter). However, the function of an enhancer is not necessarily limited to such long distances and may also function in proximity to a given promoter.

[0394] 3. Initiation signals and coupled expression Certain start signals can also be used in the expression constructs provided in the present disclosure for efficient translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal that includes the ATG start codon. One of ordinary skill in the art can readily determine this and provide the necessary signals. It is well known that in order to ensure translation of the entire insert, the start codon must be “in-frame” with the desired coding sequence's reading frame. The exogenous translation control signal and the start codon can be natural or synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements.

[0395] In certain embodiments, the use of an internal ribosome entry site (IRES) element is used to create a polygenic, or polycistronic message. The IRES element bypasses the ribosome scanning model of 5'-methylated Cap-dependent translation and can initiate translation at an internal site. IRES elements from two members of the picornavirus family (poliovirus and encephalomyocarditis), as well as IRESs from mammalian messages, have been reported. The IRES element can be ligated to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, to create a polycistronic message. The IRES element makes each open reading frame accessible to ribosomes for efficient translation. Using a single promoter / enhancer, multiple genes can be efficiently expressed to transcribe a single message.

[0396] As detailed elsewhere in this specification, certain 2A sequence elements can be used to create linked or co-expression of genes in the constructs provided by this disclosure. For example, a cleavage sequence can be used to co-express genes by linking 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., Thosea asigna virus 2A; T2A) or porcine teschovirus-1 (P2A). In a specific embodiment, in a single vector, multiple 2A sequences are non-identical, but in an alternative embodiment, the same vector utilizes two or more of the same 2A sequences. Examples of 2A sequences are provided in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety.

[0397] 4. Origin of replication To propagate the vector within a host cell, the vector can contain one or more origin of replication sites (often referred to as "ori"), for example, a nucleic acid sequence corresponding to oriP of EBV as described above, or a genetically engineered oriP having a similar or enhanced function in programming, which is a specific nucleic acid sequence where replication is initiated. Alternatively, an origin of replication of an episomal replication virus as described above, or an autonomous replication sequence (ARS) can be employed.

[0398] 5. Selection markers and screenable markers In some embodiments, NK cells comprising the CD70-targeted receptor constructs of the disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers an identifiable change to the cell that allows for easy identification of cells containing the expression vector. Generally, a selection marker confers a property that enables selection. A positive selection marker enables selection by the presence of the marker, and a negative selection marker prevents selection by the presence of the marker. An example of a positive selection marker is a drug resistance marker.

[0399] Generally, including a drug selection marker facilitates the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype enabling the identification of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are contemplated. Alternatively, enzymes that can be screened as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can also be utilized. Those skilled in the art also know methods of employing immunological markers, optionally in combination with FACS analysis. As long as it can be co-expressed with the nucleic acid encoding the gene product, the marker used is considered unimportant. Further examples of selection and screenable markers are well known to those skilled in the art.

[0400] B. Multicistronic vectors In certain embodiments, an antigen-targeted receptor, any suicide gene, any cytokine, and / or any therapeutic gene are expressed from a multicistronic vector (as used herein, the term "cistron" refers to a nucleic acid sequence from which a gene product can be produced). In certain embodiments, the multicistronic vector encodes an antigen-targeted receptor, a suicide gene, and at least one cytokine, and / or an engineered receptor such as a T cell receptor and / or an additional non-antigen-targeted CAR. In some cases, the multicistronic vector encodes at least one antigen-targeted CAR, at least one TNF-alpha mutant, and at least one cytokine. The cytokine can be of a specific type such as human or mouse or any species. In specific cases, the cytokine is IL-15, IL-12, IL-2, IL-18, and / or IL-21.

[0401] In certain embodiments, the present disclosure provides a flexible modular system that utilizes a polycistronic vector having the ability to express multiple cistrons at substantially the same level (as used herein, the term "modular" refers to cistrons or components of cistrons that allow for their interchangeability, for example, by using standard recombinant techniques to remove and replace entire cistrons or components of cistrons, respectively). This system can be used in cell engineering to enable combinatorial expression (including overexpression) of multiple genes. In specific embodiments, one or more of the genes expressed by the vector include one, two, or more antigen receptors. The multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include (1) one or more reporters, such as fluorescent or enzyme reporters for cell assays and animal imaging; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0402] In a specific case, the vector may contain at least four cistrons separated by any type of cleavage site, such as a 2A cleavage site. The vector may or may not be based on Moloney murine leukemia virus (MoMLV or MMLV) containing 3' and 5' LTRs with a psi packaging sequence in the pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene swapping. This system allows for combinatorial overexpression of multiple genes (seven or more) flanked by restriction site(s) for rapid integration by subcloning, and this system also includes at least three 2A self-cleavage sites in some embodiments. Thus, this system enables the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. This system can also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0403] Also, due to the modularity of this system, genes can be efficiently subcloned into each of the four cistrons in the polycistronic expression vector, enabling gene swapping, such as for rapid testing. The restriction sites strategically placed in the polycistronic expression vector allow for efficient gene swapping.

[0404] Embodiments of the present disclosure include systems that utilize polycistronic vectors, where at least a portion of the vector is modularized by enabling the 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 whose identity and location are specifically selected, for example, to facilitate modular use of the vector. The vector also has embodiments where multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby conferring the advantage that the vector expresses separate gene products at substantially equimolar concentrations.

[0405] The vectors of the present disclosure are modularly configured such that one or more cistrons of the vector can be altered and / or one or more components of one or more specific cistrons can be altered. The vectors can be designed to utilize unique restriction enzyme sites that flank the ends of one or more cistrons and / or the ends of one or more components of a specific cistron.

[0406] Embodiments of the present disclosure include polycistronic vectors that include each of at least two, at least three, or at least four cistrons flanked by one or more restriction enzyme sites, where 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 cleaved into separate polypeptides, while in other cases, multiple cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on the vector can be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on the vector are separated by an IRES element.

[0407] In certain embodiments, the present disclosure provides a system for cell engineering that enables combinatorial expression of multiple cistrons, which may include, for example, one, two, or more antigen receptors, including overexpression. In certain embodiments, the use of the polycistronic vectors described herein enables the vector to generate equimolar levels of multiple gene products from the same mRNA. The multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include one or more fluorescent or enzyme reporters for, e.g., cell assays and animal imaging. The vector can also include a suicide gene product for terminating the cells carrying the vector when the cells carrying the vector are no longer needed or become harmful to the host to which the vector was provided.

[0408] In certain embodiments of the present disclosure, at least one of the cistrons on the vector includes two or more modular components, and each of the modular components within the cistron is flanked by one or more restriction enzyme sites. The cistron can include, for example, three, four, or five modular components. In at least some cases, the cistron encodes a receptor having different portions of the antigen receptor encoded by the corresponding modular components. The first modular component of the cistron can encode the antigen-binding domain of the receptor. Further, the second modular component of the cistron can encode the hinge region of the receptor. Further, the third modular component of the cistron can encode the transmembrane domain of the receptor. Further, the fourth modular component of the cistron can encode the first co-stimulatory domain. Further, the fifth modular component of the cistron can encode the second co-stimulatory domain. Further, the sixth modular component of the cistron can encode the signaling domain.

[0409] In certain embodiments of the present disclosure, two different cis - trons on a vector each encode a non - identical antigen receptor. Both antigen receptors can be encoded by cis - trons containing two or more modular components, where the cis - trons are separate and each contain two or more modular components. The antigen receptor can be, for example, a chimeric antigen receptor (CAR) and / or a T - cell receptor (TCR).

[0410] 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 can include the 5’LTR, 3’LTR, and / or psi packaging element of Moloney murine leukemia virus (MMLV). In a specific case, the psi packaging is incorporated between the 5’LTR and the antigen receptor coding sequence. The vector may or may not contain the pUC19 sequence. In some embodiments of the vector, at least one cis - tron 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.

[0411] When a 2A cleavage site is utilized in the vector, the 2A cleavage site can include the P2A, T2A, E2A, and / or F2A sites.

[0412] In addition to one cistron encoding a CD70-targeted CAR, 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 CD70-targeted CAR, 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 CD70-targeted CAR, the third cistron encodes a second CAR or another antigen receptor, and the fourth cistron encodes a cytokine. In a specific embodiment, different portions of the CD70-targeted CAR and / or another receptor are encoded by corresponding modular components, the first component of the second cistron encodes an antigen-binding domain, the second component encodes a hinge and / or transmembrane domain, the third component encodes a co-stimulatory domain, and the fourth component encodes a signaling domain.

[0413] 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 cistron encodes an antigen-targeted 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., a CAR and / or a TCR). A cistron may or may not encode a reporter gene.

[0414] In certain configurations of the target gene cargo, a single vector may contain a cis - tron encoding an antigen - targeted CAR and a cis - tron encoding a second antigen receptor that is not identical to the antigen - targeted receptor. In a specific embodiment, the first antigen receptor encodes an antigen - targeted CAR and the second antigen receptor encodes a TCR, or vice versa. In certain embodiments, a vector containing separate cis - trons encoding an antigen - targeted CAR and a second antigen receptor also contains a third cis - tron encoding a cytokine or chemokine and a fourth cis - tron encoding a suicide gene. However, the suicide gene and / or cytokine (or chemokine) may not be present on the vector.

[0415] In certain embodiments, at least one cis - tron contains a plurality of modular components themselves. For example, one cis - tron can encode a multi - component gene product such as an antigen receptor having multiple parts; in a specific case, the antigen receptor is encoded from a single cis - tron, thereby ultimately generating one polypeptide. A cis - tron encoding a plurality of components can have a plurality of 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 that are unique to the vector containing the cis - tron. In a specific embodiment, a cis - tron having a plurality of components encodes an antigen receptor having a plurality of corresponding parts each attributing a receptor - specific function. In a specific embodiment, each or most of the components of a multi - component cis - tron are separated by one or more restriction enzyme digestion sites that are unique to the vector, allowing for the exchangeability of separate components if desired.

[0416] In a specific embodiment, each component of the multi-component cistron corresponds to a different part of a coded antigen receptor such as an antigen-targeted CAR. In an exemplary embodiment, component 1 may code for the antigen-binding domain of the receptor; component 2 may code for the hinge domain of the receptor; component 3 may code for the transmembrane domain of the receptor; component 4 may code for the co-stimulatory domain of the receptor; component 5 may code for the signaling domain of the receptor. In a specific embodiment, the antigen-targeted CAR may include one or more co-stimulatory domains separated by unique restriction enzyme digestion sites, respectively, with respect to the exchangeability of the co-stimulatory domain(s) within the receptor.

[0417] In a specific embodiment, there is a polycistronic vector having four separate cistrons separated by 2A cleavage sites, but in a specific embodiment, instead of the 2A cleavage site, there is an element (such as an IRES sequence) that directly or indirectly generates a separate polypeptide from the cistron. For example, four separate cistrons may be separated by three 2A peptide cleavage sites, and each cistron has restriction sites (such as X1, X2, etc.) flanking each end of the cistron to allow for the exchangeability of a particular cistron, such as when using standard recombinant techniques, with another cistron or other type of sequence. In a specific embodiment, the restriction enzyme sites flanking each cistron are unique to the vector to facilitate recombination, but in an alternative embodiment, the restriction enzyme sites are not unique to the vector.

[0418] In certain embodiments, the vector provides a unique second level of modularity by enabling exchangeability within a particular cistron that includes within a plurality of components of that particular cistron. The plurality of components of a particular cistron can be separated by one or more restriction enzyme sites that include those unique to the vector, to enable exchangeability of one or more components within the cistron. As an example, cistron 2 can include five separate components, although there may be 2, 3, 4, 5, 6 or more components per cistron. As an example, the vector can include cistron 2 having five components separated by unique enzyme restriction sites X9, X 10 , X 11 , X 12 , X 13 , and X 14 respectively, to enable standard recombination for exchanging different components 1, 2, 3, 4, and / or 5. In some cases, there may be multiple restriction enzyme sites between different components (which are unique, although one or more may instead not be unique), and there may be a sequence between the multiple restriction enzyme sites (which may instead not be present). In certain embodiments, all components encoded by a cistron are designed for the purpose of being exchangeable. In certain cases, one or more components of a cistron are designed to be exchangeable, while one or more other components of the cistron may not be designed to be exchangeable.

[0419] In a specific embodiment, the cistron encodes an antigen-targeting CAR molecule having multiple components. For example, cistron 2 can be composed of sequences encoding an antigen-targeting CAR molecule having distinct components represented by component 1, component 2, component 3, etc. The CAR molecule can include 2, 3, 4, 5, 6, 7, 8 or more exchangeable components. In a specific example, component 1 encodes a scFv; component 2 encodes a hinge; component 3 encodes a transmembrane domain; component 4 encodes a co-stimulatory domain (however, there may also be a component 4' encoding a second or more co-stimulatory domains flanked by restriction sites for exchange); component 5 encodes a signaling domain. In a particular example, component 1 encodes a scFv; component 2 encodes an IgG1 hinge and / or transmembrane domain; component 3 encodes CD28; component 4 encodes CD3 zeta.

[0420] One of ordinary skill in the art will recognize that in the design of vectors, various cistrons and components must be configured to remain in-frame if necessary.

[0421] In a particular 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; component 4 encodes CD3 zeta.

[0422] The restriction enzyme site can be of any kind and can contain any number of bases, for example, 4 to 8 bases, in its recognition site; the number of bases in the recognition site can be at least 4, 5, 6, 7, 8 or more. The site when cleaved can yield a blunt cut or sticky ends. The restriction enzyme can be, for example, type I, type II, type III, or type IV. The restriction enzyme site can be obtained from available databases such as the Integrated Relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0423] Exemplary vectors can be circular or conventional, with position 1 (the 12 o'clock position at the top of the circle, and the rest of the sequence in a clockwise direction) set as the starting point of the 5' LTR.

[0424] In embodiments where a self-cleaving 2A peptide is utilized, the 2A peptide can be a viral oligopeptide 18 - 22 amino acids (aa) in length that mediates "cleavage" of the polypeptide during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viral 2As are generally named after the virus from which they are derived. The first 2A discovered was F2A (foot-and-mouth disease virus), and subsequently, E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A) have also been identified. It has been discovered that the mechanism of "self-cleavage" mediated by 2A is that the ribosome skips the formation of the glycyl-prolyl peptide bond at the C-terminus of 2A.

[0425] In a specific case, the vector can be a γ-retroviral transduction vector. The retroviral transduction vector can include a backbone based on a plasmid such as the pUC19 plasmid (a large fragment (2.63 kb) between the HindIII and EcoRI restriction enzyme sites). The backbone can carry viral components from Moloney murine leukemia virus (MoMLV) including the 5’ LTR, the psi packaging sequence, and the 3’ LTR. The LTR is a long terminal repeat sequence found on both sides of the provirus of the retrovirus and, in the case of the transduction vector, brackets the gene cargo of interest such as the antigen-targeted CAR and related components. The psi packaging sequence, which is the target site for packaging by the nucleocapsid, is also incorporated in cis between the 5’ LTR and the CAR coding sequence. Thus, the basic structure of an example of a transduction vector can be configured as, for example, pUC19 sequence - 5’ LTR - psi packaging sequence - gene cargo of interest - 3’ LTR - pUC19 sequence. This system can also be applied to other viral and non-viral vectors including, but not limited to, lentivirus, adenovirus AAV, and non-viral plasmids.

[0426] A. Pharmaceutical composition Also provided herein are pharmaceutical compositions and formulations comprising transduced NK cells and a pharmaceutically acceptable carrier. The transduced cells can be constituted in a medium suitable for transfer into an individual and / or a medium suitable for storage such as cryopreservation including prior to transfer into an individual.

[0427] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., cells) having the desired purity, in the form of a lyophilized formulation or an aqueous solution, 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 dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; 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 further include depot 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 sHASEGP containing rHuPH20 and methods of use thereof are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more glycosaminoglycanases, such as chondroitinase.

[0428] B. Combination Therapy In certain embodiments, the compositions and methods of this embodiment include an immune cell population (including NK cell populations) that is combined with at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination thereof. The additional therapy can take the form of adjuvant therapy or neoadjuvant therapy.

[0429] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor(s) or anti-metastatic agent(s). In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of the side effects of a treatment such as an anti-nausea agent). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy that targets the PBK / ACT / mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and / or chemopreventive agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0430] Immune cell therapy can be administered before, during, after, or in various combinations with additional cancer therapies such as immune checkpoint therapy. Administration can be done simultaneously or at intervals ranging from several minutes to several weeks. In embodiments where immune cell therapy is provided to a patient separately from an additional therapeutic agent, generally, a significant period of time is not allowed to pass between each administration so that the two compounds can still exert a beneficially combined effect on the patient. In such cases, it is contemplated to provide antibody therapy and anti-cancer therapy to the patient within about 12 to 24 hours or within 72 hours of each other, more specifically, within about 6 to 12 hours of each other. Depending on the situation, it may be desirable to significantly extend the treatment period such that several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between each administration.

[0431] Various combinations can be employed. In the following examples, immune cell therapy is designated as "A" and anti-cancer agent therapy as "B": A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0432] The administration of any compound or therapy of the present embodiment to a patient can follow the general protocol for the administration of such a compound, taking into account the toxicity of the agent if any. Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy.

[0433] 1. Chemotherapy According to this embodiment, a wide variety of chemotherapeutic agents can be used. The term "chemotherapy" means the use of drugs for treating cancer. The term "chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These drugs or medications are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at which stage. Alternatively, the drugs can also be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0434] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially, bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoproteins edeine antibiotics chromophore, actinomycin chromophore, actinomycin, australinomycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; metabolic inhibitors such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone; antiadrenal drugs such as mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diacodone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; risoxacin; schizophyllan;Spirogyrma; tenuazonic acid; triazicon; 2,2’,2’’-trichloroethylamine; trichothecenes (especially, T-2 toxin, verruculogen, lolitrem A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids 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; daunomycin; aminopterin; ZERODA; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitor, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0435] 2. Radiation Therapy Other factors that cause DNA damage and have been widely used generally include those known as gamma rays, X-rays, and / or the targeted delivery of radioisotopes to tumor cells. Other DNA-damaging factors are also conceivable, such as microwave, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and ultraviolet irradiation. All of these factors are likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays is from a daily dose of 50 to 200 roentgens over a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of radioisotopes varies and depends on the half-life of the isotope, the intensity and type of radiation, and the uptake by tumor cells.;

[0436] 3. Immunotherapy One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and molecules that target and destroy cancer cells. An example is rituximab (RITUXAN®). Immune effectors can be, for example, antibodies specific for markers on the surface of tumor cells. Antibodies can function as therapeutic effectors alone or recruit other cells to actually affect cell killing. Antibodies can also bind to drugs and toxins (such as chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as targeting agents. Alternatively, the effector can be a lymphocyte with surface molecules that directly or indirectly interact with targets on tumor cells. Various effector cells include cytotoxic T cells and NK cells.

[0437] Antibody-drug conjugates have emerged as an epoch-making approach in the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. An antibody-drug conjugate (ADC) is a monoclonal antibody (MAb) covalently linked to a cytotoxic drug. In this approach, the high specificity of the MAb for the antigen target and the potent cytotoxic agent are combined to obtain an "armed" MAb that delivers the payload (drug) to tumor cells where the antigen concentration is concentrated. Targeted drug delivery also minimizes drug exposure in normal tissues, reduces toxicity, and improves the therapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013 by the FDA has proven the effectiveness of this approach. Currently, there are more than 30 ADC drug candidates in various clinical trial stages for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly rely on the identification and validation of new targets suitable for this approach and the generation of target MAbs. Two criteria for ADC targets are increased / high levels of expression in tumor cells and robust internalization.

[0438] In one aspect of immunotherapy, tumor cells must have some marker suitable for targeting, i.e., a marker that is not present in most other cells. There are many tumor markers, and any of these may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, p155, and the like. Another aspect of immunotherapy is to combine anti-cancer 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.

[0439] Examples of immunotherapies currently under research or in use are immunoadjuvants 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 all types of interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 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. Pat. No. 5,824,311). In conjunction with the antibody therapy described herein, it is contemplated that one or more anti-cancer agent therapies will be employed.

[0440] In some embodiments, the immunotherapy is an immune checkpoint inhibitor. An immune checkpoint is either to raise a signal (e.g., a co-stimulatory molecule) or to lower a signal. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibitors 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 suppressor of T cell activation (VISTA), etc. In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0441] The immune checkpoint inhibitor can be a drug, e.g., a small molecule, a recombinant form of a ligand or a receptor, or especially an antibody, e.g., a human antibody (e.g., International Patent Application Publication No. WO2015 / 016718; Nat Rev Cancer, Vol. 12(4):252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs can be used, especially chimeric, humanized, or human forms of antibodies. As will be understood by those skilled in the art, certain antibodies referred to in this disclosure may be used with alternative and / or equivalent names. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, pembrolizumab is known to also be known by the alternative and equivalent names MK-3475 and lambrolizumab.

[0442] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific embodiment, 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. 8,735,553, 8,354,509, and 8,008,449, 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, such as those described in U.S. Patent Application Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369 (all of which are incorporated herein by reference).

[0443] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or the PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, OPDIVO®, and is an anti-PD-1 antibody described in International Publication No. WO2006 / 121168. Pembrolizumab is also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, and is an anti-PD-1 antibody described in International Publication No. WO2009 / 114335. CT-011 is also known as hBAT or hBAT-1, and is an anti-PD-1 antibody described in International Publication No. WO2009 / 101611. AMP-224 is also known as B7-DCIg, and is a PDL2-Fc fusion soluble receptor described in International Publication Nos. WO2010 / 027827 and WO2011 / 066342.

[0444] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as an “off” switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily, is expressed on the surface of helper T cells, and transmits an inhibitory signal to T cells. CTLA4 is similar to the co-stimulatory protein of T cells, CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, and CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 results in increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0445] 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.

[0446] An anti-human CTLA-4 antibody (or VH and / or VL domains derived therefrom) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can also be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, International Publication Nos. WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; old ticilimumab), U.S. Patent 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 can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these technically 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 of which are incorporated herein by reference.

[0447] Exemplary anti-CTLA-4 antibodies include ipilimumab (also known as 10D1, MDX-010, MDX-101, and YERVOY®) or antigen-binding fragments and variants thereof (see, e.g., International Publication No. 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 with and / or binds to the same epitope on CTLA-4 as the above-described antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0448] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Patent Applications Nos. WO1995001994 and WO1998042752 (each incorporated herein by reference), and immunoadhesins such as those described in U.S. Patent No. 8,329,867 incorporated herein by reference.

[0449] 4. Surgery Approximately 60% of cancer patients undergo some form of surgery, which includes prophylactic surgery, diagnostic or staging surgery, therapeutic surgery, and palliative surgery. Therapeutic surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancer tissue, and can be used in combination with other therapies such as the therapies of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physically removing at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrocautery, and microscopically controlled surgery (Mohs’ surgery).

[0450] When removing some or all of cancer cells, tissues, or tumors, cavities may form in the body. Treatment can be performed by perfusing, directly injecting, or locally applying anti-cancer therapy to the site. Such treatment can be repeated, for example, every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. These treatments also vary in dosage.

[0451] 5. Other agents It is contemplated that other agents can be used in combination with certain aspects of the present embodiment to improve the effectiveness of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cell activators and differentiation promoters, cell adhesion inhibitors, agents that enhance the sensitivity of proliferating cells to apoptosis inducers, or other biological agents. An increase in intercellular signaling by increasing the number of GAP junctions will increase the anti-proliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell activator or differentiation promoter can be used in combination with an aspect of the present embodiment to improve the anti-proliferative effect of the treatment method. Cell adhesion inhibitors are contemplated to improve the effectiveness of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinases (FAKs) inhibitors and lovastatin. It is further contemplated that other agents, such as antibody c225, that increase the sensitivity of hyperproliferative cells to apoptosis, be used in combination with certain aspects of the present embodiment to improve the therapeutic effect.

[0452] IX. Kits of the present disclosure Any of the compositions described herein may be included in a kit. In non-limiting examples, cells having reduced or inhibited expression levels of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, reagents for generating the cells, vectors, and reagents for generating the vectors and / or their components may be included in the kit. In certain embodiments, NK cells may be comprised in the kit and they may be modified in any manner or may not yet be modifiable. Such a kit may or may not have one or more reagents for manipulating the cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding CRISPR reagents for KO of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, and / or CREB1, suicide gene products, receptors, and / or cytokines may be included in the kit. Proteins such as cytokines or antibodies including monoclonal antibodies may be included in the kit. Nucleotides encoding components of an engineered CAR receptor or TCR receptor may be included in the kit, including the reagents for generating it.

[0453] In certain aspects, the kit includes the NK cell therapy of the present disclosure and yet another cancer therapy. In some cases, the kit also includes a second cancer therapy such as, for example, chemotherapy, hormonal therapy, and / or immunotherapy in addition to the cell therapy embodiment. The kit(s) can be modified according to an individual's specific cancer and includes each respective second cancer therapy for the individual.

[0454] The kit can be composed of appropriately dispensed compositions of the present disclosure. The components of the kit can be packaged either in an aqueous medium or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components can be placed, preferably appropriately dispensed. If more than one component is included in the kit, the kit generally includes a second, third or other additional container into which additional components can be separately placed. However, various combinations of components can be included in the vial. The kit of the present invention also typically includes means for hermetically containing the composition and any other reagent containers for commercial sale. Such containers can include injection-molded or blow-molded plastic containers in which the desired vials are held.

Example

[0455] X. Example The following examples are included to illustrate certain non-limiting aspects of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the disclosed subject matter. However, those skilled in the art should understand that, in light of the present disclosure, many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the disclosed subject matter and still obtain similar or analogous results.

[0456] Example 1 - In tumor-infiltrating immune cells of glycolytic tumors, proton-sensitive GPRS and CREM were highly expressed. Single-cell RNA sequencing (scRNA seq) data of tumor samples from patients with pancreatic cancer were examined for the expression of genes encoding enzymes involved in glycolysis. The inventors determined that glycolytic genes are significantly upregulated in tumor cells (Figure 1A). This reflects active aerobic and anaerobic glycolysis leading to the accumulation of organic and inorganic acids in the tumor microenvironment (TME), as previously shown in the literature for multiple tumor types. Next, for the corresponding tumor-infiltrating NK cells, the expression of proton-sensing G protein-coupled receptors (GPRs) such as GPR68 and GPR132, and the expression of downstream signaling mediators of the response to acidity in the cAMP pathway such as CREM and CREB1 were 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 1B). Since CREM is an important transcription factor in the cAMP pathway, shows significant upregulation in NK cells within the TME, and is involved in mediating the common downstream signaling of these proton-sensing GPRs, the CREM gene was determined to be a potential target for gene modification that can counter acidity-mediated immunosuppression.

[0457] The 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 highly glycolytic state of this tumor (see, for example, Courtney K.D. et al., 2018; associated with von Hippel-Lindau (VHL) gene deficiency, which mechanistically promotes the development of most CC-RCCs), and was confirmed by the high expression of glycolytic enzymes (Figure 2A, from TCGA data). Using scRNA seq data publicly available from the TISCH database, the expression of CREM in kidney cancer-infiltrating immune cells and peripheral blood immune cells of healthy donor was compared (see, for example, Sun D. et al., 2020). As shown in Figure 2B, significant upregulation of CREM was observed in various immune cells of the TME, compared to the rare expression in peripheral blood immune cells from healthy donors.

[0458] Example 2 - Endonuclease-Mediated Knockout of Endogenous Genes The guide RNA (gRNA) was designed to induce double-strand breaks in GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, or CREB1, while having both a high on-target activity score and a low off-target activity score. Exemplary crispr RNA (crRNA) sequences suitable for incorporation into the gRNA are provided in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0459] For each of the gene candidates for gene targeting in immune effector cells, a panel of crispr RNAs (crRNAs) was designed (see Table 2), and the crRNAs were complexed with tracr RNA (the binding scaffold for the Cas nuclease) to form the guide RNA (gRNA).

[0460] Any one or more of the gene candidates for genetic targeting in immune effector cells are knocked out and / or mutated by one or more crRNAs complexed with tracr RNA to form the guide RNA.

[0461] Example 3 - CREM KO Significantly Improved the Cytotoxicity of NK Cells in Acidic Environments and the TME As described above, CREM has been found to be consistently upregulated in TME-infiltrating immune cells in various glycolytic tumor models. Since the CREM protein is an important transcription factor in the cAMP pathway, the inventors generated a knockout (KO) CREM locus in NK cells. The KO of the CREM locus targeted the activation of the cAMP pathway in response to acidity and served as a proof of principle for attempts to abrogate acidity-mediated immunosuppression. Using the CRISPR-Cas9 system having SEQ ID NOs: 140 and 142, the CREM KO locus in NK cells was efficiently and stably generated (Figs. 3A and 3B). Fig. 3B shows CREM isoforms selected from bulk RNA sequencing data obtained from CREM WT vs. CREM KO NK cells from three umbilical cord blood donors (D1, D2, and D3). The color scale of the heatmap represents the number of expressing cells from 0 (dark blue) to 400 (red). The FDR column corresponds to the false discovery rate (significant <0.05), and the logPostFC column represents the log fold change in the expression level of WT vs. KO, with positive values indicating higher RNA isoform expression in CREM WT NK cells and negative values indicating higher RNA isoform expression in CREM KO NK cells. RNA isoform expression analysis showed that multiple CREM isoforms in mutant NK cells were effectively knocked out and / or significantly knocked down. Examples of RNA isoforms that were knocked out and / or significantly knocked down include the ICER isoform (CREM-228), CREM-207, CREM-211, CREM-213, CREM-239, CREM-201, CREM-232, and CREM-217. The RNA isoform CREM-218 was found to be upregulated in CREM KO NK cells compared to CREM WT NK cells.

[0462] To examine the killing ability of CREM WT vs. CREM KO CAR-NK cells, a long-term killing assay of NK cells against the glycolytic UMRC3 renal cell carcinoma cell line was performed using the IncuCyte® device, along with live cell imaging of tumor cell proliferation and killing by NK cells. The results showed that the activity of CAR-NK cells after CREM KO was enhanced compared to wild-type CAR-NK cells targeting UMRC3 (Figures 4A and 4B). To examine the effect of CREM KO on the anti-tumor function of NK cells under conditions mimicking in vivo solid tumor models, a killing assay of NK cells against a 3D tumor spheroid culture model of the UMRC3 cell line was performed. Tumor spheroids mimic solid tumor masses and have been previously shown in the literature to have an acidic pH (see, for example, 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 the IncuCyte® device for 48 hours before adding NK cells. 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 CREM WT CAR-NK cells targeting UMRC3 (Figures 4C and 4D).

[0463] To examine the specific effect of CREM KO in the suppression of acidity-mediated immunosuppression, a chromium release killing assay of CREM WT vs. CREM KO NK cells against the A498 cell line was performed at various effector:target (E:T) ratios under two pH conditions, typical culture medium pH (pH = 7.2) and acidic pH (pH = 5.9). In the case of CREM WT NK cells, the predicted attenuation of NK cell cytotoxicity was shown at acidic pH (Figure 5A), but when the killing assay was performed using CREM KO NK cells, the cytotoxicity was restored (Figure 5B).

[0464] As shown in FIGS. 10A-H, CAR-NK cells were able to withstand the inhibitory effect of lactate at the same concentration as observed in the tumor microenvironment (TME) by CREM KO. As shown in FIG. 10A, the results of the 51Cr release assay showed that the cytotoxicity of anti-CD70 CAR-NK (CAR.70) cells against renal cell carcinoma (e.g., UMRC3 renal cell carcinoma tumor cells) in an acidic environment was enhanced in CREM KO CAR-NK cells compared to CREM WT CAR-NK cells at various E:T ratios (e.g., 20:1, 10:1, 5:1, 2.5:1, and 1.25:1), and was equivalent to that of CREM WT CAR-NK cells at a more neutral pH level. NK cells were either CREM wild-type (WT) or CREM knockout (KO), and were incubated at either normal pH (pH Reg ) or acidic pH (pH Lac ) created using lactate. Representative bright-field microscopy images of CAR-NK cells and UMRC3 tumor cells (elongated cells) co-cultured at normal pH (pH Reg ) or acidic pH (pH Lac ) are shown in FIG. 10B. Cell clusters indicate areas where cytotoxic CAR-NK cells are actively involved with tumor cells. The results showed that CREM KO CAR.70 NK cells presented a higher level of clearance of UMRC3 cells compared to CREM WT CAR.70 NK cells. Next, the long-term cytotoxicity of CREM WT or CREM KO CAR.70 NK cells was analyzed using a long-term cytotoxicity (e.g., Incucyte®) assay of CAR-NK cells and their non-transduced (NT) counterparts in the pH Reg or pH Lac environment when challenged against UMRC3 cells (FIG. 10C). Acidic conditions (pH Lac20(Inc) ) were achieved over a 2-week period by a gradual increase (Inc) in the cumulative lactate concentration (e.g., NK cells were gradually acclimated to more acidic conditions over a 2-week incubation period by adding approximately 2.5 mM of lactate every 2-3 days; pH Lac20(Inc)resulted in a pH of approximately 6.0; all conditions were started from the normal pH of the medium (pH Reg ~7.4). The results showed that chronic lactate exposure weakens the cytotoxicity of CAR-NK cells compared to normal pH conditions, but CREM KO CAR-NK cells can maintain strong antitumor killing despite this relatively extreme condition that mimics the low pH of the glycolytic tumor microenvironment. Figure 10D shows 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 acidic (pH Lac20(Inc) ) condition at an E:T ratio of 1:1 or 2:1.

[0465] After confirming the enhanced cytotoxicity of CREM KO CAR.70 NK cells compared to CREM WT CAR.70 NK cells when analyzed at acidic pH, phenotypic analysis of NK cells was performed. As shown in Figures 10E - 10H, NK cells (e.g., CREM WT or CREM KO) co-cultured with UMRC3 tumor cells under either pH Reg or pH Lac conditions were characterized using multiparametric spectral flow cytometry. viSNE analysis was performed for various conditions, followed by FlowSOM clustering. Figure 10E shows a colored FlowSOM clustering plot showing metaclusters 1 - 4 for the composite of all four test conditions. Figure 10F shows the distribution of four FlowSOM metaclusters (MC) across different test conditions. The results showed that in CREM WT CAR.70 NK cells co-cultured with UMRC3 cells, MC2 (orange) increased at pH Lac . However, CREM KO CAR.70 NK cells co-cultured with UMRC3 cells at pH Lac showed that at pH RegThe composition of MC2 phenotype cells similar to NK cells co-cultured under conditions was shown. Next, the underlying phenotypes related to the metaclusters were analyzed. Figure 10G presents the expression of various markers in four MCs, locally normalized to the minimum and maximum expression over various conditions, and presented on a color scale from blue (minimum) to red (maximum). The percentage of expression was overlaid by circles of various sizes proportional to the % of expression. MC2 was 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). Contour tSNE plots underlying various test conditions (pH Reg or pH Lac , and CREM WT or CREM KO) are shown in Figure 10H, and differences in overall phenotype clustering in the NK cell distribution after viSNE analysis can be observed.

[0466] Example 4 - CREM KO Enhanced NK Cell Activation and Multifunctionality To further elucidate the effect of CREM KO on the functionality of NK cells, 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-gamma and TNF-alpha, as well as the expression of the degranulation marker CD107a, were assayed. The results showed that CREM KO further enhanced the ability of CAR-NK cells targeting SKOV3 to produce effective cytokines and express markers of NK cell activation (Figures 6A and 6B). Furthermore, in a larger-scale multifunctionality assay, the LUMINEX® assay was utilized to measure multiple cytokines as markers of NK cell activation. Here too, the results showed that the level of cytokine secretion by CREM KO CAR-NK cells increased overall when compared with CREM WT CAR-NK cells (Figure 6C). The genotypes of various NK cells were assayed for the expression of markers such as activation, inhibition, and cytotoxicity using mass cytometry. The data showed that CREM KO CAR-NK cells had an immunophenotype consistent with activated and cytotoxic cells (Figure 7).

[0467] Example 5 - CREM KO conferred a growth advantage to NK cells but did not lead to autonomous growth Through the aforementioned studies involving CREM KO NK cells, an increase in the proliferation rate of CREM KO NK cells was observed compared to their corresponding CREM WT counterparts. While evaluating the effect of CREM KO on NK cell proliferation and at the same time confirming that CREM KO does not result in autonomous NK cell proliferation, the proliferation of NK cells was assayed in the absence or presence of CREM KO in the absence of IL-2 stimulation (a cytokine typically essential for the survival of NK cells in vitro). It was observed that CREM WT CAR-NK cells had enhanced proliferation compared to non-transduced (NT) NK cells, which is explained by the fact that the CAR construct also secretes IL-15. The results showed that CREM KO CAR-NK cells proliferated more and persisted longer without the addition of IL-2. Nevertheless, autonomous growth over time was not observed (Figure 8).

[0468] Example 6 - Improvement of NK cell metabolic function by CREM KO Since the above results demonstrated that CREM KO enhanced the activity of NK cells in the TME and acidic environment, the inventors hypothesized that CREM KO empowers NK cells through metabolic reprogramming that allows NK cells to compete for metabolites in the TME and withstand immunosuppression caused by highly metabolically active tumors. To test this hypothesis, the glycolytic capacity of CREM KO cells was evaluated through measurement of the extracellular acidification rate (ECAR) and the oxidative phosphorylation rate by oxygen consumption rate (OCR). The results showed that CREM KO CAR-NK cells had a higher glycolytic capacity (Figure 9A) and a better OCR (Figure 9B) compared to their CREM WT CAR-NK cell counterparts. These results suggest that CREM KO may be able to reconstruct the metabolic program of NK cells and put them in a more suitable state to resist the adverse effects brought about by the acidity of the TME.

[0469] The above results were confirmed as shown in FIGS. 13A - C, and it was found that CREM KO enhanced the metabolic adaptability of NK cells at baseline, similar to when cultured at acidic pH. FIG. 13A shows the extracellular acidification rate (ECAR; as a surrogate for glycolysis; upper panel) 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 transduced with a construct expressing IL15 in the absence of CAR were included in the assay (IL15 NK cells). The assay was performed on NK cells cultured in normal medium without stimulation using a Seahorse® glycolytic stress test assay according to the manufacturer's protocol. The oxygen consumption rate (OCR; as a surrogate for oxidative phosphorylation (OXPHOS); lower panel) was analyzed using a Seahorse® mitochondrial stress test assay according to the manufacturer's protocol. FIG. 13B shows the results of ECAR (top) and OCR (bottom) of NT, CREM WT CAR - NK cells, and CREM KO CAR - NK cells when incubated in lactate. Finally, FIG. 13C shows that both the glycolysis and OXPHOS pathways were significantly upregulated in CREM KO NK cells compared to CREM WT NK cells after incubation with lactate (analyzed using unbiased GSEA performed in bulk RNA sequencing of these conditions).

[0470] Collectively, these results confirmed that CREM KO may alter the wiring of the NK cell metabolic program and render it in a more suitable state to resist the adverse effects brought about by the acidity of the TME.

[0471] Example 7 - CREM KO improved the activation and cytotoxicity of CAR - NK cells ex vivo and / or in vivo As shown in FIGS. 11A to 11F, the inventors confirmed that CREM KO improved the activated phenotype and cytotoxicity of CAR-NK cells, including in long-term assays involving multiple tumor rechallenges. FIG. 11A shows the results of a representative rechallenge cytoto...

Claims

**Claim 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-responsive element-binding protein 1 (CREB1) gene. **Claim 2** The cell according to claim 1, wherein the mutation is a partial or complete loss of function and / or a knockout (KO) mutation. **Claim 3** The cell according to claim 1 or 2, wherein the mutation reduces or inhibits the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not have a mutated gene encoding the same endogenous gene. **Claim 4** The cell according to claim 1, wherein the mutation is a neo-functional or gain-of-function mutation. **Claim 5** The cell according to claim 1 or 4, wherein the mutation increases the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to a locus that does not have a mutated gene encoding the same endogenous gene. **Claim 6** The cell according to any one of claims 1 to 5, wherein the mutation results in a modified mRNA isoform population encoded by the mutated endogenous gene as compared to a representative mRNA population encoded by a locus that does not have a mutated gene encoding the same endogenous gene. **Claim 7** The cell according to any one of claims 1 to 4, wherein the mutation results in a modified protein isoform population encoded by the mutated endogenous gene as compared to a representative protein population encoded by a locus that does not have a mutated gene encoding the same endogenous gene. **Claim 8** The cell according to any one of claims 1 to 7, wherein the mutation comprises a knockout (KO) mutation in the endogenous gene of the cell.

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

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

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

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

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

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

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

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

17. The cell according to claim 16, wherein the increase in cytokine release comprises an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

18. The cell according to claim 15 or 16, wherein the increase in cytokine release comprises an increase in 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-1 alpha (MIP-1a), macrophage inflammatory protein-1 beta (MIP-1b), TNF-a, and / or perforin in response to stimulation by tumor cells.

19. The cell according to any one of claims 1 to 18, wherein the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cell as compared to a control non-engineered cell.

20. The cell according to any one of claims 1 to 19, wherein the mutation results in an enhanced activation and / or cytotoxic phenotype for the engineered cell as compared to a control non-engineered cell, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, mTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

21. The cell according to any one of claims 1 to 20, wherein the mutation results in upregulation of G2M, E2F, MYC, mTORC1, oxidative phosphorylation, and / or TNFa signaling.

22. The cell according to any one of claims 1 to 21, wherein the mutation results in an enhanced proliferative capacity and / or persistence phenotype for the engineered cell as compared to a control non-engineered cell.

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

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

25. The cell according to any one of claims 1 to 24, wherein the mutation results in a phenotype of enhanced metabolic adaptability for the manipulated cell as compared to the unmanipulated cells of the control.

26. The cell according to claim 25, wherein the enhanced metabolic adaptability is a higher glycolytic ability and / or an improved oxygen consumption rate (OCR).

27. The cell according to any one of claims 1 to 26, wherein the mutation provides increased chromosomal proximity across the genome when the cell is contained in an acidic microenvironment.

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

29. The cell according to claim 28, wherein the CREM mutation results in a decrease in the 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 according to claim 28 or 29, wherein the CREM mutation results in an increase in the expression of the CREM RNA isoform CREM-218.

31. The cell according to any one of claims 28 to 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 according to any one of claims 28 to 31, wherein the CREM mutation results in a decrease in more than 60% of one or more CREM protein isoforms.

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

34. The cell according to any one of claims 1 to 33, wherein the cell is further acclimated to an acidic microenvironment by contacting the cell with an acidic stimulus ex vivo.

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

36. The cell according to any one of claims 34 to 35, wherein the acclimation to the acidic microenvironment is by a gradual and / or cumulative contact with the acidic stimulus.

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

38. The cell according to claim 36 or 37, wherein the acclimation of the cell comprises applying an acidic stimulus every about 48 to 72 hours, optionally every about 48 hours.

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

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

0.

41. The cell according to any one of claims 1 to 40, 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 gamma delta T cell, an MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

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

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

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

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

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

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

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

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

50. The antigen is 5T4, 8H9, α ｖ β ６ The cell according to any one of claims 45 to 49, selected from the group consisting of 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, the 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.

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

52. The cell according to any one of claims 44 to 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 according to any one of claims 1 to 52, wherein the cell comprises the expression of one or more exogenous chemokines and / or one or more cytokines.

54. The cell according to 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 according to claim 54, wherein the cytokine is IL-15.

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

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

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

59. The cell according to claim 58, wherein the endonuclease is an RNA-induced endonuclease.

60. The cell according to claim 59, wherein the RNA-induced endonuclease is CRISPR-Cas9.

61. The cell according to any one of claims 1 to 60, wherein the cell comprises one or more additional mutations in one or more genes selected from the group consisting 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.

62. A cell population according to any one of claims 1 to 61.

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

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

65. The method according to claim 64, wherein the cell is autologous, allogeneic or xenogeneic to the individual.

66. The method according to claim 64 or 65, wherein the cell is allogeneic to the individual.

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

68. The method according to any one of claims 64 to 66, wherein the cancer does not include solid tumors.

69. The method according to any one of claims 64 to 68, wherein the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, and / or cervix.

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

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

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

73. The method according to any one of claims 64 to 72, wherein the individual is subjected to additional cancer treatment.

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

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

76. A method of manipulating immune effector cells, comprising mutating the 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-responsive element-binding protein 1 (CREB1) gene in the cells.

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

78. The method according to claim 76 or 77, wherein the mutating reduces or inhibits the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to an unmutated locus encoding the same endogenous gene.

79. The method according to claim 76, wherein said mutating generates a novel functional mutation or a gain-of-function mutation.

80. The method according to claim 76 or 79, wherein said mutating increases the transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the mutated endogenous gene as compared to an unmutated locus encoding the same endogenous gene.

81. The method according to any one of claims 76 to 80, wherein said mutating generates a modified mRNA isoform population encoded by the mutated endogenous gene as compared to a representative mRNA population encoded by an unmutated locus encoding the same endogenous gene.

82. The method according to any one of claims 76 to 79, wherein said mutating generates a modified protein isoform population encoded by the mutated endogenous gene as compared to a representative protein population encoded by an unmutated locus encoding the same endogenous gene.

83. The method according to any one of claims 76 to 82, wherein said mutating generates a knockout (KO) mutation in the endogenous gene of said cell.

84. The method according to any one of claims 76 to 82, wherein said mutating generates a homozygous mutation in the endogenous gene of said cell.

85. The method according to any one of claims 76 to 82, wherein said mutating generates a heterozygous mutation in the endogenous gene of said cell.

86. The method according to any one of claims 76 to 85, wherein said mutating generates improved cytotoxicity of said engineered cell in an acidic microenvironment and / or a tumor microenvironment (TME) as compared to control non-engineered cells.

87. The method according to any one of claims 76 to 86, wherein said mutating generates improved cytotoxicity of said engineered cell in an acidic microenvironment having a pH less than or equal to about 7.0 as compared to control non-engineered cells.

88. The method according to any one of claims 76 to 87, wherein said mutating generates improved cytotoxicity of said engineered cells in an acidic microenvironment having a pH less than or equal to about 5.9 as compared to non-engineered cells of a control.

89. The method according to any one of claims 76 to 88, wherein said mutating results in improved cytotoxicity of said engineered cells in an acidic microenvironment characterized by an increased lactate level as compared to a non-acidic microenvironment.

90. The method according to any one of claims 76 to 89, wherein said mutating generates enhanced multifunctionality of said engineered cells in response to stimulation by tumor cells as compared to non-engineered cells of a control.

91. The method according to claim 90, wherein said enhanced multifunctionality is evidenced by an increase in cytokine release in response to stimulation by tumor cells.

92. The method according to claim 91, wherein said increase in cytokine release comprises an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

93. The method according to claim 90 or 91, wherein said increase in cytokine release comprises an increase in granulocyte macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

94. The method according to any one of claims 76 to 93, wherein said mutating generates an enhanced activation and / or cytotoxic phenotype for said engineered cells as compared to non-engineered cells of a control.

95. The method according to any one of claims 76 to 93, wherein said mutating results in an enhanced activation and / or a cytotoxic phenotype for said manipulated cells as compared to control unmanipulated cells, and said enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F target, p53 pathway, mitotic spindle, MYC, mTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

96. The method according to any one of claims 76 to 95, wherein said mutating results in upregulation of G2M, E2F, MYC, mTORC1, oxidative phosphorylation, and / or TNFa signaling.

97. The method according to any one of claims 76 to 96, wherein said mutating generates an enhanced proliferative capacity and / or a persistence phenotype for said manipulated cells as compared to control unmanipulated cells.

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

99. The method according to claim 97 or 98, wherein said enhanced proliferative capacity and / or persistence does not result in autonomous proliferation.

100. The method according to any one of claims 76 to 99, wherein said mutating generates an enhanced metabolic adaptability phenotype for said manipulated cells as compared to control unmanipulated cells.

101. The method according to claim 100, wherein said enhanced metabolic adaptability is a higher glycolytic capacity and / or an improved oxygen consumption rate (OCR).

102. The method according to any one of claims 76 to 101, wherein said mutating provides increased chromosomal proximity across the genome when said cells are contained in an acidic microenvironment.

103. The method according to any one of claims 76 to 102, wherein said endogenous gene is CREM.

104. The method according to claim 103, wherein mutating CREM results in a decrease in the 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 according to claim 103 or 104, wherein mutating CREM results in an increase in the expression of CREM RNA isoform CREM-218.

106. The method according to any one of claims 103 to 105, wherein mutating 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 according to any one of claims 103 to 106, wherein mutating CREM produces a decrease in more than 60% of one or more CREM protein isoforms.

108. The method according to any one of claims 103 to 107, wherein mutating CREM produces a decrease in more than 80% of one or more CREM protein isoforms.

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

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

111. The method according to claim 109 or 110, wherein the acclimation to the acidic microenvironment is by a gradual and / or cumulative contact with the acidic stimulus.

112. The method according to any one of claims 109 to 111, wherein the acclimation is carried out over a period of at least about 10 - 18 days, optionally at least about 14 days.

113. The method according to any one of claims 109 to 112, wherein the acclimation comprises applying the acidic stimulus every about 48 - 72 hours, optionally every about 48 hours.

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

115. The method according to any one of claims 109 to 114, wherein the domestication is for an acidic microenvironment with a pH less than or equal to 6.

0.

116. The method according to any one of claims 76 to 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 gamma delta T cell, an MSC, a tumor infiltrating lymphocyte, or a dendritic cell.

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

118. The method according to claim 116 or 117, wherein the NK cell is derived from umbilical cord blood.

119. The method according to any one of claims 76 to 118, wherein the cell comprises one or more engineered receptors that specifically bind to an antigen.

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

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

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

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

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

125. wherein the antigen is 5T4, 8H9, α ｖ β ６ 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, the 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, the method according to any one of claims 120 to 124.

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

127. The method according to any one of claims 119 to 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 according to any one of claims 76 to 127, wherein the cell comprises the expression of one or more exogenous chemokines and / or one or more cytokines.

129. The method according to 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 according to claim 129, wherein the cytokine is IL-15.

131. The method according to any one of claims 76 to 130, wherein the cell contains a suicide gene.

132. The method according to any one of claims 76 to 131, wherein the mutating of the endogenous gene includes homologous recombination or non-homologous recombination.

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

134. The method according to claim 133, wherein the endonuclease is an RNA-induced endonuclease.

135. The method according to claim 134, wherein the RNA-induced endonuclease is CRISPR-Cas9.

136. The method according to any one of claims 76 to 135, wherein the cell contains one or more additional mutations in one or more genes, and the genes are selected from the group consisting 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.

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

138. The cell according to claim 137, wherein the cell contains a knockout (KO) mutation of the endogenous CREM gene in the cell.

139. The cell according to claim 138, wherein the cell contains a homozygous knockout (KO) mutation of the endogenous CREM gene.

140. The cell according to claim 138, wherein the cell contains a heterozygous KO mutation of the endogenous CREM gene in the cell.

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

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

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

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

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

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

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

148. The cell according to claim 137, wherein the increase in cytokine release comprises an increase in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and / or degranulation marker CD107a in response to stimulation by tumor cells.

149. The cell according to claim 148, wherein the increase in cytokine release comprises an increase in granulocyte macrophage colony stimulating factor (GMCSF), soluble CD137 (sCD137), INF-γ, 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-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), TNF-α, and / or perforin in response to stimulation by tumor cells.

150. The cell according to claim 137, wherein the mutation results in an enhanced activation and / or cytotoxic phenotype for the manipulated cell as compared to a non-manipulated cell of the control.

151. The cell according to claim 150, wherein the mutation results in an enhanced activation and / or cytotoxic phenotype for the manipulated cell as compared to a non-manipulated cell of the control, and the enhanced activation and / or cytotoxic phenotype is associated with one or more of the pathways identified by GSEA: G2M checkpoint, E2F target, P53 pathway, mitotic spindle, MYC, mTORC1, androgen response, unfolded protein response, spermatogenesis, heme metabolism, TNF alpha signaling, protein secretion, apoptosis, oxidative phosphorylation, DNA repair, UV response, and / or early estrogen response.

152. The cell according to claim 137, wherein the mutation results in upregulation of G2M, E2F, MYC, mTORC1, oxidative phosphorylation, and / or TNFa signaling.

153. The cell according to claim 137, wherein the mutation results in an enhanced proliferation ability and / or persistence phenotype for the manipulated cell as compared to a non-manipulated cell of the control.

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

155. The cell according to claim 153 or 154, wherein the enhanced proliferation ability and / or persistence does not result in autonomous proliferation.

156. The cell according to claim 137, wherein the mutation results in an enhanced metabolic adaptability phenotype for the manipulated cell as compared to a non-manipulated cell of the control.

157. The cell according to claim 137, wherein the enhanced metabolic adaptability is a higher glycolytic ability and / or an improved oxygen consumption rate (OCR).

158. The cell according to claim 137, wherein the mutation provides increased chromosomal proximity across the genome when the cell is contained in an acidic environment.

159. The cell according to claim 137, wherein the CREM mutation results in a decrease in the 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 according to claim 137, wherein the CREM mutation is a 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 according to claim 137, wherein the CREM mutation results in a decrease of more than 60% of the CREM protein isoforms; or the CREM mutation results in a decrease of 80% or more of the CREM protein isoforms.

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

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

164. The cell according to claim 137, wherein the cell is derived from stem cells.

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

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

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

168. The operated receptor is 5T4, 8H9, α ｖ β ６ 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, the 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, the cell according to claim 165, which specifically binds to an antigen selected from the group consisting of.

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

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

171. A cell population according to any one of claims 137 to 170.

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

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

174. The method according to claim 173, wherein the cancer comprises solid tumors.

175. The method according to claim 173, wherein the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, and / or cervix.

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

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

178. A method of manipulating the cells according to any one of claims 137 to 170.

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

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