Compositions and methods for modulating immune cells in adoptive immunotherapy

Abstract

To provide compositions and methods for immune cell modulation.SOLUTION: In one aspect, a composition comprises a population or subpopulation of immune cells, and one or more modulating agents selected from the group consisting of specific compounds. The one or more modulating agents (a) improve therapeutic potential of immune cells for adoptive cell therapy; (b) improve cell expansion, maintenance, and / or differentiation properties of immune cells; (c) improve cell proliferation, cytotoxicity, persistence, cytokine response and secretion, and / or cell recall responses of immune cells; and / or (d) increase the number or ratio of a desired immune cell subpopulation.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Related applications This application claims the priority of U.S. Provisional Patent Application Serial No. 62 / 430,263, filed on December 5, 2016, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to the field of adoptive immunotherapy. More specifically, the present disclosure relates to the use of small molecules for modulating immune cells suitable for adoptive cell therapy.

Background Art

[0003] Adoptive immunotherapy generally involves administering immune cells to a patient having cancer, a tumor, or an infection, whereby the administered immune cells provide a therapeutic benefit 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, natural killer T (NKT) cells, and hematopoietic stem or progenitor cells. Mediating a complete and permanent disease response in a patient is a central goal of these cell-based immunotherapies.

[0004] Considering 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), highlights the importance of specific attributes related to introduced T cells and certain inhibitory barriers imposed by host and tumor cells, which may need to be overcome for optimal success in cancer treatment in several contexts. Among T-cell factors, the binding affinity, proliferative and viability, migration to tumor sites, and persistence of effector function within the tumor of T cell receptors (TCRs) or chimeric antigen receptors (CARs) may be important in some situations for causing the eradication of malignant cells, for example, based on information from certain correlation studies. Adding other layers of complexity, while certain desirable attributes may be recognized, the pathways or factors influencing these attributes may not be fully elucidated, which may, in some cases, limit the ability to obtain cells with the desired quantity and quality for specific therapeutic use.

[0005] In this field, there is a need for immune cell subsets with improved therapeutic effects. A manufacturing method capable of enhancing desired T, NK, or NKT cell subsets in both quantity and quality may provide a significant enhancement of their therapeutic efficacy. Methods and compositions are provided to address such needs and offer other relevant advantages in the field of immunotherapy. [Overview of the project]

[0006] For example, when using CAR-T cell therapy, the therapeutic composition may face one or more problems related to CAR-T efficacy and persistence, migration to tumors, immunosuppressive tumor microenvironment, tumor heterogeneity, and patient safety. The selection of specific T cell subsets for therapeutic use and further manipulation of CARs may be used to improve tumor targeting, CAR efficacy, and in- and out-of-tumor safety issues.

[0007] Additional methods are also needed, for example, those that can enhance the therapeutic effect of effector cells and / or improve the persistence and / or migration of manipulated effector cells after administration. For example, there is a need for compositions and methods useful for maintaining and expanding a desired immune cell subset, as well as those useful for reducing cell differentiation during expansion, and for cells containing or enriched with one or more desired immune cell subsets that have a greater ability to increase, sustain, and / or improve one or more therapeutic outcomes, for example, in various adoptive immunotherapy situations, or for manipulated cells or therapeutic cells that have enriched cells or are useful for producing enriched therapeutic compositions. Some of the embodiments provided address such needs.

[0008] Further methods and compositions are needed in the context of NK cell-based therapies. Natural killer cells are generally classified as innate immune cells characterized by their relatively short lifespan and minimal changes in response to secondary exposure to stimuli, i.e., limited targeted memory responses. However, both activating and suppressive NK cell receptors can play important roles in self-tolerance and sustained NK cell activity, among others. In some situations, NK cells can quickly adapt to their surrounding environment and build antigen-specific immunological memory, which is fundamental for responding to secondary exposure to similar antigens. For example, a subpopulation of NK cells, which may be called adaptive NK cells or memory NK cells, has a longer lifespan and less initial exposure error (Bookmark not defined. 2013). Enriching or providing compositions enriched with cells having such properties may result in more effective cell therapy strategies compared to standard NK cells. Expanding and maintaining adaptive / memory NK cells that mediate in vivo durable antigen-specific recognition would be key to improving NK cell-based adoptive immunotherapy. Some of the embodiments provided address such needs.

[0009] Furthermore, it is conceivable that improvements could be made to isolate more effective NKT cells, a type of CD1d-restricted T cell that could serve as a regulatory target to improve cell therapy, playing a role in both the innate and adaptive immune systems, similar to T cells and NK cells.

[0010] In some embodiments, 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. An object of embodiments of the present invention is to provide one or more compounds, alone or in combination, for improving the proliferation, persistence, cytotoxicity, and / or cell recall / memory of therapeutic immune cells by increasing the number or proportion of cell subpopulations that exhibit improvement in at least one of the following properties, namely migration, homing, cytotoxicity, maintenance, expansion, persistence, lifespan, and desired differentiation state, which are expected to result in better immunotherapeutic outcomes.

[0011] 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 the compounds listed in Table 1, namely dolsomorphine; hepteridine acid; 1-pyrrolidinecarboditioic acid, ammonium salt; 2-deoxyglucose (2-DG); GSK3β inhibitor; Rho kinase inhibitor; MEK inhibitor; PDK1 agonist; TGFβ inhibitor; 6-mercaptopurine; AC-93253 iodide; thiratrichol; PI-103; fulvestrant; thapsigargin; SU4312; telmisartan; cyclosporine A; 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole; BAY61-3606; protopolph The formula contains one or more modifiers selected from the group consisting of: illin IX disodium; rapamycin; HS173; LY294002; pictilisib; DCC-2036 (levastinib); 5-azacitidine; 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-octaf; nifloxazide; tosufloxacin hydrochloride; sertraline; diethylenetriaminepentaacetic acid, pentasodium; edrophonium chloride; BIX01294; terfenadine; and dmPGE2. One or more modifiers selected from the group consisting of compounds listed in Table 1 improve the therapeutic potential of immune cells or one or more subpopulations of immune cells upon contact with them. In some embodiments, the modulation of immune cells is ex vivo. In some embodiments, one or more modifiers include at least one of the compounds in Table 1, or their salts, esters, ethers, solvates, hydrates, stereoisomers, and prodrugs. In some embodiments, one or more modifiers include at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955.

[0012] In some embodiments, one or more modifiers listed in Table 1 modulate cell proliferation, maintenance, and / or differentiation, thereby improving the proliferation, cytotoxicity, cytokine response and secretion, cell recall response, and / or persistence of immune cells or one or more subpopulations. In one embodiment, one or more modifiers listed in Table 1 improve the cell viability of immune cells or one or more subpopulations both ex vivo and in vivo. In one embodiment, one or more modifiers listed in Table 1 increase the number or proportion of one or more desired cell subpopulations of immune cells. In some embodiments, one or more modifiers include at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the regulator includes DCC-2036.

[0013] In some embodiments, the present invention provides one or more selected agents of this specification for improving the therapeutic effect of a population or subpopulation of immune cells, including but not limited to T cells, NK cells, and NKT cells. In some embodiments, immune cells suitable for adoptive cell-based therapy include T cells, NKT cells, or NK cells. In some embodiments, immune cells treated with one or more modulants include T cells, and therefore one or more desired cell subpopulations include naive T cells, stem cell memory T cells, and / or central memory T cells, having an increased number or relative ratio. In some embodiments, immune cells subjected to drug-based therapy include NKT cells, and therefore one or more desired cell subpopulations include type I NKT cells, having an increased number or relative ratio. In some other embodiments, immune cells subjected to drug-based therapy include NK cells, and one or more desired cell subpopulations include adaptive NK cells, having an increased number or relative ratio.

[0014] In some embodiments, a composition comprising one or more modifiers selected from the group consisting of compounds listed in Table 1 comprises derivatives, analogues, or pharmaceutically acceptable salts thereof selected from the group consisting of salts, esters, ethers, solvates, hydrates, stereoisomers, and prodrugs of the drugs in Table 1. In some embodiments, one or more modifiers include at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier includes DCC-2036.

[0015] In some embodiments, a composition for improving the therapeutic potential of immune cells comprises at least one agent selected from group I, and one or more modifiers selected from group II, group III, group IV, and / or group V.

[0016] Group I includes dorsomorphine, hepteridine acid, 1-pyrrolidinecarboditioic acid, and 2-DG. Without limiting themselves to theory, Group I drugs affect cellular metabolism and nutrient sensing, among other potential roles.

[0017] Group II includes GSK3β inhibitors, ROCK inhibitors, TGFβ receptor inhibitors, MEK inhibitors, PDK1 agonists, 6-mercaptopurine AC-93253 iodide, tilatrichol, PI-103, fulvestrant, tapsigargin, SU 4312, U0126, telmisartan, cyclosporine A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY 61-3606, protoporphyrin IX disodium, rapamycin, TWS119, HS173, LY294002, pictilisib, and DCC-2036 (levastinib). Not limited to theory, among other potential roles, Group II drugs influence signaling in various functional pathways.

[0018] Group III includes 5-azacitidine, fludarabine, roscovitine, and PAC-1. Among other potential roles, not limited to theory, Group III drugs affect cell proliferation and apoptosis.

[0019] Group IV includes 5,7-dichloro-8-quinolinol, 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octaf, nifloxazide, and tosufloxacin hydrochloride. Among other potential roles, not limited to theory, Group IV drugs may influence cellular properties related to the infection process.

[0020] Group V includes sertraline, diethylenetriaminepentaacetic acid, edrophonium chloride, BIX01294, terfenadine, and dmPGE2. While not limited to theory, among other potential roles, drugs in Group V generally affect expansion, maintenance, cell differentiation, and other cellular properties related to proliferation, cytotoxicity, cell recall responses, and / or persistence.

[0021] In some other embodiments, a composition for improving the therapeutic potential of immune cells comprises at least one agent selected from group II and one or more modifiers selected from group I, group III, group IV, and / or group V.

[0022] In yet another embodiment, a composition for improving the therapeutic potential of immune cells comprises at least one agent selected from group III and one or more modifiers selected from group I, group II, group IV, and / or group V.

[0023] In yet another embodiment, a composition for improving the therapeutic potential of immune cells comprises at least one agent selected from group IV, and one or more modifiers selected from group I, group II, group III, and / or group V.

[0024] In yet another embodiment, a composition for improving the therapeutic potential of immune cells comprises at least one agent selected from group V and one or more modifiers selected from group I, group II, group III, and / or group IV.

[0025] In some embodiments, one or more modifiers include at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier includes DCC-2036. In some embodiments, compositions for improving the therapeutic potential of immune cells include combinations 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 group consisting of compounds listed in Table 1, including at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some specific embodiments, compositions include synergistic combinations of two or more agents selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG.

[0026] In some embodiments, a composition comprising one or more modifiers selected from the group consisting of compounds listed in Table 1 further comprises at least one organic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof.

[0027] Another aspect of the present invention provides a composition comprising a population or subpopulation of immune cells and one or more modifiers selected from the group consisting of compounds listed in Table 1. In some embodiments, the one or more modifiers include at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier includes DCC-2036. In some embodiments, a population or subpopulation of immune cells is contacted with one or more of the modifiers to improve the therapeutic potential of the immune cells for adoptive cell therapy compared to such uncontacted immune cells. In some embodiments, immune cells are contacted with one or more modifiers to improve cell proliferation, maintenance, differentiation, and / or viability compared to immune cells that have not received the same treatment. In some other embodiments, immune cells are brought into contact with one or more of the modifiers to improve cell proliferation, cytotoxicity, persistence, and / or recall compared to immune cells that have not received the same treatment.

[0028] In some embodiments, immune cells exposed to one or more of the modifiers exhibit an increased number or relative ratio of a desired subpopulation of immune cells compared to immune cells that have not received the same treatment. In some embodiments, the immune cells include T, NK, or NKT cells. In one embodiment, the composition includes a population of T cells, and therefore the desired subpopulation of immune cells after contact with the drug 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 therefore the desired subpopulation of immune cells after contact with the drug includes type I NKT cells. In several other embodiments, the immune cells include a population of NK cells, and therefore the desired subpopulation of immune cells after contact with the drug includes adaptive NK cells. In other embodiments, the adaptive NK cells include 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.

[0029] In some embodiments, the population or subpopulation of immune cells in the composition is isolated from or contained in the subject's peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue derived from the site of infection, ascites, pleural fluid, splenic tissue, or tumor. The subject may be healthy, have an autoimmune disorder, hematopoietic malignancy, viral infection, or solid tumor, or may have been previously administered genetically modified immune cells. In some embodiments, the subject may be CMV serologically positive. In some other embodiments, the immune cells isolated for regulation are genetically modified (either genetically modified or naturally induced from rearrangement, mutation, gene imprinting, and / or epigenetic modification). In some embodiments, the immune cells isolated for regulation include at least one genetically modified form. In some embodiments, the isolated immune cell population is genomically engineered and includes insertions, deletions, and / or nucleic acid substitutions. In some embodiments, the immune cells include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modification using the disclosed compositions and methods. In some embodiments, after modification, the genetically modified immune cells isolated from the subject can be administered to the same donor or to different patients.

[0030] In yet another embodiment, immune cells differentiate in vitro from stem cells, hematopoietic stem cells or progenitor cells, or from progenitor cells; or are transdifferentiated in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the immune cells of the composition are genomically engineered and include insertions, deletions, or nucleic acid substitutions (substitutions or indels). In some embodiments, the immune cells of the composition include exogenous nucleic acids encoding T cell receptors (TCRs) and / or chimeric antigen receptors (CARs). In some embodiments, immune cells isolated from the subject's tissue are genetically engineered and may include TCRs or CARs. In some embodiments, immune cells isolated from tissue or subject are CAR-T cells.

[0031] In several other embodiments, the immune cells of the composition differentiate in vitro from stem cells, hematopoietic stem cells 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+ hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK progenitor cells, or NKT progenitor cells. In some embodiments, the stem cells, hematopoietic stem cells or progenitor cells, or progenitor cells are genomically engineered and include insertions, deletions, or nucleic acid substitutions, or include at least one mode of genetic modification. In a particular embodiment, the stem cells, hematopoietic stem cells or progenitor cells, or progenitor cells include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 overexpression. In several other embodiments, the immune cells of the composition transdifferentiate in vitro from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the desired subpopulation of regulated immune cells comprises immune cells having at least one genetically modified form. In some embodiments, the recombination form comprises at least one of the following: safety switch proteins, targeting forms, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or survival of immune cells. In some other embodiments, the genetically modified form includes (i) deletion or reduction of expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any HLA gene in the chromosome 6p21 region, and (ii) introduction or increase of expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, or surface-induced receptors for binding to bi- or multi-specific or universal engagers.

[0032] In some embodiments, a composition comprising immune cells and one or more of the modifiers further comprises a vector containing peptides, cytokines, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or substitution factors, one or more polynucleic acids of interest; antibodies or antibody fragments; and one or more additional agents or other additives / agents selected from the group consisting of chemotherapeutic agents, radioactive moieties, or immunomodulatory agents (IMiDs). 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 one or more of chemotherapeutic agents, radioactive moieties, and immunomodulatory agents (IMiDs). A chemotherapeutic agent refers to a cytotoxic antitumor agent, i.e., a chemical used therapeutically to prevent or suppress the proliferation of neoplastic cells, that is, a chemical found to preferentially kill neoplastic cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells. Chemotherapy agents are also called antitumor agents or cytotoxic agents or drugs, and are well known in the art.

[0033] In a particular embodiment, the composition comprises a population or subpopulation of immune cells and a mixture of (i) at least one of the compounds in Table 1 or their salts, esters, ethers, solvates, hydrates, stereoisomers, and prodrugs, or (ii) one or more modifiers comprising at least one of GSK3β inhibitors, TGFβ receptor inhibitors, ROCK inhibitors, MEK inhibitors, PDK1 agonists, and rapamycin, wherein the immune cells include NK cells. In some embodiments, the one or more modifiers comprise at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier comprises DCC-2036.

[0034] In another embodiment, a composition comprising a mixture of a population or subpopulation of immune cells and one or more of the modifiers further comprises one or more additives selected from the group consisting of peptides, antibodies, antibody fragments, cytokines, mitogens, growth factors, small RNAs, dsRNAs, mononuclear blood cells, feeder cells, feeder cell components or substitution factors, vectors comprising one or more polynucleic acids of interest, chemotherapeutic agents or radioactive moieties, and immunomodulatory agents (IMiDs). In yet another embodiment, a composition comprising a mixture of a population or subpopulation of immune cells and one or more of the modifiers also comprises at least one organic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof. In one embodiment, the population or subpopulation of immune cells in the composition comprises T cells. In some embodiments, the T cells in the cell population comprises CAR-T cells.

[0035] In some aspects, compositions are provided that include a population of regulated immune cells in contact with a composition containing one or more modifiers selected from the group consisting of compounds listed in Table 1. In some embodiments, the regulated immune cells are in contact with one or more modifiers including DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the regulated immune cells are in contact with DCC-2036. In some embodiments, the provided composition is a therapeutic composition having a population or subpopulation of regulated immune cells, including but not limited to T, NK, and NKT cells. In some embodiments, the therapeutic composition can be washed with a buffer substantially free of modifiers.

[0036] In some embodiments, the regulated cell population contains immune cells that have improved therapeutic potential for adoptive cell therapy compared to the unregulated cell population. In some embodiments, the isolated immune cell population exhibits improved cell proliferation, maintenance, differentiation, dedifferentiation, and / or viability compared to immune cells that have not been treated with one or more regulators. In some embodiments, the isolated immune cell population exhibits improved cell proliferation, cytotoxicity, cytokine response and secretion, cell recall response, and persistence compared to immune cells that have not been treated with one or more regulators, and otherwise incubated or treated under similar conditions but without one or more regulators, etc. In some other embodiments, the isolated immune cell population exhibits an increased number or relative ratio of one or more desired subpopulations of immune cells compared to immune cells that have not received the same treatment.

[0037] In some embodiments, an isolated population of immune cells treated with one or more of the modifiers selected from the group consisting of compounds listed in Table 1 includes T cells, and therefore one or more desired subpopulations of immune cells obtained include naive T cells, stem cell memory T cells, and / or central memory T cells. In some embodiments, an isolated population of immune cells treated with one or more modifiers includes NKT cells, and therefore one or more desired subpopulations of immune cells obtained include type I NKT cells. In some other embodiments, an isolated population of immune cells treated with one or more modifiers includes NK cells, and therefore one or more desired subpopulations of immune cells include adaptive NK cells. In some embodiments, regulated immune cells are in contact with one or more regulators, including DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, regulated immune cells are in contact with DCC-2036.

[0038] In some embodiments of the provided composition, the isolated immune cell population may be isolated from the subject's peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, or tumor. The subject may be healthy and may have autoimmune disorders, hematopoietic malignancies, viral infections, or solid tumors, or may have been previously administered genetically modified immune cells. In some embodiments, the subject may be CMV serologically positive. In some other embodiments, the immune cells isolated for regulation are genetically modified (either genetically modified or spontaneously induced from rearrangements, mutations, gene imprinting, and / or epigenetic modifications). In some embodiments, the immune cells isolated for regulation include at least one genetically modified form. In some embodiments, the isolated immune cell population is genomically engineered and includes 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 CD16 or variants thereof. Thus, genetically modified immune cells are isolated for ex vivo modification using the compositions and methods disclosed herein. In some embodiments, after modification, the genetically modified immune cells isolated from the subject can be administered to the same donor or to different patients. In some embodiments, the population of immune cells may be isolated before drug treatment. In some embodiments, the isolated immune cell population is genomically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the immune cells include exogenous nucleic acids encoding T cell receptors (TCRs) and / or chimeric antigen receptors (CARs).

[0039] In some embodiments of the provided composition, an isolated immune cell population can be differentiated from stem cells, hematopoietic stem cells or progenitor cells, or from progenitor cells. In some embodiments, an isolated immune cell population can be differentiated from stem cells, hematopoietic stem cells or progenitor cells, or from progenitor cells, before or during drug therapy. 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, pluripotent progenitor cells, T cell progenitor cells, NK progenitor cells, or NKT progenitor cells. In some further embodiments, the stem cells, hematopoietic stem cells or progenitor cells, precursors, regulated induced immune cells, or regulated induced immune cells are genomically engineered to include, for example, insertions, deletions, and / or nucleic acid substitutions. In a particular embodiment, the stem cells, hematopoietic stem cells or progenitor cells, or precursors include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 overexpression.

[0040] In some other embodiments of the provided composition, the isolated immune cell population may be converted from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the isolated immune cell population may be converted from hematopoietic or non-hematopoietic non-pluripotent cells before or during drug treatment.

[0041] One aspect of the present invention provides a method for modulating a population of immune cells for adoptive cell therapy, which generally involves contacting a population of immune cells with a sufficient amount of a composition comprising one or more modifiers listed in Table 1 to obtain a population of modulated immune cells having improved therapeutic potential for adoptive cell therapy compared to unmodulated immune cells that have not been contacted with the agents of Table 1. In some embodiments, the modulated immune cells for adoptive therapy are autologous. In some embodiments, the modulated immune cells for adoptive therapy are allogeneic. In some embodiments, the modifier comprises at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier comprises DCC-2036.

[0042] In some embodiments of this method, contacting a population of immune cells with one or more modifiers improves proliferation, cytotoxicity, cytokine response, cytokine release, cell recall response, and / or persistence compared to immune cells that have not been treated with one or more of the modifiers in Table 1; and / or improves cell proliferation, maintenance, differentiation, dedifferentiation, and / or viability. In some embodiments of this method, contacting a population of immune cells with one or more of the modifiers in Table 1 increases the number or proportion of one or more desired subpopulations of immune cells compared to immune cells that have not been treated with the same one or more modifiers. In some embodiments, the above method further includes isolating one or more desired subpopulations of immune cells that have been contacted with one or more of the modifiers in Table 1. In some embodiments, the modifier includes at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier includes DCC-2036.

[0043] In some embodiments, the method further comprises administering a population or subpopulation of treated immune cells, or one or more isolated subpopulations of treated immune cells, or a therapeutic composition thereof, to a subject in need of cell therapy. In some embodiments, the subject suffers from an autoimmune disorder, hematological malignancy, solid tumor, or infection. In some embodiments, the subject has received, is receiving, or is being treated with chemotherapy or radiotherapy.

[0044] In some embodiments, the immune cell population includes T cells, NKT cells, or NK cells. In one embodiment of this method, the immune cell population includes T cells, and one or more desired subpopulations after treatment include naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment of this method, the immune cell population includes NKT cells, and one or more desired subpopulations after treatment include type I NKT cells. In one embodiment of this method, the immune cell population includes NK cells, and one or more desired subpopulations after treatment include adaptive NK cells.

[0045] In some embodiments of the general method described above, immune cells for regulation are isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue derived from the site of infection, ascites, pleural fluid, splenic tissue, or tumors. In some embodiments, immune cells for regulation are isolated from healthy subjects; subjects with autoimmune disorders, hematopoietic malignancies, viral infections, or solid tumors; subjects previously administered genetically modified immune cells; or subjects positive for CMV serotestancy. In some other embodiments, the immune cells isolated for regulation are genetically modified (either genetically modified or spontaneously induced from rearrangement, mutation, gene imprinting, and / or epigenetic modification). In some embodiments, the immune cells isolated for regulation include overexpression of at least one genetically modified form, a chimeric antigen receptor (CAR), and / or CD16 or a variant thereof. Thus, in some embodiments, genetically modified immune cells are isolated for ex vivo regulation using the compositions and methods of the Disclosure. In some embodiments, modified immune cells isolated from the subject after adjustment may be administered to the same donor or to different patients.

[0046] In some embodiments, the immune cells for regulation are differentiated in vitro from stem cells, hematopoietic stem cells or progenitor cells, or from progenitor cells. In some embodiments, the immune cells for regulation 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 are CD34+ hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK progenitor cells, or NKT progenitor cells. In some further embodiments, the stem cells, hematopoietic stem cells or progenitor cells, or progenitor cells are genomically engineered and include insertions, deletions, or nucleic acid substitutions and / or include at least one mode of genetic modification. A desired subpopulation of regulated immune cells, either itself or derived therefrom, includes immune cells having at least one mode of genetic modification.

[0047] In some embodiments, the genetically modified mode includes at least one of the following: safety switch proteins, targeting modes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, immune response regulation and regulation, and / or survival of immune cells. In some other embodiments, the genetically modified mode includes deletion or reduction of expression of one or more B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any HLA genes in the chromosome 6p21 region. In some other embodiments, the genetically modified form includes the introduction or increase of the expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptors, or surface-evoked receptors for binding to bi- or multi-specific or universal engagers.

[0048] In some embodiments of the method for regulating immune cells, the “sufficient time” or “length of sufficient time” is 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 0.5 hours, 0.1 hours or more, or any length in between. Thus, the length of sufficient time is, for example, at least 15, 13, 11, 9, 7, 5, 3, 1, 0.5, or 0.1 hours. In some other embodiments of the method, the length of sufficient time is 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours or more, or any length in between. Thus, the length of sufficient time is, for example, 30, 42, 54, 66, 78, or 90 hours or more.

[0049] In some embodiments of the method described above, immune cells are in a feeder-free environment during and / or after regulation. Feeder-free conditions include being free of feeder cells and feeder-conditioned medium. In some embodiments of the method described above, immune cells are co-cultured with feeder cells during regulation.

[0050] In some embodiments, the subject may be a candidate for adoptive cell transplantation. In some embodiments, the subject may be a candidate for bone marrow or stem cell transplantation. In some embodiments, the subject has previously undergone bone marrow or stem cell transplantation. In some embodiments, the subject has undergone bone marrow resection or non-myeloablative chemotherapy or radiotherapy.

[0051] Aspects of the present invention provide a method for producing a therapeutic composition for cell therapy by any of the above methods for regulating a population of immune cells.

[0052] Aspects of the present invention provide the use of the above-described immunotherapy method for producing therapeutic compositions comprising modified immune cells for cell therapy. In some embodiments, the modified immune cells include T cells, NK cells, and / or NKT cells. In some embodiments, the modified NK cells include adaptive NK cells. Further embodiments of the present invention provide a population of modified immune cells comprising selectively proliferated NK cells produced by the method provided herein.

[0053] In some embodiments, the present invention provides therapeutic compositions comprising regulatory cells obtained using the methods and compositions disclosed herein and a therapeutically acceptable medium. In some embodiments of the therapeutic compositions, 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 substitution factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, and immunomodulatory agents (IMiDs).

[0054] In some embodiments, a method is provided for treating a subject in need of adoptive cell therapy by administering a therapeutically sufficient amount of the therapeutic composition to the subject. 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 treatment has an autoimmune disorder, hematological malignancy, solid tumor, cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. In some embodiments, the method of treating a subject using modified immune cells is carried out by administering the therapeutic composition in combination with an antibody, chemotherapeutic agent, or radiotherapy, where the antibody, chemotherapeutic agent, or radiotherapy is administered before, concurrently with, or after the administration of the therapeutic composition.

[0055] The various purposes and advantages of this use will become apparent from the following description, in conjunction with the accompanying drawings, which are described as specific embodiments of the present invention as examples and illustrations. [Brief explanation of the drawing]

[0056] [Figure 1A] Figure 1 is a graphical representation of the Z-scores of the percentage of CCR7 and CD62L co-expressing cells and the Z-scores of the absolute number of CCR7 and CD62 double-positive T cells in (A) the viable CD8+ cell population and (B) the viable CD4+ cell population. [Figure 1B] Same as above.

[0057] [Figure 2] Figure 2 shows the various CAR structures used in the study.

[0058] [Figure 3-1]Figure 3 shows that DCC-2036 can (A) maintain CAR-T cell viability and (B) enhance CAR-T cell expansion. A serial restimulation assay was performed to show that DCC-2036 improves CAR-T cell expansion compared to the vehicle. (C) Relative proliferation per round in a 4-round serial killing assay using irradiated target cells; (D) Total proliferation of CAR-T cell numbers throughout the 4 rounds of the serial killing assay. [Figure 3-2] Same as above.

[0059] [Figure 4] Figure 4 shows the phenotypic characteristics of CD8+ T cells after compound treatment. A: T cell subset with CCR7+CD62L+ surface expression. B: CD27 expression level reflecting the Tcm phenotype.

[0060] [Figure 5] Figure 5 shows gene expression related to T cell differentiation, and several Tcm-related genes, including CD62L and CCR7, showed increased expression after treatment with DCC-2036 (FT-61314).

[0061] [Figure 6] Figure 6 shows the expression of the depletion markers TIM-3 (A) and PD-1 (B) in CD8 CAR-T cells treated with DMSO, TWS119, and DCC-2036 during CAR-T cell generation.

[0062] [Figure 7] Figure 7 shows the improved in vivo efficacy of CAR-T cells treated with DCC-2036. CAR-T cells treated with DCC-2036 were able to eliminate tumors from the majority of mice, while untransduced T cells, or CAR-T cells treated with DMSO or TWS119, showed minimal tumor control.

[0063] [Figure 8]Figure 8 shows the in vivo tumor clearance and persistence of CAR-T cells treated with (A) DMSO, B) TWS119, and (C) DCC-2036. [Modes for carrying out the invention]

[0064] The in vivo efficacy of T cell therapy can be strongly influenced by the manufacturing process, which depends on both the initial population of T cells entering the processing or supply chain, and the ex vivo proliferation and activation methods used. The differentiation state of 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 the expression of CCR7 and CD62L markers, mediate superior antitumor activity in mouse models (Sommermeyer et al., 2015) and non-human primate models (Berger et al., 2008).

[0065] During the manufacturing process, therapeutic cells (or cell populations) are typically activated, possibly transduced, or otherwise manipulated to express recombinant receptors, and then proliferated. This process generally promotes cell differentiation, resulting in an increased proportion of cells in a more differentiated state—in the case of T cells, these more differentiated cells are phenotypically characterized as effector memory or effector T cells. Once injected into a patient, these more differentiated cells have lower proliferative capacity and are less likely to survive as a long-lived or sustained population compared to cells in a less differentiated state.

[0066] Furthermore, the importance of the manufacturing process cannot be overstated, as the final state of cells entering a patient's body, specifically the cell subtype, can largely be defined by that process. Preferentially maintaining or promoting cell subpopulations with desired differentiation states and / or adaptive immune cell characteristics during cell culture and proliferation can be extremely beneficial in enhancing the effectiveness of cell-based therapies.

[0067] Improved cell manufacturing processes offer several potential advantages, including shorter administration times, increased cell uniformity, and a higher percentage of patients reaching the desired dose. In addition, functional improvements to cells during the manufacturing process, such as increased persistence and reduced toxicity, can also lead to improvements in cell therapy.

[0068] Compositions and methods for modulating immune cells to obtain populations or subpopulations of cells having improved therapeutic capacity for adoptive immunotherapy are provided herein. Compositions comprising modulated immune cells having improved therapeutic potential are also provided. Methods for using modulated immune cells having improved therapeutic capacity to treat diseases and conditions are also provided. In some embodiments, immune cells having improved therapeutic potential exhibit at least one, at least two, or at least three of the following: namely, improved proliferation, viability, persistence, cytotoxicity, and / or improved cell recall / memory. Methods and compositions for improving the therapeutic capacity of immune cells by increasing the number or relative ratio of cells exhibiting at least one of the above properties or by enriching subpopulations are also provided, as in therapeutic cell compositions.

[0069] definition

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0071] It should be understood that the present invention is not limited to and is therefore subject to change the specific methodologies, protocols, and reagents described herein. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0072] As used herein, the articles "a," "an," and "the" refer to one or more of the grammatical objects of the article. For example, T cell means one T cell or two or more T cells.

[0073] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to the major type of leukocyte that completes maturation in the thymus and has various roles in the immune system, including the identification of specific foreign antigens in the body, as well as the activation and inactivation of other immune cells. T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and may be at any stage of development, and are not limited to, but include CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), T cells in peripheral blood mononuclear cells (PBMCs), T cells in peripheral blood leukocytes (PBLs), T cells in tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, and gamma delta T cells (γδ T cells). Further types of helper T cells include cells such as Th3, Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also differentiate from stem cells or progenitor cells.

[0074] As used herein, the term “naive T cell” or Tn refers to a mature T cell that has not encountered its alloantigen in the periphery, unlike activated T cells or memory T cells. Naive T cells are typically characterized by surface expression of L-selectin (CD62L), absence of the activation markers CD25, CD44, or CD69, and absence of the memory CD45RO isoform. They also express a functional IL-7 receptor consisting of the subunits IL-7 receptor α, CD127, and the common gamma chain, CD132. In the naive state, T cells are thought to be quiescent and non-dividing, requiring the common gamma chain cytokines IL-7 and IL-15 for homeostatic survival mechanisms.

[0075] As used herein, the term “central memory T cells” or Tcm refers to a subgroup or subpopulation of T cells that express CD45RO and CD25 but not CD45RA. Tcm also express genes associated with transport to secondary lymphoid organs, including CD62L, CXCR3, and CCR7, and are distinct from effector memory T cells, or TEM, which lose the expression of these gene products.

[0076] As used herein, the terms “stem memory T cells” or “stem cell memory T cells” or Tscm refer to a subgroup or subpopulation of T cells capable of self-renewing and generating Tcm, Tem, and Teff (effector T cells). Tscm have a similar expression pattern to Tn, but unlike Tn, they also express CD95.

[0077] As used herein, the term “NK cells” or “natural killer cells” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3).

[0078] As used herein, the terms “adaptive NK cells” and “memory NK cells” refer to a subset of NK cells that are interchangeable, phenotypically express CD3- and CD56+, and have at least one of NKG2C and CD57, and optionally CD16, but lack one or more of the following: low PLZF, low SYK, FceRγ, and low FcεRγ, low EAT-2, low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. In some embodiments, an isolated subpopulation of CD56+ NK cells includes the expression of NKG2C and CD57. In some other embodiments, isolated subpopulations of CD56+ NK cells include expression of CD57, CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and repressing KIR, NKG2A, and / or DNAM-1. CD56+ may be weakly expressed or strongly expressed.

[0079] As used herein, the term “NKT cells” or “natural killer T cells” refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells that 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 recognized: Invariant or Type I NKT cells are associated with a very limited TCR repertoire—expressing the regular α-chain (Vα24-Jα18 in humans) and a restriction spectrum of the β-chain (Vβ11 in humans). Non-classical or non-invariant Type II NKT cells, also called Type II NKT cells, exhibit more heterologous TCR αβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified as expressing at least one of the following markers: TCR Va24-Ja18, VB11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0080] As used herein, the terms “isolated,” etc., refer to cells or populations of cells separated from their original environment; that is, the environment of isolated cells substantially does not contain at least one component as found in the environment in which “unisolated” standard cells exist. This term includes cells that have been removed from some or all components when they are found in their natural environment, for example, when they are isolated from a tissue or biopsy sample. This term also includes cells that have been removed from at least one, some or all components, so that they are isolated from a non-naturally occurring environment, for example, from a cell culture or cell suspension. Thus, isolated cells are partially or completely separated from at least one component, including other substances, cells or cell populations, as they are found in nature or as they grow, store or survive in a non-natural environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in media that do not exist in nature. Isolated cells may be obtained by separating desired cells or populations from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0081] As used herein, the term "purify," etc., refers to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0082] As used herein, the term “population” means, when used in relation to T, NK, or NKT cells, a group of cells containing two or more T, NK, or NKT cells, respectively. Using T cells as an example, an isolated or enriched T cell population may contain only one type of T cell or a mixture of two or more types of T cells. An isolated T cell population may be an homogeneous population of one type of T cell or a heterogeneous population of two or more types of T cells. An isolated T cell population may also be a heterogeneous population containing T cells and at least non-T cell cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. A heterogeneous population may have 0.01% to about 100% T cells. Thus, an isolated T cell population may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells. An isolated T cell population may include, but is not limited to, one or more, or all different types of T cells, including those disclosed herein. In an isolated T cell population containing two or more types of T cells, the relative proportion of each type of T cell may range from 0.01% to 99.99%. An isolated population may also be a clonal population of T cells, where all T cells in that population are clones of a single T cell.

[0083] Isolated populations of T, NK, or NKT cells can be obtained from natural sources such as human peripheral blood or umbilical cord blood. Various methods have been developed in the art to dissociate cells from tissue or cell mixtures and separate various cell types. In some cases, these operations result in relatively homogeneous cell populations. T cells can be isolated by sorting or selection processes as described herein, or by other methods known in the art. The proportion of T cells in the isolated population may 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 proportion of T cells in the natural source. The isolated T cell population may be T cells in general, or one or more specific types of T cells.

[0084] As used herein, the term “subpopulation,” when used in reference to T, NK, or NKT cells, refers to a population containing fewer T, NK, or NKT cell species than all of the species found in nature combined.

[0085] As used herein, the term “pluripotency” means the ability of a cell to form all lineages of body or somatic cells (i.e., the 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: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental capacity ranging from incomplete or partial pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and pluripotent cells that can produce a complete organism (e.g., embryonic stem cells).

[0086] As used herein, the term “induced pluripotent stem cell” or “iPSC” means a stem cell produced from differentiated adult cells that have been induced or altered (i.e., reprogrammed) into cells capable of differentiating into all tissues of the three germ layers or cortex, namely the mesoderm, endoderm, and ectoderm.

[0087] As used herein, the term “embryonic stem cells” refers to the naturally occurring pluripotent stem cells in the inner cell mass of an embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, give rise to all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membrane or placenta and are not totipotent.

[0088] As used herein, the term “progenitor cell” means a cell with greater developmental potential, i.e., a cellular phenotype that is more primitive (e.g., in an early stage along a developmental pathway or progression) compared to the cells that can be produced by differentiation. Often, progenitor cells have significant or very high proliferative capacity. Depending on the developmental pathway and the environment in which the cell develops and differentiates, progenitor cells may give rise to multiple different cells with lower developmental potential, i.e., differentiated cell types, or a single differentiated cell type.

[0089] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased cellular efficacy,” and “increased developmental potential” refer to methods of increasing the efficacy of a cell or differentiating a cell into a less differentiated state. For example, a cell with increased cellular efficacy has greater developmental flexibility (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is less differentiated than the same cell in an unreprogrammed state.

[0090] As used herein, the term “differentiation” refers to the process by which undifferentiated (“uncommitted”) or poorly differentiated cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated cells or differentiation-inducing cells are cells that occupy a more specialized (“committed”) position within a cell lineage. When applied to the differentiation process, the term “committed” refers to a cell that has progressed in the differentiation pathway to the point where, under normal circumstances, it will continue to differentiate into a particular cell type or a subset of cell types, and under normal circumstances, it cannot differentiate into a different cell type or revert to a less differentiated cell type.

[0091] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of macromolecules and macromolecules in a biological process having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, are referred to as coding for the protein or other product of that gene or cDNA.

[0092] As used herein, the term “exogenous” is intended to mean that the molecule or activity mentioned is introduced into the host cell or is unnatural to the host cell. The molecule may be introduced, for example, by introducing the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as non-chromosomal genetic material such as a plasmid. Therefore, as used in relation to the expression of coding nucleic acids, the term refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term “endogenous” refers to the molecule or activity mentioned that is present in the host cell. Similarly, as used in relation to the expression of coding nucleic acids, this term refers to the expression of the coding nucleic acid that is present in the cell and not introduced exogenously.

[0093] As used herein, the term “polynucleotide” means a polymeric form of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. A polynucleotide sequence consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), where thymine is replaced by uracil (U) if the polynucleotide is RNA. Polynucleotides may include genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0094] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable 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 called peptides, oligopeptides, and oligomers in the art, and long chains, also commonly called polypeptides or proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0095] As used herein, the term “ex vivo” refers to activities that occur outside of living organisms, such as experiments or measurements performed in or on living tissues within an artificial environment outside of living organisms, preferably with minimal modification of natural conditions. “Ex vivo” procedures involve living cells or tissues taken from living organisms and cultured in an experimental apparatus, usually under sterile conditions, typically for several hours or up to about 24 hours, but depending on the circumstances, up to 2 to 28 days. Such tissues or cells may also be collected and frozen and subsequently thawed for further experiments, either in vitro, ex vivo, or in vivo. Tissue culture experiments or procedures that continue to use living cells or tissues for longer than several days are typically considered “in vitro,” although in certain embodiments, this term may be used interchangeably with “ex vivo.” “In vivo” activity, on the other hand, occurs in living organisms, such as mice, and such activity may include cell engraftment, cell homing, cell self-renewal, and cell expansion.

[0096] As used herein, the term “in vitro” means activity performed or occurring outside of a test tube, culture dish, or any other location outside of an organism.

[0097] As used herein, the terms “drug,” “modulator,” and “modulator” are interchangeable herein and refer to compounds or molecules that can alter gene expression profiles or the biological properties of immune cells. A drug may be a single compound or molecule, or a combination of multiple compounds or molecules. Exemplary drugs include, for example, compounds that can stimulate the prostaglandin pathway, such as prostaglandin pathway agonists, glucocorticoids, or combinations thereof.

[0098] As used herein, the terms “contact,” “treat,” or “modulate” are interchangeable when used in reference to actions performed on immune cells and mean culturing, incubating, or exposing immune cells with one or more of the agents disclosed herein.

[0099] As used herein, “uncontacted” or “untreated” cells are cells that have not been treated, for example, cells that have not been cultured, in contact with, or incubated with any agent other than a control agent or vehicle. Cells that have been in contact with a control agent such as DMSO, or with any other medium, are examples of uncontacted cells.

[0100] As used herein, “feeder cells” or “feeder” is a term referring to one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can proliferate, expand, or differentiate, by providing stimuli, growth factors, and nutrients for the support of the second cell type. Feeder cells may sometimes originate from a different species than the cells they support. For example, certain types of human cells, including stem cells, may 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 proliferation and maturation of natural killer cells. Feeder cells can usually be inactivated when co-cultured with other cells by irradiation or treatment with antimitotic agents such as mitomycin to prevent the supporting cells from proliferating. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular type of feeder cell may be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be partially used to maintain pluripotency, induce differentiation into specific strains, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0101] As used herein, “feeder-free” (FF) environment means an environment such as a culture condition, cell culture, or medium that is essentially free of feeder cells or stromal cells and / or has not been pre-conditioned by feeder cell culture. “Pre-conditioned” medium refers to a medium harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. Pre-conditioned mediums contain 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 cells and stromal cells and is also not pre-treated by feeder cell culture.

[0102] As used herein, the term “analog” means a chemical molecule that is similar in structure and function to another chemical substance and is structurally different by one single element or group, or by two or more groups (e.g., two, three, or four) if they retain the same chemical scaffold and function similarly to the parent chemical substance. Such modifications are commonplace to those skilled in the art and include, for example, additional or substituted chemical moieties such as esters or amides of acids, benzyl groups for alcohols or thiols, and protecting groups such as the tert-butoxycarbonyl group for amines. Also include modifications to alkyl side chains such as alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.), halogenation, modification of the level of saturation or unsaturation of the side chain, and addition of modifying groups such as substituted phenyl and phenoxy. Analogues may also include conjugates such as biotin or avidin moieties, enzymes such as horseradish peroxidase, and radiolabeled, bioluminescent, chemiluminescent, or fluorescent moieties. Furthermore, moiety can be added to drugs described herein to modify their pharmacokinetic properties, such as increasing their half-life in vivo or ex vivo, or increasing their cell permeability, among other desirable properties. Prodrugs are also included, which are known to improve a number of desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.).

[0103] As used herein, the term “increase” means the ability of an active agent to produce or induce a greater physiological response (i.e., downstream effect) in cells compared to the response caused by either the vehicle or the control molecule / composition. For example, an increase in the production of interleukin-2 or TNF by an isolated T cell population. An increase may be an increase in gene expression as a result of increased signaling through a particular cellular signaling pathway. An “increase” is typically a statistically significant amount and may include increases of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals greater than or equal to 1, e.g., 1.5, 1.6, 1.7, 1.8) compared to the response caused by the vehicle (absence of the drug) or the control composition.

[0104] As used herein, the term “reduce” means the ability of an active agent to produce or induce a lower physiological response (i.e., a downstream effect) in cells compared to the response caused by either the vehicle or the control molecule / composition. The reduction may be a decrease in gene expression, a decrease in cell signaling, or a decrease in cell proliferation. The amount of “reduce” is typically a “statistically significant” amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals greater than or equal to 1, e.g., 1, 5, 1, 6, 1.7, 1.8, etc.) of the response caused by the vehicle (absence of the agent) or the control composition.

[0105] As used herein, the terms “synergistic” or “synergistic” refer to a combination of two or more entities with an enhanced effect, in contrast to “antagonistic,” which is used when two or more entities in a combination cancel out or neutralize each other’s effects, and “additive,” which is used when two or more entities in a combination produce an effect that is approximately equal to the sum of their individual effects.

[0106] As used herein, the term “substantially free” means, when used to describe a composition such as a cell population or culture medium, that it does not contain any particular substance of any origin, for example, that it is 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable by conventional means. The same meaning may apply to the term “not present” when referring to the absence of a particular substance or component of a composition.

[0107] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. The range of quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length may be ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0108] As used herein, the term “subject” refers to a mammal. The subject may be a human or a non-human mammal, such as a dog, cat, cow, horse, mouse, rat, rabbit, or a transgenic species thereof.

[0109] When used in relation to subjects requiring the therapeutic compositions or methods used herein, the terms “treat,” “treatment,” and “treated” mean a desired pharmacological and / or physiological effect, including, but not limited to, improvement or elimination or recovery or prevention of one or more signs or symptoms of a disease. The effect may be preventive and / or may include completely or partially preventing the disease or its symptoms and / or achieving improvement or elimination of symptoms, or providing a partial or complete cure for the disease and / or adverse effects resulting from the disease. The term “treatment” includes any treatment of a disease in mammals, in particular humans, and in some embodiments includes (a) preventing the onset of the disease in subjects susceptible to the disease but not yet diagnosed with the disease, (b) suppressing the disease or stopping its onset, (c) reducing the disease or inducing disease regression or completely or partially eliminating the symptoms of the disease, and (d) restoring the individual to a pre-disease state, such as by reconfiguring the hematopoietic system.

[0110] As used herein, “genetic modification” means gene editing and may include (1) modifications derived from natural rearrangements, mutations, gene imprinting and / or epigenetic modifications, or (2) modifications obtained by genetic manipulation by insertions, deletions or substitutions in the genome of a cell. As used herein, genetic modification may also include one or more retainable therapeutic attributes of a source-specific immune cell that are donor-specific, disease-specific, or treatment response-specific.

[0111] As used herein, the term “genetic imprinting” means genetic or epigenetic information that contributes to a preferred therapeutic characteristic in the source cells. In particular, in the context of source cells obtained from a selected donor, disease, or treatment context, genetic imprinting contributing to a preferred therapeutic attribute may include any context-specific genes or epigenetic modifications that manifest a retainable phenotype, i.e., a preferred therapeutic attribute, regardless of whether the underlying molecular events have been identified. Donor-specific, disease-specific, or treatment-response-specific source cells may include retainable genetic imprintings in iPSCs and iPSC-derived hematopoietic lineage cells. Such genetic imprintings include, but are not limited to, single-specific TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells; trackable desired genetic polymorphisms, e.g., homozygosity for point mutations encoding high-affinity CD16 receptors in selected donors; and specified HLA requirements, i.e., selected HLA-matched donor cells exhibiting a common haplotype. When used herein, preferred therapeutic properties include improved engraftment, transport, homing, viability, self-renewal, persistence, regulation and modulation of the immune response, survival, and cytotoxicity of induced cells. Preferred therapeutic properties are also associated with antigen-targeted receptor expression; HLA presentation or absence; resistance to immunosuppressive effects of the tumor microenvironment; induction of bystander immune cells and desired immunomodulation; improved target specificity with reduced extratumor effects; and resistance to treatments such as chemotherapy.

[0112] As used herein, the term “safety switch protein” refers to an artificial protein designed to prevent potential toxicity or otherwise adverse effects of cell therapy. In some examples, safety switch protein expression is conditionally controlled to address safety concerns in transplanted cells whose genomes permanently incorporate a gene encoding the safety switch protein. This conditional regulation may be variable and may include control by small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional regulation. Safety switches may mediate the induction of apoptosis, inhibition of protein synthesis or DNA replication, growth arrest, transcription and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, safety switch proteins are activated by exogenous molecules, such as prodrugs, which, when activated, induce apoptosis and / or cell death in therapeutic cells. 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 response to an adverse event is activated by a suicide gene product, killing the transduced cells.

[0113] As used herein, “therapeutically sufficient amount” includes, in its meaning, a sufficient and / or effective amount of a particular therapeutic and / or pharmaceutical composition referred to in order to provide the desired therapeutic effect. The exact amount may vary from subject to subject depending on factors such as the patient’s general health, the patient’s age, and the stage and severity of the disease. In particular embodiments, a therapeutically sufficient amount is sufficient and / or effective to improve, alleviate, and / or improve at least one symptom associated with the disease or condition being treated.

[0114] As used herein, “sufficient amount” refers to an amount of the drug or composition provided herein that is sufficient to achieve a particular outcome or result.

[0115] Modifiers to improve the effectiveness of cell-based adoptive immunotherapy In some embodiments, the present invention provides compositions comprising one or more modifiers in an amount sufficient to improve one or more properties of immune cells, such as modulated immune cells suitable for adoptive cell-based therapy, including therapeutic potential. In some embodiments, immune cells having improved therapeutic potential exhibited improvements in proliferation, persistence, cytotoxicity, and / or cell recall / memory compared to cells produced or maintained under similar conditions, such as those without one or more modifiers. In some embodiments, by modulating immune cells with an active substance or composition comprising modifiers, the resulting immune cells exhibit one or more such improvements, including improvements in at least one attribute. In some embodiments, phenotypic distortions including, but not limited to, at least one attribute, and / or at least one or more (e.g., Teff or Tem to Tn, Tcm, and / or Tscm and / or skew phenotypes compared to one or more other T cell subpopulations, including increased cell expansion, increased cell viability; and / or improved ability in tumor clearance and persistence. In some embodiments, immune cells, such as those suitable for adoptive cell-based therapy, are contacted, treated, or modified with one or more classes of modifiers classified by their respective targets. In some embodiments, the cells are manipulated in at least one step by a process carried out in the presence of the composition or modifier or drug. For each class of modifiers provided, several non-limiting and exemplary compounds are listed in Table 1.

[0116] Where used interchangeably herein, “modulator” or “modulator” refers to an inhibitor or activator that has the ability to modulate the expression or activity of a particular target (such as a protein or coding polynucleotide). “Modulator” or “modulator” includes inhibitors and activators, e.g., ligands, agonists, and antagonists. Modulators or modulators as used herein may be organic compounds (e.g., small chemical molecules), polypeptides (e.g., peptides or antibodies), nucleic acids (e.g., DNA, RNA, double-stranded, single-stranded, oligonucleotides, antisense RNA, small inhibitory RNA, microRNA, ribozymes, etc.), oligosaccharides, or lipids, whether synthetic or spontaneously occurring; or similarly functional homologs, mimes, derivatives, analogs, or salts thereof (e.g., inhibitors or activators for the same target).

[0117] Inhibitors are agents that, for example, reduce or eliminate the expression of the target protein or other targets described; or partially or completely block the stimulating or protease inhibitory activity of the target protein; or reduce, prevent, delay the activation of, inactivate, desensitize, or downcontrol the activity of the target protein, e.g., an antagonist. Activators can, for example, induce or activate the expression of the target protein described; or stimulate, increase, activate, promote, enhance protease inhibitor activity; or sensitize or upcontrol the activity of the target protein described, e.g., an agonist. Assays for inhibitors and activators involve, for example, applying a putative modulator to cells expressing the target protein described; and then determining the functional effect and degree of effect on the expression and / or activity of the target protein described. Generally, control samples (untreated with the modulator or treated with the vehicle alone) are assigned a specific activity value of 100%. Inhibition of the target protein described is achieved when the activity level is approximately 90% of the control, and possibly 80%, 70%, 60%, 50%, 25%, 10%, 5%, or 1% or less. Activation of the target protein described is achieved when the activity level is 110% of the control, and possibly 150%, 200%, 300%, 400%, 500%, or 1000-3000% or more.

[0118] To improve the therapeutic potential of immune cells, a certain improvement in the quality of the immune cells is generally involved. Treatment with modifiers of several embodiments provided herein has been shown to enhance certain biological properties of therapeutic immune cells, for example, under certain diseases or in response to certain diseases, by modulating, or modulating, at least one of the following: phenotypic distortion, expansion, maintenance, differentiation, dedifferentiation, survival, proliferation, cytotoxicity, persistence, and / or cellular recall / memory, or the potential of at least one of these, thereby improving the therapeutic potential of immune cells. In a T cell population, for example, phenotypic distortion to naive, stem cell memory, or central memory T cells results in an increase in the number or relative ratio of naive, stem cell memory, or central memory T cell subpopulations, and / or a decrease in the number or relative ratio of effector memory or effector T cell subpopulations by modulating maintenance, expansion, differentiation, and / or their dedifferentiation, indicating better quality of T cells for improved in vivo adoptive therapeutic capacity. In one embodiment, the number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells is increased in the T cell population by treatment with one or more regulators containing at least one compound listed in the table. In one embodiment, phenotypic distortion, or an increase in the number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells, is indicated by increased expression of surface markers after drug treatment. In one embodiment, phenotypic distortion, or an increase in the number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells, is indicated by cytokine profiles associated with naive or memory T cells rather than effector cells after drug treatment.

[0119] In some other embodiments, at least one compound in Table 1 enhances the therapeutic potential of T cells by improving their cellular killing ability. In yet another embodiment, at least one compound in Table 1 enhances the therapeutic potential of T cells by increasing cell viability and proliferation. In yet another embodiment, at least one compound in Table 1 enhances the therapeutic potential of T cells by enhancing cell persistence and tumor clearance capabilities. In some embodiments, the modifier includes at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In some embodiments, the modifier includes DCC-2036.

[0120] Similarly, in NK cell populations, for example, an increase in the number or relative ratio of adaptive NK cells due to maintenance, subtype shifting, proliferation, differentiation, and / or dedifferentiation indicates better quality NK cells for improved in vivo adoptive therapeutic potential. With respect to NKT cell populations, for example, an increase in the number or relative ratio of type I NKT cells due to maintenance, subtype switching, proliferation, differentiation, and / or dedifferentiation indicates better quality NKT cells for improved in vivo adoptive therapeutic potential.

[0121] The classes of modifiers classified / identified by their respective targets in Table 1 were discovered based on their potential to improve the therapeutic potential of immune cells for adoptive therapy. Without being limited by theory, they modulate and improve therapeutic immune cells by regulating cellular metabolism, nutrient sensing, proliferation, apoptosis, signaling, cytokine production, properties related to infection processes, and / or other aspects of cellular function.

[0122] Immune cells suitable for adoptive cell-based therapy are exposed to one or more modifiers listed in Table 1. Treatment under the influence of drugs can modify the biological properties of cells by modulating cell proliferation, maintenance, and differentiation characteristics, and / or increasing proliferation, cytotoxicity, persistence, and / or cell recall / memory, and subsequently increasing the therapeutic potential of the treated cells. For example, treatment can improve the viability of therapeutic immune cells ex vivo and in vivo. Furthermore, treatment can alter the proportion of different subpopulations in the treated cell population. For example, in one embodiment, the number and proportion of naive T cells, stem cell memory T cells, and / or central memory T cells increase in the isolated T cell population compared to the vehicle upon ex vivo treatment with one or more drugs selected from Table 1. In another embodiment, the number and proportion of adaptive NK cells in an NK cell population increase upon ex vivo treatment of the NK cell population with one or more drugs selected from Table 1.

[0123] Table 1 - Immunomodulators in adoptive cell therapy [Table 1-1] [Table 1-2] [Table 1-3]

[0124] Without being limited by theory, the agents in Table 1 improve the therapeutic potential of immune cells for adoptive therapy by regulating cell growth, metabolism, and / or cell differentiation through modulating properties related to cell metabolism, nutrient sensing, proliferation, apoptosis, signal transduction, characteristics of the infection process, and / or other aspects of cell function. As understood by those skilled 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, exemplary examples of analogs and derivatives of the agent in Table 1, dmPGE2 (16,16-dimethyl prostaglandin E2, 16,16-dimethyl PGE2), include, but are not limited to, PGE 2、 16,16-dimethyl PGE2 P-(p-acetamidobenzamide) 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 PGE 2、 17-phenyl-omega-trinor PGE 2、 PGE2 serinol amide, PGE2 methyl ester, 16-phenyltetranor PGE 2、 15(S)-15-methyl PGE 2、 15(R)-15-methyl PGE 2、 8-iso-15-keto PGE 2、 8-iso PGE2 isopropyl ester, 8-iso 16 cyclohexyltetranor PGE 2、 20-hydroxy PGE 2、 20-ethyl PGE2, 11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19(R) hydroxy PGE2 are included. PG analogs or derivatives having a structure similar to PGE2 substituted with a halogen at the 9-position (see, for example, WO2001 / 12596, the entire disclosure of which is incorporated herein by reference), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives such as those described in US Patent Application Publication No. 2006 / 0247214, the entire disclosure of which is incorporated herein by reference, are also included.

[0125] GSK-3β (glycogen synthase kinase 3-beta) inhibitors suitable for use in the compositions intended herein include, but are not limited to, kempawornone, 1-azakempawornone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazole-3-yl)-(2-phenylquinazoline-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, TDZD-84-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, α-4-dibromoa-cetofe Non, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridine-3-yl]-4-pyrazine-2-ylpyrrole-2,5-dione; TWS119, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophene-2-yl)-etanone; GF1099203X; RO318220; TDZD-8; TIBPO and OTDZT are included. In one embodiment, the GSK-3β inhibitor is CHIR99021, BIO, TWS119, or Kaempaulon. 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.

[0126] Suitable ERK / MEK inhibitors for use in the compositions intended herein include PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSK120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEAI19, AZD6244, FR180204, PTK787, and 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-MEK inhibitors. Tyl-3H-benzimidazole-e-5-carboxylic acid (2,3-dihydroxy-propoxy)amide; 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-(tetrahydropyran-2-yl-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)amide, 1-[6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chlorophenylamino)-7 -Fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-1,1-dimethylethoxy)amide, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-(tetrahydrofuran-2-yl-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxyethoxy)amide, 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxyethoxy)amide, 6-(2, 4-dichlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxyethoxy)-amide, 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide, hereafter referred to as MEK inhibitor 2;This includes 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide or pharmaceutically acceptable salts thereof. Further exemplary MEK / ERK inhibitors include compounds disclosed in international publication applications WO99 / 01426, WO02 / 06213, WO03 / 077914, WO05 / 051301 and WO2007 / 044084. In one embodiment, the MEK inhibitor is PD0325901. In another embodiment, the MEK inhibitor is U0126.

[0127] Suitable ROCK (Rho-related kinase) inhibitors for use in the compositions envisioned herein include, but are not limited to, thiazovibin, Y27632, fasudil, AR122-86, Y27632H-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 ROCK inhibitors disclosed in U.S. Patent No. 8,044,201 (the entirety of which is incorporated herein by reference). In one embodiment, the ROCK inhibitor is thiazovibin, Y27632, or pyrintegrine. In one embodiment, the ROCK inhibitor is thiazovibin.

[0128] Suitable TGFβ receptor / ALK5 inhibitors for use in the compositions intended herein include SB431542;A-83-01(3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbocioamide;2-(3-(6-methylpyridine-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine,Wnt3a / BIO,GW788388(-4-[3-(pyridin-2-yl)-1H-pyrazole-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl) Benzamide), SM16, IN-1130 (3-((5-(6-methylpyridine-2-yl)-4-(quinoxaline-6-yl)-1H-imidazole-2-yl)methyl)benzamide), GW6604 (2-phenyl-4-(3-pyridine-2-yl-1H-pyrazole-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazole-4-yl)-6-methylpyridine hydrochloride; SU5416; 2-(5-benzo[1,3]dioxol)-5-yl-2- tert-butyl-3H-imidazole-4-yl)-6-methylpyridine hydrochloride (SB-505124); rederimubam (CAT-152); meterimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY2109761; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P1 44; soluble TBR2-Fc; and pyrimidine derivatives (see, for example, those listed in Stiefl et al., WO2008 / 00653 (the whole of which is incorporated herein by reference)), are examples, but are not limited thereto. Furthermore, “ALK5 inhibitors” are not intended to encompass nonspecific kinase inhibitors, but should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, e.g., SB-431542. (e.g., Inman et al., J.Mol.Pharmacol.)62(1):65-74 (see 2002). Furthermore, inhibition of the TGFβ / activin pathway is thought to have a similar effect to inhibition of ALK5. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with, or instead of, the ALK5 inhibitors described in each paragraph herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, SMAD2 / 3 phosphorylation inhibitors, SMAD2 / 3 and SMAD4 interaction inhibitors, and SMAD6 and SMAD7 activators / agonists. Furthermore, the classifications described below are for organizational purposes only, and those skilled in the art know that a compound may affect one or more points in the pathway, and therefore a compound may function in two or more defined categories. In one embodiment, a TGFβ receptor inhibitor includes SB431542.

[0129] PDK1, or 3'-phosphoinositide-dependent kinase-1, is a master kinase involved in the activation of AKT / PKB and many other AGC kinases, including PKC, S6K, and SGK. PDK1 plays a crucial role in signaling 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). Examples of allosteric activators of PDK1 include PS48((Z)-5-(4-chlorophenyl)-3-phenylpenta-2-enoic acid), PS08((Z)-5-(4-bromo-2-fluorophenyl)-3-phenylpenta-2-enoic acid), 1-(2-(3-(4-chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid; 3,5-diphenylpenta-2-enoic acid, e.g., compound 12Z(2-)(3-(4-chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(naphthalene-2-yl)-3-phenylpenta-2-enoic acid), and compound 13Z((Z))-5-(1H-indole-3-yl)-3-phenylpenta-2-enoic acid). In one embodiment, the PDK1 agonist comprises PS48.

[0130] BCR-ABL tyrosine kinase inhibitors (TKIs) inhibit the enzyme BCR-ABL tyrosine kinase. Suitable BCR-ABL tyrosine kinase inhibitors for use in the compositions intended herein include, but are not limited to, DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), and PD173955. In one embodiment, the Bcr-Abl tyrosine kinase inhibitor is DCC-2036.

[0131] 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 combination of any number, of agents selected from Table 1.

[0132] In one embodiment, a 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 dimethyl sulfoxide (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.

[0133] 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 I: dolsomorphine, hepteridine acid, 1-pyrrolidinecarboditioic acid, and 2-DG. Among other potential roles, not limited to theory, Group I agents may affect cellular metabolism and nutrient sensing.

[0134] 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: GSK3β inhibitors, ROCK inhibitors, TGFβ receptor inhibitors, MEK inhibitors, PDK1 agonists, 6-mercaptopurine, AC-93253 iodide, thiratrichol, 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 (mTOR inhibitor), TWS119, HS173, LY294002, pictilisib, and DCC-2036 (levastinib). Among other potential roles, not limited to theory, Group II agents may influence signaling in various functional pathways.

[0135] 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 III: 5-azacitidine, fludarabine, roscovitine, and PAC-1. Among other potential roles, not limited to theory, Group III agents may affect cell proliferation and apoptosis.

[0136] 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 IV: 5,7-dichloro-8-quinolinol, 2-naphthacenecarboxamide, 7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octaf, nifloxazide, and tosufloxacin hydrochloride. Among other potential roles, not limited to theory, the Group IV agents may influence cellular properties related to the infection process.

[0137] 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 V: sertraline, diethylenetriamine pentaacetic acid, edrophonium chloride, BIX01294, terfenadine, and dmPGE2. Among other potential roles, though not limited to theory, agents of Group V can generally influence expansion, maintenance, cell differentiation, and other cellular properties relating to in vivo proliferation, cytotoxicity, cell recall response, and / or persistence.

[0138] In yet another embodiment, 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 at least one modifier selected from groups II, III, IV, and V.

[0139] In some other embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one substance selected from group II and at least one modifier selected from group I, group III, group IV and / or group V.

[0140] In yet another embodiment, 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 at least one modifier selected from groups I, II, IV, and V.

[0141] In yet another embodiment, 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 at least one modifier selected from groups I, II, III, and V.

[0142] In yet another embodiment, 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 at least one modifier selected from groups I, II, III, and IV.

[0143] 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 the group consisting of GSK3β inhibitors, MEK inhibitors, ROCK inhibitors, TGFβ inhibitors, PDK1 agonists, and BCR-ABL tyrosine kinase inhibitors.

[0144] 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 the combination. As defined, “additive” refers to a combination where two or more agents produce an effect 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.

[0145] In some embodiments, the composition includes 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 acting together to produce an effect greater than the sum of their individual effects. In one embodiment, the composition including the synergistic combination includes at least one agent selected from the group consisting of TWS119, HS173, LY294002, pictilisib, and 2-DG. In one embodiment, the composition includes 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 of compounds listed in Table 1. In one embodiment, the composition including TWS119 further includes two or more additional agents selected from Table 1. In one embodiment, the composition including HS173 further includes two or more additional agents selected from Table 1. In one embodiment, the composition including LY294002 further includes two or more additional agents selected from Table 1. In one embodiment, the composition containing pictilisib further comprises two or more additional agents selected from Table 1. In one embodiment, the composition containing 2-DG further comprises two or more additional agents selected from Table 1.

[0146] In some embodiments, a composition comprising one or more modifiers selected from the group consisting of compounds listed in Table 1 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 substitution factors, and / or vectors comprising one or more polynucleic acids of interest.

[0147] In some embodiments, cytokines and growth factors include one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia suppressor 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 (IL-1 to IL-18, etc.), various colony-stimulating factors (granulocyte / macrophage colony-stimulating factor (GM-CSF), etc.), various interferons (IFN-γ, etc.), 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 can be commercially available, for example, from R&D Systems (Minneapolis, Minnesota), and may be either natural or recombinant. In certain embodiments, the growth factors and cytokines may be added at concentrations intended herein. In certain embodiments, the growth factors and cytokines may be added at concentrations determined empirically or as guided by established cytokine techniques.

[0148] In some embodiments, the mitogen as an additive to the composition comprises concanavalin A. In some other embodiments, the feeder cells of the composition are genetically modified. In some embodiments, the feeder cells of the composition comprise one or more of mononuclear hematopoiesis, thymic epithelial cells, endothelial cells, fibroblasts, leukemia cells K562, Raji cells, or their feeder cell components or their replacement factors.

[0149] In some embodiments, the small RNA of the composition comprises one or more of siRNA, shRNA, miRNA, and antisense nucleic acids. In some other embodiments, the small RNA of the composition comprises one or more of miR-362~5p, miR-483~3p, miR-210, and miR-598.

[0150] In some embodiments, the vector of the composition containing one or more polynucleic acids of interest is either embedded or non-embedded. In some embodiments, the vector of the composition containing one or more polynucleic acids of interest further comprises the backbone of an adenovirus vector, plasmid vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and episomal vector. In some embodiments, plasmid vectors for expression in animal cells include, for example, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. In some embodiments, one or more polynucleic acids contained in the vector encode one or more proteins or polypeptides. In some embodiments, one or more polynucleic acids encode delta-like 1 (DLL1), delta-like 3 (DLL3), delta-like 4 (DLL4), Jagged 1 (Jag1), or Jagged 2. In some embodiments, one or more polynucleic acids encode Jagged 1.

[0151] In some embodiments, the composition further comprises at least one therapeutic agent. In one embodiment, the therapeutic agent comprises an antibody or 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 some embodiments, the antibodies in the composition include, but are not limited to, anti-CD20 (retiximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), anti-Her2 (trastuzumab), anti-CD52 (aremtuzumab), anti-EGFR (cetuximab), and anti-CD38 (daratumumab), as well as their humanized and Fc-modified variants. Furthermore, the design of bi- and tri-specific antibodies, in which the Fab region of an antibody targeting tumor cell antigens such as anti-CD19, CD20, and CD33 antigens is fused with another Fab region that recognizes surface proteins of immune cells, stimulates cells and subsequently kills tumor cells.

[0152] In some embodiments, additional additives to the composition include one or more of the following: chemotherapeutic agents, radioactive moieties, and immunomodulatory agents (IMiDs). Immunomodulatory agents such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. Chemotherapeutic agents refer to cytotoxic anti-cancer agents, which are found to preferentially kill neoplastic cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the proliferation of neoplastic cells. Chemotherapeutic agents are also called antineoplastic agents, cytotoxic agents, or drugs, and are well known in the art.

[0153] In some embodiments, the chemotherapeutic agent includes anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamine, nitrogen mustard, nitrosourea, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins. Examples of chemotherapeutic agents include, but are not limited to, drugs (cyclophosphamide, mechloretamine, mephalin, chlorambucil, hemamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxyuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisanthren, actinomycin D, plicamycin, promycin, and gramicidin D), paclitaxel, colchicine, cytochalacin B, emetine, mytansine, tansine, and amsacrine.Additional medications 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, and carsterone. Capecitabine, celecoxib, cetuximab, cladribine, cloflavin, cytarabine, dacarbazine, denileukin-diffitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinylestradiol, exemestane, floxyuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, Ibritumomab, idarubicin, ifosfamide, imatinib, interferon alfa (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, rebamizole, mechloretamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pe Examples include gademase, pegaparagus, pentostatin, pipobromane, plicamycin, polyfeprosan, porfimer, procarbazine, quinacrine, rituximab, salglamostim, streptozosin, tamoxifen, temozolomide, teniposide, testactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinorelbine, and zoledronate.

[0154] Additional appropriate therapeutic agents are those approved for use in humans, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art. Such agents can be referenced in numerous standard physician and oncologist references (e.g., Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, McGraw-Hill, NY, 1995) or the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time.

[0155] II. Immune cells for regulation and regulated immune cells The present invention provides a composition comprising an isolated population or subpopulation of immune cells contacted in vitro or ex vivo with one or more modifiers selected from Table 1. In one embodiment, the isolated population or subpopulation of immune cells is contacted ex vivo with one or more modifiers 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 modifiers selected from the agents listed in Table 1, wherein treatment by contact of the isolated population or subpopulation of immune cells with one or more of the said agents improves the therapeutic potential of the immune cells for therapeutic adoptive therapy. Such treatment can modify the biological properties of the immune cells to improve cell proliferation, cytotoxicity, persistence, and / or reduce the relapse rate of cell therapy.

[0156] In some embodiments, one or more modifiers include at least one compound selected from Table 1, and derivatives or analogs thereof. In some embodiments, the regulated immune cell population includes T cells. In some embodiments, the regulated immune cell population includes NK cells. In some embodiments, the regulated immune cell population includes NKT cells.

[0157] In some embodiments, a population or subpopulation of T cells exposed to one or more modifiers exhibits an increase in the number and relative ratio of naive T cells (Tn), stem cell memory T cells (Tscm), and / or central memory T cells (Tcm), and / or improvements in cell proliferation, cytotoxicity, cell recall response, and / or persistence, compared to T cells that have not received the same treatment. In some embodiments, the number of Tn, Tscm, and / or Tcm increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or by at least 2, 3, 4, 5, 10, 15, or 20 times or more, compared to the number of Tn, Tscm, and / or Tcm in a cell population that has not received the same treatment with one or more modifiers selected from Table 1.

[0158] In some embodiments, a population or subpopulation of NK cells exposed to one or more of the modifiers selected from Table 1 exhibits an increase in the number or relative ratio of adaptive (or memory) NK cells, and / or improvements in cell proliferation, cytotoxicity, cell recall response, and / or persistence, compared to NK cells that have not received the same treatment. In some embodiments, the number of adaptive NK cells increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or by at least 2, 3, 4, 5, 10, 15, or 20 times or more, compared to the number of adaptive NK cells in a cell population that has not received the same treatment. In one embodiment, a population or subpopulation of NK cells exposed to one or more of the agents includes adaptive NK cells with an increased number or relative ratio. In one embodiment, adaptive NK cells are characterized by CD3- and CD56+, as well as at least one of 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, adaptive NK cells are at least two of 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, adaptive NK cells may be CD57+ and NKG2C+. In some embodiments, adaptive NK cells are at least three 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. For example, adaptive NK cells may be SYK-, FcεRγ-, and EAT-2-. In one embodiment, a population or subpopulation of NK cells contacted with one or more of the aforementioned agents is further contacted with a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, a TGFβ inhibitor, a PDK1 agonist, and / or rapamycin. In one embodiment, the GSK3 inhibitor is CHIR99021, BIO, TWS119, or Kaempaulon. In one embodiment, the GSK3 inhibitor is TWS119.In another embodiment, the GSK3 inhibitor is CHIR99021. In yet another embodiment, the GSK3 inhibitor is BIO.

[0159] In some other embodiments, a population or subpopulation of NKT cells contacted with one or more modifiers includes an increase in the number or relative ratio of type I NKT cells to type II NKT cells, and / or improvements in cell proliferation, cytotoxicity, cell recall response, and / or persistence, compared to an isolated population or subpopulation of NKT cells not treated with one or more modifiers selected from Table 1. In some embodiments, the number of type I NKT cells increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or by at least 5, 10, 15, or 20 times or more, compared to the number of type I NKT cells not treated with one or more modifiers selected from Table 1.

[0160] In some non-limiting embodiments, an increase in the number or relative ratio of naive T cells (Tn), stem cell memory T cells (Tscm), central memory T cells (Tcm), adaptive NK cells, and / or type I NKT cells in a regulated immune cell population is attributed to increased maintenance and expansion of these cell subtypes, and / or increased cell dedifferentiation / reprogramming from more mature cell subtypes to cell subtypes in a desired differentiated state, and / or phenotypic distortion from one to the other.

[0161] In some embodiments, after contacting a population of immune cells with one or more of the modifiers listed in Table 1, the number of naive T cells (Tn), stem cell memory T cells (Tscm), and central memory T cells (Tcm) in the modulated cell population increases compared to the untreated immune cell population. Here, Tn, Tscm, and Tcm are characterized by co-expression of CCR7 and / or CD62L.

[0162] In some embodiments, after contacting a population of immune cells with one or more of the modifiers listed in Table 1, the number of adaptive NK cells in the modulated cell population increases compared to the untreated immune cell population. Here, adaptive NK cells are characterized by CD3-, CD56+, CD16+, NKG2C+, and CD57+. In some other embodiments, adaptive NK cells are characterized by CD3- and CD56+, as well as at least one, two, or three of 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.

[0163] In some embodiments, after contacting a population of immune cells with one or more of the modifiers listed in Table 1, the number of type I NKT cells in the modulated cell population increases compared to the untreated immune cell population. Here, type I NKT cells are characterized by surface antigens CD3+, CD56+, TCR Vα24+, and / or TCR Vβ11+.

[0164] In some embodiments, populations or subpopulations of T, NK, or NKT cells for treatment with the modifiers disclosed herein can be isolated from human or non-human mammals. Examples of such non-human mammals include, but are not limited to, rabbits, horses, cattle, sheep, pigs, dogs, cats, mice, rats, and their transgenic species.

[0165] Populations or subpopulations of T cells for regulation can be obtained or isolated from many sources, including but not limited to peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and tissues from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. Bone marrow can be collected from the femur, iliac crest, hip joint, ribs, sternum, and other bones. In addition, T cell lines available in the art, such as Jurkat and SupT1, can also be used.

[0166] Populations or subpopulations of NK cells for regulation can be obtained or concentrated from a number of sources, including but not limited to peripheral blood, umbilical cord blood, and tumors.

[0167] Fully mature NKT cells for regulation can be obtained from or enriched in peripheral blood, using smaller populations of mature NKT cells potentially found in bone marrow, lymph node tissue, umbilical cord blood, and thymic tissue.

[0168] In certain embodiments of the present invention, isolated or concentrated populations or subpopulations of immune cells, including T, NK, and / or NKT cells for regulation, can be obtained from blood units using any number of techniques known to those skilled in the art, e.g., Ficoll® isolation. In one embodiment, T cells, NK cells, or NKT cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes cells, including T cells, monocytes, granulocytes, B cells, NK cells, NKT cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations. As will be readily apparent to those skilled in the art, the washing process can be achieved by methods known to those skilled in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other salines with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed, and the cells can be resuspended directly in the culture medium.

[0169] In another embodiment, a population of immune cells, including T, NK, or NKT cells, or a subpopulation thereof for regulation, is isolated or concentrated from peripheral blood lymphocytes by lysing erythrocytes and removing monocytes, for example, by PERCOLL® gradient or countercurrent centrifugation and washing.

[0170] In one embodiment, specific subpopulations of T cells for regulation can be further isolated or enriched by positive or negative selection techniques using, for example, 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 grown and activated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads for a sufficient period to enrich the desired T cells. In one embodiment, the period is approximately 30 minutes. In further embodiments, the period is in the range of 30 minutes to 72 hours or more, and all integer values ​​in between. In further embodiments, 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 a preferred embodiment, the incubation period is 3 to 6 days. To isolate T cells from leukemia patients, using longer incubation times, such as 24 hours, may increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or individuals with immunodeficiency. Furthermore, the use of longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, a particular population or subpopulation of T cells can be further selected at the start of culture or at other points in the process by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). Furthermore, a particular population or subpopulation of T cells can be preferentially or non-selectively selected at the start of culture or at other desired points in time by increasing or decreasing the ratio of anti-CD3 antibodies and / or anti-CD28 antibodies on the beads or other surfaces. 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 a selection procedure and use "unselected" cells in the activation and expansion processes.Unselected cells can also undergo further selection rounds.

[0171] Isolation or enrichment of a population of immune cells, including T, NK, and NKT cells, or their subpopulations, for regulation by negative selection can be achieved using a combination of antibodies against surface markers specific to negatively selected cells. One method is sorting by cell sorting and / or negative magnetic immunoadhesion or fluorescence-activated selective cell sorting, which uses a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD3+ cells by negative selection, 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 reliably select regulatory T cells, typically expressing CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugate beads or other similar selection methods. In some embodiments, a desired T cell subpopulation for immunotherapy is enriched or selected from regulated immune cells, including T cells, by CCR7 and CD62L. Alternatively, cells of interest may be selected according to physical parameters including differences in size, density, granularity, deformability, resistance, or capacitance.

[0172] In one embodiment, a population of immune cells, including adaptive NK cells, is enriched by selecting regulated immune cells containing NK cells to the phenotypic CD3- and CD56+ using identifiers such as positive expression of CD16, NKG2C, and CD57. Furthermore, negative selection of adaptive subpopulations can be based on the absence of NKG2C and / or CD57 expression, as well as the absence of expression of one or more of the following: low PLZF, low SYK, low FcεRγ, low EAT2, low TIGIT, low PDI, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA.

[0173] In one embodiment, a population or subpopulation of NKT cells for regulation is enriched by selecting within the NK cell population those that phenotypically express an immutable TCR□ chain, specifically a combination of the following markers: CD3+, CD56+, TCRVα24+, and / or TCRVβ11+. Alternatively, NKT cells can be selected based on a combination of phenotypes combined with the expression of an immutable TCR□ chain.

[0174] Blood samples or apheresis products from subjects may be collected during a period prior to the isolation of the immune cells described herein. The source of cells to be regulated itself may be collected at any number of appropriate points in time and may be desired cells such as isolated and frozen T cells, NK cells, and NKT cells for later use in cell-based immunotherapy for any number of diseases or conditions that would benefit from such cell therapy described herein. In one embodiment, blood samples or apheresis products are collected from generally healthy subjects. In a particular embodiment, blood or apheresis products are collected from generally healthy subjects who are at risk of developing a disease but have not yet developed the disease, and the cells of interest are isolated and frozen for later use. In another embodiment, blood samples or apheresis products are taken from subjects having a specific disease (e.g., cancer). In yet another embodiment, blood samples or apheresis products are taken from subjects who have previously been administered genetically modified immune cells (genetically engineered, or naturally derived from rearrangement, mutation, gene imprinting, and / or epigenetic modification). In certain embodiments, T, NK, NKT, or other immune cells may be grown, frozen, and subsequently processed and used. In certain embodiments, a sample is taken from a patient immediately after diagnosis of a particular disease but before any treatment as described herein. In some embodiments, cells are isolated from subjects that are CMV (cytomegalovirus) serologically positive. In further embodiments, cells are isolated from blood or apheresis products from subjects before treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiotherapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodestructive agents such as CAMPATH, anti-CD3 antibodies, thitoxane, fludarabine, cyclosporine, FK506, mycophenolate, steroids, FR901228, and irradiation, and any number of other related treatments, including but not limited to these.In further embodiments, cells are isolated and frozen for later use in combination with (e.g., before, concurrently with, or after) T-cell depletion therapy using bone marrow or stem cell transplantation, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, cells may be isolated and frozen for later use in treatment following B-cell depletion therapy, such as with a drug that reacts with CD20 (e.g., rituxan).

[0175] In some embodiments, immune cells for regulation, including T, NK, or NKT cells and / or subpopulations thereof, are genetically engineered, which may include insertions, deletions, or nucleic acid substitutions. Modified immune cells may express cytokine transgenes, silent inhibitory receptors, or overexpressed activating receptors, or CARs for retargeting immune cells. In some embodiments, immune cell populations isolated for regulation from a subject or donor, or immune cell populations isolated from or contained in the peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue derived from the site of infection, ascites, pleural fluid, spleen tissue, or tumors of the subject / donor, can be genetically engineered. In some embodiments, the isolated immune cell populations and the regulatory immune cells obtained therefrom are genetically engineered, including insertions, deletions, and / or nucleic acid substitutions. In some embodiments, immune cells for regulation and the regulated immune cells obtained therefrom contain exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 or variants thereof.

[0176] In one embodiment, genome-engineered immune cells for regulation and the regulated immune cells obtained therefrom include one or more genetically modified forms comprising one or more proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and regulation of the immune response, and / or the survival of immune cells. In some other embodiments, the recombination modalities include (i) deletion or reduction of expression of one or more B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and deletion or reduction of expression of any HLA gene in the chromosome 6p21 region; (ii) introduction or increase of expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, or surface-induced receptors for binding to bi- or multi-specific or universal engagers. In some embodiments, T cells, NK cells, or NKT cells contain exogenous nucleic acids for regulation or to be regulated therefrom. In some embodiments, exogenous nucleic acids are introduced into immune cells via direct genome editing of the cells. In some other embodiments, exogenous nucleic acids are introduced into immune cells by retaining immune cells from genome-modified hematopoietic stem cells or progenitor cells or iPSCs, which differentiate to produce immune cells. In some embodiments, exogenous nucleic acids for T cells can encode TCRs (T cell receptors), CARs (chimeric antigen receptors), bispecific T cell engagers (BiTEs), trispecific T cell engagers, multispecific T cell engagers, or universal engagers that adapt to multiple immune cell types.In some embodiments, the exogenous nucleic acid for NK cells may encode TCR, CAR, CD16 or their variants, NY-ESO, λ bispecific killer cell engager (BiKE), tripspecific killer cell engager (TriKE), multispecific killer cell engager, or a universal engager adapted to multiple immune cell types. In some embodiments, the exogenous nucleic acid for NKT cells may be a modified TCR or CAR. In some embodiments, the exogenous nucleic acid CAR19 is encoded. In some embodiments, the CD16 variants include high-affinity CD16 (HACD16), uncleavable CD16, and high-affinity uncleavable CD16 (hnCD16).

[0177] In some embodiments, a population or subpopulation of immune cells for regulation differentiates in vitro from stem cells or progenitor cells. In some embodiments, an isolated population or subpopulation of T, NK, or NKT cells can differentiate from stem cells, hematopoietic stem cells, or progenitor cells (HSCs) or progenitor cells. Progenitor cells may be CD34+ hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, or NKT cell precursors. Stem cells may be pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). iPSCs are reprogrammed pluripotent cells that do not exist in nature. Once the cells of interest are reprogrammed into a pluripotent state, the cells can then be programmed or differentiated into a desired cell type or subtype, such as T, NK, or NKT cells.

[0178] In some embodiments, iPSCs that can be induced to exhibit a hematopoietic phenotype are differentiated into T, NK, or NKT cells by a multi-step differentiation platform, ranging from mesodermal stem cells to fully differentiated T, NK, or NKT cells. (See, for example, U.S. Patent Applications 62 / 107,517 and 62 / 251,016, the disclosures of which are incorporated herein by reference in their entirety.) In some embodiments, iPSCs, HSCs, or precursors for T, NK, or NKT cell differentiation are genomically engineered, which may include insertions, deletions, or nucleic acid substitutions.

[0179] In some embodiments, the genomically engineered iPSCs, HSCs, or hematopoietic progenitor cells include one or more of the following: safety switch proteins, targeting modes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and modulation of immune responses, and / or survival of iPSCs, HSCs, progenitor cells, or cells derived therefrom. In some other embodiments, the recombination modalities include (i) deletion or reduction of expression of any HLA gene in the chromosome 6p21 region, such as B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and (ii) introduction or increase of expression of surface-induced receptors for binding to HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, bi- or multi-specific or universal engagers, TCR (T cell receptor), or CAR (chimeric antigen receptor). In some embodiments, the precursors for iPSC, HSC, or T, NK, or NKT cell differentiation include modified HLA class I and / or II. In some embodiments, the precursors for T, NK, or NKT cell differentiation by iPSCs, HSCs, or modified HLA class I and / or II include at least one null or low-expression of B2M, HLA-E / G, PDL1, A2AR, CD47, LAG3, TIM3, TAP1, TAP2, tapasin, NLRC5, PD1, RFKANK, CIITA, RFX5, and RFXAP. In some embodiments, the precursors for T, NK, or NKT cell differentiation have exogenous nucleic acids.In some embodiments, the exogenous nucleic acid encodes a bispecific T cell engager (BiTE), a triplicate T cell engager, a multispecific T cell engager, CD16 or its variants, NY-ESO, a bispecific killer cell engager (BiKE), a triplicate 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 precursors for T, NK, or NKT cell differentiation. In some embodiments, the exogenous nucleic acid encodes CAR19 in iPSCs, HSCs, or precursors for T, NK, or NKT cell differentiation.

[0180] In some embodiments, a population or subpopulation of immune cells transdifferentiates in vitro from non-hematopoietic fate non-pluripotent cells to hematopoietic lineage cells, or from a primary hematopoietic cell type non-pluripotent cells to another hematopoietic cell type, although this transdifferentiation can be to a specific fully differentiated type of immune cell, such as T, NK, or NKT progenitor cells or T, NK, or NKT cells. (See U.S. 9,376,664 and U.S. Application 15 / 072769, the entirety of which is incorporated herein by reference). In some embodiments, non-hematopoietic fate non-pluripotent cells are somatic cells such as cutaneous fibroblasts, adipose tissue-derived cells, and human umbilical vein endothelial cells (HUVECs). Somatic cells useful for transdifferentiation may be immortalized somatic cells.

[0181] Various strategies are being pursued in cells to induce pluripotency or increase potency (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)), and 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., Plos Bio 6, e253. doi:10.1371 / journal. Pbio0060253(2008). Lyssiotis et al., PNAS 106, 8912-8917 (2009);M. Ichida et al., Cell Stem Cell 5, 491-503 (2009);M. Mareriali, N., Hochedlinger, K. Esteban et al., Cell Stem Cell 6, 71–79 (2010);M. These disclosures are incorporated herein by reference in their entirety.

[0182] III. Methods for Regulating Immune Cells for Adoption Therapy 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 immune cells with a composition containing at least one agent selected from Table 1.

[0183] In one embodiment, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting immune cells with a composition comprising at least one agent selected from Table 1, wherein the treated immune cells exhibit increased cell proliferation, an increased number or relative ratio of one or more desired cell subpopulations, improved proliferation, cytotoxicity, or cell recall response, and improved persistence compared to immune cells not contacted with the same composition.

[0184] In some embodiments, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting immune cells with a composition comprising at least one agent selected from Table 1, where the maintenance or proliferation of one or more desired cell subpopulations is improved compared to a population of immune cells not in contact with the agents of Table 1.

[0185] In some embodiments, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting immune cells with a composition comprising at least one agent selected from Table 1, where the number or proportion of populations reprogrammed to a desired differentiation state is increased compared to a population of immune cells not contacted with the agent from Table 1.

[0186] In some embodiments, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting immune cells with a composition containing at least one agent selected from Table 1 in an amount sufficient to increase cell proliferation, resulting in an increase in the number or proportion of one or more desired immune cell subpopulations and / or improved immune cell proliferation, cytotoxicity, cell recall response, and / or persistence compared to a population of immune cells not contacted with the agents of Table 1. In one embodiment, the immunotherapy agent is about 0.1 nM to about 50 μM. In one embodiment, the immunotherapy agent 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 in between. In one embodiment, the modifier for immunotherapy is approximately 0.1 nM to approximately 5 nM, approximately 1 nM to approximately 100 nM, approximately 50 nM to approximately 250 nM, approximately 100 nM to approximately 500 nM, approximately 250 nM to approximately 1 μM, approximately 500 nM to approximately 5 μM, approximately 3 μM to approximately 10 μM, approximately 5 μM to approximately 15 μM, approximately 12 μM to approximately 20 μM, or approximately 18 μM to approximately 25 μM, or any range in between.

[0187] In some embodiments, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves exposing immune cells to a composition comprising at least one modifier selected from Table 1 for a period of time sufficient to improve at least one desired therapeutic characteristic compared to immune cells not exposed to the same composition. In one embodiment, immune cells are exposed to one or more modifiers from Table 1 for a period of at least 10 minutes, 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 in between. In one embodiment, immune cells are in contact with one or more modifiers from Table 1 for a period of 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, and about 20 days to about 30 days. In some embodiments, immune cells are in contact with one or more of the aforementioned agents for a period of 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours or more, or any length in between. Such sufficient periods of time are, for example, 15, 13, 11, 9, 7, 5, 3, or 1 hour or more. In some other embodiments of the method, sufficient periods of time are 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours or more, or any length in between. Thus, the aforementioned sufficient time is, for example, 30, 42, 54, 66, 78, 90 hours or more.

[0188] A method for 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 contact, wherein one or more desired subpopulations include naive T cells, stem cell memory T cells, central memory T cells, adaptive NK cells, or type I NKT cells.

[0189] In some embodiments, the immune cells for modulation are obtained from subjects that are CMV serologically positive or have been previously administered genetically modified immune cells. In some embodiments, after modulation, the genetically modified immune cells obtained from the subject may be autologous or allogeneic. In some embodiments, the donor-derived immune cells for modulation include exogenous nucleic acids encoding T cell receptors (TCRs) and / or chimeric antigen receptors (CARs).

[0190] The population of immune cells to be processed may be isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, tumors of the subject / donor, or pluripotent stem cell populations. The subject may be healthy or may have autoimmune disorders, hematopoietic malignancies, viral infections, or solid tumors. The population of immune cells for regulation may be genomically engineered, and the cells may include insertions, deletions, and / or nucleic acid substitutions. In some specific embodiments, the genomically engineered immune cells may include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 or its variants.

[0191] Alternatively, a population of regulatory immune cells may be differentiated in vitro from stem cells, hematopoietic stem cells or progenitor cells, or from hematopoietic or non-hematopoietic non-pluripotent cells. In some embodiments, the stem cells, hematopoietic stem cells or progenitor cells, progenitor cells, or non-pluripotent cells from which the regulatory immune cells are derived are genomically engineered to include insertions, deletions, and / or nucleic acid substitutions, which are also included in the immune cells induced therefrom. In some specific embodiments, the induced immune cells include exogenous nucleic acids encoding T cell receptors (TCRs), chimeric antigen receptors (CARs), and / or CD16 or variants thereof.

[0192] IV. Therapeutic use of modified immune cells, immune cell populations, or subpopulations The present invention provides a therapeutic composition comprising an isolated population or subpopulation of immune cells contacted, treated, or modulated with one or more modifiers selected from Table 1 in an amount sufficient to improve the therapeutic capacity of immune cells when used in cell-based adoptive therapy. In one embodiment, the contacted, treated, or modulated immune cells obtain, compared to immune cells not contacted with the same agent, at least one desirable therapeutic property, including, but not limited to, increased cell proliferation; increased number or relative ratio of a desired cell subpopulation; improved proliferation, cytotoxicity, or cell recall response; and improved persistence. In some embodiments, one or more modifiers comprise at least one compound selected from Table 1, or a derivative or analog thereof. In one embodiment, the ex vivo contacted isolated population or subpopulation of immune cells comprises naive T cells, stem cell memory T cells, and / or central memory T cells with increased numbers or ratios. In one embodiment, the ex vivo contacted isolated population or subpopulation of immune cells comprises type I NKT cells with increased numbers or ratios. In another embodiment, the ex vivo contacted isolated population or subpopulation of immune cells comprises adaptive NK cells with increased numbers or ratios.

[0193] This specification also provides combination therapeutic compositions comprising the disclosed modified immune cells and one or more therapeutic additives / agents. In some embodiments of the combination therapeutic compositions, one or more therapeutic additives include peptides, cytokines, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs).

[0194] In some embodiments, the additional therapeutic agent comprises an antibody or 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 modulated cells to better recognize and lyse target cells. In some embodiments, the antigen as an additional therapeutic agent activates regulated NK cells to utilize antibody-dependent cell-mediated cytotoxicity (ADCC). A monoclonal antibody (mAb) binds to target cells and further binds CD16 to NK cells and other cell types, killing tumor cells by ADCC both in vivo and in vitro. mAbs can also enhance ADCC by blocking NK cell inhibition and stimulate NK cells. In some embodiments, NK cell-mediated ADCC is mediated by CD16 expressed by regulated NK cells and its genetically engineered variants. Genetically engineered variants of CD16 include, but are not limited to, uncleaved CD16, high-affinity CD16 (haCD16), and high-affinity uncleaved CD16 (hnCD16). Furthermore, the design of bi- and tri-specific antibodies that fuse the Fab region of antibodies targeting tumor cell antigens such as anti-CD19, CD20, and CD33 antigens with another Fab region that recognizes CD16 on NK cells results in stimulation of NK cells, followed by killing of tumor cells. In some embodiments, antibodies suitable for combination therapy with modulated immune cells provided herein include, but are not limited to, anti-CD20 (retiximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), anti-Her2 (trastuzumab), CD52 (aremtuzumab), anti-EGFR (cerutoximab), and anti-CD38 (daratumumab), as well as their humanized and Fc-modified variants.

[0195] In some embodiments, the additional therapeutic agent includes one or more of the following: a chemotherapeutic agent, a radioactive part, or an immunomodulatory agent.

[0196] Chemotherapy agents refer to cytotoxic anti-cancer agents, that is, those found to preferentially kill newly formed cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the proliferation of newly formed cells. Chemotherapy agents are also called antitumor agents or cytotoxic agents or drugs, and are well known in the art. In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamine, nitrogen mustard, nitrosourea, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, vinca alkaloids, epipodophyllotoxins, or interleukins. Examples of chemotherapeutic agents include, but are not limited to, cyclophosphamide, mechloretamine, mephalin, chlorambucil, hemamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine, methotrexate, fluorouracil, flokithizine, cytarabine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin, vincristine, vinblastine, vindesine, etoposide, etoposide orthoquinone, teniposide, daunorubicin, doxorubicin, mitoxantrone, bisanthren, actinomycin D, plicamycin, promycin, gramicidin D, paclitaxel, colchicine, cytochalacin B, emetine, mytansine, and amsacrine.Additional medications 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, and carsterone. Capecitabine, celecoxib, cetuximab, cladribine, cloflavin, cytarabine, dacarbazine, denileukin-diffitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinylestradiol, exemestane, floxyuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, Ibritumomab, idarubicin, ifosfamide, imatinib, interferon alfa (2a,2b), irinotecan, letrozole, leucovorin, leuprolide, rebamizole, mechloretamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pe Examples include gademase, pegaparagus, pentostatin, pipobromane, plicamycin, polyfeprosan, porfimer, procarbazine, quinacrine, rituximab, salglamostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tocitumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinorelbine, and zoledronate. Other suitable agents are approved for human use, including those known in the art, that are approved as chemotherapeutic or radiotherapeutic agents.Such drugs can be referenced in numerous standard physician and oncologist references (e.g., The Pharmacological Basis of Therapeutics, 9th edition, Goodman and Gilman, McGraw-Hill, NY, 1995) or on the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time. 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 modulated therapeutic immune cells for cancer treatment.

[0197] In one embodiment, modulated immune cells can be used to treat, prevent, or improve hematological malignancies, solid tumors, precancerous conditions, autoimmune disorders, or viral infections in subjects suitable for adoptive cell therapy by introducing or administering the modulated immune cells to the subjects. In another embodiment, modulated immune cells can be used to enhance antitumor immune responses by introducing the cells into subjects requiring such treatment.

[0198] The terms “to treat” and “treatment” are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic with respect to completely or partially preventing a disease, and / or partially or completely treating a disease and / or adverse effects resulting from the disease. As used herein, “treatment” includes any treatment of a disease in mammals and includes preventing a disease from developing in a subject that is susceptible to the disease but has not yet been diagnosed with the disease, suppressing the disease, i.e., preventing its development, or reducing the disease, i.e., causing a regression of the disease. Therapeutic agents or compositions may be administered before, during, or after the onset of a disease or injury. The treatment of an ongoing disease in which the treatment stabilizes or reduces undesirable clinical symptoms in a patient is also of particular interest. In certain embodiments, a subject in need of treatment having a disease, illness, and / or injury may be cured, improved, and / or improved in at least one associated symptom by cell therapy. Certain embodiments are intended to include, but are not limited to, subjects requiring cell therapy, candidates for bone marrow or stem cell transplantation, subjects who have previously received chemotherapy or radiotherapy, subjects who have or are at risk of having hyperproliferative disorders or cancer, such as hyperproliferative disorders or cancer of the hematopoietic system, subjects who have or are at risk of developing tumors such as solid tumors, and subjects who have or are at risk of having viral infections or diseases associated with viral infections.

[0199] Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes). Solid tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, multiple sclerosis, and pemphigus. Examples of conditions that may be treated include, but are not limited to, bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, several forms of thyroiditis, several forms of uveitis, vitiligo, and granulomatous disease with polyangiitis (Wegener). Examples of viral infections that may be treated include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (herpes zoster virus associated with Kaposi's sarcoma), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, and BK polyomavirus-related disorders.

[0200] Therapeutic compositions containing the disclosed modulated immune cells may be administered to a subject before, during, and / or after other therapies. Therefore, methods of combination therapy may include administering or preparing modulated cells before, during, and / or after the use of additional therapeutic agents. As described above, one or more therapeutic additives include peptides, cytokines, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive portions, or immunomodulatory agents (IMiDs). Administration of modulated immune cells may be temporally separated from the administration of additional therapeutic agents by several hours, several days, or even several weeks. Additionally or alternatively, administration may be combined with other bioactive agents or forms of non-pharmacological therapies, such as antineoplastic agents or surgery, but not limited to these.

[0201] It can modulate both autoimmune cells and allogeneic immune cells and can be used in cell therapy as described above.

[0202] In some embodiments, the number of regulated immune cells in the therapeutic composition is at least 0.1 × 10⁶ 5 individual cells, at least 1 × 10⁶ 5 Individual cells, at least 5 × 10 5 A single cell, at least 1 × 10⁶ 6 Individual cells, at least 5 × 10 6 individual cells, at least 1 × 10⁶ 7 Individual cells, at least 5 × 10 7 A single cell, at least 1 × 10⁶ 8 Individual cells, at least 5 × 10 8 A single cell, at least 1 × 10⁶ 9 A single cell, or at least 5 × 10⁶ 9 It is an individual cell.

[0203] In some embodiments, the number of regulated immune cells in the therapeutic composition is approximately 0.1 × 10⁶ 5 ~Approx. 1×10 6Each cell is approximately 0.5 × 10⁻⁶ 6 ~Approx. 1×10 7 Each cell is approximately 0.5 × 10⁻⁶ 7 ~Approx. 1×10 8 Each cell is approximately 0.5 × 10⁻⁶ 8 ~Approx. 1×10 9 A single cell, approximately 1 x 10⁻⁶ 9 ~Approx. 5×10 9 Each cell is approximately 0.5 × 10⁻⁶ 9 ~Approx. 8×10 9 It refers to individual cells, or any range between them.

[0204] In some embodiments, the number of regulated immune cells in the therapeutic composition is approximately 0.5 × 10⁶ 6 ~Approx. 1×10 6 Each cell is approximately 0.5 × 10⁻⁶ 7 ~Approx. 1×10 7 Each cell is approximately 0.5 × 10⁻⁶ 8 ~Approx. 1×10 8 Each cell is approximately 0.5 × 10⁻⁶ 9 ~Approx. 5×10 9 A single cell, approximately 1 x 10⁻⁶ 9 ~Approx. 8×10 9 It refers to individual cells, or any range between them.

[0205] In some other embodiments, the number of regulated immune cells in the therapeutic composition is approximately 0.1 × 10⁶ 5 ~about 0.5×10 6 Each cell is approximately 0.5 × 10⁻⁶ 6 ~about 0.5×10 7 Each cell is approximately 0.5 × 10⁻⁶ 7 ~about 0.5×10 8 Each cell is approximately 0.5 × 10⁻⁶ 8 ~about 0.5×10 9 Each cell is approximately 0.5 × 10⁻⁶ 9 ~Approx. 8×10 9 It refers to individual cells, or any range between them.

[0206] In one embodiment, the number of regulated immune cells in the therapeutic composition is the number of immune cells in a partial or single umbilical cord blood, or at least 0.1 x 10 5individual cells / kg body weight, at least 0.5 x 10⁻¹⁶ cells 5 individual cells / kg body weight, at least 1 x 10⁶ 5 individual cells / kg body weight, at least 5 x 10⁻¹⁴ 5 individual cells / kg body weight, at least 10 x 10 5 Cells per kg of body weight, at least 0.75 x 10⁻¹⁶ cells. 6 Cells per kg of body weight, at least 1.25 × 10⁻⁶ 6 Cells per kg of body weight, at least 1.5 × 10⁻⁶ 6 Cells per kg of body weight, at least 1.75 × 10⁻⁶ 6 individual cells / kg body weight, at least 2 × 10⁻⁶ 6 Cells per kg of body weight, at least 2.5 × 10⁻⁶ 6 individual cells / kg body weight, at least 3 × 10⁻⁶ 6 cells / kg body weight, at least 4 × 10⁻⁶ 6 cells / kg body weight, at least 5 × 10⁻⁶ 6 Cells per kg of body weight. At least 10 x 10 6 Cells per kg of body weight, at least 15 x 10⁻¹⁴ 6 individual cells / kg body weight, at least 20 x 10 6 individual cells / kg body weight, at least 25 x 10⁻¹⁴ 6 individual cells / kg body weight, at least 30 x 10 6 individual cells / kg body weight, 1 x 10⁻⁶ 8 Cells per kg of body weight, 5 × 10⁻⁶ 8 cells per kg of body weight, or body weight 1 × 10⁶ 9 cells / kg body weight, or 8 × 10⁶ 9 It is the number of cells per kg of body weight.

[0207] The regulated immune cells provided by the present invention can be administered to a subject without being proliferated ex vivo or in vitro before administration. In certain embodiments, the regulated immune cell population can be washed to remove the regulator. In some embodiments, an isolated population of regulated immune cells derived from hematopoietic lineage cells can be recombinantly generated to express TCR, CAR or other proteins.

[0208] A therapeutic composition suitable for administration to a patient may contain one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable culture media, e.g., cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition to be administered, as well as the specific method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the present invention (see, for example, Remington's Pharmaceutical Sciences, Vol. 17, 1985, the disclosure of which is incorporated herein by reference in whole).

[0209] In certain embodiments, the therapeutic cell composition having an isolated population of regulated cells also comprises a pharmaceutically acceptable cell culture medium or a pharmaceutically acceptable carrier and / or diluent. Therapeutic compositions comprising populations of regulated immune cells, such as those disclosed herein, may be administered intravenously, intraperitoneally, enterally, or tracheally, or separately in combination with other suitable compounds that affect the desired therapeutic target.

[0210] These pharmaceutically acceptable carriers and / or diluents may be present in amounts sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Such buffers may be present in amounts of about 5% by weight of the total composition. Electrolytes, such as but not limited to sodium chloride and potassium chloride, may also be included in the therapeutic composition. In one embodiment, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH in one of the aforementioned pH ranges. In yet another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0211] The present invention also, in part, provides the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium supporting the maintenance, proliferation, and / or health of regulated immune cells of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is serum-free and / or feeder-free medium.

[0212] In various embodiments, serum-free media may be animal-free and optionally protein-free. In some cases, the medium may contain biopharmaceutically acceptable recombinant proteins. An animal-free medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially protein-free. Those skilled in the art will understand that the above examples of media are illustrative and do not limit the formulation of media suitable for use in the present invention, and that there are many suitable media available that are known to those skilled in the art. [Examples]

[0213] The following examples are provided for illustrative purposes only and not for limitation.

[0214] Example 1 - Method and Materials In vitro T cell culture. Fresh leukocytes (AllCells, Alameda, California) were obtained from a healthy donor, and T cells were negatively selected using the EasySep human T cell enrichment kit (Stem Cell Technologies, Vancouver, Canada). The newly isolated T cells were divided equally and cryopreserved. On the day screening began, the T cells were thawed and washed in an X-Vivo15 with 5% human AB serum, IL-2, pen / strep, and additional supplements. The cells were then refueled in 5 × 10⁶ cells with anti-CD3 / anti-CD28 beads. 5Cells / ml were dispensed into flat-bottomed 384-well plates. Individual compounds were added to each well from row 3 to row 22 of each plate at a final concentration of 10 μM. Positive and negative controls were added to additional wells. Cells were incubated in 5% CO2 at 37°C for approximately 6 days.

[0215] On day 6 of flow cytometry culture, cells were stained with fixed viability markers and fluorophore conjugate antibodies: CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD27, and CD122 (BD Biosciences, San Jose, California, and BioLegend, San Diego, California). Fluorescent absolute counting beads (Spherotech, Lake Forest, IL) were added immediately before acquisition. Data acquisition was performed using a BD Fortessa X-20 (BD Biosciences), and the data were analyzed using Treestar software (FlowJo, Ashland, OR) and Spotfire (Tibco, Boston, MA).

[0216] Large-scale cell culture for phenotypic and depletion marker evaluation. Isolated CD8 T cells were activated in large quantities on day 0 using anti-CD3 / anti-CD28 beads in IL-2 supplemented T cell medium. On day 1, cells were transduced with a CAR construct, and the cell density was increased to 0.5 × 10⁶. 6 Adjust to / ml, 10 6 Cells were seeded in 12-well plates in the presence of a vehicle, TWS119, or DCC-2036. On day 4, the cells were transferred to 6-well plates, and 2 ml of T cell medium was added to each well. On day 6, an additional 2 ml of medium was added to each well. On day 8, CAR T cells were analyzed by flow cytometry for surface expression of phenotypic markers (CD62L, CCR7, and CD27) and consumable markers (PD-1 and Tim-3). Example 2 - Immunotherapy

[0217] Data were analyzed to identify compounds that produce 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 co-expressing both of these identification markers were evaluated. The percentage of cells co-expressing CCR7 and CD62L within the viable CD4+ and viable CD8+ populations was determined. The expression of either CD62L or CCR7 on T cells exhibiting a desired T cell subset has been described as having favorable functional characteristics for CAR-T cell therapy and potentially other adoptive T cell therapies. Under treatment with dolsomorphine, hepteridine, GSK-3 inhibitor IX, 6-mercaptopurine, AC-93253 iodide, thiratrichol, PI-103, 5-azacitidine, 5,7-dichloro-8-quinolinol, nitrofurantoin, 5-chloro-7-iodo-8-quinolinol, or diethylenetriaminepentaacetic acid, the number or proportion of cells co-expressing CCR7 and CD62L 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-octaf, nifloxazide, and edrophonium chloride, the number or proportion of cells co-expressing CCR7 and CD62L increased, at least in the viable CD8+ population (Table 2). Under treatment with 1-pyrrolidinecarboditioic acid, ammonium salt, U0126, telmisartan, cyclosporine A, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY 61-3606, protoporphyrin IX disodium, rapamycin, roscovitine, PAC-1, tosufloxacin hydrochloride, BIX01294, and terfenadine, the number or proportion of cells co-expressing CCR7 and CD62L increased, at least in the viable CD4+ population (Table 2).

[0218] Furthermore, GSK3β (glycogen synthase kinase 3 beta) inhibitors have been shown to conserve CD37-CD19-CD56+ NK cells and increase adaptive NK cell subpopulations by influencing cell maturation and subtype skew, based on observations including but not limited to CD57+ and NKG2C+ expression.

[0219] The number of events at each of these gates relative to the absolute count beads in each sample was calculated to define a measure of the absolute number of naive, stem cell memory, or central memory T cells in the CD4+ and / or CD8+ populations. Z-scores were calculated for each of these four values ​​for the screened compound samples in each 384-well plate: 1) percentage CCR7+CD62L+ in CD4+, 2) percentage CCR7+CD62L+ in CD8+, 3) absolute relative number of CCR7+CD62L+ in CD4+, and 4) absolute number of CCR7+CD62L+ in CD8+ (Figures 1A and 1B). Z-scores were also calculated for the percentage of total cell viability in each sample and the relative absolute number of cells in each sample. A "Z-score" is a statistical measure of the relationship of a score to the mean in a group of scores. A Z-score of 0 means the score is the same as the mean. Z-scores can also be positive or negative, indicated by whether it is above or below the mean and by the number of standard deviations.

[0220] Eliminating compounds that have a detrimental effect on T cell proliferation or viability allows us to focus our efforts on the compounds most likely to be suitable for T cell production strategies. Primary hit compounds were selected based on the following criteria: a Z-score greater than -1 for "percentage viability", a Z-score greater than -1 for "absolute cell number", and a Z-score greater than +2 for one of the four values. Thirty-four compounds (Table 2) were selected because they had significantly higher Z-scores and met the above criteria for one or more of the four primary values. Five additional compounds were also included for their ability to modulate T cells (Table 3). Table 2 - Agents for T cell regulation in adoptive cell therapy [Table 2-1] [Table 2-2] [Table 2-3] Table 3 - Additional agents for T cell regulation in adoptive cell therapy [Table 3]

[0221] Example 3 - In vitro triage experiment of selected compounds In vitro experiments are conducted to optimize the method of compound exposure and to eliminate compounds that have adverse effects on T cell function. Initial tests assess whether previously observed effects on naive T cells, stem cell memory T cells, and central memory T cells are reflected in additional donors, while determining the optimal dose of individual compounds. In vitro assessments are performed for proliferative capacity, ability to polarize to Th1 and Th17, survival through cryopreservation / thawing cycles, transduction efficiency, and tumor-killing activity of CAR-transduced T cells to identify compounds that may have adverse functional effects on T cells. Compounds are tested in combination to reproducibly improve the ratio or number of proliferating naive, stem cell memory, or central memory T cells without significant adverse effects on T cell function, and additive or synergistic effects are evaluated. Through these evaluations, lead candidates or combinations are prioritized for further in vivo testing.

[0222] Example 4 - In vivo model of adoptive cell therapy using selected compounds To translate the results of in vitro screening and track in vitro triage experiments, we will apply the primary candidate compounds selected to in vivo models of adoptive cell therapy. Specifically, we will investigate the effects of small molecule modulation in adoptive cell therapy with respect to engraftment, tumor-killing activity, secondary tumor-killing response, migration, cell persistence, and graft-versus-host disease. We will also examine other information that characterizes durable adoptive cell therapy, which has been found to correlate with effective responses in the clinical setting.

[0223] These experiments are conducted in either a humanization system in which human cells are adopted into immunodeficient NSG mice with human tumors, or in a surrogate mouse model in which immunocompetent animals carry syngeneic tumors and are treated with syngeneic cell therapy.

[0224] In either the surrogate or humanized model system, mice are injected with lucifarinizing lymphoma or other tumors of interest. Immediately thereafter, adoptive cell therapy is administered, pre-treated with the vehicle or modulating compound disclosed herein. Both the cell therapy and tumor doses are optimized to allow for a range in which the favorable or adverse effects of the compound therapy may be observed. Cells treated with the vehicle or compound were characterized prior to administration, and animal body weight and tumor load were measured regularly throughout the study period.

[0225] Compounds that can improve one or more tumor-related parameters in vivo have expected effects including, but not limited to, a reduction in the cell therapy dose required for effective tumor clearance, increased persistence of adoptive cell therapy in peripheral blood, enhanced migration to tumor sites, and / or increased survival against attack by high tumor doses. Example 5 - DCC-2036 exhibits a bias towards the Tcm phenotype.

[0226] CD27 is a member of the TRA-binding TNF (tumor necrosis factor) receptor family, which also includes 4-1BB and OX-40. These transmembrane proteins are involved in regulating lymphocyte function. In humans, most naive peripheral T cells (Tn) express CD27. Once naive peripheral T cells are activated, CD27 expression increases significantly. However, terminal effector differentiation of T cells is associated with the irreversible loss of CD27 (Hintzen et al., 1994). Further elucidation of the role of CD27 has demonstrated that it is necessary for the development and long-term maintenance of T cell immunity (Hendriks et al., 2000). Furthermore, naive (Tn), stem cell memory (Tscm), and central memory (Tcm) T cells characterized by the expression of CCR7 and CD62L markers mediate superior antitumor activity in both mouse models (Sommermeye et al., 2015) and non-human primate models (Berger et al., 2008).

[0227] To determine the effect of compound treatment on distortion of the central memory phenotype, isolated CD8 T cells were collectively activated on day 0 using anti-CD3 / anti-CD28 beads in T cell medium supplemented with IL-2. On day 1, the cells were transduced with CAR construct 1 shown in Figure 2, and the cell density was increased to 0.5 × 10⁶. 6 Adjust to 10 pieces / ml. 6 Cells were seeded at the same concentration in 12-well plates in the presence of vehicle (DMSO), TWS119, or DCC-2036. On day 4, the cells were transferred to 6-well plates and 2 ml of medium was added to each well. On day 6, an additional 2 ml of medium was added to each well. On day 8, the cells were analyzed by flow cytometry for CD27 cell surface expression.

[0228] CD27 expression in compound-treated T cells and control-treated T cells is shown in Figure 4B. CD27 expression observed in vehicle treatment was defined as the mean fluorescence intensity (MFI) of 1, and the relative MFI of compound-treated cells was calculated.

[0229] Compared to the vehicle, DCC-2036 treatment resulted in a 2.3-fold increase in the frequency of CAR-T cells expressing both CD62L and CCR7, while TWS119 treatment resulted in a 1.7-fold increase in CD62L and CCR7 double-positive cells (Figure 4A). In addition to altering CD62L and CCR7 expression, DCC-2036 also caused a 2.2-fold increase in the expression of the differentiation marker CD27 compared to the vehicle (Figure 4B). In contrast, TWS119 did not affect CD27 expression.

[0230] Example 6 - DCC-2036 treatment reduces T cell depletion marker expression. T-cell dysfunction due to "waste" is a condition that can impair the proper control of cancer and infections. T-cell waste is collectively referred to as waste markers, including PD-1 and Tim-3, and is characterized by decreased effector cell function and increased expression of multiple cell surface proteins (Wherry and Kurachi 2015).

[0231] To determine the effect of compound treatment on the expression of depletion markers in CAR-T cells, cells were prepared from three different donors and treated as described in the protocol above. PD-1 and Tim-3 expression were measured using flow cytometry. TWS119 control reduced PD-1 expression 3.3-fold compared to the vehicle, while DCC-2036 treatment resulted in a 4.5-fold reduction in PD-1 surface expression (Figure 6B). Tim-3 expression was reduced 2-fold in TWS119 and 3.7-fold in DCC-2036 treatment (Figure 6A). These data indicate that T cell treatment with DCC-2036 enhances the antitumor capacity of cells by reducing T cell depletion that contributes to immune dysfunction.

[0232] Example 7 - Characterization of gene expression in CD8+ T cells treated with DCC-2036 To characterize the effects of DCC-2036 on memory cell distortion, we examined gene expression changes for a panel of key genes previously known to be highly expressed in memory T cells (i.e., CCR7, CD62L, etc.). Cryopreserved CD8+ T cells from five different donors were thawed and washed in X-Vivo 15 with 5% human AB serum, IL-2, pen / strep, and additional supplements. Cells were then refrigerated with anti-CD3 / anti-CD28 beads in a 5 × 10⁶ dialysis. 5 Cells were dispensed into flat-bottomed 384-well plates at a concentration of cells / ml. Cells were treated with either a final concentration of 1 μM DCC-2036, 10 μM TWS-119, or a control treatment with vehicle (DMSO) alone. Cells were incubated under 5% CO2 at 37°C for approximately 6 days.

[0233] After incubation, cells were lysed, RNA was isolated and fragmented, and cDNA was generated in the presence of a suitable sequencing adapter and sequenced using Illumina's next-generation sequencing technology (NGS). RNA sequencing (RNA-Seq) utilizes the capabilities of NGS to provide quantitative gene expression measurements from the transcriptome of a given sample, as well as to obtain nucleotide sequence information. Gene transcripts were normalized to counts / million reads (CPM) per sample, and the relative quantification of each gene transcript was determined in each treatment compared to the corresponding vehicle control treatment sample. Figure 5 shows the relative quantification of gene expression of several key memory T cell genes when DCC-2036 treatment induced increased expression at levels similar to or greater than that of TWS-119 (a known central memory distortion control compound). The observed results indicate that treatment with DCC-2036 promotes expression changes in the bulk CD8+ cell population that are consistent with distortion towards the central memory T cell expression pattern. Table 5 - DCC-2036 and related genes differentially expressed under control therapy [Table 4]

[0234] Example 8 - Demonstration of improvement of CAR-T cell phenotype by DCC-2036 in large-scale culture settings. To determine the effect of DCC-2036 on CD8 T cells and to demonstrate that increased expansion with DCC-2036 expands to a larger expansion format, CD8 T cells were separately activated and proliferated in vitro. Cells were transduced with a CAR-2 construct (Figure 2) one day after activation and then seeded in 24-well GREX plates with or without the compound. Half of the medium was replaced every two days thereafter until harvesting one week after activation. Cells were activated and proliferated for 6 days in the presence of the compound or vehicle. DCC-2036 was shown to improve CAR-T cell viability (Figure 3A) and proliferation (Figure 3B).

[0235] Beneficial clinical parameters that can demonstrate the potential for success of adoptive cell therapy in clinical trials in several contexts include the ability and extent of transplanted cells to expand in patients after treatment, as well as / or persistence and continued functionality after initial expansion, for example, in the context of sequential exposure to an antigen. The ability of engineered T cells to persist and continue to proliferate across multiple rounds of antigen stimulation may indicate such features and / or potential in vivo function or clinical response. In vitro serial killing / restimulation assays were used as a model system to evaluate T cells treated with each compound for various functions, including the ability to kill tumor cells expressing the target antigen in vitro and proliferation, across multiple exposures to and withdrawal from the antigen. Such assays were performed on cells treated with or without the compound to assess the effect of individual compounds on cellular function after repeated encounters with the antigen, and cell proliferation or persistence in such contexts.

[0236] CD8 T cells were activated, transduced, and proliferated in the presence of DMSO, TWS119, or DCC-2036. At the end of proliferation, the CAR-T cells were cryopreserved. The CAR-T cells were thawed and a serial restorative assay was performed by co-culturing them with irradiated K562 tumor cells expressing CD19 (the antigen recognized by CAR). Before initiating each round of killing, the cell count was adjusted so that the number of CAR-T cells was substantially the same from the cultures produced by DMSO, TWS119, and DCC-2036 treatment, respectively. The same number of irradiated CD19-expressing K562 target cells were added to the different CAR-T cultures.

[0237] Figure 3C shows that in rounds 3 and 4 of the serial restimulation assay, the cell number increase of CAR-T cells treated with DCC-2036 was significantly higher than that of the vehicle control. Vehicle-treated cells showed a significantly decreased expansion, while TWS119-treated cells showed a moderate increase in expansion compared to the vehicle (Figure 3C). Thus, treatment with DCC-2036 enables increased proliferation of CAR-T cells through multiple rounds of killing / restimulation. The total expansion of CAR-T cell numbers across all four rounds under each compound treatment was also determined, and DCC-2036-treated CAR-T cells showed a much greater expansion compared to the vehicle control (Figure 3D). In summary, these data indicate that DCC-2036 treatment can provide the desirable effect of continuously increased proliferation of treated CAR-T cells despite repeated exposure to target tumor cells. Killing capacity, along with cell proliferation shown over longer periods, is an indicator of the cellular persistence of the treated CAR-T product.

[0238] Further studies have shown that the addition of TWS119 or DCC-2036 reduces the expression of T cell depletion markers Tim-3 (Figure 6A) and PD-1 (Figure 6B) on CD8 T cells compared to DMSO, one week after activation.

[0239] Example 9 - Improved in vivo efficacy of cryopreserved DCC-2036-treated CAR-T cells To determine whether DCC-2036 treatment increases tumor clearance and CAR-T cell persistence in vivo, compound-treated CAR-T cells were evaluated in NSG mice using CD19 CAR-T cells and Nalm-6-luc tumor cells. NSG mice were injected with Nalm-6-luc, a CD19+ human tumor line engineered to express firefly luciferase. CAR-T cells were generated from separately activated and expanded CD4+ and CD8+ T cells in vitro. The cells were transduced with the CAR-2 construct (Figure 2) one day after activation and then seeded in 24-well GREX plates with or without the compound. Half of the medium was then changed every two days until collection one week after activation. The cells were cryopreserved and then thawed for use. NSG mice were injected with tumor cells and received CAR-T transplantation via retro-orbital injection one week later. Four days after IV injection of Nalm-6-luc cells, the mice were treated with 2.0×10 5 CAR-T cells (1:1 CD4:CD8) per animal. The mice were imaged periodically to determine tumor burden. Figures 7 and 8 show that mice in the DCC-2036 treatment group had a dramatic reduction in tumor burden compared to mice that received CAR-T cells grown in the presence of TWS119 or DMSO. Mice that received CAR-T cells grown in the presence of TWS119 or DMSO showed minimal tumor control.

[0240] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein represent 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 presented herein without departing from the scope and spirit of the invention.

[0241] All patents and publications referred to herein are indicative of the level of those skilled in the art to which the disclosure pertains. All patents and publications are hereby incorporated by reference into this specification to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0242] The disclosures described herein as exemplary may also be adequately implemented without any elements or limitations not specifically disclosed herein. Therefore, for example, in each example herein, the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. The terms and expressions used are for illustrative purposes only and are not intended to limit, nor is there any intention in the use of such terms and expressions to exclude equivalents of the exhibited and described features or any part thereof, and it should be recognized that various modifications are possible within the scope of the disclosure as defined in the claims. Thus, while the disclosure is specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein are frequently made by those skilled in the art, and such modifications and changes are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. A composition for adoptive immunotherapy, wherein the composition is (a) A population of immune cells, and a BCR-ABL tyrosine kinase inhibitor (TKI), (b) comprising a population of T cells derived from the immune cell population of the composition, (c) The T cell population shows increased expression of one or more of CD62L, CCR7, and CD27 compared to the immune cell population before contact with BCR-ABL TKI. (d) The T cell population is CD4 ＋ T cells or CD8 ＋ Including T cells, Composition for adoptive immune cell therapy.

2. The composition according to claim 1, wherein the T cell population comprises an increased number of naive T cells, stem cell memory T cells, and / or central memory T cells compared to the immune cell population before contact with BCR-ABL TKI.

3. The composition according to claim 1, wherein the immune cell population is isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue derived from the site of infection, ascites, pleural fluid, splenic tissue, or tumor.

4. The composition according to claim 1, wherein the immune cell population is isolated from (a) a healthy subject; or (b) a subject having an autoimmune disorder, hematopoietic malignancy, viral infection, or solid tumor.

5. The aforementioned immune cell population (a) Genomically engineered, including insertions, deletions, or nucleic acid substitutions; (b) comprising exogenous nucleic acids encoding T cell receptors (TCRs) and / or chimeric antigen receptors (CARs); (c) Stem cells, hematopoietic stem cells or progenitor cells, or cells differentiated from progenitor cells; or (d) The composition according to claim 1, which is trans-differentiated from non-hematopoietic non-pluripotent cells.

6. The composition according to claim 5, wherein the stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

7. The composition according to claim 6, wherein the progenitor cells are pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, or NKT cell progenitor cells.

8. The aforementioned stem cells, hematopoietic stem cells or progenitor cells, or progenitor cells, (a) Genomically engineered, including insertions, deletions, or nucleic acid substitutions; (b) The composition according to claim 5, comprising an exogenous nucleic acid encoding a protein including a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

9. The composition according to claim 1, wherein the BCR-ABL TKI comprises at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), PD173955, and their analogues.

10. The composition according to claim 1, wherein the BCR-ABL TKI comprises DCC-2036.

11. Compared to the immune cell population before contact with BCR-ABL TKI, (a) Increased expression of CD62L and CCR7; (b) Decreased expression of at least one of PD-1 and Tim-3; (c) Increase in the central memory T cell subpopulation; (d) Decrease in effector T cell subpopulation; (e) Improvement of proliferation and survival rate; or (f) Improvement of tumor clearance and persistence The composition according to claim 1, characterized by one or more of the following.

12. A composition according to any one of claims 1 to 11, for the treatment of a patient having an autoimmune disorder, hematological malignancy, solid tumor, cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

13. A method for preparing a T cell population for use in adoptive immunotherapy, (a) Exposing an immune cell population to a BCR-ABL tyrosine kinase inhibitor (TKI) for a sufficient amount of time to obtain a T cell population; (b) Formulating the T cell population into a pharmaceutical composition for administration to a target; Includes, (i) The T cell population shows increased expression of one or more of CD62L, CCR7, and CD27 compared to the immune cell population before contact with BCR-ABL TKI, and (ii) The T cell population shows increased expression of CD4 ＋ T cells or CD8 ＋ A method involving T cells.

14. The method according to claim 13, wherein the T cell population includes an increased number of naive T cells, stem cell memory T cells, and / or central memory T cells compared to the immune cell population before contact with BCR-ABL TKI.

15. The method according to claim 13, wherein the immune cell population is isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, or tumor.

16. The method according to claim 13, wherein the immune cell population is isolated from (a) a healthy subject; or (b) a subject having an autoimmune disorder, hematopoietic malignancy, viral infection, or solid tumor.

17. The aforementioned immune cell population (a) Genomically engineered, including insertions, deletions, or nucleic acid substitutions; (b) comprising exogenous nucleic acids encoding T cell receptors (TCRs) and / or chimeric antigen receptors (CARs); (c) Stem cells, hematopoietic stem cells or progenitor cells, or cells differentiated from progenitor cells; or (d) The method according to claim 13, wherein the cells are trans-differentiated from non-hematopoietic non-pluripotent cells.

18. The method according to claim 17, wherein the stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

19. The method according to claim 17, wherein the progenitor cell is a pluripotent progenitor cell, a T cell progenitor cell, an NK cell progenitor cell, or an NKT cell progenitor cell.

20. The aforementioned stem cells, hematopoietic stem cells or progenitor cells, or progenitor cells, (a) Genomically engineered, including insertions, deletions, or nucleic acid substitutions; (b) The method according to claim 17, comprising an exogenous nucleic acid encoding a protein including a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

21. The method according to claim 13, wherein the BCR-ABL TKI comprises at least one of DCC-2036 (levastinib), imatinib (STI571), nilotinib (AMN107), dasatinib (BMS-345825), bosutinib (SKI-606), ponatinib (AP-24534), bafetinib (INNO-406), PD173955, and their analogues.

22. The method according to claim 13, wherein the BCR-ABL TKI includes DCC-2036.

23. Compared to the immune cell population before contact with BCR-ABL TKI, (a) Increased expression of CD62L and CCR7; (b) Decreased expression of at least one of PD-1 and Tim-3; (c) Increase in the central memory T cell subpopulation; (d) Decrease in effector T cell subpopulation; (e) Improvement of proliferation and survival rate; or (f) Improvement of tumor clearance and persistence The method according to claim 13, characterized by one or more of the above.

24. The method according to claim 13, for the treatment of a patient having an autoimmune disorder, hematological malignancy, solid tumor, cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

Images