Compositions and methods for immune cell modulation in adoptive immunotherapy

Abstract

Compounds are identified that produce either a higher percentage or a greater absolute number of phenotypically identified naive, stem cell memory, central memory T cells, adoptive NK cells, and type I NKT cells. Compositions and methods for modulating immune cells, including T, NK, and NKT cells, for adoptive cell therapy with improved efficacy are provided. [Selected Figure] Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 281,064, filed January 20, 2016, and U.S. Provisional Application No. 62 / 336,339, filed May 13, 2016, the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present disclosure relates generally to the field of adoptive cellular immunotherapy. More specifically, the present disclosure relates to the use of small molecules to modulate immune cells suitable for adoptive cellular therapy. [Background technology]

[0003] Adoptive immunotherapy involves the administration of immune cells to a patient with cancer, tumor, or infectious disease, such that the administered immune cells provide a therapeutic effect to the patient. Generally speaking, immune cells suitable for immunotherapy include, but are not limited to, B cells, dendritic cells (DCs), T cells, natural killer (NK) cells, NKT (natural killer T) cells, and hematopoietic stem or progenitor cells. Mediating a complete and durable disease response in patients is the central goal of these cell-based immunotherapies.

[0004] Advances in our understanding of the biological mechanisms behind the efficacy of adoptive T cell therapy, including but not limited to CAR-T cells, TCR-T cells, virus-specific T cells (VSTs) and tumor-infiltrating T cells (TILs), have clearly demonstrated the importance of certain properties associated with the introduced T cells and revealed the complexity of the inhibitory barriers caused by the host and tumor cells that need to be overcome for successful cancer treatment. Among the T cell factors, the binding activity of the T cell receptor (TCR) or chimeric antigen receptor (CAR), proliferation and survival capacity, migration to the tumor site, and the ability to sustain effector functions within the tumor have been shown in correlation studies to be critical determinants for triggering the eradication of malignant cells. However, in addition to another layer of complexity, although some of these desirable properties have been recognized, the pathways or players that bring about these properties are still unclear, which limits one's ability to intervene and obtain cells with the desired quantity and quality for their therapeutic use.

[0005] Using CAR-T cell therapy as an example, the therapy must overcome multiple challenges, including CAR-T capacity and persistence, migration to tumors, immunosuppressive tumor microenvironment, tumor heterogeneity, and patient safety. To overcome these challenges, multiple approaches have been applied. For example, specific T cell subsets may be selected for therapeutic use, and further engineering of the CAR may be used to improve tumor targeting, CAR capacity, and on-target / off-tumor safety issues. However, improvements in CAR-T therapeutic capacity, including CAR-T persistence and migration, remain to be resolved. It has been shown that the in vivo efficacy of T cell therapy can be strongly influenced by the manufacturing process, which is dependent on both the starting population of T cells put into the process or raw material, and the ex vivo expansion and activation method utilized. It has been shown that the differentiation state of the administered T cells can significantly affect in vivo persistence and antitumor activity. T helper (CD4+ T cells) and cytotoxic T cells (CD8+), specifically naive (Tn), stem cell memory (Tscm) and central memory (Tcm) T cells, characterized by expression of CCR7 and CD62L markers, mediate potent antitumor activity in both mouse (Sommermeyer et al. 2015) and non-human primate models (Berger et al. 2008).

[0006] During the manufacturing process, therapeutic cells (or cell populations) are typically activated and expanded. This process generally results in differentiation of cells, leading to an increase in the proportion of cells in a more differentiated state, and in the case of T cells, the more differentiated cells are phenotypically characterized as effector memory or effector T cells. When infused into a patient, these more differentiated cells have a lower ability to proliferate and a lower ability to persist as a long-term surviving or persistent population compared to cells in a less differentiated state. Thus, there is an urgent need in the art for compositions and methods useful for maintaining and expanding desired immune cell subsets, as well as for reducing cell differentiation during expansion and dedifferentiating cells into less differentiated cells, thereby obtaining desired immune cell subsets that have a higher ability to proliferate and persist, in order to improve the efficacy of various adoptive immunotherapies.

[0007] Similar efficacy issues also exist in NK cell-based therapies. Natural killer cells have been characterized as living for a relatively short period of time and traditionally classified as innate immune cells that exhibit minimal changes in response to secondary exposure to stimuli, i.e., limited targeted memory responses. However, recent studies have revealed information about both activating and inhibitory NK cell receptors that play important roles, including self-tolerance and sustained NK cell activity. Data have demonstrated the ability of NK cells to easily adapt to the surrounding environment and orchestrate antigen-specific immune memory that is fundamental to respond to secondary exposure to the same antigen. A subpopulation of NK cells, now called adoptive or memory NK cells, has been identified by several groups. These cells have many functional characteristics similar to CD8+ T cells, including surviving for a longer period of time and having an enhanced response to stimuli after initial exposure. These properties may lead to more effective cell therapy strategies compared to standard NK cells. The expansion and maintenance of adoptive / memory NK cells that mediate persistent antigen-specific recognition in vivo will be key to improving NK cell-based adoptive immunotherapy.

[0008] Furthermore, it is believed that improvements can be made to isolate more effective NKT cells, a type of CD1d-restricted T cell that plays a role in both the innate and adaptive immune systems, which, like T and NK cells, can be targeted for modulation to produce improved cellular therapies.

[0009] The importance of the manufacturing process cannot be overstated, since the final state of the cells that are put into the patient, or specifically, the cell subtype, can be determined to a large extent by the manufacturing process. During cell culture and expansion, preferentially maintaining or expanding cell subpopulations with desired differentiation state and / or adoptive immune cell characteristics can be extremely beneficial to enhance the efficacy of cell-based therapy. Thus, a manufacturing approach that can enhance the desired T, NK or NKT cell subsets in both quantity and quality can result in a significant enhancement of their therapeutic efficacy.

[0010] There is a substantial need in the art for immune cell subsets with improved therapeutic efficacy.However, while certain desirable properties of therapeutic immune cells are known, the pathways and / or players involved in achieving these properties are largely unknown.The method and composition of the present invention address these needs and provide other related advantages in the field of immune cell therapy. Summary of the Invention

[0011] The present invention provides compositions and methods for modulating one or more populations or subpopulations of immune cells to improve their therapeutic potential for adoptive immunotherapy. For example, it is an object of the present invention to provide one or more compounds, either alone or in combination, for improving the proliferation, persistence, cytotoxicity, and / or cellular recall / memory of therapeutic immune cells by increasing the number or proportion of a subpopulation of cells that exhibits improvements in at least one of the following properties that are predicted to result in better immunotherapy outcomes: migration, homing, cytotoxicity, maintenance, expansion, persistence, longevity extension, differentiation to a desired state.

[0012] One aspect of the present invention provides a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy, the composition comprising a compound selected from the group consisting of dorsomorphin, hepteridic acid, 1-pyrrolidinecarbodithioic acid, ammonium salt, 2-deoxyglucose (2-DG), GSK3 inhibitors, Rho kinase inhibitors, MEK inhibitors, PDK1 agonists, TGFβ inhibitors, 6-mercaptopurine, AC-93253 iodide, tiratricol, PI-103, fulvestrant, thapsigargin, SU4312, telmisartan, cyclosporin A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY61-3606, and protease inhibitors. The present invention includes one or more agents selected from the group consisting of: toporphyrin IX disodium; rapamycin; HS173; LY294002; pictilisib; 5-azacytidine; fludarabine; roscovitine, (S)-isomer; PAC-1, 8-quinolinol, 5,7-dichloro-; nitrofurantoin; 8-quinolinol, 5-chloro-7-iodo-; 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octadecane; nifuroxazide; tosufloxacin hydrochloride; sertraline; diethylenetriaminepentaacetic acid, pentasodium; edrophonium chloride; BIX01294; terfenadine; and dmPGE2. One or more agents selected from the group consisting of the compounds listed in Table 1 improve the treatability of immune cells or one or more subpopulations thereof via modulation of the immune cells using the one or more agents. In some embodiments, modulation of the immune cells is in vitro.

[0013] In some embodiments, one or more of the compounds listed in Table 1 modulate cell expansion, maintenance, and / or differentiation, thereby improving the proliferation, cytotoxicity, cytokine response and secretion, cell recall, and / or persistence of immune cells, or one or more subpopulations thereof.

[0014] In one embodiment, one or more of the compounds listed in Table 1 improves cell viability of immune cells, or one or more subpopulations thereof, both in vitro and in vivo.

[0015] In one embodiment, one or more of the compounds listed in Table 1 increase the proportion of one or more desired cell subpopulations of immune cells.

[0016] In some embodiments, the present invention provides herein one or more selected agents for improving the therapeutic effect of a population or subpopulation of immune cells, including but not limited to T, NK and NKT cells. In some embodiments, the immune cells immune cells suitable for adoptive cell-based therapy include T cells, NKT cells, or NK cells. In some embodiments, the immune cells to be treated include T cells, and thus one or more desired cell subpopulations have an increased proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In some embodiments, the immune cells to be treated with the agent include NKT cells, and thus one or more desired cell subpopulations have an increased proportion of type I NKT cells. In some other embodiments, the immune cells to be treated with the agent include NK cells, and thus one or more desired cell subpopulations have an increased proportion of adoptive NK cells.

[0017] In some embodiments, the compositions include one or more drugs selected from the group consisting of compounds, or derivatives, analogs, or pharma- ceutically acceptable salts thereof, listed in Table 1. The compounds, or derivatives, analogs, or pharma- ceutically acceptable salts thereof, further include esters, ethers, solvates, hydrates, stereoisomers, and prodrugs of the compounds in Table 1.

[0018] In some embodiments, a composition for improving the therapeutic potential of an immune cell comprises at least one agent selected from Group I and one or more agents selected from Group II, Group III, Group IV, and / or Group V.

[0019] Group I includes dorsomorphin, hepteridic acid, 1-pyrrolidinecarbodithioic acid, and 2-DG. Without being limited by theory, Group I agents affect cellular metabolism and nutrient sensing, among other possible roles.

[0020] Group II includes GSK3 inhibitors, ROCK inhibitors, TGFβ receptor inhibitors, MEK inhibitors, PDK1 agonists, 6-mercaptopurine, AC-93253 iodide, tiratricol, PI-103, fulvestrant, thapsigargin, SU4312, U0126, telmisartan, cyclosporine A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY61-3606, protoporphyrin IX disodium, rapamycin, TWS119, HS173, LY294002, and pictilisib. Without being limited by theory, Group II agents affect signal transduction in various functional pathways, among other possible roles.

[0021] Group III includes 5-azacytidine, fludarabine, roscovitine, and PAC-1. Without being limited by theory, Group III agents affect cell proliferation and apoptosis, among other possible roles.

[0022] Group IV includes 5,7-dichloro-8-quinolinol, 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octadecane, nifuroxazide, and tosufloxacin hydrochloride. Without being limited by theory, Group IV agents may affect cellular properties related to the infectious process, among other possible roles.

[0023] Group V includes sertraline, diethylenetriaminepentaacetic acid, edrophonium chloride, BIX01294, terfenadine, and dmPGE2. Without being limited by theory, Group V agents generally affect other cellular properties related to expansion, maintenance, differentiation, dedifferentiation, viability, proliferation, cytotoxicity, cell recall, and / or persistence, among other possible roles.

[0024] In some other embodiments, a composition for improving the therapeutic potential of an immune cell comprises at least one agent selected from Group II and one or more agents selected from Group I, Group III, Group IV, and / or Group V.

[0025] In yet other embodiments, a composition for improving the therapeutic potential of an immune cell comprises at least one agent selected from Group III and one or more agents selected from Group I, Group II, Group IV, and / or Group V.

[0026] In yet other embodiments, a composition for improving the therapeutic potential of an immune cell comprises at least one agent selected from Group IV and one or more agents selected from Group I, Group II, Group III, and / or Group V.

[0027] In yet some other embodiments, a composition for improving the therapeutic potential of an immune cell comprises at least one agent selected from Group V and one or more agents selected from Group I, Group II, Group III, and / or Group IV.

[0028] In some embodiments, a composition for improving the therapeutic potential of an immune cell comprises a combination of at least one agent selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG, and one or more additional agents selected from the group consisting of the compounds listed in Table 1. In some specific embodiments, the composition comprises a synergistic combination of two or more agents selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG.

[0029] In some embodiments, the composition comprising one or more agents selected from the group consisting of the compounds listed in Table 1 further comprises at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof.

[0030] Another aspect of the invention provides compositions comprising a population or subpopulation of immune cells and one or more agents selected from the group consisting of the compounds listed in Table 1, and derivatives and analogs thereof. In some embodiments, the immune cells are contacted with the one or more agents to improve the therapeutic potential of the immune cells for adoptive cell therapy, compared to immune cells not treated with such treatment. In some embodiments, the immune cells are contacted with the one or more agents to improve cell expansion, maintenance, differentiation, dedifferentiation, and / or survival, compared to immune cells not treated with the same treatment. In yet some other embodiments, the immune cells are contacted with the one or more agents to improve cell proliferation, cytotoxicity, persistence, and / or recall, compared to immune cells not treated with the same treatment.

[0031] In some embodiments, immune cells contacted with one or more agents have an increased number or percentage of a desired subpopulation of immune cells compared to immune cells not treated with the same. In some embodiments, the immune cells include T, NK, or NKT cells. In one embodiment, the composition includes a population of T cells, and thus the desired subpopulation of immune cells after contact with the agent includes naive T cells, stem cell memory T cells, and / or central memory T cells. In some embodiments, the composition includes a population of NKT cells, and thus the desired subpopulation of immune cells after contact with the agent includes type I NKT cells. In yet some other embodiments, the immune cells include a population of NK cells. In some other embodiments, the NK cells include CD57- NK cells or CD57-NKG2C+ NK cells. Thus, the desired subpopulation of immune cells after contact with the agent includes CD57+ NK cells. In some embodiments, the CD57+ NK cells include adoptive NK cells. In other embodiments, the adoptive NK cells include CD57 + , and NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0032] In some embodiments, the population or subpopulation of immune cells of the composition is isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor of a subject. The subject may be healthy, have an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor, or may have already been administered genetically modified immune cells. In some embodiments, the subject may be CMV seropositive. In some other embodiments, the isolated immune cells for modulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications). In some embodiments, the isolated immune cells for modulation include at least one genetically modified modality. In some embodiments, the isolated population of immune cells is genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpressed CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genetically modified immune cells isolated from the subject may be administered to the same donor or to a different patient.

[0033] In yet another embodiment, the immune cells are differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells, or transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some specific embodiments, the immune cells of the composition comprise an exogenous nucleic acid encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR), and / or overexpressed CD16 or a variant thereof. In one embodiment, CD16 is hnCD16 (high affinity non-cleavable CD16).

[0034] In yet some other embodiments, the immune cells of the composition are differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells. In one embodiment, the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In one embodiment, the progenitor cells are CD34+ hemogenic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK progenitor cells, or NKT progenitor cells. In some embodiments, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells are genomically engineered, include insertions, deletions, or nucleic acid substitutions, or include at least one genetically modified modality. In one particular embodiment, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or overexpression of CD16. In some other embodiments, the immune cells of the composition are transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the desired cell subpopulation of immune cells after modulation comprises immune cells having at least one genetically modified modality. In one embodiment, the desired cell subpopulation of immune cells comprises CD57+NK cells having at least one genetically modified modality. In some embodiments, the genetically modified modality comprises at least one of a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a medicamentously effective protein and peptide, a drug target candidate; or a protein that promotes immune cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival.In some other embodiments, the genetically modified modality comprises (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region; and (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling with bi- or multispecific or universal engagers. In one particular embodiment, CD57. + In yet another embodiment, the NK cells comprise expression of hnCD16. + The NK cells are adoptive NK cells. In another embodiment, the NK cells are CD57 + NK cells are NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0035] In some embodiments, the composition comprises one or more of a GSK3 inhibitor, a TGFβ receptor inhibitor, a ROCK inhibitor, a MEK inhibitor, a PDK1 agonist, and rapamycin. In some embodiments, the composition comprises a GSK3 inhibitor. In some embodiments, the composition comprising immune cells and one or more agents selected from the group consisting of the compounds listed in Table 1 further comprises one or more additional additives selected from the group consisting of a peptide, a cytokine, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or replacement factor, a vector comprising one or more polynucleic acids of interest; an antibody, or an antibody fragment; and a chemotherapeutic agent, a radioactive moiety, or an immunomodulatory drug (IMiD). In some of these embodiments, the antibody, or antibody fragment, specifically binds to a viral antigen. In other embodiments, the antibody, or antibody fragment, specifically binds to a tumor antigen. In some embodiments, the additional additive comprises. Chemotherapeutic agent refers to cytotoxic antitumor drug, i.e., chemical agent that preferentially kills tumor cells or disrupts the cell cycle of rapidly growing cells, or that is found to eradicate cancer stem cells, and chemical agent that is used therapeutically to prevent or reduce the growth of tumor cells.Chemotherapeutic agent is also sometimes referred to as antitumor or cytotoxic drug or drug, and is well known in the art.

[0036] In one particular embodiment, the composition comprises a mixture of a population or subpopulation of immune cells and one or more of a GSK3 inhibitor, a TGFβ receptor inhibitor, a ROCK inhibitor, a MEK inhibitor, a PDK1 agonist, and an mTOR inhibitor, wherein the immune cells comprise NK cells. In some embodiments, the mTOR inhibitor is selected from rapamycin and analogs or derivatives thereof. In some embodiments, the mTOR inhibitor is selected from rapamycin and analogs or derivatives thereof, including sirolimus, sirolimus derivatives, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus), 40-O-(3-hydroxy)propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, and other O-alkylated or O-methylated rapamycin derivatives. In one embodiment, the composition comprises a GSK3 inhibitor. In another embodiment, the composition comprises a GSK3 inhibitor, a MEK inhibitor, and rapamycin. In yet another embodiment, the composition comprises a GSK3 inhibitor. In one embodiment, the composition comprising a GSK3 inhibitor further comprises at least one of stimulatory cytokines including IL2, IL15, IL12, IL18 and IL21. In one embodiment, the composition comprising a GSK3 inhibitor further comprises one or more MEK inhibitors, rapamycin, and a STING agonist. In some embodiments, the STING agonist comprises a cyclic dinucleotide (CDN), or a xanthenone, or an analog or derivative thereof. In some embodiments, the CDN may be synthetic or derived from a prokaryotic cell or a mammalian cell. In some other embodiments, the STING agonist comprises at least one molecule selected from the group consisting of cGAMP, c-di-GMP, c-di-AMP, c-di-IMP, c-di-UMP, cAMP-GMP, R,R dithio-modified CDA compounds (ML RR-S2 CDA and RR-S2 CDA), ML RR-S2 cGAMP, DMXAA (5,6-dimethylxanthenone-4-acetic acid), and analogs and derivatives thereof.

[0037] One aspect of the present invention provides a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy, comprising one or more agents selected from the group consisting of the compounds listed in Table 1 and derivatives and analogs thereof, wherein the one or more agents improve the therapeutic potential of the immune cells through modulation of the immune cells. In some embodiments of the composition, the agents are capable of modulating cell expansion, maintenance and / or differentiation; improving cell proliferation, cytotoxicity, cytokine response and secretion, recall and / or persistence; improving cell viability; and / or increasing the proportion of one or more desired cell subpopulations of immune cells. In some embodiments, the derivatives and analogs include salts, esters, ethers, solvates, hydrates, stereoisomers, and prodrugs of the agents in Table 1.

[0038] In some embodiments, immune cells suitable for modulation by the agent include T cells, NKT cells, and / or NK cells. In some embodiments, NK cells for modulation include CD57- NK cells, and / or CD57-NKG2C+ NK cells. In some embodiments, the modulated immune cells include one or more desired cell subpopulations having an increased proportion, which may be naive T cells, stem cell memory T cells, and / or central memory T cells; type I NKT cells; or CD57+ NK cells. In some embodiments, the CD57+ NK cells include adoptive NK cells. In some embodiments, the adoptive NK cells include CD57 + , and NKG2C + In some embodiments, the adoptive NK cells are characterized as at least one of: low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. + NKG2C + It can be characterized as:

[0039] In some embodiments, the immune cells suitable for modulation by the agent are isolated or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor. In one embodiment, the immune cells are isolated from a healthy subject; a subject with an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor; a subject who has already been administered genetically modified immune cells; or a subject who is CMV seropositive. In some other embodiments, the isolated immune cells for modulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications). In some embodiments, the isolated immune cells for modulation include at least one genetically modified modality. In some embodiments, the isolated population of immune cells is genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpressed CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genetically modified immune cells isolated from the subject may be administered to the same donor or to a different patient.

[0040] In some other embodiments, immune cells suitable for modulation by the agent are differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells, or transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In other embodiments, the progenitor cells are CD34 + The progenitor cells are hemogenic endothelial cells, multipotent progenitor cells, T cell progenitor cells, NK progenitor cells, or NKT progenitor cells.

[0041] In some embodiments, stem cells, hematopoietic stem or progenitor cells, or progenitor cells for differentiation into various immune cells, including T, NK, and NKT cells, are genomically engineered to include insertions, deletions, or nucleic acid substitutions, or to include at least one genetically modified modality. Thus, the desired subpopulation of regulated immune cells resulting from stem cells, hematopoietic stem or progenitor cells, or progenitor cells also includes at least one genetically modified modality. In one particular embodiment, the desired cell subpopulation of immune cells includes at least one genetically modified modality, such as CD57 + In some embodiments, the genetically modified modality comprises at least one of a safety switch protein, a targeting modality, a receptor, a signal molecule, a transcription factor, a medicamentously effective protein and peptide, a drug target candidate; or a protein that promotes immune cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival. In some other embodiments, the genetically modified modality comprises one or more of deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosome 6p21 region. In some embodiments, the genetically modified modality includes one or more induced or increased expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling with bi- or multispecific or universal engagers. In one particular embodiment, CD57 is included in the desired subpopulation resulting from immune cell modulation. + The NK cells comprise expression of hnCD16. In another embodiment, the NK cells express hnCD16 and CD57 + NK cells are NKG2C +, low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0042] In some embodiments, the immune cells are modulated by a composition comprising a GSK3 inhibitor. In some embodiments, the composition comprising a GSK3 inhibitor further comprises at least one of stimulatory cytokines comprising IL2, IL15, IL12, IL18 and IL21. In some embodiments, the composition comprising a GSK3 inhibitor further comprises one or more MEK inhibitors, rapamycin, and a STING agonist. Thus, in some embodiments, the composition comprises a population of NK cells, and a GSK3 inhibitor. In some embodiments, the composition comprises a population of NK cells, a GSK3 inhibitor, and IL15. In some embodiments, the composition comprises a population of NK cells, a GSK3 inhibitor, IL15, and a STING agonist. In some embodiments, the immune cells are modulated by a composition comprising a modulator and at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof.

[0043] Yet another aspect of the present invention provides a composition comprising an isolated population of immune cells contacted or modulated with a composition comprising one or more agents listed in Table 1, or derivatives or analogs thereof. In some embodiments, the compositions provided are therapeutic compositions having treated isolated populations or subpopulations of immune cells, including, but not limited to, T, NK and NKT cells. The therapeutic composition can be washed with a buffer that is substantially free of the modulating agent.

[0044] In some embodiments, the regulated cell population comprises immune cells with improved therapeutic potential for adoptive cell therapy compared to unregulated cell populations. In some embodiments, the isolated population of immune cells has improved cell expansion, maintenance, differentiation, dedifferentiation, and / or survival compared to immune cells not treated with one or more agents. In some embodiments, the isolated population of immune cells has improved cell proliferation, cytotoxicity, cytokine response and secretion, cell recall, and persistence compared to immune cells not treated with one or more agents. In some other embodiments, the isolated population of immune cells has an increased number or percentage of one or more desired subpopulations of immune cells compared to immune cells not treated with the same agents.

[0045] In some embodiments, the isolated population of immune cells treated with one or more agents selected from the group consisting of the compounds listed in Table 1 comprises T cells, and thus the resulting one or more desired subpopulations of immune cells comprise naive T cells, stem cell memory T cells, and / or central memory T cells. In some embodiments, the isolated population of immune cells treated with one or more agents comprises NKT cells, and thus the resulting one or more desired subpopulations of immune cells comprise type I NKT cells. In yet some other embodiments, the isolated population of immune cells treated with one or more agents comprises NK cells, and thus the resulting one or more desired subpopulations of immune cells comprise adoptive NK cells.

[0046] In some embodiments of the provided compositions, the isolated population of immune cells may be isolated from peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor of a subject. The subject may be healthy, have an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor, or may have already been administered genetically modified immune cells. In some embodiments, the subject may be CMV seropositive. In some other embodiments, the isolated immune cells for modulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications). In some embodiments, the isolated immune cells for modulation include at least one genetically modified modality. In some embodiments, the isolated population of immune cells are genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpressed CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genetically modified immune cells isolated from the subject may be administered to the same donor or to a different patient.

[0047] In some embodiments of the provided compositions, the isolated population of immune cells may be differentiated from stem cells, hematopoietic stem or progenitor cells, or progenitor cells. In some embodiments, the isolated population of immune cells may be differentiated from stem cells, hematopoietic stem or progenitor cells, or progenitor cells prior to or during treatment with the agent. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the progenitor cells are CD34 +Hematopoietic endothelial cells, multipotent progenitor cells, T cell progenitors, NK progenitors, or NKT progenitors. In some further embodiments, the stem cells, hematopoietic stem or progenitor cells, progenitor cells, immune cells derived for regulation, or regulated derived immune cells are genomically engineered, for example, by insertion, deletion, and / or nucleic acid substitution. In one particular embodiment, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpression of CD16.

[0048] In some other embodiments of the provided compositions, the isolated population of immune cells may be transdifferentiated from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the isolated population of immune cells may be transdifferentiated from hematopoietic or non-hematopoietic non-pluripotent cells prior to or during treatment with the agent.

[0049] Another aspect of the invention provides a method of modulating immune cells comprising contacting immune cells with a sufficient amount of a composition comprising at least one agent selected from the group consisting of the compounds listed in Table 1, and derivatives and analogs thereof, for a sufficient time to obtain a population of modulated immune cells having improved therapeutic potential for adoptive cell therapy compared to unmodulated immune cells. In some embodiments, the derivatives and analogs include salts, esters, ethers, solvates, hydrates, stereoisomers, and prodrugs of the agents of Table 1.

[0050] In some embodiments of the method, the modulated immune cells include cells having improved proliferation, cytotoxicity, cytokine response, cytokine release, cell recall, and / or persistence; improved cell expansion, maintenance, differentiation, dedifferentiation, and / or survival; and / or increased numbers or proportions of one or more desired subpopulations of immune cells, compared to immune cells not contacted with one or more agents of Table 1. In some embodiments of the method, the method further includes isolating one or more desired subpopulations from the modulated immune cells. In one embodiment, the immune cells for modulation include T cells, NKT cells, or NK cells. In another embodiment, the NK cells for modulation are CD57 - NK cells, or CD57 - NKG2C + Following immune cell modulation using the method, in one embodiment, one or more desired subpopulations of the modulated immune cells may include naive T cells, stem cell memory T cells, and / or central memory T cells; type I NKT cells; or CD57+ NK cells. In one embodiment, the CD57+ NK cells obtained following modulation may be selected from the group consisting of naive T cells, stem cell memory T cells, and / or central memory T cells; type I NKT cells; or CD57+ NK cells. + The NK cells include adoptive NK cells. In some embodiments, the adoptive NK cells are CD57 + , and NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0051] In some embodiments of the general method, the immune cells for regulation are isolated or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor. In some embodiments, the immune cells for regulation are isolated from a healthy subject; a subject with an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor; a subject who has already been administered genetically modified immune cells; or a subject who is CMV seropositive. In some other embodiments, the isolated immune cells for regulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications). In some embodiments, the isolated immune cells for regulation comprise at least one genetically modified modality. In some embodiments, the isolated population of immune cells is genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpressed CD16 or variants thereof. Thus, the genomically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genomically modified immune cells isolated from the subject may be administered to the same donor or to a different patient.

[0052] In some embodiments, the regulatory immune cells are differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells. In some embodiments, the regulatory immune cells are transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the progenitor cells include CD34 +The stem cells, hematopoietic stem or progenitor cells, or progenitor cells are hemogenic endothelial cells, multipotent progenitor cells, T cell progenitors, NK progenitors, or NKT progenitors. In yet some other embodiments, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells are genomically engineered to include insertions, deletions, or nucleic acid substitutions, and / or to include at least one genetically modified modality. Thus, the desired cell subpopulation of regulated immune cells resulting therefrom includes immune cells having at least one genetically modified modality. In one embodiment, the desired subpopulation of immune cells includes immune cells having at least one genetically modified modality, such as CD57 + Includes NK cells.

[0053] In some embodiments, the genetically modified modalities include at least one of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates; or proteins that promote immune cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival. In some other embodiments, the genetically modified modalities include one or more deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region. In some other embodiments, the genetically modified modality includes one or more induced or increased expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling with bi- or multispecific or universal engagers. In one embodiment, CD57 is included in the desired subpopulation of regulated immune cells. + The NK cells comprise expression of hnCD16. In another embodiment, the NK cells express hnCD16 and CD57 + NK cells are NKG2C+ , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0054] In some embodiments of the method of modulating immune cells, the composition comprises a GSK3 inhibitor as a modulator. In some embodiments, the composition further comprises at least one of stimulatory cytokines including IL2, IL15, IL12, IL18 and IL21. In some embodiments, the composition comprising a GSK3 inhibitor further comprises one or more MEK inhibitors, rapamycin, and a STING agonist. In some embodiments, the composition comprising a modulator further comprises at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof.

[0055] In some embodiments of the method of modulating immune cells, the "sufficient time" or "sufficient length of time" is 16 hours or more, 14 hours or more, 12 hours or more, 10 hours or more, 8 hours or more, 6 hours or more, 4 hours or more, 2 hours or more, or any length of time in between. Thus, the sufficient length of time is, for example, 15 hours or more, 13 hours or more, 11 hours or more, 9 hours or more, 7 hours or more, 5 hours or more, 3 hours or more, or 1 hour or more. In some other embodiments of the method, the sufficient length of time is, for example, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, or any length of time in between. Thus, the sufficient length of time is, for example, 30 hours or more, 42 hours or more, 54 hours or more, 66 hours or more, 78 hours or more, 90 hours or more.

[0056] In some embodiments of the method, the immune cells are in a feeder-free environment during and / or after conditioning. Feeder-free conditions include feeder cell-free and feeder-conditioned medium-free. In some embodiments of the method, the immune cells are co-cultured with feeder cells during conditioning.

[0057] Another aspect of the invention provides a method of modulating immune cells comprising contacting immune cells with a sufficient amount of a composition comprising a GSK3 inhibitor for a sufficient time to obtain a population of modulated immune cells having improved therapeutic potential for adoptive cell therapy compared to unmodulated immune cells.

[0058] A further aspect of the invention provides a method of modulating NK cells comprising contacting NK cells with a sufficient amount of a composition comprising a GSK3 inhibitor for a sufficient time to obtain a population of modulated NK cells having improved therapeutic potential for adoptive cell therapy compared to unmodulated NK cells. In some embodiments of this general method, the modulated NK cells have improved proliferation, cytotoxicity, cytokine response, cytokine release, cell recall, and / or persistence. In some embodiments, the modulated NK cells have improved cell expansion, maintenance, differentiation, dedifferentiation, and / or survival. In some embodiments, the modulated NK cells have an increased number or proportion of one or more desired subpopulations of NK cells compared to NK cells not contacted with a composition comprising a GSK3 inhibitor. In some embodiments, the improved properties of the modulated NK cells are in vitro. In some embodiments, the improved properties of the modulated NK cells are in vivo. In some embodiments, the NK cells for modulation are CD57 - In some embodiments, the NK cells for modulation include CD57 - NKG2C + Includes NK cells.

[0059] In one embodiment of the method of modulating NK cells with a GSK3 inhibitor, the method further comprises isolating one or more desired subpopulations from the modulated NK cells. In some embodiments, the one or more desired subpopulations of NK cells are CD57 + In some embodiments, CD57 + The NK cells include adoptive NK cells. In some embodiments, the adoptive NK cells are CD57 + , and NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0060] In some embodiments of the method of modulating NK cells, the NK cells for modulation are isolated or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, or tumor. In one embodiment, the NK cells are isolated from a healthy subject. In another embodiment, the NK cells are isolated from a subject having an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor. In one embodiment, the NK cells are isolated from a subject who has already been administered genetically modified immune cells; in yet another embodiment, the NK cells for modulation are isolated from a subject who is CMV seropositive. In some other embodiments, the isolated immune cells for modulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications). In some embodiments, the isolated immune cells for modulation comprise at least one genetically modified modality. In some embodiments, the isolated population of immune cells is genomically engineered to include insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or overexpressed CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genetically modified immune cells isolated from the subject may be administered to the same donor or a different patient.

[0061] In some other embodiments, the NK cells for regulation are genomically engineered to include insertions, deletions, or nucleic acid substitutions. In one embodiment, the NK cells for regulation include at least one genetically modified modality. In another embodiment, the NK cells for regulation are differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells, or transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the progenitor cells are CD34 + The stem cells, hematopoietic stem or progenitor cells, or progenitor cells are hemogenic endothelial cells, multipotent progenitor cells, or NK progenitor cells. In some specific embodiments, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells are genomically engineered to include insertions, deletions, or nucleic acid substitutions. In one embodiment, the stem cells, hematopoietic stem or progenitor cells, or progenitor cells include at least one genetically modified modality. The stem cells, hematopoietic stem or progenitor cells, or progenitor cells that include at least one genetically modified modality may be used to produce differentiated immune cells, including NK cells.

[0062] In one embodiment, the desired subpopulation of regulated NK cells includes NK cells having at least one genetically modified modality. In one embodiment, the desired subpopulation of regulated NK cells includes NK cells having at least one genetically modified modality, such as CD57 +In some embodiments, the genetically modified modality comprises at least one of a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a medicamentously effective protein and peptide, a drug target candidate; or a protein that promotes immune cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival. In some other embodiments, the genetically modified modality comprises (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region; and (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling with bi- or multispecific or universal engagers. In one particular embodiment, CD57 + The NK cells comprise expression of hnCD16. In another embodiment, the NK cells express hnCD16 and CD57 + NK cells are NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0063] In some embodiments of the method, the composition comprising a GSK3 inhibitor further comprises at least one of stimulatory cytokines including IL2, IL15, IL12, IL18 and IL21. In one embodiment, the composition comprising a GSK3 inhibitor further comprises one or more MEK inhibitors, rapamycin, and a STING agonist. In some embodiments of the method, the composition comprising a GSK3 inhibitor further comprises at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof.

[0064] In some embodiments, the GSK3 inhibitor is CHIR99021, BIO, TWS119, or Kenpaullone. In one embodiment, the GSK3 inhibitor is CHIR99021. In some embodiments, the time sufficient for GSK3 inhibitor modulation is 16 hours or more. In some embodiments, the modulated NK cells have at least a two-fold increased expansion. In some other embodiments, the modulated NK cells have increased production of one or more, including IFNγ and / or TNFα. In yet some other embodiments, the one or more subpopulations of NK cells having an increased number or percentage include adoptive NK cells; NK cells expressing CD57; or cells expressing CD57 and NKG2C. In one embodiment, the method is performed in a feeder-free environment. In some embodiments of the method, the immune cells are co-cultured with feeder cells during modulation.

[0065] Also provided is a method of culturing a population of immune cells, comprising contacting the population with a composition comprising a GSK3 inhibitor to obtain selectively expanded NK cells in the population. The method further comprises obtaining a population of immune cells from a subject prior to contacting the immune cells with a composition comprising a GSK3 inhibitor, wherein the immune cells comprise peripheral blood mononuclear cells, and depleting CD3 and CD19 cells from the population of peripheral blood mononuclear cells. In some embodiments, the subject may be healthy, have an autoimmune disease, hematopoietic malignancy, viral infection or solid tumor, or be CMV seropositive, or may have already been administered genetically modified immune cells. In some embodiments, the expanded NK cells have improved cytotoxicity and cytokine response and secretion, improved proliferation, cell recall, and / or persistence, improved cell expansion and / or maintenance, increased subtype bias with respect to maturation, increased number or percentage of one or more subpopulations in the cell population, and / or maintain an adoptive / memory cell-like state, when compared to NK cells not contacted with a composition comprising a GSK3 inhibitor. In some embodiments, the regulated NK cells have at least a two-fold increased expansion. In some other embodiments, the regulated NK cells have increased production of one or more cytokines, including IFNγ and / or TNFα. In yet some other embodiments, the one or more subpopulations of NK cells with increased number or percentage include adoptive NK cells; NK cells expressing CD57; or cells expressing CD57 and NKG2C. In one embodiment, the method is carried out in a feeder-free environment. In some embodiments, the method is carried out including co-culturing with feeder cells. In some other embodiments, the composition comprising a GSK3 inhibitor further comprises one or more of IL15, IL12, IL18, a MEK inhibitor, and rapamycin. In one embodiment, the GSK3 inhibitor is CHIR999021.

[0066] Further provided is a composition comprising a population of immune cells, and a composition comprising a GSK3 inhibitor, wherein the population comprises immune cells having improved therapeutic potential of the immune cells for adoptive cell therapy upon modulation of the population with a composition comprising a GSK3 inhibitor.

[0067] Yet another aspect of the application provides a method of modulating adoptive NK cells, comprising contacting a population of immune cells comprising adoptive NK cells with a sufficient amount of a composition comprising a GSK3 inhibitor for a sufficient time to obtain a population of modulated adoptive NK cells. The method may further comprise obtaining a population of immune cells from a subject, and depleting CD3 and / or CD19 cells from the obtained population of immune cells prior to contacting the immune cells with a composition comprising a GSK3 inhibitor. In some embodiments, the method further comprises activating the population of immune cells before or during the step of contacting the immune cells with a composition comprising a GSK3 inhibitor. In some embodiments, the modulated adoptive NK cells have improved cytotoxicity and cytokine response and secretion; improved proliferation, cell recall, and / or persistence; and / or improved cell expansion and / or maintenance compared to adoptive NK cells not contacted with a composition comprising a GSK3 inhibitor. In some embodiments, the modulated adoptive NK cells comprised in the population of immune cells have at least a two-fold increased expansion after contacting with a composition comprising a GSK3 inhibitor. In one embodiment, the method is carried out in a feeder-free environment. In one embodiment, the method is carried out with feeder cells. In some other embodiments, the composition comprising a GSK3 inhibitor further comprises one or more of IL15, IL12, IL18, IL21, a MEK inhibitor, rapamycin, and a STING agonist. In one embodiment, the GSK3 inhibitor is CHIR999021. Also provided is a population of regulated NK cells having increased expression in one or more of CD107a, NKG2C, NKG2D, CD16, KIR, CD2, NKp30, NKp44, and NKp46 compared to unregulated NK cells, wherein the expression is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more.

[0068] A further aspect of the invention provides the use of the immune cell modulation method described above to generate a therapeutic composition comprising regulated immune cells for cell therapy. In some embodiments, the regulated immune cells comprise T, NK / or NKT cells. In some embodiments, the regulated NK cells comprise adoptive NK cells. An additional aspect of the invention provides a population of regulated immune cells comprising selectively expanded NK cells generated by the methods provided herein.

[0069] Yet another aspect of the present invention provides a therapeutic composition comprising the regulated cells obtained using the methods and compositions disclosed herein, and a therapeutically acceptable medium.In some embodiments of the therapeutic composition, the composition further comprises one or more additional additives selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, and immunomodulatory drugs (IMiDs).

[0070] Further provided is a method of treating a subject by administering a therapeutically sufficient amount of the therapeutic composition to a subject in need of adoptive cell therapy. In some embodiments, the cell therapy is autologous. In some other embodiments, the cell therapy is allogeneic. In some embodiments, the subject in need of therapy has an autoimmune disorder, a hematological tumor, a solid tumor, a cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. In some embodiments, the method of treating a subject using regulated immune cells is carried out by administering the therapeutic composition in combination with an antibody, a chemotherapy treatment, or a radioactive treatment, where the antibody, chemotherapy treatment, or radioactive treatment is administered prior to, during, or after the administration of the therapeutic composition.

[0071] Yet another aspect of the present invention provides use of a mixture for the manufacture of a therapeutic composition for cell therapy according to the methods provided herein, the mixture for the manufacture comprising an isolated population of immune cells and a composition for immune cell modulation comprising a GSK3 inhibitor.

[0072] Various objects and advantages of this use will become apparent from the following description taken in conjunction with the accompanying drawings which set forth, by way of illustration and example, certain embodiments of the invention. [Brief description of the drawings]

[0073] [Figure 1] FIG. 1 is a graphical representation of the z-scores of the relative measurements of the percentage of cells co-expressing both CCR7 and CD62L, as well as the absolute numbers of naive, stem cell memory, or central memory T cells, in (A) the viable CD8+ cell population and (B) the viable CD4+ cell population.

[0074] [Diagram 2] Figure 2 is a graphical representation showing that a multi-day culture process selectively expands NK cells, but not T cells, derived from PBMCs. The percentages of CD56+ and CD3+ cells from the apheresis product are shown before (left) and after (middle) depletion, and after 7 days of culture with IL-15 (10 ng / mL) plus 5 μM CHIR99021 (right).

[0075] [Diagram 3] FIG. 3 is a graphical representation showing that a multi-day GSK3i culture process increases total NK cell purity, number, and number of adoptive NK cells compared to the current process.

[0076] [Figure 4]Figure 4 is a graphical representation showing that GSK inhibition is a key reagent in 7-day cultures that effectively increases NK cell expansion, biasing the population towards adoptive (NKG2C+ / CD57+) and total CD57+ subsets. A. Fold increase in cell number. B. Percentage of cells.

[0077] [Diagram 5] Figure 5 is a graphical representation showing that GSK3 inhibition results in maturation of immature NK cells. A. Gating method and cell quadrants after a 7 day GSK3i culture period. B. Cell proliferation and cell surface marker expression in each cell quadrant.

[0078] [Figure 6AB] FIG. 6 is a graphical representation showing that GSK3 inhibition enhances cytotoxic activity. A. K562 cell specific killing. B. Raji cell specific killing. C. and D. NK cells cultured with GSK3 inhibitors increased killing kinetics of SKOV-3 cells. E. GSK inhibition expanded the enhanced adoptive NK killing of SKOV3 further in cooperation with Her2 ADCC. F. and G. GSK3 inhibitor treatment for 7 days enhances ADCC mediated killing of A549 cells. [Figure 6C-D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above.

[0079] [Figure 7] Figure 7 is a graphical representation showing that GSK3 inhibition enhances NK cell cytokine responses. A. IFNγ. B. TNFα.

[0080] [Figure 8]Figure 8 is a graphical representation showing that GSK3 inhibition and combination GSK3i / MEKi / rapamycin enhance NK proliferation and increase NK subtype bias in the presence of K562 feeder cells. A. Total number of NK cells. B. Total number of CD57+ NK cells. C. Total number of NKG2C+ NK cells.

[0081] [Figure 9] Figure 9 is a graphical representation showing that NK cells cultured in vitro for 7 days with GSK3i maintain their phenotypic change in vivo for at least 1 week after introduction without the presence of small molecules. A. Population percentage of CD57+ cells. B. Population percentage of NK cell subsets.

[0082] [Figure 10] FIG. 10 is a graphical representation showing the in vivo tumor clearance ability of NK cells cultured with a GSK3 inhibitor.

[0083] [Figure 11] Figure 11 shows that GSK3 inhibitor treatment does not lead to a higher frequency of CD56+CD57+ cells in the short term. A. CD57+NK fraction in 16 hour cultures with and without GSK3 inhibitor. B. CD57+NK fraction in 0-7 day cultures with and without GSK3 inhibitor.

[0084] [Figure 12A] Figure 12 shows that NK cells treated with GSK3 inhibitors for multiple days are highly effective against SKOV-3 cells in vivo. A. NK cell ADCC killing assay using Herceptin alone, NK cells primed with Herceptin overnight, NK cells treated with vehicle for 7 days together with Herceptin, or NK cells treated with GSK3 inhibitors for 7 days together with Herceptin. B. Comparison of ADCC killing effect using cells under different treatments. [Figure 12B] Same as above.

[0085] [Figure 13]FIG. 13 shows the cytotoxicity of NK cells treated with GSK3 inhibitors for multiple days versus overnight primed NK cells against primary AML blasts: A. AML donor 1; B. AML donor 2; C. AML donor 3.

[0086] [Figure 14] FIG. 14 shows the cytotoxicity of GSK3i-modulated NK cells against malignant cells but not allogeneic PBMCs in a dual target cytotoxicity assay in which K562 tumor cells are mixed with PBMCs: A. PBMC donor 1; B. PBMC donor 2; C. PBMC donor 3; D. PBMC donor 4.

[0087] [Figure 15] FIG. 15 shows that the STING agonist ML RR-S2 CDA has limited effects on (A) total CD57+ NK numbers, and (B) CD57+NKG2C+ NK phenotype.

[0088] [Figure 16] Figure 16 shows that the addition of STING agonist in combination with GSK3i enhances NK cell production of A. IFNγ; and B. TNFα for 4 hours of stimulation with IL-12+IL-18 or K562 cells. STING agonist in combination with GSK3i also increases the frequency of CD107a+ NK cells after stimulation (C).

[0089] [Figure 17] FIG. 17 shows that addition of a STING agonist enhanced the cytotoxicity of NK treated with a GSK3 inhibitor in a direct killing assay using A.K562, B.Nalm6, and C.KG-1 as target cells.

[0090] [Figure 18A]Figure 18 shows that STING agonists synergize with GSK3 inhibitors to enhance target cell killing. The combination of GSK3 inhibitors and STING agonists enhances NK cell cytotoxicity against A.A549 and B.SKOV-3 target cells in an ADCC assay using an anti-Her2 antibody. [Figure 18B] Same as above.

[0091] [Figure 19] Figure 19 shows that the combination of a GSK3 inhibitor and a STING agonist enhances NKp30 expression on NK cells.

[0092] The present invention provides compositions and methods for modulating immune cell populations or subpopulations to obtain improved therapeutic potential for adoptive immunotherapy. The present invention also provides methods for using modulated immune cells with improved therapeutic potential. In general, immune cells with improved therapeutic potential exhibit at least one of the following: improved proliferation, persistence, cytotoxicity, and / or cell recall / memory. The present invention provides methods for improving the therapeutic potential of immune cells through improvements to immune cell properties, for example, an increase in the number or proportion of subpopulations of cells that exhibit improvements in at least one of the following properties: migration, homing, cytotoxicity, maintenance, expansion, persistence, life span, differentiation, and / or dedifferentiation of the same cells would be expected to result in better immunotherapy outcomes.

[0093] definition

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with this application will have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms will include pluralities and plural terms will include the singular.

[0095] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0096] As used herein, the articles "a," "an," and "the" refer to one or to more than one of the grammatical object of the article. For purposes of illustration, a T cell means one T cell or more than one T cell.

[0097] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, including identifying specific foreign antigens in the body, and activating and inactivating other immune cells. T cells can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells are classified into CD3 +The T cells may be any type of T cell and may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), and the like. Additional types of helper T cells include cells such as Th3, Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells may also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0098] As used herein, the term "naive T cells" or Tn refers to mature T cells that, unlike activated or memory T cells, have not encountered their cognate antigen in the periphery. Naive T cells are generally characterized by surface expression of L-selectin (CD62L); absence of activation markers CD25, CD44 or CD69; and absence of memory CD45RO isoforms. They also express functional IL-7 receptors, consisting of subunits IL-7 receptor-α, CD127, and the common gamma chain, CD132. In the naive state, T cells are considered to be quiescent and non-dividing, requiring the common gamma chain cytokines IL-7 and IL-15 for homeostatic survival mechanisms.

[0099] As used herein, the term "central memory T cells" or Tcm refers to a subgroup or subpopulation of T cells that have lower expression or pro-apoptotic signaling genes, e.g., Bid, Bnip3, and Bad, and higher expression of genes associated with trafficking to secondary lymphoid organs, including genes CD62L, CXCR3, CCR7, compared to effector memory T cells, or Tems.

[0100] As used herein, the term "stem memory T cells," or "stem cell memory T cells," or Tscm, refers to a subgroup or subpopulation of T cells that are capable of self-renewal and generating Tcm, Tems and Teffs (effector T cells) and express lymphoid homing molecules such as CD27, as well as CCR7 and CD62L, properties important for mediating long-term immunity.

[0101] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adoptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56+, and have at least one of the following: CD57+, NKG2C+, low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. In some embodiments, CD56 + The isolated subpopulation of NK cells comprises expression of NKG2C and CD57. In some other embodiments, the isolated subpopulation of CD56+ NK cells comprises expression of CD57, CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56 + may be weakly expressed or fully expressed.

[0102] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are currently recognized. Invariant or type I NKT cells express a very restricted TCR repertoire, a classical α-chain (Vα24-Jα18 in humans) associated with a restricted spectrum of β-chains (Vβ11 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, displays a more heterogeneous TCRαβ usage. Type I NKT cells are currently considered suitable for immunotherapy. Adoptive or invariant (Type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161 and CD56.

[0103] As used herein, the term "isolated" and the like refers to a cell or a population of cells separated from its native environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell is present. The term includes a cell that has been removed from some or all components found in its natural environment, e.g., tissue, biopsy. The term also includes a cell that has been removed from at least one, some, or all components found in the environment in which the cell does not naturally occur, e.g., culture medium, cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations found in nature or grown, stored, or present in the non-naturally occurring environment. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in a non-naturally occurring medium. An isolated cell may be obtained by separating the desired cell, or a population thereof, from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment. As used herein, the term "purify" or the like refers to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0104] As used herein, the term "population," when used in reference to T, NK, or NKT cells, refers to a group of cells that includes two or more T, NK, or NKT cells, respectively. Using T cells as an example, an isolated or enriched population of T cells may include only one type of T cell or may include a mixture of two or more types of T cells. An isolated population of T cells may be a homogenous population of one type of T cell, or a heterogenous population of two or more types of T cells. An isolated population of T cells may also be a heterogenous population having T cells and at least cells other than T cells, such as B cells, macrophages, neutrophils, red blood cells, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. A heterogenous population may have 0.01% to about 100% T cells. Thus, an isolated population of T cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells. The isolated population of T cells may include one or more, or all, different types of T cells, including but not limited to those disclosed herein. In an isolated population of T cells that includes more than one type of T cells, the percentage of each type of T cell may range from 0.01% to 99.99%. The isolated population may also be a clonal population of T cells, where all T cells in the population are clones of a single T cell.

[0105] The isolated population of T, NK or NKT cells may be obtained from natural sources such as human peripheral blood or umbilical cord blood. Various methods have been developed in the art to separate cells from tissues or cell mixtures to separate different cell types. In some cases, these procedures result in a relatively homogenous population of cells. T cells can be isolated by sorting or selection processes described herein or by other methods known in the art. The percentage of T cells in the isolated population can be at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% higher than the percentage of T cells in natural sources. The T cells of the isolated population can be general T cells or one or more specific types of T cells.

[0106] As used herein, the term "subpopulation," when used in reference to T, NK, or NKT cells, refers to a population of T, NK, or NKT cells that includes, but not all, types of T, NK, or NKT cells, respectively, found in nature.

[0107] As used herein, the term "pluripotency" refers to the ability of cells to form all lineages of body or somatic cells (i.e., embryonic body).For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, ectoderm, mesoderm, and endoderm.Pluripotency is a continuum of developmental potential that ranges from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0108] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to stem cells generated from differentiated adult cells that have been induced or changed (i.e., reprogrammed) into cells capable of differentiating into tissues of all three embryonic or germ layers: mesoderm, endoderm, and ectoderm.

[0109] As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cell of the inner cell mass of embryo placenta.Embryonic stem cells are pluripotent and arise during development into all derivatives of the three primitive germ layers: ectoderm, endoderm and mesoderm.They are not involved in extraembryonic membranes or placenta and are not totipotent.

[0110] As used herein, the term "progenitor cell" refers to a cell that has a phenotype that is more primitive (e.g., at an earlier stage along a developmental pathway or progression) than the cell that it can give rise to by differentiation, i.e., a progenitor cell has a significant or very high proliferation potential. A progenitor cell can give rise to multiple separate cells with lower developmental potential, i.e., differentiated cell types, or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

[0111] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to the method of increasing cell potential or dedifferentiating cell into a less differentiated state.For example, a cell with increased cell potential has more developmental flexibility (i.e., can differentiate into more cell types) compared with the same cell in a non-reprogrammed state.In other words, a reprogrammed cell is in a less differentiated state than the same cell in a non-reprogrammed state.

[0112] As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the properties of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiated cell has acquired a more specialized ("committed") position in the lineage of a cell. The term "committed" as applied to the process of differentiation refers to cells that have progressed in the differentiation pathway to a point where, under normal circumstances, they would continue to differentiate into a particular cell type or subset of cell types, and where, under normal circumstances, they are unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0113] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a predetermined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a predetermined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence of the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0114] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity is introduced into the host cell. The molecule can be introduced, for example, by integration into the host chromosome or by introduction of the encoding nucleic acid into the host genetic material, such as as non-chromosomal genetic material such as a plasmid. Thus, the term used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the cell. The term "endogenous" refers to the referenced molecule or activity present in the host cell. Similarly, the term, when used in reference to expression of an encoding nucleic acid, refers to expression of an encoding nucleic acid that is contained within the cell and not introduced exogenously.

[0115] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide consists of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and when the polynucleotide is RNA, uracil (U) is substituted for thymine. A polynucleotide can include a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), an exon, an intron, a messenger RNA (mRNA), a transfer RNA, a ribosomal RNA, a ribozyme, a cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, a nucleic acid probe, and a primer. A polynucleotide also refers to both double-stranded and single-stranded molecules.

[0116] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, also commonly referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0117] As used herein, the term "in vitro" refers to activities that occur outside of an organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside the organism with minimal changes to its natural state. "In vitro" procedures can include living cells or tissues taken from an organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 2-28 days, depending on the circumstances. Such tissues or cells can also be collected, frozen, and later thawed for processing in vitro. Tissue culture experiments or procedures that use living cells or tissues and last longer than a few days are typically considered to be "in vitro", although in certain embodiments, the term can be used interchangeably with in vitro. On the other hand, "in vivo" activities, such as cell engraftment, cell homing, cell self-renewal, and cell expansion, occur inside of an organism.

[0118] As used herein, the term "in vitro" refers to an activity carried out or occurring in a test tube, in a culture dish, or anywhere outside a living organism.

[0119] As used herein, the terms "agent", "regulatory agent" and "modulator" are used interchangeably herein and refer to a compound or molecule capable of modifying the gene expression or biological properties of a cell, including an immune cell. An agent can be a single compound or molecule, or a combination of more than one compound or molecule.

[0120] As used herein, the terms "contacting," "treating," or "modulating," when used in reference to immune cells, are used interchangeably herein and refer to culturing, incubating, or exposing immune cells with one or more of the agents disclosed herein.

[0121] As used herein, "uncontacted" or "untreated" cells are cells that have not been treated, e.g., cultured, contacted, or incubated with an agent other than a control agent. Cells that have been contacted with a control agent such as DMSO or with another vehicle are examples of uncontacted cells.

[0122] As used herein, "feeder cells" or "feeders" are terms describing one type of cell that is co-cultured with another type of cell to provide an environment in which the other type of cell can grow, as the feeder cells provide stimuli, growth factors and nutrients for the support of the other cell type. Feeder cells are optionally from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts, or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from overgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without being limited to the foregoing, one specific feeder cell type can be a human feeder, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation into certain lineages, enhance proliferation capacity, and promote maturation into specific cell types, such as effector cells.

[0123] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture or medium, that is essentially free of feeder or stromal cells and / or has not been previously conditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that has been collected after feeder cells have been cultured in the medium for a period of time, such as at least one day. A preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium. In some embodiments, a feeder-free environment is free of both feeder or stromal cells and has not been previously conditioned by culturing feeder cells.

[0124] As used herein, the term "analog" refers to a chemical molecule that resembles another chemical in structure and function, differing structurally at one single element or group, or at more than one group (e.g., 2, 3, or 4 groups), provided that it retains the same chemical backbone and function as the parent chemical. Such modifications are routine to those of skill in the art and include, for example, additional or substituted chemical moieties, such as esters or amides of acids, protecting groups such as benzyl groups for alcohols or thiols, and tert-butoxylcarbonyl groups for amines. Modifications to alkyl side chains, such as alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.), modifications to the level of saturation or unsaturation of the side chain, and the addition of modified groups such as substituted phenyl and phenoxy are also included. Analogs may also include conjugates such as biotin or avidin moieties, enzymes such as horseradish peroxidase, and include radiolabeled, bioluminescent, chemiluminescent, or fluorescent moieties. Moieties can also be added to the drugs described herein to modify their pharmacokinetic properties, such as increasing their half-life in vivo or in vitro, or increasing their cell entry properties, among other desirable properties. Also included are prodrugs, which are known to enhance many desirable properties of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.).

[0125] As used herein, the term "enhancement" refers to the ability of an agent to produce or cause a greater physiological response (i.e., downstream effect) in a cell compared to the response produced by either a vehicle or a control molecule / composition, for example, interleukin 4 or interleukin 10 production is increased by an isolated population of T cells. The increase can be an increase in gene expression as a result of increased signaling through a certain cell signaling pathway. The amount of "enhancement" is typically a statistically significant amount and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (all integers and decimals between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the response produced by a vehicle (absence of agent) or a control composition.

[0126] As used herein, the term "reduction" refers to the ability of an agent to produce or cause a smaller physiological response (i.e., downstream effect) in a cell compared to the response produced by either a vehicle or a control molecule / composition. The reduction can be a reduction in gene expression, a reduction in cell signaling, or a reduction in cell proliferation. The amount of "reduced" is typically a "statistically significant" amount and can include a reduction that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (all integers and decimals between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by vehicle (absence of agent) or a control composition.

[0127] As used herein, the term "synergistic effect" or "synergistic" refers to a combination of two or more entities for an enhanced effect such that two or more entities working together produce an effect that is greater than the sum of their individual effects, as compared to "antagonistic," which is used when two or more entities in the combination attenuate or neutralize the effect of each other, and as compared to "additive," which is used when two or more entities in the combination produce an effect that is approximately equal to the sum of their individual effects.

[0128] As used herein, the term "substantially free" when used to describe a composition such as a cell population or a medium refers to a composition that is 95% free, 96% free, 97% free, 98% free, 99% free, or free of any source of a particular substance, such as a particular substance that is not detectable as measured by conventional means. A similar meaning can be applied to the term "absence of" when referring to the absence of a particular substance or component of a composition.

[0129] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. The amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length range can be ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the approximately reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0130] As used herein, the term "subject" refers to a mammal. The subject may be a human or a non-human, such as a dog, cat, cow, horse, mouse, rat, rabbit, or a transgenic species thereof.

[0131] As used herein, the term "treat" and the like, when used in relation to a subject, refers to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, achieving an improvement or elimination of symptoms of a disease. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic, in terms of achieving an improvement or elimination of symptoms, or resulting in a partial or complete cure of a disease and / or side effects caused by a disease. The term "treatment" includes any treatment of a disease in a mammal, particularly a human, including (a) preventing a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting a disease or arresting its development; (c) relieving a disease or causing a regression of a disease or completely or partially eliminating symptoms of a disease; and (d) restoring an individual to a pre-disease state, such as restoring the hematopoietic system.

[0132] As used herein, "genetic modification" refers to gene editing, including (1) naturally resulting from rearrangements, mutations, genetic imprinting and / or epigenetic modifications, or (2) obtained through genomic engineering via insertion, deletion or substitution in the genome of a cell. As used herein, genetic modification also includes one or more retainable therapeutic traits of source-specific immune cells that are donor, disease, or treatment response specific.

[0133] As used herein, the term "genetic imprint" refers to the genetic or epigenetic information that is responsible for the preferential therapeutic trait in source cells. In the embodiment of source cells obtained from a specifically selected donor, disease or treatment situation, the genetic imprint that is responsible for the preferential therapeutic trait may include any situation-specific genetic or epigenetic modification that reveals a conservable phenotype, i.e., preferential therapeutic trait, regardless of the underlying molecular phenomenon that may or may not be identified. The donor, disease or treatment response specific source cells may include genetic imprints that are retainable in iPSCs and derived hematopoietic cells, including, but not limited to, a predetermined monospecific TCR derived from, for example, virus-specific T cells or invariant natural killer T (iNKT) cells; a trackable and desirable genetic polymorphism, for example, homozygous for a point mutation that codes for high affinity CD16 receptor in selected donors; and a predetermined HLA requirement, i.e., selected HLA-matched donor cells that present a phenotype with an expanded population. As used herein, preferential therapeutic properties include improved engraftment, transport, homing, viability, self-renewal, persistence, immune response control and regulation, survival, and cytotoxicity of the resulting cells.Preferential therapeutic properties may also relate to antigen targeting receptor expression; HLA presentation or lack thereof; resistance to the tumor microenvironment; induction and immunomodulation of bystander immune cells; improved on-target specificity with reduced off-tumor effects; and resistance to treatments such as chemotherapy.

[0134] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or otherwise side effects of cell therapy. In some cases, safety switch protein expression is conditionally regulated to address safety concerns for transplanted engineered cells that have persistently incorporated a gene encoding the safety switch protein into their genome. This conditional control can be variable and may include regulation via small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional control. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene control, and / or antibody-mediated depletion. In some cases, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, which when activated triggers apoptosis and / or cell death of the therapeutic cell. Examples of safety switch proteins include, but are not limited to, suicide genes, such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B-cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered in the event of an adverse event is activated by the suicide gene production, killing the transfected cell.

[0135] As used herein, a "therapeutically sufficient amount" includes, in its meaning, a non-toxic but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition referred to that produces the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's general health condition, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to improve, reduce, and / or ameliorate at least one symptom associated with the disease or condition of the subject being treated.

[0136] I. Agents for improving the efficacy of cell-based adoptive immunotherapy The present invention provides compositions comprising a sufficient amount of one or more agents for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy. Immune cells with improved therapeutic potential exhibit improved proliferation, persistence, cytotoxicity, and / or cell recall / memory. Immune cells may specifically have improved in vivo proliferation, in vivo persistence, in vivo cytotoxicity, and / or in vivo cell recall / memory. To improve immune cell therapeutic potential, better-qualified immune cells in the T cell population are generally required, e.g., an increase in the number or percentage of naive T cells, stem cell memory T cells, and / or central memory T cells, via their maintenance, expansion, differentiation, and / or dedifferentiation, is an indication of a better T cell property for improved in vivo adoptive therapeutic potential. In the NK cell population, an increase in the number or percentage of adoptive NK cells, e.g., via their maintenance, subtype bias, expansion, differentiation, and / or dedifferentiation, is an indication of a better NK cell property for improved in vivo adoptive therapeutic potential. With respect to NKT cell populations, an increase in the number or proportion of type I NKT cells, for example through their maintenance, subtype switching, expansion, differentiation, and / or dedifferentiation, is an indication of better properties of the NKT cells for improved in vivo adoptive therapeutic potential.

[0137] Immune cells suitable for adoptive cell-based therapy are contacted, treated, or modulated with one or more agents included in Table 1. Treatment with an agent can modify the biological properties of the cells, or subpopulations of cells, including by modulating cell expansion, maintenance, differentiation, dedifferentiation, and / or survival, and / or by increasing proliferation, cytotoxicity, persistence, and / or cell recall / memory, and thus the therapeutic potential of the treated cells. For example, treatment can improve the survival rate of therapeutic immune cells both in vitro and in vivo. Furthermore, treatment can change the proportion of different subpopulations of the treated cell population. For example, in one embodiment, the number and percentage of naive T cells, stem cell memory T cells, and / or central memory T cells are increased in an isolated T cell population upon treatment with one or more agents selected from Table 1. In another embodiment, upon treatment of an NK cell population with one or more agents selected from Table 1, the number and percentage of adoptive NK cells are increased in the population.

[0138] [Table 1-1]

[0139] [Table 1-2]

[0140] Without being limited by theory, the agents in Table 1 improve the therapeutic potential of immune cells for adoptive therapy by modulating cell expansion, metabolism, and / or cell differentiation through control of cell metabolism, nutrient sensing, proliferation, apoptosis, signal transduction, properties involved in the infectious process, and / or other aspects of cell function. As will be appreciated by one of skill in the art, the scope of the present invention also includes analogs or derivatives, including, but not limited to, salts, esters, ethers, solvates, hydrates, stereoisomers, or prodrugs of the agents listed in Table 1. For example, illustrative examples of analogs and derivatives of the drug dmPGE2 (16,16-dimethyl prostaglandin E2) in Table 1 are PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-ketofluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinolate, amide, PGE2 methyl ester, 16-phenyltetranor PGE2, 15(S)-15-methyl PGE2, 15(R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-isoPGE2 isopropyl ester, 8-iso-16-cyclohexyl-tetranor PGE2, 20-hydroxy PGE2, 20-ethyl PGE2, 11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19(R) hydroxy PGE2. Also included are PG analogs or derivatives having a structure similar to PGE2 substituted with a halogen at the 9-position (see, e.g., International Publication No. WO 2001 / 12596, the disclosure of which is incorporated herein by reference in its entirety), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives such as those described in U.S. Publication No. 2006 / 0247214, the disclosure of which is incorporated herein by reference in its entirety.

[0141] GSK3 (glycogen synthase kinase 3) inhibitors may include antibodies that bind to dominant negative variants of siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides that target GSK3. GSK3 inhibitors (GSK3i) suitable for use in the compositions contemplated herein include Kenpaullone, l-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-acetoxime, (5-methyl-lH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienyl ruthenium complex, TDZD-8 These include, but are not limited to, 4-benzyl-2-methyl-l,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(l-pyridyl)-[l,3,4]-oxadiazole, OTDZT, alpha-4-dibromoacetophenone, AR-AO144-18, 3-(l-(3-hydroxypropyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrazin-2-yl-pyrrole-2,5-dione; TWS119, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-l-(4,5-dibromo-thiophen-2-yl)-ethanone; GF109203X; RO318220; TDZD-8; TIBPO; and OTDZT. In one embodiment, the GSK-3 inhibitor is CHIR99021, BIO, TWS119, or Kenpaullone. In one embodiment, the GSK3 inhibitor is TWS119. In another embodiment, the GSK-3 inhibitor is CHIR99021. In yet another embodiment, the GSK3 inhibitor is BIO. 0140 MEK / ERK pathway inhibitors refer to inhibitors of either the MEK or ERK serine / threonine kinases that are part of the Raf / MEK / ERK pathway. ERK / MEK inhibitors suitable for use in the compositions contemplated herein include PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSKl 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEAl 19, AZD6244, FR180204, PTK787, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide;6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-yl)m- ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazol-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-yl m-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole- e-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(2,4-dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6- -oxo-1,6-dihydropyridine-3-carboxamide; hereinafter referred to as MEK inhibitor 2; and 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6- - Examples of MEK / ERK inhibitors include, but are not limited to, oxo-1,6-dihydropyridazine-3-carboxamide, or a pharma- ceutically acceptable salt thereof.Further illustrative MEK / ERK inhibitors include those compounds disclosed in WO 99 / 01426, WO 02 / 06213, WO 03 / 077914, WO 05 / 051301, and WO 2007 / 044084.In one embodiment, the MEK inhibitor is PD0325901.In another embodiment, the MEK inhibitor is U0126; 0141 ROCK (Rho-associated kinase) inhibitor refers to the inhibitor of Rho-GTPase / ROCK pathway. The pathway includes downstream protein myosin II, which is further downstream of ROCK (Rho-ROCK-myosin II forms a pathway / system). Therefore, one can use any or all of Rho GTPase inhibitor, ROCK inhibitor, or myosin II inhibitor to achieve the effects described herein. Suitable ROCK inhibitors for use in the compositions contemplated herein include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, and the ROCK inhibitors disclosed in U.S. Patent No. 8,044,201 (incorporated herein in its entirety by reference).In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegurin. In one embodiment, the ROCK inhibitor is thiazovivin.

[0142] Activin receptor-like kinase 5 (ALK5) is the main TGFβ receptor that mediates cellular responses to TGFβs. Upon ligand binding, constitutively active TβRII kinase phosphorylates ALK5 and activates downstream signaling cascades in the gastrointestinal tract. TGFβ receptor / ALK5 inhibitors can include antibodies against TGFβ / ALK5 receptor, dominant negative variants of TGFβ / ALK5 receptor, and siRNA, microRNA, antisense nucleic acid, and other polynucleotides that suppress the expression of TGFβ / ALK5 receptor.TGFβ receptor / ALK5 inhibitors suitable for use in the compositions contemplated herein include SB431542; A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl ]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16, IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl- 3H-imidazol-4-yl)-6-methylpyridine hydrochloride;SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124);lerdelimumb (CAT-152);metelimu-mab (CAT-192);GC-1008;ID11;AP-12009;AP-11014;LY550410;LY580276; These include, but are not limited to, LY364947; LY2109761; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P144; soluble TBR2-Fc; and pyrimidine derivatives (e.g., those listed in Stiefl et al., International Publication No. WO 2008 / 006583, incorporated herein by reference).Furthermore, "ALK5 inhibitor" is not intended to include non-specific kinase inhibitors, but "ALK5 inhibitor" should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542 (see, for example, Inman, et al., J. Mol. Pharmacol. 62(1):65-74 (2002)). It is further believed that inhibition of TGFβ / activin pathway will have a similar effect of inhibiting ALK5. Therefore, any inhibitor of TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of the ALK5 inhibitor described in each paragraph herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD2 / 3 phosphorylation, inhibitors of the interaction of SMAD2 / 3 with SMAD4, and activators / agonists of SMAD6 and SMAD7. Additionally, the classifications described herein are merely for organizational purposes, and one of skill in the art will know that a compound can affect one or more points in a pathway, thereby allowing the compound to function in more than one of the defined classifications. In one embodiment, the TGFβ receptor inhibitor comprises SB431542.

[0143] PDK1 or 3'-phosphoinositide-dependent kinase-1 is a prominent kinase that is associated with the activation of AKT / PKB and many other AGC kinases, including PKC, S6K, and SGK. An important role of PDK1 is in signal transduction pathways activated by several growth factors and hormones, including insulin signaling. Exemplary PDK1 agonists include sphingosine (King et al, Journal of Biological Chemistry, 275:18108-18113, 2000). Exemplary allosteric activators of PDK1 include 3,5-diphenylpent-2-enoic acids such as PS48 ((Z)-5-(4-chlorophenyl)-3-phenylpent-2-enoic acid), PS08 ((Z)-5-(4-bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid), 1-(2-(3-(4-chlorophenyl)-3-oxo-l-phenylpropylthio)acetic acid; compound 12Z (2-(3-(4-chlorophenyl)-3-oxo-l-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid), and compound 13Z ((Z)-5-(lH-indol-3-yl)-3-phenylpent-2-enoic acid). In one embodiment, the PDK1 agonist comprises PS48.

[0144] Mammalian target of rapamycin (mTOR) inhibitors block the activity of mammalian target of rapamycin. mTOR is a protein kinase that controls growth factors that stimulate cell growth and angiogenesis. mTOR inhibitors suitable for the compositions and methods of the present invention include, but are not limited to, rapamycin and its analogs and derivatives, including sirolimus, sirolimus derivatives, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus), 40-O-(3-hydroxy)propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, and other O-alkylated or O-methylated rapamycin derivatives.

[0145] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Table 1. In one embodiment, a composition for improving the therapeutic potential of immune cells comprises at least two, three, four, five, or six, or any number of combinations of the agents selected from Table 1.

[0146] In one embodiment, the composition comprising at least one agent selected from Table 1 further comprises an organic solvent. In certain embodiments, the organic solvent is substantially free of methyl acetate. In certain embodiments, the organic solvent is selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof. In some embodiments, the organic solvent is DMSO. In some embodiments, the organic solvent is ethanol. In some other embodiments, the organic solvent is a mixture of DMSO and ethanol.

[0147] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group I: dorsomorphin, hepteridic acid, 1-pyrrolidinecarbodithioic acid, and 2-DG. Without being limited by theory, Group I agents may affect cellular metabolism and nutrient sensing, among other possible roles.

[0148] In some embodiments, compositions for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy include at least one agent selected from group II: GSK3 inhibitors, ROCK inhibitors, TGFβ receptor inhibitors, MEK inhibitors, PDK1 agonists, 6-mercaptopurine, AC-93253 iodide, tiratricol, PI-103, fulvestrant, thapsigargin, SU4312, U0126, telmisartan, cyclosporine A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY61-3606, protoporphyrin IX disodium, mTOR inhibitors, TWS119, HS173, LY294002, and pictilisib. Without being limited by theory, group II agents may affect signal transduction in various functional pathways, among other possible roles.

[0149] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group III: 5-azacytidine, fludarabine, roscovitine, and PAC-1. Without being limited by theory, Group III agents may affect cell proliferation and apoptosis, among other possible roles.

[0150] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group IV: 5,7-dichloro-8-quinolinol, 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octaphenyl, nifuroxazide, and tosufloxacin hydrochloride. Without being limited by theory, Group IV agents may affect cellular properties related to the infection process, among other possible roles.

[0151] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group V: sertraline, diethylenetriaminepentaacetic acid, edrophonium chloride, BIX01294, terfenadine, and dmPGE2. Without being limited by theory, Group V agents may generally affect other cellular properties related to expansion, maintenance, differentiation, dedifferentiation, viability, proliferation, cytotoxicity, cell recall, and / or persistence, among other possible roles.

[0152] In yet some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group I, and one or more agents selected from Group II, Group III, Group IV, and / or Group V.

[0153] In some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group II and one or more agents selected from Group I, Group III, Group IV, and / or Group V.

[0154] In yet some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group III and one or more agents selected from Group I, Group II, Group IV, and / or Group V.

[0155] In yet some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group IV and one or more agents selected from Group I, Group II, Group III, and / or Group V.

[0156] In yet some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from Group IV, and one or more agents selected from Group I, Group II, Group III, and / or Group IV.

[0157] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one agent selected from the group consisting of a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, a TGFβ inhibitor, a PDK1 agonist, and an mTOR inhibitor.

[0158] In some embodiments, the composition comprises a combination of two or more agents selected from Table 1, where the agents have an additive effect in combination. As defined, "additive" refers to when two or more agents in a combination produce an effect that is approximately equal to the sum of their individual effects. In some embodiments, one or more agents in the combination are from the same group: Group I, II, III, IV, or V. In some embodiments, one or more agents in the combination are from different groups.

[0159] In some embodiments, the composition comprises a synergistic combination of two or more agents selected from Table 1. As defined, a "synergistic effect" is an enhanced effect, such as two or more agents working together to produce an effect greater than the sum of their individual effects. In one embodiment, the composition comprising the synergistic combination comprises at least one agent selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG. In one embodiment, the composition comprises a combination comprising at least one agent selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG, and one or more additional agents selected from the compounds listed in Table 1. In one embodiment, the composition comprising TWS119 further comprises two or more additional agents selected from Table 1. In one embodiment, the composition comprising HS173 further comprises two or more additional agents selected from Table 1. In one embodiment, the composition comprising LY294002 further comprises two or more additional agents selected from Table 1. In one embodiment, the composition comprising pictilisib further comprises two or more additional agents selected from Table 1. In one embodiment, the composition comprising 2-DG further comprises two or more additional agents selected from Table 1.

[0160] In some embodiments, the composition comprising one or more agents selected from the group consisting of the compounds listed in Table 1 further comprises one of the additional additives selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNA, dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies and antibody fragments thereof, and / or chemotherapeutic agents or radioactive moieties. In some embodiments, the additional additive comprises an antibody, or an antibody fragment. In some of these embodiments, the antibody, or antibody fragment, specifically binds to a viral antigen. In other embodiments, the antibody, or antibody fragment, specifically binds to a tumor antigen.

[0161] In some embodiments, the cytokines and growth factors include one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF) transferrin, various interleukins (e.g., IL-1 through IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), and erythropoietin (Epo). In some embodiments, the cytokines include at least interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15, interleukin-18 (IL-18), interleukin-21 (IL-21), or any combination thereof. In some embodiments, the growth factors of the composition include fibroblast growth factors. These cytokines may be obtained commercially, for example, from R&D Systems (Minneapolis, MN), and may be either natural or recombinant. In certain embodiments, the growth factors and cytokines may be added at concentrations contemplated herein. In certain embodiments, the growth factors and cytokines may be added at concentrations determined empirically or guided by established cytokine art knowledge.

[0162] In some embodiments, the mitogen of the composition comprises concanavalin A. In some other embodiments, the feeder cells are genetically modified. In some embodiments, the feeder cells comprise one or more of the following: mononuclear blood cells, thymic epithelial reticular cells, endothelial cells, fibroblasts, leukemia cells K562, Raji cells, or feeder cell components or replacement factors thereof.

[0163] In some embodiments, the one or more small RNAs include siRNA, shRNA, miRNA, and antisense nucleic acids. In some other embodiments, the small RNAs include one or more of the following: miR-362-5p, miR-483-3p, miR-210, and miR-598.

[0164] In some embodiments, the vector containing one or more polynucleic acids of interest is integrating or non-integrating. In some embodiments, the vector containing one or more polynucleic acids of interest further comprises a backbone such as an adenovirus vector, a plasmid vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector, an episomal vector, etc. In some embodiments, the plasmid vector for expression in animal cells includes, for example, pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. In some embodiments, the one or more polynucleic acids contained in the vector encode one or more proteins or polypeptides. In some embodiments, the one or more polynucleic acids encode Delta-like 1 (DLL1), Delta-like 3 (DLL3), Delta-like 4 (DLL4), Jagged1 (Jag1), or Jagged2. In some embodiments, the one or more polynucleic acids encode Jagged1. II. Immune Cells for Adoptive Cell Therapy

[0165] The present invention provides a composition comprising an isolated population or subpopulation of immune cells contacted with one or more agents selected from Table 1. In one embodiment, the isolated population or subpopulation of immune cells is contacted with one or more agents selected from Table 1 in an amount sufficient to improve the therapeutic potential of the immune cells. In some embodiments, the treated immune cells are used in cell-based adoptive therapy. The present invention further provides a population or subpopulation of immune cells and one or more agents selected from the agents listed in Table 1, wherein treating the population or subpopulation of immune cells with one or more agents selected from the agents listed in Table 1 improves the therapeutic potential of the immune cells in adoptive therapy. The treatment can modify biological properties of the immune cells to improve cell proliferation, cytotoxicity, and persistence, and / or reduce relapse rate of cell therapy.

[0166] In some embodiments, the population of immune cells comprises T cells. In some embodiments, the population of immune cells comprises NK cells. In some embodiments, the population of immune cells comprises NKT cells.

[0167] In some embodiments, a population or subpopulation of T cells contacted with one or more agents selected from Table 1 comprises an increased number or percentage of naive T cells (Tn), stem cell memory T cells (Tscm), and / or central memory T cells (Tcm), and / or improved cell proliferation, cytotoxicity, cell recall, and / or persistence, compared to the same untreated T cells. In some embodiments, the number of Tn, Tscm, and / or Tcm is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or is increased by at least 5, 10, 15, or 20 fold, compared to the number of Tn, Tscm, and / or Tcm in a cell population that has not been treated with the same agent or agents selected from Table 1.

[0168] In some embodiments, a population or subpopulation of NK cells contacted with one or more agents selected from Table 1 comprises an increased number or percentage of adoptive (or memory) NK cells and / or improved cell proliferation, cytotoxicity, cell recall, and / or persistence compared to NK cells that have not been treated with the same. In some embodiments, the number of adoptive NK cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or increased by at least 5, 10, 15, or 20-fold compared to the number of adoptive NK cells in a cell population that has not been treated with the same agent or agents selected from Table 1. In one embodiment, a population or subpopulation of NK cells contacted with a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, a TGFβ inhibitor, a PDK1 agonist, and / or rapamycin comprises an increased number or percentage of adoptive NK cells. In one embodiment, the adoptive NK cells are CD3 - and CD56 + , and CD57 + , NKG2C + In some embodiments, the adoptive NK cells are characterized by at least one of: low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. + , NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and at least two of low CD45RA. For example, adoptive NK cells are + And NKG2C + In some embodiments, the adoptive NK cells may be CD57 + , NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. For example, adoptive NK cells are SYK-, FcεRγ-, and EAT-2 -In one embodiment, the GSK-3β inhibitor is CHIR99021, BIO, TWS119, or Kenpaullone. In one embodiment, the GSK-3β inhibitor is TWS119. In another embodiment, the GSK-3β inhibitor is CHIR99021. In yet another embodiment, the GSK-3β inhibitor is BIO.

[0169] In some other embodiments, a population or subpopulation of NKT cells contacted with one or more agents selected from Table 1 comprises an increased number of type I NKT cells or an increased ratio of type I to type II NKT cells, and / or improved cell proliferation, cytotoxicity, cell recall, and / or persistence, compared to an isolated population or subpopulation of NKT cells that has not been treated with one or more agents selected from Table 1. In some embodiments, the number of type I NKT cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or is increased by at least 5, 10, 15, or 20 fold, compared to the number of type I NKT cells in the same cell population that has not been treated with one or more agents selected from Table 1.

[0170] In some embodiments, increased numbers or proportions of naive T cells (Tn), stem cell memory T cells (Tscm), central memory T cells (Tcm), adoptive NK cells, and / or type I NKT cells result from improved maintenance and expansion of these cell types and / or increased cell dedifferentiation / reprogramming from more mature cell subtypes to cell subtypes in a desired differentiation state.

[0171] In some embodiments, after contacting a population of immune cells with one or more of the agents included in Table 1, the number of naive T cells (Tn), stem cell memory T cells (Tscm), and central memory T cells (Tcm) in the population is increased compared to an untreated immune cell population, wherein Tn, Tscm, and Tcm are characterized by co-expression of CCR7 and / or CD62L.

[0172] In some embodiments, after contacting a population of immune cells with one or more of the agents included in Table 1, the number of adoptive NK cells in the population is increased compared to an untreated immune cell population, where the type I adoptive NK cells are characterized by CD3-, CD56+, CD16+, NKG2C+, and CD57+. In some other embodiments, the adoptive NK cells are characterized by CD3 - , CD56 + , and CD57 + , NKG2C + , low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0173] In some embodiments, after contacting a population of immune cells with one or more of the agents included in Table 1, the number of type I adoptive NKT cells in the population is increased compared to an untreated immune cell population, where the adoptive NKT cells are characterized by CD3+, CD56+, TCR Vα24+, and TCR Vβ11+.

[0174] In some embodiments, populations or subpopulations of T, NK or NKT cells for treatment with the agents disclosed herein can be isolated from humans or non-human mammals. Examples of such non-human mammals include, but are not limited to, rabbits, horses, cows, sheep, pigs, dogs, cats, mice, rats, and transgenic species thereof.

[0175] T cell population or subpopulation can be obtained or isolated from a number of sources, including, but not limited to, peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from site of infection, ascites, pleural fluid, spleen tissue, and tumor.Bone marrow can be obtained from femur, iliac crest, hip, rib, and other bones.In addition, T cell lines available in the art, such as Jurkat, SupT1, and others, can also be used.

[0176] Populations or subpopulations of NK cells can be obtained or enriched from a number of sources, including, but not limited to, peripheral blood, umbilical cord blood, and tumors.

[0177] Fully mature NKT cells can be obtained or enriched from peripheral blood, including bone marrow, lymph node tissue and umbilical cord blood, a smaller population of mature NKT cells potentially found in thymic tissue.

[0178] In certain embodiments of the invention, isolated or enriched populations or subpopulations of T, NK, NKT cells can be obtained from a blood unit using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In one embodiment, T, NK or NKT cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains cells, including T cells, monocytes, granulocytes, B cells, NK cells, NKT cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and the cells placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution can lack calcium, lack magnesium, or lack many, but not all, divalent cations. As one of skill in the art would readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other buffers with or without saline solutions. Alternatively, undesirable components of the apheresis sample are removed and the cells are resuspended directly in culture medium.

[0179] In another embodiment, a population or subpopulation of T, NK or NKT cells is isolated or enriched from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation.

[0180] In one embodiment, a specific subpopulation of T cells can be further isolated or enriched by positive or negative selection, such as with CD3, CD28, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD27, and / or CD122 antibodies. For example, in one embodiment, the isolated or enriched population or subpopulation of T cells is expanded and activated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection of the desired T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 72 hours or more and all integer values ​​therebetween. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 72 hours. In one preferred embodiment, the incubation period is 24 hours. For isolating T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are underrepresented compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Additionally, longer incubation times can be used to isolate CD8 +The efficiency of T cell capture can be increased. Thus, by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads and / or increasing or decreasing the ratio of beads to T cells (as described further herein), specific populations or subpopulations of T cells can be further selected for or against at the beginning of culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, specific populations or subpopulations of T cells can be preferentially selected for or against at the beginning of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform the selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subject to additional rounds of selection.

[0181] Isolation or enrichment of populations or subpopulations of T, NK or NKT cells by negative selection can be achieved with a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunobinding or fluorescence activated cell sorting using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to isolate or enrich for CD3 +To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, and HLA-DR. In certain embodiments, it may be desirable to enrich or positively select for regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25-conjugated beads or other similar methods of selection. In some embodiments, the desired T cell subpopulation for immunotherapy is enriched or selected from regulated immune cells, including T cells, by CCR7 and CD62L. Alternatively, the cells of interest may be selected according to physical parameters, including differences in size, density, granularity, deformability, resistance, or volume.

[0182] In one embodiment, a population or subpopulation of adoptive NK cells is enriched within regulated immune cells, including NK cells, by selection using identifiers such as CD3- and CD56+ on their phenotype, including positive expression of CD16, NKG2C, and CD57. Additionally, negative selection of the adoptive subpopulation can be based on lack of expression of NKG2C and / or CD57, as well as lack of expression of one or more of the following: low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0183] In one embodiment, a population or subpopulation of NKT cells is enriched within a population of NK cells by selecting for those that phenotypically express the invariant TCR α chain, specifically a combination of the following markers: CD3+, CD56+, TCR Vα24+, and / or TCR Vβ11+. Alternatively, NKT cells can be selected based on a combination of phenotypes coupled with expression of the invariant TCR α chain.

[0184] Blood samples or apheresis products from subjects can be collected at a time prior to when immune cells described herein are isolated. Thus, the source of cells to be regulated can be collected at any time point required, and desired cells such as T cells, NK cells and NKT cells can be isolated and frozen for later use in cell-based immunotherapy for any number of diseases or conditions that benefit from such cell therapy, such as those described herein. In one embodiment, blood samples or apheresis products are collected from normal healthy subjects. In certain embodiments, blood or apheresis products are collected from normal healthy subjects that are at risk of developing disease but have not yet developed disease, and cells of interest are isolated and frozen for later use. In other embodiments, blood samples or apheresis products are collected from subjects that have already been administered genetically modified immune cells (genetically engineered or naturally resulting from rearrangements, mutations, gene imprinting and / or epigenetic modifications). In certain embodiments, T, NK, NKT or other immune cells can be expanded, frozen, and later processed and used. In certain embodiments, samples are collected from patients immediately after diagnosis of certain diseases described herein, but prior to any treatment. In some embodiments, cells are isolated from subjects presenting CMV (cytomegalovirus) seropositivity. In further embodiments, cells are isolated from blood or apheresis products from subjects prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral drugs, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.In a further embodiment, cells are isolated from a patient and frozen for use following bone marrow or stem cell transplantation, T cell depleting therapy using either chemotherapy drugs such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (e.g., before, simultaneously, or after) . In another embodiment, cells may be isolated prior to treatment and frozen for use following treatment following B cell depleting therapy, such as an agent reactive with CD20, e.g., Rituxan.

[0185] In some embodiments, the population or subpopulation of T, NK or NKT cells is genomically engineered and includes insertions, deletions, or nucleic acid substitutions. The modified immune cells may express a cytokine transgene, a silenced inhibitory receptor; or overexpress an activating receptor, or a CAR for retargeting immune cells. In some embodiments, the population of immune cells isolated from a subject or donor for conditioning, or isolated or contained in the peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumor of the subject / donor, may be genetically modified. In some embodiments, the isolated population of immune cells is genomically engineered and includes insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells include an exogenous nucleic acid encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR), and / or overexpressed CD16 or a variant thereof.

[0186] Genomically engineered immune cells include genetically modified modalities including one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; proteins that promote immune cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival. In some other embodiments, the genetically modified modality comprises: (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosome 6p21 region; (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling with bi- or multispecific or universal engagers. In some embodiments, the T, NK, or NKT cells comprise an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid is introduced into the immune cell via direct genome editing of the cell. In some other embodiments, the exogenous nucleic acid is introduced into the immune cells via carrying the same from genomically engineered hematopoietic stem or progenitor cells, or iPSCs, which give rise to the immune cells through differentiation. In some embodiments, the exogenous nucleic acid for T cells can encode a TCR (T cell receptor), a CAR (chimeric antigen receptor), a bispecific T cell engager (BiTE), a trispecific T cell engager, a multispecific T cell engager, or a universal engager compatible with multiple immune cell types. In some embodiments, the exogenous nucleic acid for NK cells can encode a TCR, a CAR, CD16 or a variant thereof, NY-ESO, a bispecific killer cell engager (BiKE), a trispecific killer cell engager (TriKE), a multispecific killer cell engager, or a universal engager compatible with multiple immune cell types.In some embodiments, the foreign nucleic acid for the NKT cells can be an altered TCR or CAR. In some embodiments, the foreign nucleic acid encodes CAR19. In some embodiments, the CD16 variants include high affinity CD16 (HACD16), non-cleavable CD16, and high affinity non-cleavable CD16 (hnCD16). Thus, in view of methods and compositions for regulating immune cells using GSK3i, some aspects of the invention provide genetically engineered T, NK, or NKT cells regulated by GSK3i. One aspect of the invention provides NK cells regulated by GSK3i that include hnCD16. Another aspect of the invention provides immune cells regulated by GSK3i that have modified HLA class I and / or II. In some embodiments, GSK3i-regulated immune cells having modified HLA class I and / or II include no or low expression of at least one of B2M, HLA-E / G, PDL1, A2AR, CD47, LAG3, TIM3, TAP1, TAP2, tapasin, NLRC5, PD1, RFKANK, CIITA, RFX5, and RFXAP.

[0187] In some embodiments, the immune cell population or subpopulation for regulation is differentiated in vitro from stem cells or progenitor cells. In some embodiments, the isolated population or subpopulation of T, NK or NKT cells can be differentiated from stem cells, hematopoietic stem cells or progenitor cells (HSC), or progenitor cells. The progenitor cells can be CD34+ hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, or NKT cell progenitor cells. The stem cells can be pluripotent stem cells, such as induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC). iPSC is a reprogrammed pluripotent cell that does not exist in nature. Once the cells of interest are reprogrammed to a pluripotent state, the cells can then be programmed or differentiated into the desired cell type or subtype, such as T, NK, or NKT cells. In some embodiments, iPSCs can be differentiated into T, NK or NKT cells through a multi-stage differentiation platform in which cells from various stages of development can be induced to assume hematopoietic phenotypes ranging from mesodermal stem cells to fully differentiated T, NK or NKT cells (see, e.g., U.S. Patent Application Nos. 62 / 107,517 and 62 / 251,016, the disclosures of which are incorporated herein in their entireties).

[0188] In some embodiments, iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation are genomically engineered to include insertions, deletions, or nucleic acid substitutions. In some embodiments, the genomically engineered iPSCs, HSCs, or hematopoietic progenitor cells include genetically modified modalities including one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of the iPSCs, HSCs, progenitor cells, or their derived cells. In some other embodiments, the genetically modified modality comprises: (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region; (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, surface triggering receptors for coupling with bi- or multispecific or universal engagers, TCRs (T cell receptors), or CARs (chimeric antigen receptors). In some embodiments, iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation comprise modified HLA class I and / or II. In some embodiments, the iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation with modified HLA class I and / or II comprise no or low expression of at least one of B2M, HLA-E / G, PDL1, A2AR, CD47, LAG3, TIM3, TAP1, TAP2, tapasin, NLRC5, PD1, RFKANK, CIITA, RFX5, and RFXAP. In some embodiments, the iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation comprise exogenous nucleic acid.In some embodiments, the exogenous nucleic acid can encode a bispecific T cell engager (BiTE), a trispecific T cell engager, a multispecific T cell engager, CD16 or a variant thereof, NY-ESO, a bispecific killer cell engager (BiKE), a trispecific killer cell engager (TriKE), a multispecific killer cell engager, or a universal engager compatible with multiple immune cell types. In some embodiments, the exogenous nucleic acid encodes hnCD16 in iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation. In some embodiments, the exogenous nucleic acid encodes CAR19 in iPSCs, HSCs, or progenitor cells for T, NK, or NKT cell differentiation.

[0189] In some embodiments, the population or subpopulation of immune cells can be a specific type of fully differentiated immune cell, such as T, NK, or NKT progenitor cells, or T, NK, or NKT cells, transdifferentiated in vitro from non-pluripotent cells of non-hematopoietic fate to hematopoietic cells, or from non-pluripotent cells of a first hematopoietic cell type to a different hematopoietic cell type (see, e.g., U.S. Patent No. 9,376,664 and U.S. Patent Application No. 15 / 072,769, the disclosures of which are incorporated herein in their entirety). In some embodiments, the non-pluripotent cells of non-hematopoietic fate are somatic cells, such as skin fibroblasts, cells derived from adipose tissue, and human umbilical vein endothelial cells (HUVEC). Somatic cells useful for transdifferentiation can be immortalized somatic cells.

[0190] Various strategies have been pursued to induce pluripotency or increase capacity in cells (Takahashi, K., and Yamanaka, S., Cell 126, 663-676 (2006); Takahashi et al., Cell 131, 861-872 (2007); Yu et al., Science 318, 1917-1920 (2007); Zhou et al., Cell Stem Cell 4, 381-384 (2009); Kim et al., Cell Stem Cell 4, 472-476 (2009); Yamanaka et al., 2009; Saha, K., Jaenisch, R., Cell Stem Cell 5, 584-595 (2009)), as well as to improve the efficiency of reprogramming (Shi et al., Cell Stem Cell 2,525-528(2008a);Shi et al.,Cell Stem Cell 3,568-574(2008b);Huangfu et al.,Nat Biotechnol 26,795-797(2008a);Huangfu et al.,Nat Biotechnol 26,1269-1275(2008b);Silva et al. al.,Plos Bio 6,e253.Doi:10.1371 / journal.Pbio.0060253(2008);Lyssiotis et al.,PNAS 106,8912-8917(2009);Ichida et al.,Cell Stem Cell 5,491-503(2009);Maherali, N., Hochedlinger, K., Curr Biol 19, 1718-1723 (2009b); Esteban et al., Cell Stem Cell 6, 71-79 (2010); and Feng et al., Cell Stem Cell 4, 301-312 (2009)), the disclosures of which are incorporated herein by reference in their entireties. III. Methods for Modulating Immune Cells for Adoptive Therapy

[0191] The present invention provides a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy, the method comprising contacting an immune cell with a composition comprising at least one agent selected from Table 1.

[0192] In one embodiment, a method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy comprises contacting immune cells with a composition comprising at least one agent selected from Table 1, wherein the contacted immune cells have increased cell expansion, increased numbers or percentages of one or more desired cell subpopulations, and / or improved proliferation, cytotoxicity, cell recall, and / or persistence compared to immune cells not contacted with an agent of Table 1.

[0193] In some embodiments, a method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy comprises contacting immune cells with a composition comprising at least one agent selected from Table 1, wherein maintenance and expansion of one or more desired subpopulations of cells is improved compared to immune cells not contacted with an agent of Table 1.

[0194] In some embodiments, a method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy comprises contacting immune cells with a composition comprising at least one agent selected from Table 1, wherein the number or percentage of immune cells in the population reprogrammed to a desired state of differentiation is increased compared to immune cells not contacted with an agent of Table 1.

[0195] In some embodiments, a method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy includes contacting immune cells with a composition comprising at least one agent selected from Table 1 in an amount sufficient to increase cell expansion, increase the number or percentage of one or more desired immune cell subpopulations, and / or improve immune cell proliferation, cytotoxicity, cell recall, and / or persistence compared to immune cells not contacted with an agent of Table 1. In one embodiment, the agent for immune cell treatment is about 0.1 nM to about 50 μM. In one embodiment, the agent for immune cell treatment is about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, or 25 μM, or any concentration therebetween. In one embodiment, the agent for immune cell treatment is about 0.1 nM to about 5 nM, about 1 nM to about 100 nM, about 50 nM to about 250 nM, about 100 nM to about 500 nM, about 250 nM to about 1 μM, about 500 nM to about 5 μM, about 3 μM to about 10 μM, about 5 μM to about 15 μM, about 12 μM to about 20 μM, or about 18 μM to about 25 μM.

[0196] In some embodiments, a method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy comprises contacting immune cells with a composition comprising at least one agent selected from Table 1 for a time sufficient to increase cell expansion, increase the number or percentage of one or more desired immune cell subpopulations, and / or improve immune cell proliferation, cytotoxicity, cell recall, and / or persistence compared to immune cells not contacted with an agent of Table 1. In one embodiment, immune cells are contacted with one or more agents of Table 1 for a period of at least 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, 16 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days, 20 days, 25 days, 30 days, or any length of time in between. In one embodiment, the immune cells are contacted with one or more agents of Table 1 for about 0.5 hours to about 2 hours, about 1 hour to about 12 hours, about 10 hours to about 2 days, about 1 day to about 3 days, about 2 days to about 5 days, about 3 days to about 6 days, about 5 days to about 8 days, about 7 days to about 14 days, about 12 days to about 22 days, about 14 days to about 25 days, about 20 days to about 30 days. In some embodiments, the immune cells are contacted with one or more agents of Table 1 for 16 hours or more, 14 hours or more, 12 hours or more, 10 hours or more, 8 hours or more, 6 hours or more, 4 hours or more, 2 hours or more, or any length of time in between. Thus, the sufficient length of time can be, for example, 15 hours or more, 13 hours or more, 11 hours or more, 9 hours or more, 7 hours or more, 5 hours or more, 3 hours or more, or 1 hour or more. In some other embodiments of the methods, the sufficient length of time is 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, or any length of time in between. Thus, the sufficient length of time can be, for example, 30 hours or more, 42 hours or more, 54 hours or more, 66 hours or more, 78 hours or more, 90 hours or more.

[0197] The method of modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy, comprising contacting immune cells with a composition comprising at least one agent selected from Table 1, further comprises enriching or isolating one or more desired subpopulations from the immune cells after contacting, wherein the one or more desired subpopulations are selected from the group consisting of naive T cells, stem cell memory T cells, central memory T cells, adoptive NK cells, and type I NKT cells. The adoptive NK cells may have a phenotype of CD57+NKG2C+, or a more mature population of CD57+. In some embodiments, a sufficient amount of a composition comprising a GSK3 inhibitor and at least one of a stimulatory cytokine comprising IL2, IL15, IL12, IL18, and IL21, or one or more of a MEK inhibitor, rapamycin, and a STING agonist may be used for treatment / modulation. In some embodiments, the composition comprising a GSK3 inhibitor may further comprise one or more additional additives selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNA, dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors, vectors comprising one or more polynucleic acids of interest, and antibodies or fragments thereof. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the additional additive comprises an antibody or antibody fragment. In some of these embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen.

[0198] The population of immune cells to be treated or modulated may be isolated from a subject or donor, or may be isolated or contained in the peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, tumor of a subject / donor. The subject may be healthy or have an autoimmune disease, hematopoietic malignancy, viral infection or solid tumor. In some embodiments, the subject may be CMV seropositive or may have already been administered genetically modified immune cells. In some embodiments, the subject may be CMV seropositive. In some other embodiments, the immune cells isolated for modulation are genetically modified (genetically engineered or naturally resulting from rearrangements, mutations, genetic imprinting and / or epigenetic modifications). In some embodiments, the immune cells isolated for modulation include at least one genetically modified modality. In some embodiments, the isolated population of immune cells is genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells comprise exogenous nucleic acids encoding T cell receptors (TCR), chimeric antigen receptors (CAR), and / or overexpressed CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modulation using the disclosed compositions and methods. In some embodiments, after modulation, the genetically modified immune cells isolated from the subject may be administered to the same donor or to a different patient.

[0199] Alternatively, the population of immune cells for regulation may be differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells; or transdifferentiated from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the stem cells, hematopoietic stem or progenitor cells, progenitor cells, or non-pluripotent cells that provide the immune cells for regulation are genomically engineered to include insertions, deletions, and / or nucleic acid substitutions, such that the immune cells provided for regulation include the same genetic modalities introduced by genomic engineering in the source cells.

[0200] In a method of modulating a population of NK cells using a GSK3 inhibitor, the population of NK cells is cultured with a sufficient amount of a composition comprising a GSK3 inhibitor and for a sufficient time to improve the therapeutic potential of the NK cells for adoptive cell therapy. To obtain a population of NK cells for modulation, peripheral blood mononuclear cells (PBMCs) may be isolated from a subject. PBMCs consist of lymphocytes (T cells, approximately 30%-70%; B cells, approximately 10%-20%; NK cells, approximately 5%-20%) and monocytes. The subject may be healthy, may have an autoimmune disease, hematopoietic malignancy, viral infection or solid tumor, may be CMV seropositive, or may have already been administered genetically modified immune cells.

[0201] In some embodiments of the method, the isolated PBMCs may be depleted of T cells and B cells, for example, using CD3 and CD19 antibodies, or other sorting methods known in the art. In some embodiments, the isolated PBMCs may have no extended culture or expansion, or minimal prior depletion culture or expansion. By "no extended culture or expansion, or minimal prior depletion culture or expansion," it means that the PBMCs are pre-depleted for less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 8 hours, less than 6 hours, or less than 2 hours. In some embodiments, the PBMCs are pre-depleted for less than 2 hours, less than 4 hours, less than 6 hours, less than 8 hours, less than 10 hours, less than 12 hours, less than 24 hours, less than 48 hours. In one embodiment, the PBMCs are pre-depleted for less than overnight. In some embodiments, the PBMCs are not cultured at all prior to depletion. By "pre-depletion culture", it is meant that PBMCs are cultured without the small molecules disclosed herein, but with cytokines, feeder cells, blocking or activating antibodies, recombinant activating ligands, and / or autologous leukocytes prior to depletion of T and B cells to maintain or expand the PBMCs. Under typical cytokine culture conditions, T and B cells may expand more quickly than other subpopulations, such as NK cells, particularly some NK cell subsets, including but not limited to CD57+ and CD57+NKG2C+ NK cells. This may be because T and B cells may grow more easily under these culture conditions, may have a faster growth rate, may have a larger percentage in the starting cell population, and / or the conditions may not be suitable for these NK cell subsets to grow and expand. Thus, the expanded pre-depletion culture may result in a smaller percentage of NK cell subsets in the cultured cell population, as it overgrows these NK cell subsets. CD57+ cells and adoptive NK cells have been shown to be less proliferative than other NK subpopulations even under conventional cytokine culture and without growth pressure / competition from T and B cells.Thus, culturing PBMCs with less than about 0.1%, about 1%, about 5%, or about 10% CD57+ or adoptive NK cells to start with, along with T and B cell depletion, will cause the CD57+ and CD57+NKG2C+ NK subsets to be reduced or eliminated from the PBMC population.

[0202] Thus, one aspect of the present invention provides for culturing a sample containing PBMCs with GSK3i for multiple days to selectively expand NK subsets in the PBMC sample. In some embodiments, the PBMCs are depleted of T cells and B cells. In some embodiments, the PBMCs have not undergone pre-depletion culture prior to culturing with the GSK3 inhibitor. In other embodiments, the pre-depletion culture is minimal, less than 24 hours, less than 16 hours, less than 8 hours, or less than 2 hours, or any length of time in between. In one embodiment, the multi-day culture in the presence of the GSK3 inhibitor is under feeder-free conditions. In one embodiment, the multi-day culture in the presence of the GSK3 inhibitor is with feeder cells.

[0203] According to the present disclosure, in one embodiment, the cell population has not undergone extended prior depletion culture, and after depletion of T cells and B cells, the cell population comprises CD57-, CD57-NKG2C+, CD57+ and / or CD57+NKG2C+ NK cells. In some embodiments, the percentage of NK cells in the population after depletion but without prior depletion culture is about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%. In some embodiments, the percentage of NK cells in the population after depletion but without prior depletion culture is at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 40%, at most about 50%, at most about 60%, or at most about 70%. In some embodiments, the percentage of CD57+ NK cells or CD57+NKG2C+ cells in the population after depletion, but without pre-depletion culture, is about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, 30%, 35%, 40%, 45%, 50%, and any percentage therebetween.

[0204] The NK cell populations isolated from PBMCs that have been depleted of T and B cells and have undergone minimal pre-depletion culture, referred to as "overnight stimulated" NK cells, are then subject to modulation in the presence of small molecules provided herein for a time sufficient to modulate the phenotype and functionality of the NK cells and subpopulations thereof. In some embodiments, the modulation of NK cells comprises biasing NK cells towards maturation. In one embodiment, the modulation of NK cells comprises biasing CD57- NK cells towards CD57+ NK cells, resulting in NK cell maturation. In some embodiments, the modulation of NK cells comprises biasing CD57-NKG2C+ NK cells towards CD57+NKG2C+ NK cells. In some embodiments, the modulation of NK cells comprises increasing NK cell cytotoxicity and / or improving cytokine response. In some embodiments, the modulation of NK cells comprises biasing CD57-NK cells towards CD57+ NK cells, resulting in NK cell maturation, increasing cytotoxicity, and improving cytokine response. In some other embodiments, the modulation of NK cells comprises increasing cellular expression of one or more of CD107a, NKG2C, NKG2D, CD16, KIR, CD2, and natural cytotoxicity receptors (NCRs). NCRs include NKp30, NKp44, and NKp46. In some other embodiments, the modulation of NK cells comprises increasing NKp30 expression. In some other embodiments, the modulation of NK cells comprises increasing CD107a. Thus, the present invention also provides a population of regulated NK cells having increased expression of one or more of CD107a, NKG2C, NKG2D, CD16, KIR, CD2, NKp30, NKp44, and NKp46 compared to non-regulated NK cells, where the expression is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more. In yet some other embodiments, modulating NK cells comprises improving the proliferation, cytokine release, cell recall, and / or persistence of NK cells or a subpopulation thereof.In yet some other embodiments, modulating NK cells includes improving cell expansion, maintenance, differentiation, dedifferentiation, and / or survival of NK cells or a subpopulation thereof.

[0205] A time sufficient to modulate NK cell phenotype and / or functionality can include, for example, at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 21 days, 28 days, or any length of time in between. In some embodiments, the NK cells obtained in the methods of the invention are modulated in the presence of a GSK3 inhibitor for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 21 days, 28 days, or any length of time in between. In some embodiments, a time sufficient to modulate NK cells is 12 hours or more, 16 hours or more, 24 hours or more, 48 hours or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. In some embodiments, the NK cells obtained in the methods of the invention are modulated in the presence of a GSK3 inhibitor for 24 hours or more, 48 hours or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. In some embodiments, the time sufficient to modulate the NK cells is 1-28 days, 1-21 days, 1-14 days, 1-7 days, 2-12 days, 3-11 days, 4-10 days, 5-9 days, 6-8 days, 7-14 days, 14-21 days, 21-28 days, or any range therein. In some embodiments, the NK cells obtained in the methods of the invention are modulated in the presence of a GSK3i for 1-28 days, 1-21 days, 1-14 days, 1-7 days, 2-12 days, 3-11 days, 4-10 days, 5-9 days, 6-8 days, 7-14 days, 14-21 days, 21-28 days, or any range therein. In some embodiments, the time sufficient to modulate the NK cells is about 3 days, about 5 days, about 7 days, or about 9 days. In some embodiments, the NK cells obtained in the methods of the invention are modulated in the presence of a GSK3i for about 3 days, about 5 days, about 7 days, or about 9 days.

[0206] In some embodiments, the modulation of immune cells for a sufficient time is in the presence of a small molecule that is less than 30 μM, less than 25 μM, less than 20 μM, less than 15 μM, less than 10 μM, or less than 1 μM. In some embodiments, the small molecule used in modulating immune cells for a sufficient time is less than 25 μM, less than 20 μM, less than 15 μM, less than 10 μM, less than 5 μM, or less than 1 μM. In some embodiments, the population of NK cells modulated in the presence of a GSK3 inhibitor for a sufficient time has an NK cell expansion that is increased in number or percentage by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold. In some embodiments, the population of NK cells modulated in the presence of a GSK3 inhibitor has an NK cell purity of at least about 80%, 85%, 90%, 95%, or at least about 99%. In some embodiments, the population of NK cells modulated in the presence of a GSK3 inhibitor has an increased CD57+ NK cell expansion in number or percentage by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold. In some embodiments, the percentage of CD57+ NK cells or CD57+NKG2C+ cells in the population after modulation is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or 80%, and any percentage therebetween. In some embodiments, the population of NK cells modulated in the presence of a GSK3 inhibitor has an increased CD57+NKG2C+ NK cell expansion in number or percentage by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold.

[0207] Following the processes disclosed herein, methods of modulating populations of NK cells using GSK3 inhibitors result in modulated NK cells for cell therapy with increased numbers or percentages of CD57+ NK cells, and NK subpopulations such as CD57+NKG2C+ NK cells; improved cell expansion, maintenance and maturation; enhanced adoptive / memory-like state of NK cell subsets leading to improved proliferation, cell recall, and / or persistence; and improved cytotoxicity and cytokine response and secretion. Thus, different aspects of the present invention further provide methods for selectively expanding NK cells and subpopulations thereof comprising contacting a depleted PBMC sample without prior depletion culture with a composition comprising a sufficient amount of a GSK3 inhibitor for modulation and for a sufficient time to obtain modulated cells with improved cell therapy potential; methods for expanding and modulating adoptive NK cells; methods for improving NK cell expansion, maintenance or maturation; methods for enhancing the adoptive / memory-like state of NK cells; methods for improving the therapeutic potential of NK cells, including but not limited to cell proliferation, cell recall, and cell persistence; methods for improving NK cell cytotoxicity; and methods for increasing NK cell cytokine secretion and response.

[0208] In another embodiment, the method comprises obtaining a sample of PBMCs from a subject; depleting the PBMC sample without prior depletion culture of the sample, for example using antibodies comprising CD3 and CD19; and contacting the depleted PBMC sample with a composition comprising a sufficient amount of GSK3i for a sufficient time to obtain regulated cells with improved potential for cell therapy.

[0209] In yet another embodiment, the method comprises obtaining a sample of PBMCs from a subject; depleting the PBMC sample without prior depletion culture using antibodies comprising CD3 and CD19 or other non-NK specific markers; contacting the depleted PBMC sample with a composition comprising a sufficient amount of GSK3i for modulation for a sufficient time to obtain regulated cells; and isolating CD57+ NK cells from the regulated cells.

[0210] In some embodiments of the above methods, the methods may further comprise activating the population of NK cells prior to or during the step of contacting the NK cells with a composition comprising a GSK3 inhibitor.

[0211] In some embodiments of the above methods, contacting with a composition comprising a sufficient amount of GSK3i for a sufficient period of time may be performed under feeder-free conditions, i.e., without feeder cells or components thereof and without feeder cell conditioned medium.

[0212] In some embodiments of the above methods, contacting with a composition comprising a sufficient amount of GSK3i for a sufficient period of time may be performed in the presence of feeder cells, in some embodiments, the feeder cells are modified by genome editing to express an exogenous surface protein. IV. Therapeutic Uses of Treated Immune Cells, Immune Cell Populations or Subpopulations

[0213] The present invention provides compositions comprising an isolated population or subpopulation of immune cells contacted with one or more agents selected from Table 1 in an amount sufficient to improve the therapeutic potential of the immune cells when used in cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of treated immune cells comprises an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of contacted immune cells comprises an increased number or proportion of type I NKT cells. In another embodiment, the isolated population or subpopulation of contacted immune cells comprises an increased number or proportion of adoptive NK cells. Combination therapies using NK cell therapy products together with other agents that target tumor cells or modulate the cytotoxic activity of NK cells are contemplated herein. In some embodiments of the composition, the composition further comprises one or more additional additives selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNA, dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, and immunomodulatory drugs (IMiDs).

[0214] The present invention also provides compositions and methods of combination therapy comprising immune cells modulated with one or more agents, including the compounds listed in Table 1, and additional therapeutic agents. In some embodiments, the additional therapeutic agent comprises an antibody, or an antibody fragment. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody, or antibody fragment, specifically binds to a viral antigen. In other embodiments, the antibody, or antibody fragment, specifically binds to a tumor antigen. In some embodiments, the tumor or virus specific antigen activates the modulated NK cells to lyse target cells using antibody-dependent cellular cytotoxicity (ADCC). The monoclonal antibody (mAb) binds to mobilized CD16 on target cells plus NK cells and other cell types, resulting in tumor cell killing by ADCC both in vivo and in vitro. The mAb can also enhance ADCC by blocking NK cell inhibition and stimulate NK cells. In some embodiments, the ADCC mediated by NK cells is via CD16 expressed by the modulated NK cells and genetically engineered variants thereof. Genetically engineered variants of Cd16 include, but are not limited to, uncleavable CD16, high affinity CD16 (haCD16), and high affinity uncleavable CD16 (hnCD16). Thus, the above aspects of the present invention provide GSK3i-regulated NK cells capable of performing ADCC in antibody combination cancer therapy. In some embodiments, antibodies suitable for combination treatment with anti-cancer NK cells provided herein include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-Her2 (trastuzumab), anti-CD52 (alemtuzumab), anti-EGFR (certuximab), and anti-CD38 (daratumumab), and their humanized and Fc-modified variants.In addition, the design of bi- and trispecific antibodies fusing the Fab region of an antibody targeting a tumor cell antigen, such as anti-CD19, CD20, and CD33 antigens, combined with another Fab region that recognizes CD16 on NK cells, leads to stimulation of NK cells and subsequent tumor killing.

[0215] In some embodiments, the additional therapeutic agent comprises one or more chemotherapeutic agents or radioactive moieties.Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents that preferentially kill tumor cells or disrupt the cell cycle of rapidly growing cells, or chemical agents that have been found to eradicate cancer stem cells, and chemical agents that are used therapeutically to prevent or reduce the growth of tumor cells.Chemotherapeutic agents are also sometimes referred to as antitumor or cytotoxic drugs or agents, and are well known in the art.

[0216] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethylenimines, methylmelamine, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing drugs, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechlorethamine, mephalin, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisanthrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include aminoglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calcitabine, capecitabine, celecoxib, and cetaxel. Mab, cladribine, clofarabine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab , idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegadome , pegaspargase, pentostatin, pipobroman, plicamycin, porifeprozan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiotherapeutic agents and those known in the art.Such agents can be found through any of a number of standard physician and oncologist reference works, both of which are updated from time to time (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, NY, 1995), or through the National Cancer Institute's website (fda.gov / cder / cancer / druglistfrarne.htm).

[0217] Immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, stimulate both NK cells and T cells. As provided herein, IMiDs may be used in conjunction with regulated therapeutic immune cells for cancer treatment.

[0218] Various diseases may be ameliorated by introducing the cells of the present invention into a subject suitable for adoptive cell therapy. Examples of diseases include, but are not limited to, various autoimmune diseases, including alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis myositis, diabetes mellitus (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's). autoimmune disorders; hematopoietic malignancies, including but not limited to acute and chronic leukemia, lymphoma, multiple myeloma and myelodysplastic syndromes; solid tumors, including but not limited to tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, pharynx, or esophagus; and infectious diseases, including but not limited to HIV- (human immunodeficiency virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), adenovirus- and BK polyomavirus-associated disorders. Working Example

[0219] The following examples are given by way of illustration and not by way of limitation.

[0220] Example 1 - Methods and Materials In vitro cell culture. Fresh leukopaks (AllCells, Alameda, CA) were obtained from healthy donors and then T cells were negatively selected using the EasySep Human T cell Enrichment Kit (Stem Cell Technologies, Vancouver, Canada). Freshly isolated T cells were aliquoted and cryopreserved. Freshly isolated T cells were aliquoted and cryopreserved. On the day that screening began, T cells were thawed and poured into X-Vivo 15 containing 5% human AB serum, IL-2, penicillin / streptomycin, and additional supernatant. Cells were plated at 5 × 10 5 Cells were dispensed at 1000 x 10 cells / ml with anti-CD3 / anti-CD28 dynabeads (Thermo Fisher, Waltham, MA) at a bead to cell ratio of 3:1. Individual compounds were added to each well from column 3 to column 22 of each plate at a final concentration of 10 μM. Positive and negative controls were added to additional wells. Cells were incubated for approximately 6 days at 37° C., 5% CO2.

[0221] Flow cytometry. On day 6 of culture, cells were stained with fixable viability markers and fluorophore-conjugated antibodies: CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD27, and CD122 (Becton Dickinson Biosciences, San Jose, CA; and BioLegend, San Diego, CA). Fluorescent absolute counting beads (Spherotech, Lake Forest, IL) were added immediately before acquisition. Data acquisition was performed on a BD Fortessa X-20 (Becton Dickinson Biosciences) and analyzed using Treestar software (FlowJo, Ashland, OR) and Spotfire (Tibco, Boston, MA).

[0222] NK cell isolation. Healthy adult donor blood was obtained from commercial sources or from the GCRC core laboratory at Scripps Research Institute, La Jolla, CA. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare: Life Sciences), and then depleted for B and T cells using CD19 Microbeads (Miltenyi Biotec, no. 130-050-301) and CD3 Microbeads (Miltenyi Biotec, no. 130-050-101) according to the manufacturer's instructions, without culturing the PBMCs for 24–36 h.

[0223] NK cell culture: CD3 / CD19-depleted PBMCs (0.5–1 × 10E6 / mL) were cultured in B0 medium (a 2:1 mixture of DMEM, (Corning Cell-Gro, no. 10-017-CV) and Ham's F12 (Corning, 30-002-CI) containing 10% human AB serum (Valley Biomedical, no. HP1022), 100 U / mL penicillin and 100 μg / mL streptomycin (Corning, 30-002-CI), 20 μM 2-mercaptoethanol (Sigma, M3148), 50 μM ethanolamine (Sigma, E0135), 10 μg / mL ascorbic acid (Sigma, A4544), and 1.6 ng / mL sodium selenate (Sigma, S5261). Cells were cultured in 1000-mL 10-mL 10-mL 10-mL 10-mL 15 ... The cells were analyzed using the ELISA software. For flow cytometry, commercially available antibodies (Becton Dickinson Biosciences, Biolegend, or R&D Systems) against the following surface markers were used: CD3, CD14, CD16, CD19, CD56, CD57, and NKG2C. K562 feeder cells were used where indicated. Other modulators and culture reagents were added where indicated.

[0224] NK cell sorting: CD3 / CD19-depleted PBMCs were labeled with 10 μM e450 proliferation dye (eBioscience) and stained with FITC-conjugated anti-CD57, PE-conjugated anti-NKG2C, APC-conjugated anti-CD56, and Percp-Cy5.5-conjugated anti-CD3 antibodies (Becton Dickinson Biosciences, and R&D Systems). Four populations of CD3-CD56+ NK cells were sorted according to CD57 and NKG2C expression; CD57+NKG2C-, CD57+NKG2C+, CD57-NKG2C+, and CD57-NKG2C-. Sorted NK cell populations were cultured at a 2:1 NK:monocyte ratio in B0 medium containing 10 ng / ml IL-15 and CHIR99021 or vehicle control. Stained but unsorted cells were cultured in parallel as controls.

[0225] NK cell cytotoxicity assay: Cytotoxicity was determined using a flow cytometry-based method based on a previously published method (Kim, et al. J Immunol Methods. 2007 Aug 31;325(1-2):51-66). NK cells were co-cultured with e450-proliferation dye-labeled targets for 4 hours. CellEvent™ Caspase-3 / 7 Green Flow Cytometry Assay Kit (Life Technologies) was used to assay for cell death among target cells and samples were read on a BD LSRFortessa cytometer. Killing of SKOV-3 targets was quantified using an Incucyte Zoom instrument. SKOV-3 cells were transiently transduced with NucLight-Red BacMam reagent (Essen Biosciences) and co-cultured with the indicated NK cell populations for 74 hours, with images acquired at 2-hour intervals. Caspase-3 / 7 activity was monitored using CellEvent™ Caspase-3 / 7 Green Detection Reagent to indicate target cells undergoing apoptosis. Target cell numbers were normalized to SKOV-3 cells cultured alone, and targets undergoing apoptosis were quantified based on caspase-3 / 7 activity.

[0226] Intracellular cytokine staining of NK cells: CD3 / CD9 depleted PBMCs were cultured for 7 days with 10ng / ml IL-15 and 5μM CHIR99021 or vehicle control. On day 7 of culture, cells were washed and cultured separately with IL-12 and IL-18 (10ng / ml and 50ng / ml, respectively) (Life Technologies) or with K562 cells at a 1:1 ratio. Golgistop (Becton Dickinson Biosciences) was included in the medium to facilitate intracellular detection of cytokines. After a 4-h culture period, cells were stained for cell surface antigens and fixed prior to intracellular staining with antibodies against IFN-gamma and TNF-alpha (Becton Dickinson Biosciences). Staining was performed using the BD Cytofix / Cytoperm™ Kit according to the manufacturer's instructions.

[0227] Screening of NK cell modulating compounds: CD3 / CD19 depleted PBMCs were cultured with 10ng / ml IL-15 and 5μM CHIR99021 with or without feeder cells for 7 days. Various small molecules and / or cytokines were added on day 0 in multiple rounds to assess their effects on proliferation and phenotype. Cultures were monitored for medium acidification or cell overgrowth throughout the 7 day period. After culture, cells were harvested and stained for phenotypic analysis by flow cytometry. Data were analyzed for changes in proliferation and marker expression. Flow cytometry was performed on a BD LSRFortessa and results were analyzed with FlowJo software. For flow cytometry, commercially available antibodies against the following surface markers were used: CD3, CD14, CD16, CD19, CD56, CD57 and NKG2C (Becton Dickinson Biosciences, Biolegend, or R&D Systems).

[0228] In vivo tumor elimination using regulated NK cells: 2×10 5 NSG mice were irradiated with 300 rads one day prior to intraperitoneal implantation of luciferase-expressing SKOV-3 cells. Five days after implantation of ovarian cancer cells, mice were left untreated, treated with Herceptin alone, or treated with Herceptin, IL-15, and NK cells cultured with IL-15 and CHIR99021 for 7 days. Luciferase-expressing SKOV-3 cells were imaged on day 21 using the IVIS imaging system.

[0229] Example 2 - Agents for immune cell modulation Data was analyzed to identify compounds that produced either a higher percentage or a higher absolute number of phenotypically identified naive, stem cell memory, or central memory T cells. These cells are characterized by the expression of CCR7 and CD62L. Therefore, cells that co-express both of these identification markers were evaluated. The percentage of cells that co-express CCR7 and CD62L was determined within the live CD4+ and live CD8+ populations. As an indication of the desired T cell subset, the expression of either CD62L or CCR7 on T cells was described as having favorable functional characteristics for CAR-T cell therapy, and potentially other adoptive T cell therapy. Under treatment with dorsomorphin, hepteridic acid, the GSK3 inhibitor, 6-mercaptopurine, AC-93253 iodide, tiratricol, PI-103, 5-azacytidine, 5,7-dichloro-8-quinolinol, nitrofurantoin, 5-chloro-7-iodo-8-quinolinol, or diethylenetriaminepentaacetic acid, the number or percentage of cells coexpressing CCR7 and CD62L was increased in both the viable CD4+ and viable CD8+ populations (Table 2). Under treatment with fulvestrant, thapsigargin, SU4312, fludarabine, 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy, nifuroxazide, and edrophonium chloride, the number or percentage of cells coexpressing CCR7 and CD62L was increased, at least in the viable CD8+ population (Table 2). Under treatment with 1-pyrrolidinecarbodithioic acid, ammonium salt, U0126, telmisartan, cyclosporine A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY61-3606, protoporphyrin IX disodium, rapamycin, roscovitine, PAC-1, tosufloxacin hydrochloride, BIX01294, and terfenadine, the number or percentage of cells coexpressing CCR7 and CD62L was increased, at least in the viable CD8+ population (Table 2).

[0230] In addition, GSK3 (glycogen synthase kinase 3) inhibitors have been shown to preserve CD3-CD19-CD56+ NK cells and expanded adoptive NK cell subpopulations by influencing cell maturation and subtype bias based on observations including, but not limited to, CD57+ and NKG2C+ expression.

[0231] The number of events in each of these gates, which correlates with the absolute number of bead counts in each sample, was calculated, defining a relative measure of the absolute number of naive, stem cell memory, or central memory T cells within the CD4+ and / or CD8+ populations. A z-score was calculated for each of these four values: 1) percent CCR7+CD62L+ in CD4+, 2) percent CCR7+CD62L+ in CD8+, 3) absolute relative number of CCR7+CD62L+ in CD4+, and 4) absolute relative number of CCR7+CD62L+ in CD8+ (Figures 1A and 1B). Z-scores were calculated for the percent viability of all cells in each sample, and for the relative absolute number of cells in each sample. A "Z-score" is a statistical measure of the association of a score with the mean of the group of scores. A Z-score of 0 means that the score is the same as the mean. A Z-score can also be positive or negative, indicating whether it is above or below the mean and by how many standard deviations.

[0232] Elimination of compounds with adverse effects on T cell proliferation or viability has focused efforts on compounds that are most likely suitable for T cell manufacturing methods. First hit compounds were selected by the following criteria: "percent viability" Z-score greater than -1, "relative absolute number" of cells Z-score greater than -1, and Z-score of one of four values ​​greater than +2. Thirty-four compounds (Table 2) were selected in that they had much higher Z-scores and met the above criteria for more than one of the four first values. Five more compounds are also included in terms of their ability to modulate T cells (Table 3).

[0233] [Table 2-1]

[0234] [Table 2-2]

[0235] [Table 2-3]

[0236] [Table 3]

[0237] Example 3 - In vitro triage experiments of selected compounds In vitro experiments are performed to optimize compound exposure and triage compound methods that have a definitive effect on T cell function. The first test determines the optimal dose of each compound while evaluating whether the effects already observed on naive, stem cell memory, and central memory T cells are repeated in additional donors. In order to triage compounds that have a possible definitive functional effect on T cells, in vitro evaluations are performed on the proliferation capacity, ability to polarize Th1 and Th17, survival through cryopreservation / thawing cycles, transduction efficiency, and tumoricidal activity of CAR-transduced T cells. Compounds that reproducibly improve the proportion or number of naive, stem cell memory, or central memory T cells during expansion without significant negative effects on T cell function are tested in combination and evaluated for additive or synergistic effects. Through these evaluations, promising candidates or combinations are prioritized for further testing in vivo.

[0238] Example 4 - In vivo model of adoptive cell therapy using selected compounds To interpret the results of the in vivo screening and follow-up in vitro triage experiments, selected potential compounds are applied to an in vivo model of adoptive cell therapy. Specifically, the impact of small molecule modulation on adoptive cell therapy is examined with respect to engraftment, tumoricidal activity, secondary tumoricidal responses, migration, cell persistence, and graft-versus-host disease. Other readouts that are characteristics of persistent adoptive cell therapy that have been found to correlate with effective responses in the clinic are also examined.

[0239] These experiments are performed either in a humanized system in which human cells are adoptively transplanted into immunodeficient NSG mice that develop human tumors, or in an alternative mouse model in which immune-competent animals develop syngeneic tumors and are treated with syngeneic cell therapy.

[0240] In either alternative or humanized model systems, mice are injected with luciferase-labeled lymphoma or other tumors of interest. Immediately thereafter, adoptive cell therapy is administered, pretreated with vehicle or regulatory compound as disclosed in the present invention. The doses of both cell therapy and tumor are optimized to allow a window of observation of the positive or adverse effects of compound treatment. Animal weight, plasma cytokine concentration, tumor volume, and adoptive cell therapy in peripheral blood, secondary lymphoid organs and tumor mass; tumor burden, tumor metastasis, and cell therapy phenotype are monitored for the duration of the experiment.

[0241] Compounds capable of improving one or multiple tumor-associated parameters in vivo have anticipated effects including, but not limited to, lowering the dose of cell therapy required for effective tumor clearance, increasing the persistence of adoptive cell therapy in peripheral blood, enhancing migration to tumor sites, and / or increased survival against high tumor dose challenges.

[0242] Example 5 - Selective expansion of NK cells and subpopulations using GSK3 inhibitors One aspect of the present invention is to provide methods and systems for culturing, expanding, and / or modulating NK cells for adoptive cell therapy. In some embodiments, the methods and systems for culturing, expanding, and / or modulating NK cells do not use feeder cells or feeder cell conditioned medium. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult donors. The isolated apheresis product (day 0, pre-depletion; Figure 2A) was T and B cells depleted using anti-CD19 and CD3 antibodies without pre-depletion culture (day 0, post-depletion; Figure 2B). The post-depletion cell population was then cultured in feeder-free conditions in the presence of recombinant human IL-15 (10 ng / mL) and 5 μM CHIR99021, a GSK3 inhibitor, for approximately 3-7 days. As shown, culture with IL-15 and GSK3 inhibitor appears to selectively and significantly result in NK cell (CD56+CD3-) expansion from approximately 20% (Figure 2B) to approximately 99% (Figure 2C) at culture day 7. In contrast, the remaining T cells (CD56-CD3+) in the post-depletion population (approximately 1%; Figure 2B) are not expanded, resulting in a reduced percentage in the population (down to 0.25%; Figure 2C).

[0243] After depletion and overnight culture (day 0, post-depletion; overnight culture with IL15 only; also referred to as "priming"), the cell population contains approximately 10-40% (due to donor variability) NK cells. Selective and significant expansion of NK cells after 7 days of culture with cytokines and a GSK3 inhibitor resulted in highly improved NK cell purity from about 80% to greater than about 99% (Figure 3A), representing an average of about 8-fold expansion in NK cell numbers (Figure 3B), with some samples having a larger expansion of about 18-fold. Note that donor variation and process variation may contribute to variability.

[0244] The NK cell subpopulations in the overnight primed cell population and in the cell population cultured with GSK3i on day 7 were further analyzed and compared. These analyses illustrate that the cell numbers of CD57+NKG2C+ and CD57+NK subsets increased by an average of about 5-6 fold after culture with GSK3 inhibitors (Figures 3C and 3D). Typically, CD57+ and CD57+NKG2C+NK subsets are present in relatively small numbers in donor-derived cells. Furthermore, CD57+ and CD57+NKG2C+NK subsets were not well maintained when cultured under cytokines alone (7 days of culture without GSK3i; Figure 4A), which was further represented by a reduced percentage of CD57+NK cells in the NK population (Figure 4B). In comparison, when cultured with GSK3 inhibitors, the number of CD57+ and CD57+NKG2C+ NK cells was significantly increased by an average of 7-8 fold compared to the number of cells cultured without GSK3i (Figure 4A). Furthermore, 7 days of culture with GSK3i also resulted in an increased percentage of CD57+ cells in the total NK population compared to 7 days of culture without GSK3i (Figure 4B). The data herein showed that traditional culture processes of NK cell populations (i.e., using cytokines without GSK3 inhibitors) would reduce or even eliminate the CD57+ and CD57+NKG2C+ NK subsets from the population, especially when NK cells are included in a donor-derived PBMC population without T and / or B cell depletion.

[0245] Example 6 - GSK3 inhibition leads to NK cell maturation CD3 / CD19-depleted PBMCs were labeled with e450 proliferation dye and CD56+CD3- NK cells were sorted in bulk or into four populations based on CD57 and NKG2C expression (Figure 5A). Total unfractionated NK cells were cultured in parallel with CD57+NKG2C-(Q1), CD57+NKG2C+(Q2), CD57-NKG2C+(Q3), and CD57-NKG2C-(Q4) sorted NK cells with autologous CD14+ monocytes at a 1:1 ratio of NK cells:monocytes in either (1) cytokines (10 ng / ml IL15) and 5 μM CHIR99021, or (2) cytokines alone, including vehicle control (e.g., DMSO only) (Figure 5B). After a 7-day culture period, cultures of each sorted population were stained for flow cytometry and analyzed for proliferation and cell surface marker expression. It was shown that after 7 days of culture with GSK3 inhibitor, there was a significant increase in CD57 expression in total NK cells (Figure 5B, unsorted lower panel). When analyzing each cell quadrant, it was clear that the increased CD57 expression was not simply accounted for by the observed enhanced proliferation of CD57+ cells (Figure 5B, Q1 and Q2). It was further found that the upregulated expression of CD57 in Q3 and Q4 sorted cells, which were CD57- before culture with GSK3 inhibitor, had undergone maturation and was largely responsible for the increased CD57 levels (Figure 5B, Q3 and Q4). Therefore, we conclude that GSK3 inhibitors result in NK cell maturation as indicated by upregulation of CD57 expression in CD57- cells and also contribute to a significantly increased percentage of CD57+ and CD57+NKG2C+ NK cells in the GSK3i-modulated population.

[0246] It was further observed that when the same CD3 / CD19 depleted PBMC samples underwent treatment for a short period, e.g., 16 hours, with a GSK3 inhibitor, the GSK3 inhibitor did not result in a higher frequency of CD57+ cells in the cultures (Figure 11A), which is consistent with vehicle treatment. Quantification of the CD57+ NK cell fraction in cultures with or without GSK3i is further shown in Figure 11B, where it was shown that GSK3i resulted in the creation of a regulated NK cell population with a higher frequency of the CD57+ fraction when the NK cell population was treated with GSK3i for a sufficient period of time, longer than 16 hours.

[0247] The highly stable expression of CD57 probably represents a late stage in NK cell maturation. - Compared with cells, CD57 + NK cells are thought to potentially proliferate less in response to IL-2 and IL-15, and produce little IFN-γ in response to IL-12 and IL-18. It is therefore surprising to find that modulation of NK cells for a sufficient length of time with a composition comprising a GSK3 inhibitor results in a CD57− bias towards CD57+, while both the number and percentage of CD57+ NK cells improve following modulation, which is accompanied by enhanced NK cytotoxic activity and cytokine responses, as further described below.

[0248] Example 7 - GSK3 inhibition enhances NK cytotoxic activity and cytokine responses The ability of GSK3 inhibition to enhance the cytotoxicity of NK cells directly isolated from donors was evaluated using an in vitro killing assay. C3 / C19-depleted PBMC populations were treated overnight with 10 ng / mL IL-15 alone (priming) or with 10 ng / mL IL-15 and 5 μM CHIR99021 in combination culture for 7 days. Target cells for the killing assay included K562 cells, CD20+ Raji cells, HER2+ SKOV3 cells and HER2+ A549 cells. Effector:target (E:T) ratios applied in each assay ranged from 0.1, 0.5, 1, 5-10. After 4 h of incubation of target cells with differently treated NK cells, the respective percentages of specific killing (target cell lysis) were calculated. E:T ratios for NK cells treated with IL15 alone in the K562 killing assay were 0.1, 0.5, 1, 0.5, 1.0, 0.5 ... 50 (E:T ratio at which 50% of target cell lysis occurs) was approximately 10, whereas the E:T for NK cells treated with GSK3i and IL15 was approximately 10. 50 The E:T ratio was reduced to approximately 1, which represents a greatly improved cytotoxic activity (Figure 6A). Raji cells are less prone to NK-mediated cytotoxicity. Figure 6B shows that Raji cells suppressed NK cells with IL15 in the presence of GSK3i for 7 days, resulting in an E:T ratio almost as high as 100. 50 , while still being fairly resistant to NK cells treated overnight with IL-15, E:T 50 however, was dramatically reduced to 1-2, indicating a significant effect of the combination of IL15 and GSK3 inhibitors in enhancing NK cell cytotoxicity even killing resistant cells.

[0249] In a similar manner, SKOV3 cells were treated with NK cells resulting from overnight culture with IL15, 7-day culture with IL15, and 7-day culture with IL15 and GSK3i. The data show that NK cells treated with GSK3i were the most effective in killing SKOV3 cells, both in terms of killing rate and killing efficiency, based on cell killing as indicated by elimination of SKOV3 cells (Figure 6C) and the first maximum peak of caspase 3 / 7 event (apoptosis) (Figure 6D).

[0250] Antibody-dependent cellular cytotoxicity (ADCC) was also shown to be enhanced in NK cells cultured with IL15 and GSK3i for 7 days when HER2 antibody (Herceptin) was used to mediate killing of SKOV3, compared to ADCC killing using NK cells resulting from overnight culture with IL15 and 7 days culture with IL15 (Figure 6E). For further proof, A549 cell line (small cell lung cancer line) with high expression of HER2 antigen, which can be targeted by Herceptin antibody, was applied to test NK cell killing to test ADCC-mediated killing. Similar enhanced ADCC-mediated cell killing by NK cells cultured with GSK3i for 7 days was observed in both killing rate (Figure 6F) and efficiency (Figure 6G) of A549 cell killing. Together, the data show that by conditioning NK cultures for multiple days in IL15+GSK3i, adoptive and CD57+ mature cells are maintained and expanded, resulting in a significant increase in the cytotoxic effect of NK cells.

[0251] The effect of GSK3 inhibitors on NK cell cytokine responses was also analyzed. PBMCs obtained directly from donors were cultured with 10 ng / ml IL-15 and 5 μM CHIR99021 or with IL15 alone for 7 days after CD3 / CD9 depletion. On day 7, cells were washed and cultured separately with IL-12 (10 ng / ml) and IL-18 (50 ng / ml) or with K562 cells at a 1:1 ratio for stimulation. After a 4-h culture period, cells were stained for cell surface antigens and fixed prior to intracellular staining with antibodies against IFNγ and TNFα, respectively. Figure 7 shows that IFNγ and TNFα production in NK cells treated with GSK3i and IL15 was enhanced under both IL12+IL18 and K562 stimulation compared to primed (depleted and cultured overnight with IL15) or IL15 only treated samples for 7 days. Enhanced cytokine production in NK cells treated with GSK3i and IL15 is shown by the significant percentage increase shown in Figures 7A (IFNγ) and 7B (TNFα) compared to control NK cells (IL15 overnight) and NK cells cultured with IL15 for 7 days. Furthermore, since responses to IL-12 and IL-18 are typically associated with immature NK cells, the enhanced IFNγ and TNFα responses observed above support that the compositions and methods disclosed herein lead to enhanced function associated with both mature NK (cytotoxicity) and immature NK (cytokine responsiveness) in CD3 / 19 depleted PBMCs obtained directly from donors.

[0252] Example 8 - GSK3 inhibitors have an effect on NK proliferation and subtype bias in the presence of modified K562 feeder cells It was observed that co-culture of NK cells with K562 feeder cells using the process disclosed herein, i.e., depleting PBMCs on day 0 and then culturing NK cells in the presence of GSK3i for multiple days, allowed a significant increase in the expansion of selective NK cell subsets with enhanced functionality over feeder-free culture. Unmodified and modified K562 cells (e.g., with transgene-mediated surface expression of certain proteins) were tested and showed similar results in selective NK expansion and subtype bias by immediate depletion of donor samples without prior depletion culture followed by GSK3i culture for multiple days.

[0253] CD3 / 19 depleted PBMCs were cultured for 7 days with modified K562 feeder cells in the presence of DMSO (vehicle), 0.1 μM GSK3i CHIR99021 alone (C), or a combination of 0.1 μM GSK3i CHIR99021, 40 nM MEKi PD0325901, and 1 nM rapamycin (CPR). Figure 8A shows that the addition of small molecules (C or CPR) overall increased the proliferation of activated NK cells compared to culture alone with K562 lines modified to express IL21 and 41BBL. In comparison, the increase in the number of CD57+ NK cells was greatest with GSK3i, while CPR resulted in a lower number of CD57+ NK cells than the vehicle control (Figure 8B). However, the number of NKG2C+ NK cells was increased by treatment with CPR when NK cells were cultured with modified K562 feeder cells (Figure 8C).

[0254] The data show that the presence of feeder cells, a unique combination of small molecules, when applied according to the processes disclosed herein, can increase various properties of the expanding NK population, including a bias towards a mature state, as indicated by an increase in the CD57+ population, or towards an adoptive state, as indicated by an increase in the NKG2C+ population.

[0255] Example 9 - Maintenance of a regulated NK cell phenotype in vivo NK cells cultured overnight with IL15, NK cells cultured with IL15 and vehicle for 7 days, and NK cells cultured with IL15 and GSK3i for 7 days were transferred into NSG mice, respectively. After 7 days, NK cells from the peripheral blood of the mice were analyzed for phenotype (n=3 per group). Mice received only IL-15 and no GSK3i. It was shown that NK cells cultured in vitro with GSK3i for 7 days maintained their phenotypic changes in vivo for at least 1 week after transfer without the presence of small molecules. CD57 remained high, which represents the maturity of NK cells, which was not the case when cells were cultured without GSK3i (Figures 9A and 9B). Furthermore, CD16, a marker for antibody-dependent killing, and NKG2C+ / CD57+ were maintained in vivo (Figure 9B).

[0256] In addition, the process provided herein was used to test the in vivo tumor clearance ability of NK cells cultured with GSK3i. One day before intraperitoneal implantation of luciferase-expressing SKOV-3 cells, NSG mice were irradiated with 300 rads. Five days after ovarian cancer cell implantation, mice were either left untreated, treated with Herceptin alone, or treated with Herceptin, IL-15, and NK cells cultured with IL-15 and CHIR99021 for 7 days. Luciferase-expressing SKOV-3 cells were imaged 21 days after implantation (or 16 days after treatment) using an IVIS imaging system. Figure 10 shows that the addition of GSK3i-treated NK cells enhances the tumor suppressive effect of Herceptin antibody treatment, suggesting that GSK3i-treated NK cells are capable of mediating antibody-dependent cellular cytotoxicity in vivo. These data suggest that the effects of multi-day culture with IL-15 and GSK3i are long-lasting and remain imprinted on NK cells for at least 1 week after cessation of the small molecule, CHIR99021.

[0257] Next, the in vivo tumor clearance ability of NK cells cultured with GSK3i using the process provided herein was compared to CD3 / CD19-depleted PBMCs primed overnight and CD3 / CD19-depleted PBMCs cultured with IL-15 and DMSO for 7 days. SKOV-3 cells expressing luciferase were injected intraperitoneally into NSG mice. After 5 days, mice were treated with 100 μg of Herceptin antibody and 2.5×10 6 cells derived from either CD3 / CD19-depleted cells primed overnight, CD3 / CD19-depleted PBMCs cultured with IL-15 and DMSO (vehicle) for 7 days, or CD3 / CD19-depleted PBMCs cultured with IL-15 and GSK3i for 7 days (GSK3i-regulated NK cells). 6 NK cells were treated with IL-15 and GSK3i. Luciferase-expressing SKOV-3 cells were quantified by measuring luciferase activity using an IVIS imaging system. Figure 12A shows that NK cells cultured for multiple days with IL-15 and GSK3i were highly and consistently effective against SKOV-3 cells in vivo. Figure 12B shows a quantitative comparison of the in vivo SKOV-3 ADCC killing effect by differently treated NK cells, which further confirmed the observation in Figure 12A below.

[0258] The cytotoxicity of overnight primed NK cells versus CD3 / CD19 depleted PBMCs cultured with IL-15 and GSK3i for multiple days (GSK3i conditioned NK cells) against primary AML blasts was also compared. Percent specific killing of primary AML blasts from multiple patients was measured by a flow cytometry based cytotoxicity assay. Figure 13 showed that NK cells cultured with IL-15 and GSK3i for multiple days were highly and consistently effective against AML blasts.

[0259] Cytotoxicity of GSK3i-modulated NK cells against allogeneic PBMCs was assessed in comparison to K562 tumor cells in a dual target cytotoxicity assay. Percent specific killing was measured in PBMCs+K562+overnight primed NK cells or PBMCs+K562+GSK3i-modulated NK cells. Data representing the mean ± SEM of four (FIGS. 14A-C) or three (FIG. 14D) NK cell donors per healthy PBMC target donor demonstrate the ability of GSK3i-modulated NK cells to discriminate against malignant cells and selectively target them among normal cells.

[0260] Example 10 - STING agonists enhance GSK3i-regulated NK cell cytokine production and cytotoxicity The STING (stimulator of interferon genes) complex plays a key role in detecting the presence of tumor cells and promoting aggressive anti-tumor responses by the body's innate immune system. The STING complex acts as both a direct cytoplasmic DNA sensor (CDS) and an adaptor protein in type I interferon signaling through different molecular mechanisms. STING is expressed in hematopoietic cells in peripheral lymphoid tissues, including T lymphocytes, NK cells, myeloid cells, and monocytes. Detection of cytoplasmic DNA initiates a series of interactions that lead to STING pathway activation. Pathway activation triggers the production of interferon-β and interferon-α. STING agonists activate the STING pathway. STING agonists include STING ligands that bind and activate STING. Known STING agonists include cyclic dinucleotides (CDNs) and xanthenones, as well as their analogs and derivatives. CDNs may be synthetic or derived from prokaryotic or mammalian cells. Exemplary STING agonists include, but are not limited to, cGAMP, c-di-GMP, c-di-AMP, c-di-IMP, c-di-UMP, cAMP-GMP, R,R dithio-modified CDA compounds (ML RR-S2 CDA and RR-S2 CDA), ML RR-S2 cGAMP, DMXAA (5,6-dimethylxanthenone-4-acetic acid). Additional CDN analogs, derivatives, and bacterial CDN products will continue to be identified as STING ligands.

[0261] CD3 / CD19 depleted PBMCs were cultured for 7 days in the presence of 10ng / ml IL15 and (1) DMSO (vehicle); (2) 5μM GSK3i (CHIR99021); (3) 1μM STING agonist (ML RR-S2 CDA); or (4) 5μM GSK3i+1μM STING agonist. On day 7, NK cell function and phenotype were assessed by intracellular cytokine staining, cytotoxicity assay, and flow cytometry. The STING agonist ML RR-S2 CDA was shown to have limited effects on adoptive NK phenotype and total NK numbers. Figure 15 shows that addition of STING agonist to GSK3i+IL-15 NK cell cultures resulted in continued maintenance of the CD57+ fraction, with a slight increase during IL-15+GSK3i culture. However, as shown in Figure 16, the addition of STING agonist in combination with GSK3i enhances NK cell production of IFNγ (Figure 16A) and TNFα (Figure 16B) for 4 hours of stimulation with either IL-12+IL-18 or an equal number of K562 cells. The frequency of CD107a+ NK cells after incubation with K562 cells was increased for cells cultured with IL-15+GSK3i+STING agonist for 7 days compared to cells cultured with IL-15+GSK3i only (Figure 16C). Increased cell surface CD107a expression is associated with enhanced degranulation and cytotoxic potential of NK cells in the presence of STING agonist compared to GSK3i.

[0262] In vitro killing assays were used to evaluate the ability of STING agonists to enhance the cytotoxicity of NK cells modulated for multiple days with GSK3i. CD3 / C19-depleted PBMC populations were treated with 10 ng / mL IL-15 alone overnight (priming), cultured for 7 days with 10 ng / mL IL-15 alone, cultured for 7 days with a combination of 10 ng / mL IL-15 and 5 μM CHIR99021, or cultured for 7 days with a combination of 10 ng / mL IL-15, 5 μM CHIR99021, and 1 μM STING agonist. Target cells for the killing assays included K562, Nalm6, and KG-1 cells. Effector:target ratios applied in each assay ranged from 0.1, 0.3, 1, and 3–10. After incubation of target cells with the differently treated NK cells for 4 hours, the respective percentages of specific killing (target cell lysis) were calculated and shown in Figure 17. In all assays, addition of STING agonists enhanced GSK3i-modulated NK cell cytotoxicity, albeit to different extents.

[0263] Antibody-dependent cellular cytotoxicity (ADCC) was also shown to be enhanced in NK cells cultured with STING antagonist and GSK3i for 7 days when Herceptin was used to mediate killing of SKOV3 or A549 compared to ADCC killing using NK from 7-day culture with GSK3i. NK cells were added to targets at a ratio of 3:1. Figure 18 showed that the combination of GSK3i and STING agonist in multi-day culture enhanced NK cell cytotoxicity against A549 (Figure 18A) and SKOV-3 (Figure 18B) target cells in ADCC assays using anti-Her2 antibodies. Figure 18B also shows that the combination of STING agonist and GSK3i was more effective in enhancing NK cell cytotoxicity than either modulator used alone.

[0264] Natural killer (NK) cells recognize and destroy tumor and virus-infected cells in an antibody-dependent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs), including NKp30, NKp44, and NKp46. NCRs initiate tumor targeting by recognition of their specific ligands on cancer cells. As shown in Figure 19, the combination of GSK3i and STING agonist synergistically enhances NKp30 expression on modulated NK cells compared to NK cells modulated by STING agonist or GSK3i alone, which may be involved in enhanced NK cell function, among other mechanisms.

[0265] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0266] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0267] The present disclosure, as properly described herein as an explanation, may be implemented in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, in each instance herein, the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions that have been utilized are used as terms of description and not as terms of limitation, and it is recognized that there is no limitation in the use of such terms and expressions to exclude any equivalents of the shown and described characteristics or portions thereof, but various modifications are possible within the scope of the present disclosure as claimed. Thus, although the present disclosure has been specifically disclosed by preferred embodiments, it should be understood that the characteristics, modifications and variations of the concepts disclosed herein may be reclassified by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

Claims

1. contacting the population of immune cells with at least one agent comprising a GSK3 inhibitor for a time sufficient to obtain a population of modulated immune cells having improved therapeutic potential compared to non-modulated immune cells; formulating the population comprising the regulated immune cells into a pharmaceutical composition; The population of immune cells before modulation includes NK cells, A method for producing a pharmaceutical composition comprising a regulated population of immune cells, wherein the regulated population of immune cells comprises NK cells, and said improved therapeutic potential comprises an increased proportion of CD56+CD57+ adoptive NK cells. (a) the immune cells have at least one of the following characteristics prior to contact with the at least one agent: (i) (1) CD57-NK cells; or (2) CD57-NKG2C+ NK cells; Including, (ii) isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor; (iii) (1) Healthy subjects; (2) A subject with an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor; (3) a subject who has already been administered genetically modified immune cells; or (4) CMV seropositive subjects or (iv) is genomically engineered, including insertions, deletions, or nucleic acid substitutions; (v) differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells; or (vi) transdifferentiation in vitro from hematopoietic or non-hematopoietic non-pluripotent cells; (b) said composition comprising said at least one pharmaceutical agent further comprising one or more additives; and / or (c) (i) activating said immune cells prior to the step of contacting with a sufficient amount of the modulating composition; and, optionally, prior to the activating step; (ii) depleting said immune cells from CD3 and CD19 cells for activation and regulation; The method of claim 1 , comprising:

3. 3. The method of claim 2 having at least one of the following features: (a) the stem cells comprise induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); (b) the progenitor cells are CD34+ hemogenic endothelial cells, multipotent progenitor cells, or NK progenitor cells; (c) the stem cells, hematopoietic stem or progenitor cells, or progenitor cells are genomically engineered to contain insertions, deletions, or nucleic acid substitutions; (d) the immune cells are (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region; and (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling to bi- or multispecific or universal engagers; (e) the one or more additives comprise: (i) at least one of a peptide, an antibody, an antibody fragment, a cytokine, a mitogen, a growth factor, a small RNA, a dsRNA, a mononuclear blood cell, a feeder cell, a feeder cell component or replacement factor, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, and an immunomodulatory drug (IMiD); (ii) at least one of a stimulatory cytokine, including IL2, IL15, IL12, IL18, and IL21, or (iii) one or more of a MEK inhibitor, a rapamycin, and a STING agonist; or (f) the composition comprises: (i) one or more of a TGFβ receptor inhibitor, a ROCK inhibitor, a MEK inhibitor, a PDK1 agonist, and rapamycin; (ii) IL15; (iii) an IL15 and STING agonist; or (iv) further comprising at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof; (g) the regulated immune cell adoptive NK cell is: (A) and adoptive NK cells comprising at least one of NKG2C+, low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA; (B) hnCD16-expressing NK cells; or (h) the method further comprises isolating adoptive NK cells from the modulated immune cells; (i) the sufficient time is 16 hours or more; (j) the immune cells are in a feeder-free environment; (k) the regulated NK cells (i) increasing the expansion by at least two-fold; and / or (ii) The method, wherein the improved cytokine response comprises increased production of one or more cytokines, including IFNγ and / or TNFα.

4. 1. A method for producing a pharmaceutical composition comprising a population of NK cells, comprising: (a) A population of immune cells (i) Healthy subjects; (ii) a subject with an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor; (iii) a subject who has already received genetically modified immune cells; or (iv) a subject who is CMV seropositive; (b) depleting CD3 and CD19 cells from said population of immune cells; and (c) contacting the population of immune cells resulting from step (b) with a sufficient amount of a composition comprising a GSK3 inhibitor for a sufficient time to obtain a population of modulated NK cells having improved therapeutic potential for adoptive cell therapy compared to unmodulated NK cells, Prior to depletion of CD3 and CD19 cells, the population of immune cells is not cultured with cytokines or GSK3 inhibitors; and The modulated NK cells increase NK cell expansion by at least 2-fold, and / or the improved cytokine response includes increased production of one or more cytokines, including IFNγ and / or TNFα, The improved therapeutic potential comprises an increased proportion of CD56+CD57+ adoptive NK cells.

5. 1. A pharmaceutical composition comprising a population of immune cells and one or more agents, including a GSK3 inhibitor, (i) the population of immune cells comprises regulated immune cells, including regulated NK cells; (ii) the modulated immune cells are modulated by one or more agents, including a GSK3 inhibitor, and have improved therapeutic potential compared to immune cells that are not modulated by the one or more agents, including a GSK3 inhibitor; (iii) the improved therapeutic potential comprises an increased proportion of CD56+CD57+ adoptive NK cells; and (iv) The pharmaceutical composition, wherein the pharmaceutical composition comprises adoptive NK cells.

6. 6. The composition of claim 5, (a) the immune cell has at least one of the following characteristics: (i) (1) CD57-NK cells; or (2) Prepared from an immune cell population containing CD57-NKG2C+ NK cells; (ii) isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumor; (iii) (1) Healthy subjects; (2) A subject with an autoimmune disease, hematopoietic malignancy, viral infection, or solid tumor; (3) a subject who has already been administered genetically modified immune cells; or (4) CMV seropositive subjects isolated from (iv) is genomically engineered, including insertions, deletions, or nucleic acid substitutions; (v) differentiated in vitro from stem cells, hematopoietic stem cells, or hematopoietic progenitor or precursor cells; or (vi) transdifferentiating in vitro from hematopoietic or non-hematopoietic non-pluripotent cells; or (b) the composition further comprises one or more additives; composition.

7. (a) the regulated immune cell adoptive NK cell is: (i) adoptive NK cells comprising CD57+ and at least one of NKG2C+, low PLZF, low SYK, low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA; (ii) hnCD16-expressing CD57+ NK cells; (b) the stem cells comprise induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); (c) the progenitor cells are CD34+ hemogenic endothelial cells, multipotent progenitor cells, or NK progenitor cells; (d) the stem cells, hematopoietic stem or progenitor cells, or progenitor cells are genomically engineered to contain insertions, deletions, or nucleic acid substitutions; (e) the immune cells are (i) deleted or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes in the chromosome 6p21 region; and (ii) induced or increased expression of one or more of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface triggering receptors for coupling to bi- or multispecific or universal engagers; (f) the one or more additives are: (i) at least one of a peptide, an antibody, an antibody fragment, a cytokine, a mitogen, a growth factor, a small RNA, a dsRNA, a mononuclear blood cell, a feeder cell, a feeder cell component or replacement factor, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, and an immunomodulatory drug (IMiD); (ii) at least one of the stimulatory cytokines, including IL2, IL15, IL12, IL18, and IL21; (iii) one or more of a MEK inhibitor, rapamycin, and a STING agonist; or (h) the composition comprises: (i) one or more of a TGFβ receptor inhibitor, a ROCK inhibitor, a MEK inhibitor, a PDK1 agonist, and rapamycin; (ii) IL15; (iii) IL15 and STING agonists; or (iv) at least one organic solvent selected from the group consisting of dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof; The composition of claim 6.

8. The method of any one of claims 1 to 4, wherein the NK cells are genetically modified.

9. A pharmaceutical composition comprising a regulated population of NK cells produced according to any one of claims 1 to 4.

10. The pharmaceutical composition of claim 9 , wherein the regulated NK cell population comprises genetically modified NK cells.

11. 11. The pharmaceutical composition of claim 9 or 10, further comprising one or more additional additives optionally selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNA, dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, and immunomodulatory drugs (IMiDs).

12. 12. The pharmaceutical composition of claim 11, wherein the adoptive cell therapy is autologous or allogeneic; and the subject of the adoptive cell therapy has an autoimmune disorder, a hematological tumor, a solid tumor, cancer, or an infectious disease associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

13. 13. A pharmaceutical composition according to claim 12 for use in medicine, said use comprising administering a therapeutically sufficient amount of the composition in combination with an antibody, a chemotherapeutic agent, or a radioactive treatment, said antibody, chemotherapeutic agent, or radioactive treatment prior to, during, or after administration of the composition.

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