Prepared natural killer cells and uses thereof

Abstract

Provided herein are methods for producing an isolated population of prepared NK cells. In particular embodiments, prepared NK cells are expanded for a certain duration of time. In particular embodiments, prepared NK cells are expanded in one or more certain media(s). Further provided herein are compositions for preparing NK cells, compositions comprising prepared NK cells, and methods for the treatment of disease by administering the prepared NK cells.

Description

PREPARED NATURAL KILLER CELLS AND USES THEREOFCLAIM OF PRIORITY

[0001] This Application claims the benefit of U.S. Provisional Patent Application No. 63 / 631,838, filed on April 9, 2024, and U.S. Provisional Patent Application No. 63 / 648,920, filed on May 17, 2024, the contents of both of which are incorporated by reference herein in their entirety.TECHNICAL FIELD

[0002] The present disclosure concerns at least the fields of cell biology, molecular biology, medicine, and immunology.BACKGROUND

[0003] Natural killer (NK) cells have been studied as potential anti-tumor effectors, yet a number of barriers limit their therapeutic exploitation, such as their small numbers, requiring ex vivo expansion for adoptive immunotherapy. Ex vivo NK cell expansion is often poor compared to ex vivo expansion of other kinds of immune cells, and NK cells are susceptible to telomere shortening and senescence after only a few passages. There is an unmet need for improved strategies to produce highly functional prepared NK cells in sufficient number for in vivo therapeutic applications.SUMMARY

[0004] The present disclosure provides, among other things, compositions and methods useful for preparing (such as but not limited to, pre-activation, expansion, culturing, etc.) NK cells with superior characteristics, including, e.g., improved longevity, increased transgene transduction efficiency, improved metabolic profiles, improved NK cell engraftment, and enhanced anti-tumor cytotoxicity, exemplified both in vitro and in vivo using various cancer models. The present disclosure also includes, in some embodiments, compositions and methods comprising the NK cells with superior characteristics. The present disclosure is based, in part, on the observation that in certain embodiments, superior NK cells result from sequentially culturing NK cells in different media (such as but not limited to, pre-activation media, activation, and expansion media), each supplemented with cytokines (such as but not limited to, IL-2, IL-12, IL-15, and / or IL-18), growth factors (such as but not limited to, tumor growth factor beta TGFP), antigen presenting cells (such as but not limited to, artificial antigen presenting cells [aAPCs], universal antigen presenting cells [uAPCs], and / or feeder cells) oractivating beads, inhibitors (such as but not limited to, mTOR inhibitors, PI3K inhibitors, AKT inhibitors, IDH1 inhibitors, IDH2 inhibitors, and / or IDH1 / 2 inhibitors) for different lengths of time. Specifically, the some embodiments of the present disclosure are based, in part, on the insight that superior NK cells are produced by culturing naive NK cells in (i) pre-activation media (e.g., overnight) including IL-12, IL-15, and IL18, and subsequent culture in (ii) activate and expand media (e.g., for 7 to 14 days), including TGFp, IL-2, IL-12, IL-18, and antigen presenting cells (e.g., universal antigen presenting cells), and which can optionally comprise mTOR inhibitors (e.g., rapamycin), and PI3K inhibitors (e.g., Idelalisib). Among other things, the methods described herein solve multiple problems associated with producing high quantities of immunologically activated NK cells with in vitro and / or in vivo anti-tumor cytotoxicity, high functionality, and longevity.

[0005] Certain embodiments of the present disclosure provide methods and compositions concerning the production of natural killer (NK) cells and the use thereof for cell therapies. Certain embodiments of the present disclosure provide methods and compositions concerning the production of pre-activation (pre-activation may be optional) and expansion of NK cells and the use thereof for cell therapies. Certain embodiments of the present disclosure provide methods and compositions concerning the production of NK cells and the use thereof for cell therapies. In specific embodiments, certain aspects of the methods of the disclosure concern particular durations in time for one or more steps of the production methods. In specific embodiments, the present disclosure concerns the impact of days in culture on the in vivo function of CAR NK cells. In certain embodiments, the duration of an expansion step for the NK cells is of a particular length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, and in particular embodiments the duration of an expansion step is 9 days. In certain embodiments, an expansion step comprises at least two culturing steps, a first culturing step, and a second culturing step. In certain embodiments, a first culturing step comprises exposure of NK cells to APCs and / or feeder cells. In certain embodiments, a second culturing step does not comprise exposure of NK cells to APCS and / or feeder cells. In certain embodiments, an expansion step may comprise one or more transformation and / or transduction steps. In specific embodiments, NK cells are exposed to APCs for a particular length in time, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, and in particular embodiments the duration is exposure to APCs is 4, 5, 6, 7, 8, or 9 days. In particular embodiments, the duration of exposure to APCs is about 5-7 days, or about 6 days. In certain embodiments, the duration of exposure to APCs is less than or equal to about 7 days. In certain embodiments, during and following transformation and / or transduction of NK cells, the NK cells are not exposed to APCs and / or feeder cells.Compositions of Day 9-frozen NK cells, including that express one or more engineered antigen receptors, are encompassed herein.

[0006] In particular embodiments, there is an in vitro method for preparing natural killer (NK) cells comprising optionally pre-activating a population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; expanding NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said culture comprising: (1) IL-2; (2) antigen presenting cells (APCs); (3) one or more of IL-12, IL-15, and IL-18; (4) activating beads; or (5) a combination thereof; optionally transducing the cells with an engineered construct; culturing the preactivated NK cells in a culture comprising IL-2 and optionally aAPCs to produce prepared NK cells; and optionally exposing the cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated NK cell. In specific embodiments, the expanding step is for about 8-10 days, such as for about 9 days. In some embodiments, the culturing step is for 1, 2, 3, 4, or more days. In some embodiments, the culturing step is for less than about 3, 4, or 5 days.

[0007] In particular embodiments, no further aAPCs are provided in a culture following the expanding step. The aAPCs may be removed from the culture following the expanding step. In certain embodiments, the aAPCs are removed from the culture following the first culturing step. In certain embodiments, the aAPCs are removed from the culturing through purification / selection of NK cells. The aAPCs may be removed from the culture by about 5, 6, 7, 8, or 9 days from the beginning of the expanding step. In certain embodiments, the first and / or second culturing step is in the absence of aAPCs. In particular embodiments, the method includes the pre-activation step, transforming the cells with an engineered construct and / or exposing the cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated NK cell. In specific embodiments, the expansion culture step one and / or culture step two comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 U / mL, or any range or value derivable therein, of IL-2. In specific embodiments, the expansion culture step one and / or culture step two comprises about 200 U / mL of IL-2. In some embodiments, the culturing step occurs in the expansion culture. The culturing step may lack aAPCs. In some embodiments, the exposing to one or more deactivating agents occurs in the presence of IL-2. The exposing step may occur for about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours or any range or value derivable therein. The pre-activating step may be about 12, 14, 16, 18, 24, or 28 hours or any range or value derivable therein. In specific embodiments, the pre-activating step is about 16 hours.

[0008] In some embodiments, the method further comprises obtaining the prepared NK cells, freezing the prepared NK cells, or both. In specific embodiments, the method further comprises the step of administering the cells to an individual in need thereof. The prepared NK cells may be cryopreserved and are thawed prior to administering the cells to an individual in need thereof.

[0009] In specific embodiments, the engineered construct comprises a sequence encoding an engineered antigen receptor, such as a chimeric antigen receptor, a T cell receptor, or both. In addition, or alternatively, the engineered construct can comprise a sequence that results in expression of a cytokine, such as IL-15, IL-21, IL-4, IL-7, IL-9, IL-10, IL-27, or combinations thereof (e.g., a combination of two cytokines). The transducing step may occur at any time during the method, but in specific embodiments it occurs during the expansion step. It may occur on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of an expansion step. In certain embodiments, the transducing step occurs on or about day 4, day 5, day 6, or day 7 of the expansion step. In certain embodiments, the transducing step occurs in the absence of aAPCs and / or feeder cells. In certain embodiments, the NK cells are exposed to the transducing agent for less than or equal to about 5 days, 4 days, 3 days, 2 days, or 1 day.

[0010] In specific embodiments, the NK cells are exposed to one or more deactivating agents. In some embodiments, the NK cells are exposed to one or more deactivating agents prior to freezing. Any method encompassed herein may include treating an NK cell with an effective amount of one or more deactivating agents under conditions to produce a deactivated NK cell. The deactivating agent may be a kinase inhibitor. The deactivating agent may be a mechanistic target of rapamycin (mTOR) inhibitor, such as rapamycin, everolimus, and / or temsirolimus. The deactivating agent may be a tyrosine kinase (TK) inhibitor, such as Lorlatinib, Brigatinib, Ceritinib, Alectinib, Crizotinib, Bosutinib, Ponatinib, Nilotinib, Dasatinib, Imatinib, Zanubrutinib, Acalabrutinib, Ibrutinib, Capmatinib, Pexidartinib, Dacomitinib, Osimertinib, Erlotinib, Gefitinib, Lapatinib, Afatinib, Pemigatinib, Erdafitinib, Nintedanib, Gilteritinib, Midostaurin, Tucatinib, Neratinib, Baricitinib, Ruxolitinib, Fedratinib, Tofacitinib, Ripretinib, Selumetinib, Binimetinib, Cobimetinib, Trametinib, Upadacitinib, Avapritinib, Selpercatinib, Cabozantinib, Fostamatinib, Larotrectinib, Entrectinib, Axitinib, Regorafenib, Pazopanib, Sorafenib, Lenvatinib, Vandetanib, and / or Sunitinib. The TK inhibitor may be a BCR-Abl inhibitor. The TK inhibitor may be Bosutinib, Ponatinib, Nilotinib, Dasatinib, Asciminib and / or Imatinib.

[0011] The treatment of the NK cells with one or more deactivating agents may be at any point during production of the NK cell. The treatment may be for about 24 to about 96 hours, about 36 to about 84 hours, or about 48 to about 72 hours. The treatment may be for about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cell is treated with the deactivating agent at a concentration of about 1 to about 1000 nM, 5 to about 500 nM, 20 to about 200 nM, 30 to about 100 nM. In certain embodiments, the deactivated NK cell has an increased expression of one or more of C-kit, CCR-5, CD62L and / or CXCR4, and / or decreased expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95 relative to an activated NK cell. Following treatment with one or more deactivating agents for the NK cells may be followed by one or more washing steps to wash off the one or more deactivating agents off of the cells.

[0012] In some embodiments, NK cells are removed from a culture comprising IL-2, APCs, or a combination thereof. In some cases, the cells are added to a vessel, such as a flask or plate that may or may not include one or more compounds for the cells. In specific embodiments, the vessel comprises media and may or may not comprise one or more cytokines, such as IL-2. In specific embodiments, the vessel lacks APCs. In specific embodiments, the vessel is a RetroNectin plate. In specific embodiments, the vessel comprises media with or without IL-2. In some embodiments, one or more deactivating agents are provided to the cells in the vessel.

[0013] At any point in time of the production methods of the disclosure, the cells may be cryopreserved. In some embodiments, upon thawing of the cells, they are used substantially immediately, such as provided to an individual in need thereof. They may or may not be further modified prior to use following thawing, such as formulated in a therapeutic formulation.

[0014] In a first embodiment, there is provided an in vitro method for expanding NK cells comprising obtaining a population of NK cells; pre-activating the population of NK cells in a pre-activation culture comprising an effective concentration of IL-12, IL-15, and IL-18 to obtain pre-activated NK cells; and expanding the pre-activated NK cells in an expansion culture comprising artificial antigen presenting cells (aAPCs) expressing CD 137 ligand, thereby producing prepared NK cells.

[0015] In some aspects, the population of NK cells is obtained from cord blood (CB), peripheral blood (PB), stem cells, or bone marrow. In particular aspects, the stem cells are induced pluripotent stem cells. In specific aspects, the isolated population of NK cells is obtained from CB, such as pooled CB. In some aspects, the CB is pooled from 2 or more (e.g.,3, 4, 5, 6, 7, 8, or more) individual cord blood units. In certain aspects, the isolated populationof NK cells are CB mononuclear cells (CBMCs). In some aspects, the isolated population of NK cells are further defined as CD56+NK cells.

[0016] In certain aspects, the aAPCs further express a membrane-bound cytokine. In some aspects, the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membranebound IL-15 (mIL-15). In some aspects, the membrane -bound cytokine is mIL-21. In certain aspects, the aAPCs further express a heterologous CD137 ligand. In certain aspects, the aAPCs further express a heterologous CD48 protein. In certain embodiments, the aAPCs further express a heterologous CS1 protein, membrane-bound cytokine. In some aspects, the aAPCs have essentially no expression of endogenous HLA class I, II, or CDld molecules. In certain aspects, the aAPCs express ICAM-1 (CD54) and LFA-3 (CD58). In some aspects, the aAPCs are further defined as leukemia cell-derived aAPCs. In certain aspects, the leukemia-cell derived aAPCs are further defined as K562 cells engineered to express CD 137 ligand and / or mIL-21. In some aspects, the K562 cells are engineered to express CD137 ligand and mIL-21. In some aspects, the K562 cells are engineered to express CD 137 ligand, CD48, and mIL-21. In certain aspects, engineered is further defined as retroviral transduction. In particular aspects, the aAPCs are irradiated. In particular aspects, the aAPCs are irradiated such that they lose proliferative advantage relative to NK cells.

[0017] In some aspects, the pre-activating step is for 10-20 hours, such as 14-18 hours (e.g., about 14, 15, 16, 17, or 18 hours), particularly about 16 hours. In certain aspects, the preactivation culture comprises IL- 18 and / or IL- 15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, particularly about 50 ng / mL. In some aspects, the pre-activation culture comprises IL-12 at a concentration of 0.1-150 ng / mL, such as 1-20 ng / mL, particularly about 10 ng / mL.

[0018] In certain aspects, the method further comprises washing the pre-activated NK cells prior to expanding. In some aspects, washing is performed multiple times, such as 2, 3, or 4 times.

[0019] In some aspects, expanding is for 5-20 days, such as 12-16 days (e.g., 12, 13, 14, 15, or 16 days), particularly about 14 days. In certain aspects, the pre-activated NK cells and aAPCs are present in the expansion culture at a ratio of 3: 1 to 1:3. In specific aspects, the pre-activated NK cells and aAPCs are present in the expansion culture at a ratio of about 1:2.

[0020] In additional aspects, the expansion culture further comprises IL-2. In some aspects, the IL-2 is present at a concentration of 10-500 U / mL, such as 100-300 U / mL, particularly about 200 U / mL. In some aspects, the IL-12, IL-18, IL-15, and / or IL-2 is recombinant human IL-2. In some aspects, the IL-2 is replenished in the expansion culture every 2-3 days. In someaspects, the aAPCs are added to the expansion culture at least a second time. In some aspects, the method is performed in serum-free media.

[0021] In further aspects, the NK cells are engineered to express a chimeric antigen receptor (CAR) or TCR. In some aspects, the CAR or TCR comprises a CD19, CD123, mesothelin, CD5, CD47, CLL-1, CD33, CD99, U5snRNP200, CD200, CS1, BAFF-R, ROR-1, TROP2, GPC3, B7-H3, GD2, HLA-G, CD70, uPAR, CD20, HER2, EGFR, MUC1, PRAME, NYESO1, KRAS, HPV, CMV or BCMA antigen-binding domain. In some aspects, the CAR comprises a humanized antigen-binding domain. In some aspects, the CAR comprises IL- 15. In some aspects, the CAR comprises a suicide gene. In specific aspects, the suicide gene is CD20, CD52, EGFRv3, or inducible caspase 9.

[0022] Further embodiments provide populations of expanded NK cells that are prepared and produced according to the embodiments (e.g., obtaining an isolated population of NK cells; pre-activating the isolated population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; and expanding the pre-activated NK cells in an expansion culture comprising artificial antigen presenting cells (aAPCs) expressing CD137 ligand, thereby producing expanded NK cells). Also provided herein is a pharmaceutical composition comprising the population of expanded NK cells of the embodiments and a pharmaceutically acceptable carrier.

[0023] In another embodiment, there is provided a composition comprising an effective amount of the expanded NK cells of the embodiments (e.g., obtaining an isolated population of NK cells; pre-activating the isolated population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; and expanding the pre-activated NK cells in an expansion culture comprising artificial antigen presenting cells (aAPCs) expressing CD137 ligand, thereby producing expanded NK cells) for use in the treatment of a disease or disorder in a subject.

[0024] Further provided herein is method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of the expanded NK cells of the embodiments to the subject.

[0025] In some aspects of the above embodiments, the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection. In some aspects, the cancer is a leukemia. In certain aspects, the leukemia is an acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), or a chronic myelogenous leukemia (CML).

[0026] In some aspects, the NK cells are allogeneic. In other aspects, the NK cells are autologous. In certain aspects, the subject is a human. For any NK cells encompassed herein, the NK cells may or may not be primary NK cells, and may or may not be derived from stem cells and / or induced pluripotent stem cells (iPSCs). Any NK cells may or may not be complexed to one or more antibodies, and the one or more antibodies may be one or more bispecific or multi- specific antibodies, wherein at least one of the bispecific or multi- specific antibodies comprises an anti-CD3 antibody linked. Examples of antibodies includes Blinatumomab, Tebentafusp, Mosunetuzumab, Teclistamab, Glofitamab, Epcoritamab, Flotetuzumab, APV0436, and / or TNB383B.

[0027] In certain aspects, the disorder is graft versus host disease (GVHD). In some aspects, the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythrematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0028] In some aspects, the method further comprises administering at least a second therapeutic agent. In certain aspects, the at least a second therapeutic agent is a therapeutically effective amount of an anti-cancer agent, immunomodulatory agent, or an immunosuppressive agent. In some aspects, the anti-cancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy. In specific aspects, the immunosuppressive agent is a calcineurin inhibitor, an mTOR inhibitor, an antibody, a chemotherapeutic agent irradiation, a chemokine, an interleukins or an inhibitor of a chemokine or an interleukin.

[0029] In certain aspects, the NK cells and / or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

[0030] In some aspects, the second therapeutic agent is an antibody. In particular aspects, the antibody if a monoclonal, bispecific, or trispecific antibody. In some aspects, the antibody is a monoclonal antibody. In one specific aspect, the antibody is rituximab.

[0031] In some embodiments, provided herein are in vitro methods for preparing natural killer (NK) cells comprising: optionally pre-activating a population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; an expanding step comprising: A) a first culturing of NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous: (i) IL-2; (ii) antigen presenting cells (APCs) and / or feeder cells;(iii) one or more of IL- 12, IL- 18, and TGFb; (iv) activating beads; (v) one or more mTOR inhibitor; or (vi) any combination thereof; B) optionally enriching for the NK cells; C) optionally transforming and / or transducing the cells with an engineered construct; and D) a second culturing of the NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous IL-2, IL-12, IL- 18, and TGFb, and optionally one or more mammalian target of rapamycin (mTOR) inhibitor, optionally one or more phosphoinositide 3-kinase (PI3K) inhibitor, optionally one or more isocitrate dehydrogenase 2 (IDH2) inhibitor, optionally one or more isocitrate dehydrogenase 1 (IDH1) inhibitor, optionally one or more isocitrate dehydrogenase 1 / 2 (IDH1 / 2) inhibitors, optionally one or more protein kinase B (PKB, AKT) inhibitors, optionally one or more additional cytokines, optionally one or more non-metabolizable glucose analogs, optionally one or more metabolizable sugars, and / or optionally aAPCs, to produce prepared NK cells; and optionally a deactivating step comprising exposing the prepared NK cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated prepared NK cell.

[0032] In some embodiments, an expanding step is for about 7-14 days. In some embodiments, an expanding step is for about 7-10 days. In some embodiments, an expanding step is for about 8, or about 9 days. In some embodiments, an expanding step is for 9 days.

[0033] In some embodiments, a second culturing step is for 1, 2, 3, 4, or more days. In some embodiments, a second culturing step is for about 8, or about 9 days. In some embodiments, a second culturing step is for 3 days. In some embodiments, a second culturing step is for equal to or less than about 5 days. In some embodiments, a second culturing step is for equal to or less than about 4 days. In some embodiments, a second culturing step is for equal to or less than about 3 days.

[0034] In some embodiments, a first culturing step is for 1, 2, 3, 4, 5, 6, or 7, or less days. In some embodiments, a first culturing step is for equal to 6 days. In some embodiments, a first culturing step is for equal to or less than about 5, 6, or 7 days. In some embodiments, a first culturing step is for less than about 6 days. In some embodiments, a first culturing step is for equal to or less than about 5 days.

[0035] In some embodiments, a aAPCs are provided in the second culturing step. In some embodiments, no further aAPCs are provided in a culture following the first culturing step. In some embodiments, aAPCs are removed from the culture following the first culturing step. In some embodiments, a first and second culturing step are separated by an NK cell enrichment step. In some embodiments, aAPCs are removed from the culture by about 5, 6, 7, 8, or 9 daysfrom the beginning of the first culturing step. In some embodiments, aAPCs are removed from the culture by about 5, 6 or 7 days from the beginning of the expanding step. In some embodiments, a first and / or second culturing step is in the absence of aAPCs. In some embodiments, a second culturing step is in the absence of aAPCs.

[0036] In some embodiments, a first culturing step comprises a culture media comprising exogenous IL-2, IL- 12, IL- 18, and TGFb. In some embodiments, a first culturing step comprises a culture media comprising aAPCs, activating beads, and / or feeder cells. In some embodiments, a first culturing step comprises a culture media comprising IL-2, IL- 12, IL- 18, and TGFb, wherein the concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 10 ng / mL particularly about 0.3 ng / mL. In some embodiments, a first culturing step comprises a culture media comprising IL-2, IL- 12, IL- 18, and TGFb, wherein the concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL.

[0037] In some embodiments, a first culturing step comprises an mTOR inhibitor. In some embodiments, a first culturing step comprises an mTOR inhibitor that consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku- 0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'- substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI-027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25. In some embodiments, a first culturing step comprises rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM. In some embodiments, a first culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

[0038] In some embodiments, methods of preparing natural killer cells includes a preactivation step. In some embodiments, methods of preparing natural killer cells includes transforming and / or transducing the cells with an engineered construct. In some embodiments, transforming comprises transient introduction of one or more ribonucleoproteins, polynucleotides, and / or polypeptides. In some embodiments, transducing comprises stabletransgene introduction and maintenance in the NK cell. In some embodiments, transducing comprises stable transgene introduction into the genome of the NK cell. In some embodiments, transducing comprises contacting the NK cells with one or more RetroNectin bound retroviral constructs.

[0039] In some embodiments, an mTOR inhibitor in the first and / or second culturing step comprises, consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE-687, WYE-354, GDC- 0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3- d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI-027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25. In some embodiments, an mTOR inhibitor comprises, consists essentially of, or consists of rapamycin. In some embodiments, rapamycin is at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM or 10 nM. In some embodiments, a first and / or second culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

[0040] In some embodiments, a PI3K inhibitor comprises, consists essentially of, or consists of one or more of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, and / or umbrasilib. In some embodiments, a PI3K inhibitor comprises, consists essentially of, or consists of Idelalisib. In some embodiments, Idelalisib is at a concentration of about 0.1 nM to 100 nM, particularly about 10 nM. In some embodiments, Idelalisib is at a concentration of about 0.1 nM to 10 pM, particularly about 1 pM.

[0041] In some embodiments, methods of preparing natural killer cells include use of a dual action mTOR and PI3K inhibitor. In some embodiments, a dual action mTOR and PI3K inhibitor comprises, consists essentially of, or consists of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), and / or voxtalisib (XL765).

[0042] In some embodiments, methods of preparing natural killer cells include use of an AKT inhibitor. In some embodiments, an AKT inhibitor comprises, consists essentially of, or consists of capivasertib (AZD-5363), ipatasertib, miltefosine, uprosertib (GSK2141795), miransertib (ARQ 092), afuresertib (GSK2110183), palomid 529 (P529), and / or perifosine (KRX-0401).

[0043] In some embodiments, methods of preparing natural killer cells include use of a non- metabolizable glucose analog. In some embodiments, a non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), and / or 6-Deoxy-D-Glucose (6DG). In some embodiments, a non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-DG at a concentration of about 0.1 mM to 20 mM particularly about 2 mM.

[0044] In some embodiments, methods of preparing natural killer cells include use of a metabolizable sugar. In some embodiments, a metabolizable sugar comprises, consists essentially of, or consists of sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum lycopersici (E-FOL).

[0045] In some embodiments, methods of preparing natural killer cells include use of an isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, an IDH2 inhibitor comprises, consists essentially of, or consists of Enasidenib and / or Ivosidenib. In some embodiments, an IDH2 inhibitor is at about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

[0046] In some embodiments, a first and / or second expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, or any range or value derivable therein, of IL- 2. In some embodiments, a first expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 U / mL, or any range or value derivable therein, of IL-2, and the second expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, or any range or value derivable therein, of IL-2. In some embodiments, a transduction and / or transfection step takes place in the presence of about 200 U / mL or 600 U / mL of IL-2.

[0047] In some embodiments, a transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, and TGEb. In some embodiments, a transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, and rapamycin. In some embodiments, a transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, rapamycin, and idelalisib. In some embodiments, a transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, rapamycin, and 2- DG. In some embodiments, a transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, rapamycin, and Enasidenib.

[0048] In some embodiments, methods provided herein includes exposing the cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated prepared NK cell. In some embodiments, a deactivating agent comprises or is Dasatinib. In some embodiments, a deactivating agent comprises or is Dasatinib at a concentration of equalto or about 1 pM. In some embodiments, exposing to the deactivating agent(s) occurs in the presence of IL-2. In some embodiments, exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, and TGFb. In some embodiments, exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, and rapamycin. In some embodiments, exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and idelalisib. In some embodiments, exposing to the deactivating agent occurs in the presence of IL-2, IL-12, IL-18, TGFb, rapamycin, and 2-DG. In some embodiments, exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

[0049] In some embodiments of methods provided herein, a concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, a concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, a concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and a concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL. In some embodiments, an exposing and / or expansion step takes place in an incubator set at 37 °C with 5% CO2. In some embodiments, exposing to the deactivating agent step takes place in the presence of one or more mTOR inhibitors, one or more PI3K inhibitors, one or more metabolizable sugars, and / or one or more non-metabolizable glucose analogs. In some embodiments, exposing to the deactivating agent step occurs for about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours. In some embodiments, exposing to the deactivating agent step occurs for about 24 hours.

[0050] Also provided herein are methods comprising obtaining prepared NK cells or freezing deactivated prepared NK cells. In some embodiments, prepared NK cells are further suspended in a pharmaceutically acceptable media. In some embodiments, prepared NK cells are suspended in PlasmaLyte-A containing 0.5% HSA. In some embodiments, prepared NK cells are cryopreserved and are thawed prior to administering the cells to an individual in need thereof.

[0051] In some embodiments, NK cells are engineered to comprise an engineered construct that comprises a sequence encoding an engineered antigen receptor. In some embodiments, an engineered antigen receptor is a chimeric antigen receptor, a T cell receptor, or both. In some embodiments, an engineered antigen receptor targets one or more of CD5, CD19, CD20, CD30, CD70, TROP2, PRAME, KRAS, IL13, EGVRv3, BCMA, GPRC5D, HER2, c-MET, GPC3, B7_H3, GD2, NY-ESO1, CD40, KRAS, EGFR, HPV, CMV, and / or FRa. In some embodiments, engineering, such as transfection and / or transduction comprises introduction of an engineered mutation in one or more of endogenous NK cell genes. In some embodiments,an engineered mutation is in one or more of GR, TGFBR2, CISH, CD38, TIGIT, and / or ADAM 17 genes. In some embodiments, an engineered construct comprises a sequence that encodes one or more cytokines. In some embodiments, a cytokine is IL- 15, IL-21, IL-4, IL-7, IL-9, IL- 10, IL- 27, or combination thereof (e.g., a combination of two or more cytokines). In some embodiments, a construct comprises regulatory regions and / or coding sequences that result in a cytokine being autonomously secreted.

[0052] In some embodiments, NK cells are obtained from cord blood (CB), peripheral blood (PB), stem cells, or bone marrow. In some embodiments, NK cells are obtained from CB. In some embodiments, CB is pooled from 2, 3, 4, 5, 6, 7, or 8 or more individual cord blood units. In some embodiments, a population of NK cells are further defined as CD56+NK cells, CD 16+ NK cells, or CD56+CD16+ NK cells.

[0053] In some embodiments, aAPCs further express a membrane-bound cytokine. In some embodiments, a membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membranebound IL-15 (mIL-15). In some embodiments, aAPCs have essentially no expression of endogenous HLA class I, II, or CD Id molecules. In some embodiments, aAPCs express ICAM- 1 (CD54) and LFA-3 (CD58). In some embodiments, aAPCs are further defined as leukemia cell-derived aAPCs. In some embodiments, leukemia-cell derived aAPCs are K562 cells engineered to express CD137 ligand, CD48, CS1, and / or mIL-21. In some embodiments, aAPCs have been engineered by retroviral transduction, are irradiated, or both.

[0054] In some embodiments, a pre-activating step is for about 10-20 hours. In some embodiments, a pre-activating step is for 14-18 hours. In some embodiments, a pre-activating step is for 16 hours. In some embodiments, a pre-activation culture comprises IL- 18 and / or IL- 15 at a concentration of 10-100 ng / mL. In some embodiments, a pre-activation culture comprises IL- 18 and / or IL- 15 at a concentration of 40-60 ng / mL. In some embodiments, a pre- activation culture comprises IL- 18 and / or IL- 15 at a concentration of 50 ng / mL. In some embodiments, a pre-activation culture comprises IL-12 at a concentration of 0.1-150 ng / mL. In some embodiments, a pre-activation culture comprises IL- 12 at a concentration of 1-20 ng / mL. In some embodiments, a pre-activation culture comprises IL- 12 at a concentration of 10 ng / mL.

[0055] In some embodiments, pre-activated NK cells and aAPCs are present in an expansion culture at a ratio of 3:1 to 1:3. In some embodiments, pre-activated NK cells and aAPCs are present in an expansion culture at a ratio 1:2. In some embodiments, IL-2 in the first or second expansion culture is present at a concentration of 10-1000 U / mL. In some embodiments, IL-2is present at a concentration of 100-300 U / mL. In some embodiments, IL-2 is present at a concentration of 200 U / mL.

[0056] Also provided herein are populations of prepared NK cells produced using methods described herein. In some embodiments, prepared NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared utilizing other methods. In some embodiments, prepared NK cells have a shifted chemokine profile compared to NK cells prepared utilizing other methods. In some embodiments, a population of prepared NK cells has increased expression levels of CD62L, CXCR4, and / or CCR7 relative to NK cells prepared utilizing other methods. In some embodiments, a population of the prepared NK cells includes at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the NK cells being positive for CD62L. In some embodiments, a population of the prepared NK cells includes at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, of the NK cells being positive for CXCR4. In some embodiments, a population of the prepared NK cells includes at least about 20%, 25%, 30%, 35%, 40%, or 45% of the NK cells being positive for CCR7. In some embodiments, a population of prepared NK cells has an increased level of transduction and / or transformation relative to NK cells prepared utilizing other methods. In some embodiments, a population of prepared NK cells has a transduction efficiency level of at least 50%, 55%, or 60%. In some embodiments, a population of prepared NK cells has a transfection efficiency level of at least 80%, 85%, 90%, or 95%.

[0057] In some embodiments, prepared NK cells provided herein are comprised in a composition that includes one or more antibodies. In some embodiments, antibodies are complexed to the NK cells. In some embodiments, prepared NK cells provided herein are comprised in a pharmaceutical composition comprising the population of prepared NK cells and a pharmaceutically acceptable carrier.

[0058] In some embodiments, provided herein are compositions comprising an effective amount of prepared NK cells for use in the treatment of a disease or disorder in a subject. In some embodiments, a disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection. In some embodiments, a cancer is a leukemia. In some embodiments, a leukemia is an acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), or a chronic myelogenous leukemia (CML). In some embodiments, a cancer is a solid tumor. In some embodiments, a cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovariancancer. In some embodiments, a disorder is graft versus host disease (GVHD). In some embodiments, a disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0059] Also provided herein, in some embodiments, are methods of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of prepared NK cells described. In some embodiments, a disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection. In some embodiments, a cancer is a leukemia. In some embodiments, a cancer is a solid tumor. In some embodiments, a cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer. In some embodiments, a disorder is graft versus host disease (GVHD). In some embodiments, a disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0060] Also provided herein are compositions for expansion of NK cells, comprising, exogenously provided IL-2, IL- 12, IL- 18, and TGFb. In some embodiments, a composition comprises one or more mTOR inhibitor, one or more PI3K inhibitor, one or more dual action mTOR and PI3K inhibitor, one or more non-metabolizable glucose analogs, one or more metabolizable sugars, one or more IDH2 inhibitors, one or more IDH1 inhibitors, one or more dual IDH1 / 2 inhibitors, and / or aAPCs. In some embodiments, a composition comprises a concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL. In some embodiments, TGFb comprises or is TGFbl. In some embodiments, a mTOR inhibitor comprises, consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, non-rapalog allosteric inhibitor R1-R5, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSL 027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25. In some embodiments, a mTOR inhibitor comprises, consists essentially of, or consists of rapamycin. In some embodiments, rapamycin is at a concentration of about 0.1 nM to 100 nM particularly about 1 nM or 10 nM. In some embodiments, a PI3K inhibitor comprises, consists essentially of, orconsists of one or more of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, and / or umbrasilib. In some embodiments, a PI3K inhibitor comprises, consists essentially of, or consists of Idelalisib. In some embodiments, Idelalisib is at a concentration of about 0.1 nM to 100 nM particularly about 10 nM. In some embodiments, Idelalisib is at a concentration of about 0.1 nM to 10 pM particularly about 10 pM. In some embodiments, a comprising a dual action mTOR and PI3K inhibitor. In some embodiments, a dual action mTOR and PI3K inhibitor comprises, consists essentially of, or consists of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), and / or voxtalisib (XL765). In some embodiments, a non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O-Methyl-D- Glucose (20MG), and / or 6-Deoxy-D-Glucose (6DG). In some embodiments, a non- metabolizable glucose analog comprises, consists essentially of, or consists of 2-DG at a concentration of about 0.1 mM to 20 mM particularly about 2 mM. In some embodiments, one or more metabolizable sugar comprises, consists essentially of, or consists of sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum lycopersici (E-FOL). In some embodiments, an isocitrate dehydrogenase 2 (IDH2) inhibitor comprises, consists essentially of, or consists of Enasidenib and / or Ivosidenib. In some embodiments, an IDH2 inhibitor comprises Enasidenib. In some embodiments, an IDH1 inhibitor comprises Ivosidenib. In some embodiments, a dual IDH1 and IDH2 inhibitor comprises Vorasidenib. In some embodiments, a concentration of IDH2 inhibitor is at about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

[0061] In some embodiments, also provided herein are compositions comprising NK cells and exogenously provided IL-2, IL- 12, IL- 18, and TGFb, and optionally: one or more mTOR inhibitor, one or more PI3K inhibitor, one or more dual action mTOR and PI3K inhibitor, one or more non-metabolizable glucose analogs, one or more metabolizable sugars, one or more IDH1 inhibitors, one or more IDH2 inhibitors, one or more dual IDH1 and IDH2 inhibitors, and / or aAPCs. In some embodiments, NK cells are NK cells pre-activated by exposure to IL12, IL15, and IL18. In some embodiments, also provided herein are compositions comprising NK cells and exogenously provided IL-2, IL- 12, IL- 18, and TGFb, and optionally: one or more mTOR inhibitor, one or more PI3K inhibitor, one or more dual action mTOR and PI3K inhibitor, one or more non-metabolizable glucose analogs, one or more metabolizable sugars, one or more IDH2 inhibitors, and / or aAPCs, and one or more retroviral particles and / or polynucleotide constructs. In some embodiments, compositions comprise one or moredeactivating agents. In some embodiments, aAPCs that have been stably engineered to transgenically express CD 137 ligand, CS1, CD48, and / or mIL21; optionally wherein the aAPCs have been irradiated.

[0062] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0063] Aspect 1A is an in vitro method for preparing natural killer (NK) cells comprising: optionally pre-activating a population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; an expanding step comprising: A) a first culturing of NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous: (i) IL-2; (ii) antigen presenting cells (APCs) and / or feeder cells; (iii) one or more of IL- 12, IL- 18, and TGFb; (iv) activating beads; (v) one or more mTOR inhibitor; or (vi) any combination thereof; B) optionally enriching for the NK cells, C) optionally transforming and / or transducing the cells with an engineered construct; and optionally a deactivating step comprising exposing the prepared NK cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated prepared NK cell.

[0064] Aspect 2A is the method of aspect 1A, further including a second culturing step of the NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous IL-2, IL-12, IL-18, and TGFb, and optionally one or more mammalian target of rapamycin (mTOR) inhibitor, optionally one or more phosphoinositide 3-kinase (PI3K) inhibitor, optionally one or more isocitrate dehydrogenase 2 (IDH2) inhibitor, optionally one or more protein kinase B (PKB, AKT) inhibitors, optionally one or more additional cytokines, optionally one or more non- metabolizable glucose analogs, optionally one or more metabolizable sugars, and / or optionally aAPCs, to produce prepared NK cells.

[0065] Aspect 3A is an in vitro method for preparing natural killer (NK) cells comprising: optionally pre-activating a population of NK cells in a pre-activation culture comprising an effective concentration of IL- 12, IL- 15, and IL- 18 to obtain pre-activated NK cells; an expanding step comprising: A) a first culturing of NK cells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous: (i) IL-2; (ii) antigen presenting cells (APCs) and / or feeder cells; (iii) one or more of IL- 12, IL- 18, and TGFb; (iv) activating beads; (v) one or more mTOR inhibitor; or (vi) any combination thereof; B) optionally enriching for the NK cells, C) optionally transforming and / or transducing the cells with an engineered construct; and D) a second culturing of the NKcells or the pre-activated NK cells in an expansion culture for a duration of time of about 1-14 days, said expansion culture comprising exogenous IL-2, IL- 12, IL- 18, and TGFb, and optionally one or more mammalian target of rapamycin (mTOR) inhibitor, optionally one or more phosphoinositide 3-kinase (PI3K) inhibitor, optionally one or more isocitrate dehydrogenase 2 (IDH2) inhibitor, optionally one or more protein kinase B (PKB, AKT) inhibitors, optionally one or more additional cytokines, optionally one or more non- metabolizable glucose analogs, optionally one or more metabolizable sugars, and / or optionally aAPCs, to produce prepared NK cells; and optionally a deactivating step comprising exposing the prepared NK cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated prepared NK cell.

[0066] Aspect 4A is the method of any one of aspects 1A-3A, wherein the expanding step is for about 7-14 days.

[0067] Aspect 5A is the method of any one of aspects 1A-4A, wherein the expanding step is for about 7-10 days.

[0068] Aspect 6A is the method of any one of aspects 1A-5A, wherein the expanding step is for about 8, or about 9 days.

[0069] Aspect 7A is the method of aspect 6A, wherein the expanding step is for 9 days.

[0070] Aspect 8A is the method of any one of aspects 1A-7A, wherein the second culturing step is for 1, 2, 3, 4, or more days.

[0071] Aspect 9A is the method of any one of aspects 1A-4A, wherein the second culturing step is for about 8, or about 9 days.

[0072] Aspect 10A is the method of any one of aspects 1A-8A, wherein the second culturing step is for 3 days.

[0073] Aspect 11A is the method of any one of aspects 1A-10A, wherein the second culturing step is for equal to or less than about 5 days.

[0074] Aspect 12A is the method of any one of aspects 1A-11A, wherein the second culturing step is for equal to or less than about 4 days.

[0075] Aspect 13A is the method of any one of aspects 1A-12A, wherein the second culturing step is for equal to or less than about 3 days.

[0076] Aspect 14A is the method of any one of the preceding aspects, wherein the first culturing step is for 1, 2, 3, 4, 5, 6, or 7, or less days.

[0077] Aspect 15A is the method of any one of the preceding aspects, wherein the first culturing step is for equal to 6 days.

[0078] Aspect 16A is the method of any one of the preceding aspects, wherein the first culturing step is for equal to or less than about 5, 6, or 7 days.

[0079] Aspect 17A is the method of any one of the preceding aspects, wherein the first culturing step is for less than about 6 days.

[0080] Aspect 18A is the method of any one of the preceding aspects, wherein the first culturing step is for equal to or less than about 5 days.

[0081] Aspect 19A is the method of any one of aspects 1A-18A, wherein aAPCs are provided in the second culturing step.

[0082] Aspect 20A is the method of any one of aspects 1A-18A, wherein no further aAPCs are provided in a culture following the first culturing step.

[0083] Aspect 21 A is the method of any one of aspects 1A-20A, wherein the aAPCs are removed from the culture following the first culturing step.

[0084] Aspect 22A is the method of any one of aspects 1A-21A, wherein the first and second culturing step are separated by an NK cell enrichment step.

[0085] Aspect 23A is the method of aspect 21A, wherein the aAPCs are removed from the culture by about 5, 6, 7, 8, or 9 days from the beginning of the first culturing step.

[0086] Aspect 24A is the method of aspect 23A, wherein the aAPCs are removed from the culture by about 5, 6 or 7 days from the beginning of the expanding step.

[0087] Aspect 25A is the method of any one of aspects 1 A-24A, wherein the first and / or second culturing step is in the absence of aAPCs.

[0088] Aspect 26A is the method of any one of aspects 1A-25A, wherein the second culturing step is in the absence of aAPCs.

[0089] Aspect 27 A is the method of any one of aspects 1A-26A, wherein the first culturing step comprises a culture media comprising exogenous IL-2, IL- 12, IL- 18, and TGFb.

[0090] Aspect 28 A is the method of aspect 27 A, wherein the first culturing step comprises a culture media comprising aAPCs, activating beads, and / or feeder cells.

[0091] Aspect 29A is the method of any one of aspects 1A-28A, wherein the first culturing step comprises a culture media comprising IL-2, IL- 12, IL- 18, and TGFb, wherein the concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL.

[0092] Aspect 30A is the method of any one of aspects 1A-29A, wherein the first culturing step comprises an mTOR inhibitor.

[0093] Aspect 31A is the method of any one of aspects 1A-30A, wherein the first culturing step comprises an mTOR inhibitor that consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE- 687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4- morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4- hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI-027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25.

[0094] Aspect 32A is the method of aspect 31 A, wherein the first culturing step comprises rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM.

[0095] Aspect 33A is the method of aspect 32A, wherein the first culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

[0096] Aspect 34A is the method of any one of aspects 1A-25A, wherein the method includes the pre-activation step.

[0097] Aspect 35A is the method of any one of aspects 1A-34A, wherein the method includes transforming and / or transducing the cells with an engineered construct, optionally wherein the engineered construct comprises a polynucleotide.

[0098] Aspect 36A is the method of any one of aspects 1A-35A, wherein the transforming comprises transient introduction of one or more ribonucleoproteins, polynucleotides, and / or polypeptides.

[0099] Aspect 37 A is the method of any one of aspects 1A-36A, wherein the transducing comprises stable transgene introduction into the genome of the NK cell.

[0100] Aspect 38A is the method of aspect 37A, wherein the transducing comprises contacting the NK cells with one or more RetroNectin bound retroviral constructs.

[0101] Aspect 39A is the method of any one of aspects 1A-38A, wherein the mTOR inhibitor in the first and / or second culturing step comprises, consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY- 600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI- 027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25.

[0102] Aspect 40A is the method of aspect 39A, wherein the mTOR inhibitor comprises, consists essentially of, or consists of rapamycin.

[0103] Aspect 41A is the method of aspect 39A or 40A, wherein the rapamycin is at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM or 10 nM.

[0104] Aspect 42A is the method of aspect 41 A, wherein the first and / or second culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

[0105] Aspect 43A is the method of any one of aspects 1A-42A, wherein the PI3K inhibitor comprises, consists essentially of, or consists of one or more of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, and / or umbrasilib.

[0106] Aspect 44A is the method of aspect 43A, wherein the PI3K inhibitor comprises, consists essentially of, or consists of Idelalisib.

[0107] Aspect 45A is the method of aspect 43A or 44A, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM, particularly about 10 nM.

[0108] Aspect 46A is the method of any one of aspects 1A-45A, comprising a dual action mTOR and PI3K inhibitor.

[0109] Aspect 47A is the method of aspect 46A, wherein the dual action mTOR and PI3K inhibitor comprises, consists essentially of, or consists of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), and / or voxtalisib (XL765).

[0110] Aspect 48A is the method of any one of aspects 1A-47A, comprising an AKT inhibitor.

[0111] Aspect 49A is the method of aspect 48A, wherein the AKT inhibitor comprises, consists essentially of, or consists of capivasertib (AZD-5363), ipatasertib, miltefosine, uprosertib (GSK2141795), miransertib (ARQ 092), afuresertib (GSK2110183), palomid 529 (P529), and / or perifosine (KRX-0401).

[0112] Aspect 50A is the method of any one of aspects 1A-49A, comprising a non- metabolizable glucose analog.

[0113] Aspect 51 A is the method of aspect 50A, wherein the non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D- Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), and / or 6-Deoxy-D-Glucose (6DG).

[0114] Aspect 52A is the method of aspect 51 A, wherein the non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-DG at a concentration of about 0.1 mM to 20 mM particularly about 2 mM.

[0115] Aspect 53A is the method of any one of aspects 1A-52A, comprising a metabolizable sugar.

[0116] Aspect 54A is the method of aspect 53A, wherein the metabolizable sugar comprises, consists essentially of, or consists of sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum ly coper sici (E-FOL).

[0117] Aspect 55A is the method of any one of aspects 1A-54A, comprising an isocitrate dehydrogenase 2 (IDH2) inhibitor.

[0118] Aspect 56A is the method of aspect 55A, wherein the IDH2 inhibitor comprises, consists essentially of, or consists of Enasidenib and / or Ivosidenib.

[0119] Aspect 57A is the method of aspect 56A, wherein the concentration of IDH2 inhibitor is at about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

[0120] Aspect 58A is the method of any one of aspects 1-57A, wherein the first and / or second expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, or any range or value derivable therein, of IL-2.

[0121] Aspect 59A is the method of any one of aspects 1A-58A, wherein the first expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 U / mL, or any range or value derivable therein, of IL-2, and the second expansion culture comprises about 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, or any range or value derivable therein, of IL-2.

[0122] Aspect 60A is the method of any one of aspects 1A-59A, wherein the transduction and / or transfection step takes place in the presence of about 200 U / mL or 600 U / mL of IL-2.

[0123] Aspect 61 A is the method of any one of aspects 1A-60A, wherein the transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, and TGEb.

[0124] Aspect 62A is the method of any one of aspects 1A-61A, wherein the transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, and rapamycin.

[0125] Aspect 63A is the method of any one of aspects 1A-62A, wherein the transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, rapamycin, and idelalisib.

[0126] Aspect 64A is the method of any one of aspects 1A-63A, wherein the transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGEb, rapamycin, and 2-DG.

[0127] Aspect 65A is the method of any one of aspects 1A-64A, wherein the transduction and / or transfection step takes place in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

[0128] Aspect 66A is the method of any one of aspects 1A-65A, wherein the method includes exposing the cells to an effective amount of one or more deactivating agents under conditions to produce a deactivated prepared NK cell.

[0129] Aspect 67A is the method of any one of aspects 1 A-66A, wherein the deactivating agent comprises or is Dasatinib.

[0130] Aspect 68A is the method of any one of aspects 1 A-67A, wherein the deactivating agent comprises or is Dasatinib at a concentration of equal to or about 1 pM.

[0131] Aspect 69A is the method of any one of aspects 1A-68A, wherein the exposing to the deactivating agent(s) occurs in the presence of IL-2.

[0132] Aspect 70A is the method of any one of aspects 1A-69A, wherein the exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, and TGFb.

[0133] Aspect 71A is the method of any one of aspects 1A-70A, wherein the exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, and rapamycin.

[0134] Aspect 72A is the method of any one of aspects 1A-71A, wherein the exposing to the deactivating agent occurs in the presence of IE-2, IE- 12, IL- 18, TGFb, rapamycin, and idelalisib.

[0135] Aspect 73A is the method of any one of aspects 1A-72A, wherein the exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and 2-DG.

[0136] Aspect 74A is the method of any one of aspects 1A-74A, wherein the exposing to the deactivating agent occurs in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

[0137] Aspect 75 A is the method of any one of aspects 1A-74A, wherein the concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL.

[0138] Aspect 76A is the method of any one of aspects 1A-75A, wherein the exposing step takes place in an incubator set at 37 °C with 5% CO2.

[0139] Aspect 77A is the method of any one of aspects 1A-76A, wherein the exposing step takes place in the presence of one or more mTOR inhibitors, one or more PI3K inhibitors, one or more metabolizable sugars, and / or one or more non-metabolizable glucose analogs.

[0140] Aspect 78 A is the method of any one of aspects 66A or 69A, wherein the exposing step occurs for about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

[0141] Aspect 79A is the method of aspect 78A, wherein the exposing step occurs for about 24 hours.

[0142] Aspect 80A is the method of any one of aspects 1A-58A, further comprising obtaining the prepared NK cells or freezing the prepared NK cells.

[0143] Aspect 81 A is the method of any one of aspects 1 A-80A, wherein the prepared NK cells are further suspended in a pharmaceutically acceptable media.

[0144] Aspect 82A is the method of any one of aspects 1 A-82A, wherein the prepared NK cells are suspended in PlasmaLyte-A containing 0.5% HSA.

[0145] Aspect 83A is the method of any one of aspects 1 A-82A, wherein the prepared NK cells are cryopreserved and are thawed prior to administering the cells to an individual in need thereof.

[0146] Aspect 84A is the method of any one of aspects 1A-83A, wherein the engineered construct comprises a sequence (e.g., a nucleic acid sequence) encoding an engineered antigen receptor.

[0147] Aspect 85A is the method of aspect 84A, wherein the engineered antigen receptor is a chimeric antigen receptor, a T cell receptor, or both.

[0148] Aspect 86A is the method of aspect 84A or 85A, wherein the engineered antigen receptor targets one or more of CD5, CD19, CD20, CD30, CD70, TROP2, PRAME, KRAS, IL13, EGVRv3, BCMA, GPRC5D, HER2, and / or c-MET.

[0149] Aspect 87A is the method of any one of aspects 1A-86A, wherein the transfection and / or transduction step comprises introduction of an engineered mutation in one or more of endogenous NK cell genes.

[0150] Aspect 88 A is the method of aspect 87 A, wherein the engineered mutation is in one or more of GR, TGFBR2, CISH, and / or CD38 genes.

[0151] Aspect 89A is the method of any one of aspects 1A-88A, wherein the engineered construct comprises a sequence encoding one or more cytokines.

[0152] Aspect 90A is the method of aspect 89A, wherein the cytokine is IL-15, IL-21, or both.

[0153] Aspect 91 A is the method of aspect 89A or 90A, wherein the cytokine is autonomously secreted.

[0154] Aspect 92A is the method of any one of aspects 1A-91A, wherein the NK cells are obtained from cord blood (CB), peripheral blood (PB), stem cells, or bone marrow.

[0155] Aspect 93 A is the method of aspect 92A, wherein the NK cells is obtained from CB.

[0156] Aspect 94A is the method of aspect 93A, wherein the CB is pooled from 2, 3, 4, 5, 6, 7, or 8 or more individual cord blood units.

[0157] Aspect 95A is the method of any one of aspects 1 A-94A, wherein the population of NK cells are further defined as CD56+NK cells, CD16+ NK cells, or CD56+CD16+ NK cells.

[0158] Aspect 96A is the method of any one of aspects 1A-95A, wherein the aAPCs further express a membrane -bound cytokine.

[0159] Aspect 97 A is the method of aspect 96 A, wherein the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membrane-bound IL- 15 (mIL-15).

[0160] Aspect 98A is the method of any one of aspects 1A-97A, wherein the aAPCs have essentially no expression of endogenous HLA class I, II, or CDld molecules.

[0161] Aspect 99A is the method of any one of aspects 1A-98A, wherein the aAPCs express ICAM-1 (CD54) and LFA-3 (CD58).

[0162] Aspect 100A is the method of any one of aspects 1A-99A, wherein the aAPCs are further defined as leukemia cell-derived aAPCs.

[0163] Aspect 101 A is the method of aspect 100A, wherein the leukemia-cell derived aAPCs are K562 cells engineered to express CD137 ligand, CD48, CS1, and / or mIL-21.

[0164] Aspect 102A is the method of aspect 101A, wherein the aAPCs have been engineered by retroviral transduction, are irradiated, or both.

[0165] Aspect 103A is the method of any one of aspects 1A-102A, wherein the pre-activating step is for about 10-20 hours.

[0166] Aspect 104A is the method of any one of aspects 1A-103A, wherein the pre-activating step is for 14-18 hours.

[0167] Aspect 105A is the method of any one of aspects 1A-104A, wherein the pre-activating step is for 16 hours.

[0168] Aspect 106A is the method of any one of aspects 1A-105A, wherein the pre-activation culture comprises IL-18 and / or IL-15 at a concentration of 10-100 ng / mL.

[0169] Aspect 107A is the method of any one of aspects 1A-106A, wherein the pre-activation culture comprises IL- 18 and / or IL- 15 at a concentration of 40-60 ng / mL.

[0170] Aspect 108A is the method of any one of aspects 1A-107A, wherein the pre-activation culture comprises IL- 18 and / or IL- 15 at a concentration of 50 ng / mL.

[0171] Aspect 109A is the method of any one of aspects 1A-108A, wherein the pre-activation culture comprises IL- 12 at a concentration of 0.1-150 ng / mL.

[0172] Aspect 110A is the method of any one of aspects 1A-109A, wherein the pre-activation culture comprises IL- 12 at a concentration of 1-20 ng / mL.

[0173] Aspect 111A is the method of any one of aspects 1A-110A, wherein the pre-activation culture comprises IL- 12 at a concentration of 10 ng / mL.

[0174] Aspect 112A is the method of any one of aspects 1-A111A, wherein the pre-activated NK cells and aAPCs are present in the expansion culture at a ratio of 3:1 to 1:3.

[0175] Aspect 113A is the method of any one of aspects 1A-112A, wherein the pre-activated NK cells and aAPCs are present in the expansion culture at a ratio 1:2.

[0176] Aspect 114A is the method of any one of aspects 1 A-l 13 A, wherein the IL-2 in the first or second expansion culture is present at a concentration of 10-1000 U / mL.

[0177] Aspect 115A is the method of aspect 114A, wherein the IL-2 is present at a concentration of 100-300 U / mL.

[0178] Aspect 116A is the method of aspect 115A, wherein the IL-2 is present at a concentration of 200 U / mL.

[0179] Aspect 117 A is a population of prepared NK cells produced according to the method of any one of aspects 1A-116A.

[0180] Aspect 118A is the population of aspect 117A, wherein the prepared NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared utilizing other methods.

[0181] Aspect 119A is the population of aspect 117A or 118A, wherein the prepared NK cells have a shifted chemokine profile compared to NK cells prepared utilizing other methods.

[0182] Aspect 120A is the population of aspect 117A, wherein a population of the prepared NK cells has increased expression levels of CD62L, CXCR4, and / or CCR7 relative to NK cells prepared utilizing other methods.

[0183] Aspect 121 A is the population of any one of aspects 117A-120A, wherein a population of the prepared NK cells includes at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the NK cells being positive for CD62L.

[0184] Aspect 122A is the population of any one of aspects 117 A- 121 A, wherein a population of the prepared NK cells includes at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, of the NK cells being positive for CXCR4.

[0185] Aspect 123 A is the population of any one of aspects 117A-122A, wherein a population of the prepared NK cells includes at least about 20%, 25%, 30%, 35%, 40%, or 45% of the NK cells being positive for CCR7.

[0186] Aspect 124A is the population of any one of aspects 117A-123A, wherein a population of prepared NK cells has an increased level of transduction and / or transformation relative to NK cells prepared utilizing other methods.

[0187] Aspect 125A is the population of any one of aspects 117A-124A, wherein a population of prepared NK cells has a transduction efficiency level of at least 50%, 55%, or 60%.

[0188] Aspect 126A is the population of any one of aspects 117A-125A, wherein a population of prepared NK cells has a transfection efficiency level of at least 80%, 85%, 90%, or 95%.

[0189] Aspect 127A is the population of aspect 117A, further comprising one or more antibodies.

[0190] Aspect 128 A is the population of aspect 127 A, wherein the antibodies are complexed to the NK cells.

[0191] Aspect 129A is a pharmaceutical composition comprising the population of prepared NK cells of any one of aspects 117A-128A and a pharmaceutically acceptable carrier.

[0192] Aspect 130A is a composition comprising an effective amount of the prepared NK cells of aspect 129A for use in the treatment of a disease or disorder in a subject.

[0193] Aspect 131 A is the composition of aspect 130A, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

[0194] Aspect 132A is the composition of aspect 131 A, wherein the cancer is a leukemia.

[0195] Aspect 133 A is the composition of aspect 132A, wherein the leukemia is an acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), or a chronic myelogenous leukemia (CML).

[0196] Aspect 134A is the composition of aspect 131 A, wherein the cancer is a solid tumor.

[0197] Aspect 135A is the composition of aspect 131 A, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

[0198] Aspect 136A is the composition of aspect 131 A, wherein the disorder is graft versus host disease (GVHD).

[0199] Aspect 137A is the composition of aspect 131 A, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0200] Aspect 138A is a method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of the prepared NK cells of any one of aspects 117A-128A to the subject.

[0201] Aspect 139A is the method of aspect 138A, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

[0202] Aspect 140A is the method of aspect 139A, wherein the cancer is a leukemia.

[0203] Aspect 141A is the method of aspect 139A, wherein the cancer is a solid tumor.

[0204] Aspect 142A is the composition of aspect 141A, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

[0205] Aspect 143A is the method of aspect 139A, wherein the disorder is graft versus host disease (GVHD).

[0206] Aspect 144A is the method of aspect 139A, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0207] Aspect 145 Ais a composition for expansion of NK cells comprising, IL-2, IL-12, IL- 18, and TGFb.

[0208] Aspect 146A is the composition of aspect 145A, further comprising one or more mTOR inhibitor, one or more PI3K inhibitor, one or more dual action mTOR and PI3K inhibitor, one or more non-metabolizable glucose analogs, one or more metabolizable sugars, one or more IDH2 inhibitors, and / or aAPCs.

[0209] Aspect 147A is the composition of aspect 145A or 146A, comprising a concentration of IL-2 is equal to or about 100 to 300 U / mL particularly about 200 U / mL, the concentration of IL- 12 is equal to or about 1-100 ng / mL particularly about 10 ng / mL, the concentration of IL- 18 is equal to or about 1-200 ng / mL particularly about 20 ng / mL, and the concentration of TGFb is equal to or about 0.01 to 1 ng / mL particularly about 0.3 ng / mL.

[0210] Aspect 148A is the composition of any one of aspects 145A-147A, wherein the TGFb comprises or is TGFbl.

[0211] Aspect 149A is the composition of any one of aspects 145A-148A, wherein the mTOR inhibitor comprises, consists essentially of, or consists of one or more of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, non-rapalog allosteric inhibitor R1-R5, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI- 027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25.

[0212] Aspect 150A is the composition of any one of aspects 146A-149A, wherein the mTOR inhibitor comprises, consists essentially of, or consists of rapamycin.

[0213] Aspect 151 A is the composition of aspect 149A or 150A, wherein the rapamycin is at a concentration of about 0.1 nM to 100 nM particularly about 1 nM or 10 nM.

[0214] Aspect 152A is the composition of any one of aspects 146A-151A, wherein the PI3K inhibitor comprises, consists essentially of, or consists of one or more of idelalisib,wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, and / or umbrasilib.

[0215] Aspect 153A is the composition of any one of aspects 146A-152A, wherein the PI3K inhibitor comprises, consists essentially of, or consists of Idelalisib.

[0216] Aspect 154A is the composition of aspect 152A or 153A, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM particularly about 10 nM.

[0217] Aspect 155A is the composition of any one of aspects 146A-154A, comprising a dual action mTOR and PI3K inhibitor.

[0218] Aspect 156A is the composition of aspect 155A, wherein the dual action mTOR and PI3K inhibitor comprises, consists essentially of, or consists of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), and / or voxtalisib (XL765).

[0219] Aspect 157A is the composition of any one of aspects 146A-156A, wherein the non- metabolizable glucose analog comprises, consists essentially of, or consists of 2-Deoxy-d- Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), and / or 6- Deoxy-D-Glucose (6DG).

[0220] Aspect 158A is the composition of aspect 157A, wherein the non-metabolizable glucose analog comprises, consists essentially of, or consists of 2-DG at a concentration of about 0.1 mM to 20 mM particularly about 2 mM.

[0221] Aspect 159A is the composition of any one of aspects 146A-158A, wherein the one or more metabolizable sugar comprises, consists essentially of, or consists of sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum ly coper sici (E-FOL).

[0222] Aspect 160A is the composition of any one of aspects 146A-159A, wherein the isocitrate dehydrogenase 2 (IDH2) inhibitor comprises, consists essentially of, or consists of Enasidenib and / or Ivo sidenib.

[0223] Aspect 161A is the composition of any one of aspects 146A-160A, wherein the concentration of IDH2 inhibitor is at about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

[0224] Aspect 162A is the composition of any one of aspects 145A-161A, further comprising NK cells.

[0225] Aspect 163 A is the composition of aspect 162A, wherein the NK cells are NK cells preactivated by exposure to IL12, IL15, and IL18.

[0226] Aspect 164A is the composition of any one of aspects 145A-163A, further comprising one or more retroviral particles and / or polynucleotide constructs.

[0227] Aspect 165A is the composition of any one of aspects 145A-164A, further comprising one or more deactivating agents.

[0228] Aspect 166A is the composition of any one of aspects 145A-165A, comprising aAPCs that have been stably engineered to transgenically express CD137 ligand, CS1, CD48, and / or mIL21; optionally wherein the aAPCs have been irradiated.

[0229] Aspect IB is an in vitro method comprising: an expanding step comprising: a culturing step comprising culturing NK cells for about 1-14 days in an expansion culture media comprising exogenous: (i) IL-2, IL- 12, IL- 18, and TGFb; and (ii) antigen presenting cells (APCs), artificial antigen presenting cells (aAPCs), universal antigen presenting cells (uAPCs), activating beads, and / or feeder cells.

[0230] Aspect 2B is the method of aspect IB, wherein the expanding step comprises a second culturing step comprising: culturing the NK cells for 1-14 days in a second expansion culture media comprising: (i) exogenous IL-2, IL- 12, IL- 18, and TGFb, and (ii) one or more mammalian target of rapamycin (mTOR) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, dual mTOR and PI3K inhibitor, isocitrate dehydrogenase 2 (IDH2) inhibitor, isocitrate dehydrogenase 1 (IDH2) inhibitor, dual IDH1 and IDH2 (IDH1 / 2) inhibitor, protein kinase B (PKB, AKT) inhibitor, additional cytokines, non-metabolizable glucose analogs, metabolizable sugars, APCs, aAPCs, uAPCs, activating beads, or a combination thereof.

[0231] Aspect 3B is the method of aspect IB or 2B, further comprising a pre-activation step comprising culturing the NK cells in pre-activation media comprising IL- 12, IL- 15, and IL- 18 prior to the expanding step.

[0232] Aspect 4B is the method of any one of aspects 1B-3B, wherein the expansion culture media and / or second expansion culture media comprises an mTOR inhibitor.

[0233] Aspect 5B is the method of any one of aspects 1B-4B, wherein the expansion culture media and / or second expansion culture media comprise exogenous IL-2 at a concentration of about 100 to 300 U / mL, IL- 12 at a concentration of about 1-100 ng / mL, IL- 18 at a concentration of about 1-200 ng / mL, and TGFb at a concentration of about 0.01 to 1 ng / mL.

[0234] Aspect 6B is the method of any one of aspects 1B-5B, wherein the expanding step comprises culturing NK cells for 1-9 days.

[0235] Aspect 7B is the method of any one of aspects 1B-6B, wherein the expanding step comprises culturing NK cells for about 1-4 days in the expansion culture media.

[0236] Aspect 8B is the method of any one of aspects 2B-7B, wherein the second culturing step comprises culturing NK cells for about 4 days in the expansion culture media.

[0237] Aspect 9B is the method of any one of aspects 2B-8B, wherein the second culturing step comprises culturing NK cells for 1-9 days in the second expansion culture media.

[0238] Aspect 10B is the method of any one of aspects 2B-9B, wherein the second culturing step comprises culturing NK cells for about 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 4 days, or 3 days in the second expansion culture media.

[0239] Aspect 11B is the method of any one of aspects 1B-10B, wherein the expanding step comprises culturing NK cells for or for less than 1, 2, 3, 4, 5, 6, or 7 days in the expansion culture media, and comprises culturing NK cells for or for less than 1, 2, 3, 4, 5, 6, or 7 days in the second expansion culture media.

[0240] Aspect 12B is the method of any one of aspects 1B-11B, wherein the expanding step comprises culturing NK cells for 3-6 days in the expansion culture media.

[0241] Aspect 13B is the method of any one of aspects 1B-12B, wherein the expanding step comprises or consists of culturing the NK cells for 3-5 or 4 days in the expansion culture media and culturing the NK cells for 2-5 or 3 days in the second expansion culture media

[0242] Aspect 14B is the method of any one of aspects 3B-13B, wherein the pre-activation step comprises culturing the NK cells for 12-24 hours, and the expanding step comprises culturing the NK cells for 3-5 or 4 days in the expansion culture media, and the second culturing step comprises culturing the NK cells for 2-5 or 3 days in the second expansion culture media.

[0243] Aspect 15B is the method of any one of aspects 2B-14B, wherein APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are provided in the second culturing step.

[0244] Aspect 16B is the method of any one of aspects 2B-15B, wherein no APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are provided in the second culturing step.

[0245] Aspect 17B is the method of any one of aspects 2B-16B, further comprising removing the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells from the expansion culture media before the second culturing step.

[0246] Aspect 18B is the method of any one of aspects 2B-17B, wherein the first culturing step and second culturing step are separated by an NK cell enrichment step.

[0247] Aspect 19B is the method of any one of aspects 1B-18B, wherein the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are removed from the expansion culture media or second expansion culture media by about 5, 6, 7, 8, or 9 days from the beginning of the expanding step.

[0248] Aspect 20B is the method of any one of aspects 1B-19B, wherein the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are removed from the expansion culture media by about 5, 6 or 7 days from the beginning of the expanding step.

[0249] Aspect 2 IB is the method of any one of aspects 1B-20B, wherein the second culturing step comprises culturing the NK cells in the absence of APCs, aAPCs, uAPCs, activating beads, and / or feeder cells.

[0250] Aspect 22B is the method of any one of aspects 1B-22B, further comprising an enrichment step comprising removing the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells from the expansion culture media immediately before the second culturing step.

[0251] Aspect 23B is the method of any one of aspects 1B-23B, wherein the expansion culture media and / or second expansion culture media comprises:

[0252] an exogenous IL-2 concentration equal to or about 100 to 600 U / mL, IL- 12 concentration equal to or about 1-100 ng / mL, and IL- 18 concentration equal to or about 1-200 ng / mL.

[0253] Aspect 24B is the method of any one of aspects 1B-23B, wherein the expansion culture media and / or second expansion culture media comprises an exogenous TGFb concentration of less than 10 ng / mL, less than or about 5 ng / mL, or about 0.01 to 1 ng / mL, or about 0.1 to 0.5 ng / mL, or about 0.3 ng / mL.

[0254] Aspect 25B is the method of any one of aspects 1B-24B, wherein the expansion culture media and / or second expansion culture media comprises:

[0255] an exogenous TGFb concentration of less than 10 ng / mL, less than or about 5 ng / mL, or about 0.01 to 1 ng / mL, or about 0.1 to 0.5 ng / mL, or about 0.3 ng / mL.

[0256] Aspect 26B is the method of any one of aspects 1B-25B, wherein the expansion culture media and / or second expansion culture media comprises a TGFb concentration of less than about 1 ng / mL or about 0.3 ng / mL.

[0257] Aspect 27B is the method of any one of aspects 1B-26B, wherein the expansion culture media and / or second expansion culture media comprises an mTOR inhibitor selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI- 027, OXA-01, XL388, CC214-1, CC-223, CC-115, DHM25, or combinations thereof.

[0258] Aspect 28B is the method of any one of aspects 1B-27B, wherein the expansion culture media comprises rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM.

[0259] Aspect 29B is the method of any one of aspects 1B-28B, wherein the first culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

[0260] Aspect 30B is the method of any one of aspects 1B-29B, wherein the expansion culture media and / or second expansion culture media comprises the mTOR inhibitor rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM 1 nM.

[0261] Aspect 3 IB is the method of any one of aspects 1B-30B, wherein the expansion culture media and the second expansion culture media comprise rapamycin.

[0262] Aspect 32B is the method of any one of aspects 1B-31B, wherein the method includes transforming and / or transducing the cells with an engineered construct.

[0263] Aspect 33B is the method of any one of aspects 1B-32B, wherein the transforming comprises transient introduction of one or more ribonucleoproteins, polynucleotides, and / or polypeptides.

[0264] Aspect 34B is the method of any one of aspects 1B-33B, wherein the transducing comprises stable transgene introduction into the genome of the NK cell.

[0265] Aspect 35B is the method of any one of aspects 1B-34B, wherein the transducing comprises contacting the NK cells with one or more RetroNectin bound retroviral constructs.

[0266] Aspect 36B is the method of any one of aspects 1B-35B, wherein the PI3K inhibitor is selected from the group consisting of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, umbrasilib, or combinations thereof.

[0267] Aspect 37B is the method of any one of aspects 1B-36B, wherein the PI3K inhibitor is Idelalisib.

[0268] Aspect 38B is the method of any one of aspects 1B-37B, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM, or about 10 nM.

[0269] Aspect 39B is the method of any one of aspects 1B-38B, wherein the second expansion culture media comprises Idelalisib at about 10 nM concentration.

[0270] Aspect 40B is the method of any one of aspects 2B-39B, wherein the dual mTOR and PI3K inhibitor is selected from the group consisting of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), voxtalisib (XL765), or combinations thereof

[0271] Aspect 41B is the method of any one of aspects 2B-40B, wherein the dual mTOR and PI3K inhibitor is at a concentration of about 0.1 nM to 100 nM, or about 10 nM.

[0272] Aspect 42B is the method of any one of aspects 2B-41B, wherein the AKT inhibitor is selected from the group consisting of capivasertib (AZD-5363), ipatasertib, miltefosine, uprosertib (GSK2141795), miransertib (ARQ 092), afuresertib (GSK2110183), palomid 529 (P529), perifosine (KRX-0401), or combinations thereof.

[0273] Aspect 43B is the method of any one of aspects 2B-42B, wherein the second expansion culture media comprises the AKT inhibitor capivasertib.

[0274] Aspect 44B is the method of any one of aspects 2B-43B, wherein the non-metabolizable glucose analog is selected from the group consisting of 2-Deoxy-d-Glucose (2-DG), 3-O- Methyl-D-Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), 6-Deoxy-D-Glucose (6DG), or combinations thereof.

[0275] Aspect 45B is the method of any one of aspects 2B-44B, wherein the non-metabolizable glucose analog comprises consists of 2-DG at a concentration of about 0.1 mM to 20 mM or about 2 mM.

[0276] Aspect 46B is the method of any one of aspects 2B-45B, wherein the second expansion culture media comprises the non-metabolizable glucose analog 2-DG at a concentration of about 2 mM.

[0277] Aspect 47B is the method of any one of aspects 2B-46B, wherein the metabolizable sugar is selected from the group consisting of sucrose, palatinose, turanose, elicitor preparation of F. oxysporum ly coper sici (E-FOL), or combinations thereof.

[0278] Aspect 48B is the method of any one of aspects 2B-47B, wherein the second expansion culture media comprises the metabolizable sugar sucrose.

[0279] Aspect 49B is the method of any one of aspects 2B-48B, wherein the IDH2 inhibitor is selected from the group consisting of Enasidenib, Ivosidenib, or combinations thereof.

[0280] Aspect 50B is the method of any one of aspects 2B-49B, wherein the concentration of IDH2 inhibitor is about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

[0281] Aspect 5 IB is the method of any one of aspects 1B-50B, wherein the expansion culture media and / or second expansion culture media comprises about 200, 100, 150, 175, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, of IL-2.

[0282] Aspect 52B is the method of any one of aspects 1B-51B, wherein the expansion culture media and second expansion culture media comprises about 200, 100, 150, 175, 225, 250, 275, 300, 325, 350, 375, or 400 U / mL, of IL-2.

[0283] Aspect 53B is the method of any one of aspects 32B-52B, wherein the transforming and / or transducing takes place in the presence of about 200 U / mL or 600 U / mL of IL-2.

[0284] Aspect 54B is the method of any one of aspects 32B-53B, wherein the transforming and / or transducing takes place in the presence of IL-2, IL- 12, IL- 18, and TGFb.

[0285]

[0286] Aspect 55B is the method of any one of aspects 32B-54B, wherein the transforming and / or transducing takes place in the presence of IL-2, IL- 12, IL- 18, TGFb, and rapamycin.

[0287] Aspect 56B is the method of any one of aspects 32B-55B, wherein the transforming and / or transducing takes place in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and idelalisib.

[0288] Aspect 57B is the method of any one of aspects 32B-56B, wherein the transforming and / or transducing takes place in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and 2- DG.

[0289] Aspect 58B is the method of any one of aspects 32B-57B, wherein the transforming and / or transducing takes place in the presence of IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

[0290] Aspect 59B is the method of any one of aspects 1B-58B, further comprising a deactivating step comprising exposing the NK cells to an effective amount of one or more deactivating agents after the expanding step.

[0291] Aspect 60B is the method of aspect 59, wherein the deactivating agent is Dasatinib.

[0292] Aspect 6 IB is the method of aspect 60, wherein the Dasatinib concentration is about 1 pM.

[0293] Aspect 62B is the method of any one of aspects 59B-61B, wherein the deactivating step further comprises exposing the NK cells to IL-2.

[0294] Aspect 63B is the method of any one of aspects 59B-62B, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, and TGFb.

[0295] Aspect 64B is the method of any one of aspects 59B-63B, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, and rapamycin.

[0296] Aspect 65B is the method of any one of aspects 59B-64B, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and idelalisib.

[0297] Aspect 66B is the method of any one of aspects 59B-65B, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and 2-DG.

[0298] Aspect 67B is the method of any one of aspects 59B-66B, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

[0299] Aspect 68B is the method of any one of aspects 59B-67B, wherein the deactivating step further comprises exposing the NK cells to one or more mTOR inhibitors, PI3K inhibitors, metabolizable sugars, and / or non-metabolizable glucose analogs.

[0300] Aspect 69B is the method of any one of aspects 59B-68B, wherein the deactivating step occurs for about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

[0301] Aspect 70B is the method of any one of aspects 59B-69B, wherein the deactivating step occurs for about 24 hours.

[0302] Aspect 7 IB is the method of any one of aspects 59B-70B, further comprising cry opreserving the prepared NK cells.

[0303] Aspect 72B is the method of any one of aspects 59B-71B, further comprising suspending the NK cells in a pharmaceutically acceptable media.

[0304] Aspect 73B is the method of any one of aspects 59B-72B, further comprising suspending the NK cells in PlasmaLyte-A containing 0.5% HSA.

[0305] Aspect 74B is the method of aspect 71B-73B, further comprising thawing the cryopreserved NK cells prior to administering the NK cells to an individual in need thereof.

[0306] Aspect 75B is the method of any one of aspects 32B-74B, wherein the engineered construct comprises a nucleic acid sequence encoding an engineered antigen receptor.

[0307] Aspect 76B is the method of aspect 75B, wherein the engineered antigen receptor is a chimeric antigen receptor, a T cell receptor, or both.

[0308] Aspect 77B is the method of aspect 75B or 76B, wherein the engineered antigen receptor targets one or more antigenic peptide derived from and / or antigen selected from the group consisting of: CD19, CD5, CD20, CD30, CD70, TROP2, PRAME, KRAS, IL13, EGVRv3, BCMA, GPRC5D, HER2, c-MET, NYESO1, HPV, CMV, or combinations thereof.

[0309] Aspect 78B is the method of any one of aspects 32B-77B, wherein the transfection and / or transduction step comprises introduction of an engineered mutation in one or more of endogenous NK cell genes.

[0310] Aspect 79B is the method of aspect 78B, wherein the engineered mutation is in one or more of GR, TGFBR2, CISH, and / or CD38 genes.

[0311] Aspect 80B is the method of any one of aspects 32B-79B, wherein the engineered construct comprises a nucleic acid sequence encoding one or more cytokines.

[0312] Aspect 8 IB is the method of aspect 80B, wherein the nucleic acid sequence encodes IL- 15, IL-21, or both IL- 15 and IL-21.

[0313] Aspect 82B is the method of aspect 80B, wherein the nucleic acid sequence encodes one or more autonomous secretory signal.

[0314] Aspect 83B is the method of any one of aspects 1B-82B, wherein the NK cells are obtained from cord blood (CB), peripheral blood (PB), stem cells, or bone marrow.

[0315] Aspect 84B is the method of aspect 83B, wherein the NK cells is obtained from CB.

[0316] Aspect 85B is the method of aspect 84B, wherein the CB is pooled from 2, 3, 4, 5, 6, 7, or 8 or more individual cord blood units.

[0317] Aspect 86B is the method of any one of aspects 1B-85B, wherein the NK cells are CD56+NK cells, CD16+ NK cells, or CD56+CD16+ NK cells.

[0318] Aspect 87B is the method of any one of aspects 1B-86B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells express a membrane-bound cytokine.

[0319] Aspect 88B is the method of aspect 87B, wherein the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membrane-bound IL- 15 (mIL-15).

[0320] Aspect 89B is the method of any one of aspects 1B-88B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells have essentially no expression of endogenous HLA class I, II, or CD Id molecules.

[0321] Aspect 90B is the method of any one of aspects 1B-89B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells express ICAM-1 (CD54) and LFA-3 (CD58).

[0322] Aspect 9 IB is the method of any one of aspects 1B-90B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells are derived from leukemia cells.

[0323] Aspect 92B is the method of any one of aspects 1B-91B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells are engineered to express CD 137 ligand, CD48, CS1, and / or mlL- 21.

[0324] Aspect 93B is the method of any one of aspects 1B-93B, wherein the APCs, aAPCs, uAPCs, and / or feeder cells have been engineered by retroviral transduction, are irradiated, or both.

[0325] Aspect 94B is the method of any one of aspects 3B-93B, wherein the pre-activation step is for about 10-20 hours.

[0326] Aspect 95B is the method of any one of aspects 3B-94B, wherein the pre-activation step is for 14-18 hours.

[0327] Aspect 96B is the method of any one of aspects 3B-95B, wherein the pre-activation step is for 16-24 hours.

[0328] Aspect 97B is the method of any one of aspects 3B-96B, wherein the pre-activation media comprises IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, 40-60 ng / mL, or 50 ng / mL.

[0329] Aspect 98B is the method of any one of aspects 3B-97B, wherein the pre-activation media comprises IL-12 at a concentration of 0.1-150 ng / mL, 1-20 ng / mL, or 10 ng / mL.

[0330] Aspect 99B is the method of any one of aspects 1B-98B, wherein the NK cells and APCs, aAPCs, uAPCs, and / or feeder cells are present in the expansion culture media and / or second expansion culture media at a ratio of 3:1 to 1:3, or 1:2.

[0331] Aspect 100B is the method of any one of aspects 1B-99B, wherein the IL-2 in the expansion culture media and / or second expansion culture media is present at a concentration of 10-1000 U / mL, 100-300 U / mL, or 200 U / mL.

[0332] Aspect 101. Is a kit for performing the method of producing NK cells and / or producing a medicament according to the method of any one of aspects 1B-100B.

[0333] Aspect 102. NK cells produced according to the method of any one of any one of aspects 1B-100B.

[0334] Aspect 103B is the NK cells of aspect 102B, wherein the NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared utilizing other methods.

[0335] Aspect 104B is the NK cells of aspect 102B or 103B, wherein the NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared in the absence of exogenous TGFb.

[0336] Aspect 105B is the NK cells of any one of aspects 102B-104B, wherein the NK cells have a shifted chemokine profile compared to NK cells prepared utilizing other methods.

[0337] Aspect 106B is the NK cells of any one of aspects 102B-105B, wherein the NK cells exhibit increased expression levels of CD62L, CXCR4, and / or CCR7 relative to NK cells prepared utilizing other methods.

[0338] Aspect 107B is the NK cells of any one of aspects 102B-106B, wherein at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the NK cells are positive for CD62L.

[0339] Aspect 108B is the NK cells of any one of aspects 102B-107B, wherein at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, of the NK cells are positive for CXCR4.

[0340] Aspect 109B is the NK cells of any one of aspects 102B-108B, wherein at least about 20%, 25%, 30%, 35%, 40%, or 45% of the NK cells are positive for CCR7.

[0341] Aspect HOB is the NK cells of any one of aspects 102B-109B, wherein the NK cells exhibit an increased level of transduction and / or transformation relative to NK cells prepared utilizing other methods.

[0342] Aspect 11 IB is the NK cells of any one of aspects 102B-110B, wherein the NK cells exhibit a transduction efficiency level of at least 50%, 55%, or 60%.

[0343] Aspect 112B is the NK cells of any one of aspects 102B-111B, wherein the NK cells exhibit a transfection efficiency level of at least 80%, 85%, 90%, or 95%.

[0344] Aspect 113B is the NK cells of any one of aspects 102B-112B, wherein the NK cells have a phenotype as described in FIGs 21B, 39A, 39B, and / or 39C.

[0345] Aspect 114B is the NK cells of any one of aspects 102B-113B, wherein the NK cells have a reduced baseline SMAD2 / 3 phosphorylation level relative to NK cells prepared using concentrations of TGFb greater than or equal to 5, 6, 7, 8, 9, or 10 ng / ml.

[0346] Aspect 115B is the NK cells of any one of aspects 102B-114B, wherein the NK cells are characterized by higher expression of TRAIL, DNAM-1, CD25, CLA, and / or CD16 relative to NK cells prepared using other methods.

[0347] Aspect 116B is the NK cells of any one of aspects 102B-115B, wherein the NK cells can be characterized by higher expression of DNAM and TRAIL, similar or lower levels of SMAD2 / 3, reduced TIGIT expression, comparable CD38 levels, and / or decreased perforin and granzyme B, relative to NK cells prepared using other methods.

[0348] Aspect 117B is the NK cells of any one of aspects 102B-116B, wherein the NK cells can be characterized by altered SMAD3, CD38, perforin, granzyme, and FasL mRNA / protein levels, and increased TRAIL mRNA / protein levels relative to NK cells prepared using other methods.

[0349] Aspect 118. A composition comprising the NK cells of any one of aspects 102B- 117B and one or more antibodies.

[0350] Aspect 119B is the composition of aspect 118B, wherein the one or more antibodies are complexed to the NK cells.

[0351] Aspect 120. A pharmaceutical composition comprising NK cells according prepared according to the method of any one of aspects 1B-100B, and a pharmaceutically acceptable carrier.

[0352] Aspect 121B is the pharmaceutical composition of aspect 120B for use in the treatment of a disease or disorder in a subject.

[0353] Aspect 122B is the pharmaceutical composition of aspect 121B, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

[0354] Aspect 123B is the pharmaceutical composition of aspect 122B, wherein the cancer is a leukemia.

[0355] Aspect 124B is the pharmaceutical composition of aspect 123B, wherein the leukemia is an acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), or a chronic myelogenous leukemia (CML).

[0356] Aspect 125B is the pharmaceutical composition of aspect 122B, wherein the cancer is a solid tumor.

[0357] Aspect 126B is the pharmaceutical composition of aspect 122B, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

[0358] Aspect 127B is the pharmaceutical composition of aspect 122B, wherein the disorder is graft versus host disease (GVHD).

[0359] Aspect 128B is the pharmaceutical composition of aspect 122B, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0360] Aspect 129. A method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of the prepared NK cells of aspect 101B-117B to the subject.

[0361] Aspect 130B is the method of aspect 129B, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

[0362] Aspect 13 IB is the method of aspect 130B, wherein the cancer is a leukemia.

[0363] Aspect 132B is the method of aspect 130B, wherein the cancer is a solid tumor.

[0364] Aspect 133B is the method of aspect 130B, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

[0365] Aspect 134B is the method of aspect 130B, wherein the disorder is graft versus host disease (GVHD).

[0366] Aspect 135B is the method of aspect 130B, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

[0367] Aspect 136B is an in vitro composition for expansion of NK cells comprising, IL-2, IL- 12, IL- 18, and TGFb.

[0368] Aspect 137B is the composition of aspect 136B, further comprising one or more mTOR inhibitor, PI3K inhibitor, dual action mTOR and PI3K inhibitor, non-metabolizable glucose analogs, metabolizable sugars, IDH1 inhibitors, IDH2 inhibitors, IDH1 / 2 inhibitors, APCs, aAPCs, uAPCs, activating beads, and / or feeder cells, or a combination thereof.

[0369] Aspect 138B is the composition of aspect 136B or 137B, comprising a concentration of IL-2 of or of about 100 to 300 U / mL, a concentration of IL- 12 of or of about 1-100 ng / mL, a concentration of IL- 18 of or about 1-200 ng / mL, and the concentration of TGFb of or of about 0.01 to 5 ng / mL or 0.1 to 1 ng / mL.

[0370] Aspect 139B is the composition of any one of aspects 136B-138B, wherein the TGFb is TGFbl.

[0371] Aspect 140B is the composition of any one of aspects 136B-139B, wherein the composition comprises an mTOR inhibitor, and the mTOR inhibitor is selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, non-rapalog allosteric inhibitor R1-R5, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE- 555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3- d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI-027, OXA-01, XL388, CC214-1, CC-223, CC-115, DHM25, or combinations thereof.

[0372] Aspect 141B is the composition of aspect 140B, wherein the mTOR inhibitor is rapamycin.

[0373] Aspect 142B is the composition of aspect 141B, wherein the rapamycin is at a concentration of about 0.1 nM to 100 nM.

[0374] Aspect 143B is the composition of any one of aspects 136B-142B, wherein the composition comprises a PI3K inhibitor, and the PI3K inhibitor is selected from the group consisting of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, umbrasilib, or combinations thereof.

[0375] Aspect 144B is the composition of aspect 143B, wherein the PI3K inhibitor is Idelalisib.

[0376] Aspect 145B is the composition of aspect 144B, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM, or about 0.1 nM to 10 pM.

[0377] Aspect 146B is the composition of any one of aspects 136B-146B, wherein the composition comprises a dual action mTOR and PI3K inhibitor, and the dual action mTOR and PI3K inhibitor is selected from the group consisting of dactolisib (NVP-BEZ235), gedatolisib (PKL587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), voxtalisib (XL765), or combinations thereof.

[0378] Aspect 147B is the composition of any one of aspects 136B-146B, wherein the composition comprises a non-metabolizable glucose analog, and the non-metabolizable glucose analog is selected from the group consisting of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), 6-Deoxy-D-Glucose (6DG), or combinations thereof.

[0379] Aspect 148B is the composition of aspect 147B, wherein the non-metabolizable glucose analog is 2-DG at a concentration of about 0.1 mM to 20 mM.

[0380] Aspect 149B is the composition of any one of aspects 136B-148B, wherein the composition comprises one or more metabolizable sugar, and the metabolizable sugar is selected from the group consisting of sucrose, palatinose, turanose, elicitor preparation of F. oxysporum lycopersici (E-FOL), or combinations thereof.

[0381] Aspect 150B is the composition of any one of aspects 137B-149B, wherein the isocitrate dehydrogenase 2 (IDH2) inhibitor is Enasidenib and / or Ivosidenib.

[0382] Aspect 151B is the composition of any one of aspects 137B-149B, wherein the concentration of IDH2 inhibitor is about 1 nM to 100 pM, or about 100 nM to 10 pM, or about 1 pM.

[0383] Aspect 152B is the composition of any one of aspects 136B-151B, further comprising NK cells.

[0384] Aspect 153B is the composition of aspect 152B, wherein the NK cells are NK cells preactivated by exposure to IL12, IL15, and IL18.

[0385] Aspect 154B is the composition of any one of aspects 136B-153B, further comprising one or more retroviral particles and / or polynucleotide constructs.

[0386] Aspect 155B is the composition any one of aspects 136B-154B, further comprising one or more deactivating agents.

[0387] Aspect 156B is the composition any one of aspects 136B-155B, further comprising APCs, aAPCs, uAPCs, or feeder cells, optionally wherein the APCs, aAPCs, uAPCs, or feeder cells have been stably engineered to transgenically express CD137 ligand, CS1, CD48, and / or mIL21; optionally wherein the APCs, aAPCs, uAPCs, or feeder cells have been irradiated.

[0388] Aspect 157B is an in vitro method for activating and expanding NK cells comprising: (i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the preactivation step comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL- 12, IL- 15, and IL- 18; and (ii) an expansion step comprising a) a first culturing step of the NK cells in a first expansion culture media, wherein the first culturing is for about 2-8 days, wherein the first expansion culture media comprises: IL-2, IL- 12, IL- 18, TGEb; and APCs, aAPCs, uAPCs, feeder cells, or activating beads; and b) a second culturing step of the NK cells in a second expansion culture media, wherein the second culturing is for about 2-8 days, wherein the second expansion media comprises: IL-2, IL- 12, IL- 18, TGEb; and whereinthe second expansion media may further comprise one or more of: APCs, aAPCs, uAPCs, feeder cells or activating beads, an mTOR inhibitor, a PI3K inhibitor, a dual mTOR and PI3K inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a dual IDH1 and IDH2 inhibitor, an additional cytokine, a non-metabolizable glucose analog, a metabolizable sugar, or a combination thereof.

[0389] Aspect 158B is the method of aspect 157B, wherein the first and / or second expansion culture media comprises a TGFb concentration of about 0.3-10 ng / ml.

[0390] Aspect 159B is the method of aspect 157B or 158B, wherein the first and / or second expansion culture media comprises a TGFb concentration of about 0.3-5.0 ng / ml.

[0391] Aspect 160B is the method of any one of aspects 157B-159B, wherein the first and / or second expansion culture media comprises an mTOR inhibitor.

[0392] Aspect 161B is the method of any one of aspects 157B-160B, wherein the first and / or second expansion culture media comprises the mTOR inhibitor rapamycin.

[0393] Aspect 162B is the method of any one of aspects 157B-161B, wherein the second expansion culture media comprises the PI3K inhibitor Idelalisib.

[0394] Aspect 163B is the method of any one of aspects 157B-162B, wherein the first expansion culture media and / or the second expansion culture media comprises an exogenous IL-2 concentration of about 100-600 lU / ml, an IL- 12 concentration of about 5-25 ng / ml, an IL- 18 concentration of about 5-40 ng / ml, and a TGFb concentration of about 0.3-5.0 ng / ml.

[0395] Aspect 164B is an in vitro method comprising: (i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step is or comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL- 18, (ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-8 days, and wherein the first expansion media comprises (a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin; and (b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-3 days, and wherein the second expansion culture media comprises: IL-2, IL- 12, IL- 18, TGFb, and rapamycin, and (v) optionally deactivating and cry opreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

[0396] Aspect 165B is an in vitro method comprising: (i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step is or comprisesabout 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL- 18, (ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-8 days, and wherein the first expansion media comprises (a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin; (b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-3 days, and wherein the second expansion culture media comprises: IL-2, IL- 12, IL- 18, TGFb, rapamycin and Idelalisib, and (v) optionally deactivating and cry opreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

[0397] Aspect 166B is an in vitro method comprising: (i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL-18, (ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-13 days, and wherein the first expansion media comprises (a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin; (b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and (iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-5 days, and wherein the second expansion culture media comprises: IL-2, IL-12, IL- 18, TGFb, and rapamycin, and (v) optionally deactivating and cryopreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

[0398] Aspect 167B is the method of aspect 166B, wherein the second expansion culture media comprises Idelalisib.

[0399] Aspect 168B is the method of any one of aspects 164B-167B, wherein the pre- activation media comprises about 10 ng / ml IL-12, about 50 ng / ml IL15, and about 50 ng / ml IL- 18; wherein the first expansion media comprises about 200 lU / ml of IL-2, about 10 ng / ml IL- 12, about 20 ng. ml IL- 18, about 0.3 ng / ml TGFb, about 1 nM rapamycin, and uAPCs; wherein the second expansion culture media comprises about 200 lU / ml of IL-2, 10 ng / ml IL- 12, about 20 ng / ml IL- 18, about 0.3 ng / ml TGFb, and about 1 nM rapamycin; and / or wherein the Dasatinib concentration is about 1 pM.

[0400] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0401] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0402] FIGs. 1A-1C show how the duration of ex vivo NK cell expansion can significantly impact in vivo efficacy. FIG. 1A is a schematic outlining an experimental model for testing efficacy of prepared NK cells against tumor cells in vivo (e.g., MM1S cells, a murine model of multiple myeloma, mice were irradiated and received 250,000 MM Is cells administered intravenously through the tail vein). FIG. IB provides representative bioluminescence imaging (BLI) of 7 cohorts of tumor bearing mice that were prepared as described in FIG. 1A. The results demonstrated increased tumor control when increased amounts of thawed engineered prepared NK cells (comprising an anti-CD70 CAR and IL 15 expressing construct; see PCT application publication WO 2022 / 15979 Al, published July 28, 2022, which is incorporated herein by reference in its entirety) were provided to the animals (e.g., less BLI signal in 10 million (M) and 5M dosed animals relative to IM dosage). The results also showed that thawed CD70 CAR / IL15 NK cells that were expanded until Day 8, deactivated for 24 hours, and frozen on Day 9, displayed superior in vivo tumor control relative to thawed CD70 CAR / IL15 NK cells expanded until Day 13, deactivated for 24 hours, and frozen on Day 14. FIG. 1C is a quantitation of fluorescence shown in FIG IB, with individual animals shown.

[0403] FIGs. 2A-2B provide additional characterization of the in vivo experiments described in FIG. 1. FIG. 2A shows persistence of CD56+CD16+ NK cells in the blood of mice treated as described in FIG. 1. The results showed increased levels of engraftment and persistence for the Day 9 cohorts, particularly at 20 days post injection. FIG. 2B shows a survival graph of the MM IS engrafted mice treated with different amounts of the prepared NK cells that were frozen on Day 9 or Day 14, respectively. The results indicate that animals receiving NK cells frozen on Day 9 exhibited significantly longer survival compared to those receiving NK cells frozenon Day 14. Moreover, survival was significantly extended in animals administered 5 million or 10 million CD70 / IL-15 engineered NK cells compared to those receiving only 1 million cells.

[0404] FIG. 3 provides additional characterization of the in vivo experiments described in FIG. 1. Shown is a schematic representation of CB NK cell preparation, with cells being expanded and frozen on Day 9, or on Day 14 (top), with those frozen on Day 14 undergoing a second round of uAPC stimulation during the second expansion step following transduction, and a comparison of the in vivo efficacy of said cells upon thawing and in vivo tumor challenge (bottom). The results demonstrated that the expansion time for CAR NK cell manufacturing significantly impacted the potency of CAR NK cells in vivo.

[0405] FIG. 4 provides an example of a clinical trial evaluating the safety and efficacy of engineered anti-CD5 CAR NK cells (for example, see PCT application publication WO 2022 / 221548 Al, published April 14, 2022, which is incorporated herein by reference in its entirety) for cancer immunotherapy.

[0406] FIGs. 5A-5C provide schematic diagrams showing illustrated exemplary workflows for preparing NK cells and in vitro functionality testing. FIG. 5A depicts an exemplary workflow comprising NK cells (e.g., cord blood NK cells) that are isolated, pre-activated, and expanded using methods and compositions provided herein (e.g., pre-activation comprising 16 hours in the presence of IL- 12 (e.g., lO ng / ml), IL-18 (e.g., 50 ng / ml), and IL15 (e.g., lO ng / ml); e.g., as described in US patent application publication US 2020 / 0390816 Al published December 17, 2020, which is incorporated here by reference in its entirety for any purpose described herein), co-cultured with Antigen Presenting Cells (e.g., universal APCs (uAPCs); e.g., expressing mbIL-21, 41BBL, CD48, and / or CS1; e.g., as described in US patent application publication 2020 / 0390815 Al published December 17, 2020, which is incorporated herein by reference in its entirety for any purpose described herein) in the presence of a cytokine mix comprising IL-2 (e.g., 200 lU / ml), IL-12 (e.g., 10 ng / ml), IL-18 (e.g., 20 ng / ml), and TGF-Beta (“TGFb” or “TGFP”, e.g., 0.3-10 ng / ml), optionally the APCs are removed and / or the NK cells are enriched for and resuspended in media lacking APCs, and optionally then the cells are transduced and / or transformed with one or more constructs and / or particles. After a period of time (e.g., on day 8), the prepared NK cells can then be collected, deactivated and frozen (e.g., using Dasatinib at 1 pM / ml), or utilized fresh. Cell and / or media characteristics (e.g., purity, proliferation rates, etc.) can be measured at any point during the methodology. Cell viability and efficacy against various cell lines (e.g., cancer cell lines, e.g., SKOV3, PATC148, OVCAR5, K562, Raji, etc.) can be measured using functional assays (e.g.,IncuCyte®, xCELLigence®, etc.). FIG. 5B depicts an exemplary NK cell preparation workflow, comprising NK cells that are isolated, pre-activated, and expanded using methods and compositions provided herein (e.g., comprising pre-activation (start Day -1) for about 16 hours in the presence of IL-12 (e.g., 10 ng / ml), IL-18 (e.g., 50 ng / ml), and IL-15 (e.g., 10 ng / ml), and co-cultured (start Day 0) with Antigen Presenting Cells (APCs) in the presence of IL-2 (e.g., 200 lU / ml), IL-12 (e.g., 10 ng / ml), IL-18 (e.g., 20 ng / ml), and TGL-Beta (e.g., 0.3- 10 ng / ml), and transduced with one or more constructs. After a period of time (e.g., on Day 8 of expansion), prepared NK cells can then be collected, deactivated and frozen, or utilized fresh. Cell viability and efficacy against various cell lines (e.g., cancer cell lines, e.g., SKOV3, PATC148, OVCAR5, K562, Raji, etc.) can be measured using functional assays (e.g., Incucyte®, xCELLigence®, etc.). FIG. 5C depicts an exemplary workflow for a preactivation, expansion with transduction, deactivation, and cryopreservation methodology for production of prepared NK cells as described herein.

[0407] FIG. 6 shows proliferation rates in control and experimental NK cell expansion conditions. NK cells were isolated from cord blood and pre-activated with IL- 12 (10 ng / ml), IL-15 (50 ng / ml), and IL-18 (50 ng / ml) for 16 hours, the cells were then washed and expanded in a first culture condition with irradiated uAPC cells with IL-2 (200 U / ml) alone (“P+E”), or irradiated uAPC cells with a combination of IL-2 (200 U / ml), IL- 12 (10 ng / ml), IL- 18 (20 ng / ml), and TGEb (0.3-10 ng / ml) (“IL2+IL12+IL18+TGEb”) for 5 days, refreshed every 2-3 days. On day 5, NK cells were transduced with an anti-TROP2 CAR and expanded in a second culture condition for another 4 days without uAPCs, but in the presence of the IL-2 or the IL- 2, IL- 12, IL- 18, and TGEb cytokine mix. Acridine orange / Propidium iodide (AO / PI) staining was used to count NK cells every two to three days. These data demonstrate that NK cells cultured in IL2+IL12+IL18+TGPb exhibited similar proliferation rates to P+E, and that lower TGEb concentrations resulted in a slight but appreciable proliferative advantage for expansion of NK cells.

[0408] FIGs. 7A-7B shows that inclusion of IL- 12, IL- 18, and TGEb in NK cell expansion culture significantly enhanced transduction efficiency. NK cells were derived from cord blood (CB) and pre-activated with IL-12 (10 ng / ml), IL-15 (50 ng / ml), and IL-18 (50 ng / ml) for 16 hours, the cells were then washed and expanded using irradiated uAPC cells with either IL-2 (200 U / ml) alone (“P+E”), or a combination of IL-2 (200 U / ml), IL-12 (10 ng / ml), IL-18 (20 ng / ml), and TGEb (0.3-10 ng / ml) (“IL2+IL12+IL18+TGEb”), refreshed every 2-3 days for 5 days. On day 5, NK cells were enriched from the culture and added to a transduction culture media where they were transduced with an exemplary anti-TROP2 CAR(“1C9 / TROP2CAR / IL-15”; e.g., as described in PCT publication WO 2023 / 283644 A2 published January 12, 2023, which is incorporated herein by reference in its entirety for any purpose) in the presence of IL-2 (200 U / ml) (“P+E Transduced”), or IL-2 (200 U / ml), IL- 12 (10 ng / ml), IL-18 (20 ng / ml), andTGEb (0.3-10 ng / ml) (“IL2+IL12+IL18+TGEb transduced”). RetroNectin was used to enhance transduction efficiency (e.g., as described in US patent application publication 2022 / 0389383 Al, published December 8, 2022, which is incorporated herein by reference in its entirety for any purpose). NK cells were maintained in the transduction culture media, and forty eight hours post transduction, the transduction efficiency was measured by checking the surface expression of CAR in the NK cells using flow cytometry where percentage (%) corresponded to the transduction efficiency. Cells from non-transduced (NT) NK cells were used as control. NK cells (e.g., CB-NK cells) expanded in the presence of IL2+IL12+IL18+TGEb showed significantly higher transduction efficiency (greater than -80% average) when compared to P+E CB-NK cells (-40% average). FIG. 7A shows representative flow plots of the CAR staining of CAR transduction efficiency, which was quantified from three different donors and presented in FIG. 7B.

[0409] FIGs. 8A-8C shows how IL2+IL12+IL18+TGFb expanded TROP2 CAR NK Cells (iC9 / TROP2CAR / IL-15 NK Cells) demonstrated enhanced in vitro antitumor activity against tumor GIST cells (OVCAR5), PDAC cells (PATC148), and Ovarian Cancer cells (SKOV3) when compared to control P+E expanded iC9 / TROP2CAR / IL-15 NK cells in a representative long-term xCELLigence® cytotoxicity assays. NK cells were expanded with either uAPC + IL-2 (200 U / ml) alone (“P+E”), or a combination of uAPC + IL-2 (200 U / ml), IL-12 (10 ng / ml), IL-18 (20 ng / ml), and TGEb (0.3-10 ng / ml) after being preactivated with IL-12 (10 ng / ml), IL- 15 (50 ng / ml), and IL-18 (50 ng / ml) for 16 hours. The media was replaced every 2-3 days and supplemented with cytokines. On day 5, NK cells were enriched for and removed from the uAPCs, and transferred to transduction culture as described in EIG. 7 and transduced with a construct comprising a CAR targeting TROP2 and secreted IL- 15 (e.g., iC9 / TROP2CAR / IL- 15), and cultured either with cytokine IL-2 (200 U / ml) alone (in P+E conditions), or a combination of IL-2 (200 U / ml), IL- 12 (10 ng / ml), IL- 18 (20 ng / ml), and TGEb (0.3-10 ng / ml. On Day 8, NK cells were treated with Dasatinib (1 pM / ml) for 24 hours and then frozen on Day 9. NK cells were thawed and cocultured with tumor cells at a 1:1 ratio. Compared to IL-2 alone controls, NK cells expanded with IL2+IL12+IL18+TGEb showed increased cytotoxic activity over time against OVCAR5 (GIST) cell lines (FIG. 8A), PATC148 (PDAC) cell lines (FIG. 8B), and SKOV3 (Ovarian Cancer) cell lines (FIG. 8C).

[0410] FIGs. 9A-9F shows how IL2+IL12+IL18+TGFb expanded TROP2 CAR NK cells, prepared as described in FIG. 8, demonstrated enhanced antitumor activity against Ovarian cancer (SKOV3), Pancreatic cancer (PATC148), and GIST (OVCAR5) tumor cell spheroids in long-term IncuCyte® spheroid assays relative to tumor alone or control P+E expanded TROP2 CAR NK cells. FIG. 9A shows representative images of SKOV3 (ovarian cancer) spheroids (tumor cells were transduced with GFP). NK cells were prepared and thawed as described in FIG. 8, and cocultured with SKOV3 spheroid at a 1:1 ratio. The data showed a readily observable decrease in SKOV3 spheroid size in the wells treated with NK cells expanded in IL2+IL12+IL18+TGFb culture conditions relative to P+E and tumor controls. FIG. 9B provides quantification of the total integrated green intensity over time for the test groups as represented in FIG. 9A. The data showed a significant decrease in total integrated green intensity when SKOV3 spheroids were exposed to NK cells expanded with IL2+IL12+IL18+TGFb, relative to controls. FIG. 9C shows representative images of PATC148 (pancreatic cancer) spheroids (tumor cells transduced with GFP). NK cells were prepared and thawed as described in FIG. 8, and cocultured with PATC148 spheroid at a 1:1 ratio. The data showed a readily observable decrease in PATC148 spheroid size in the wells treated with NK cells expanded in IL2+IL12+IL18+TGFb culture conditions relative to P+E and tumor controls. FIG. 9D provides quantification of the total integrated green intensity over time for the test groups as represented in FIG. 9C. The data showed a significant decrease in total integrated green intensity when PATC148 spheroids were exposed to NK cells expanded with IL2+IL12+IL18+TGFb, relative to controls. FIG. 9E shows representative images OVCAR5 (GIST) spheroids (tumor cells transduced with GFP). NK cells were prepared and thawed as described in FIG. 8, and cocultured with OVCAR5 spheroid at a 1:1 ratio. The data showed a readily observable decrease in OVCAR5 spheroid size in the wells treated with NK cells expanded in IL2+IL12+IL18+TGFb culture conditions relative to P+E and tumor controls. FIG. 9F provides quantification of the total integrated green intensity over time for the test groups as represented in FIG. 9E. The data showed a significant decrease in total integrated green intensity when OVCAR5 spheroids were exposed to NK cells expanded with IL2+IL12+IL18+TGFb, relative to controls.

[0411] FIGs. 10A-10B show how IL2+IL12+IL18+TGFb expanded non-transduced (NT) NK Cells (unaltered, including no transformations) demonstrated enhanced antitumor activity against GCS267 (GBM) spheroids when compared to P+E NK Cells (e.g., without addition of IL12+IL18+TGFb) in a long-term IncuCyte® spheroid assay. FIG. 10A provides representative images of GCS267 spheroids (tumor cells transduced with mCherry). NK cellswere preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml), and IL-18 (50 ng / ml) for 16 hours, and then expanded with either uAPC + IL-2 (200 U / ml) alone (“P+E”), or uAPC + a combination of IL-2 (200 U / ml), IL-12 (10 ng / ml), IL-18 (20 ng / ml), and TGFb (0.3-10 ng / ml). The media was replaced every 2-3 days and supplemented with the noted cytokines. Cells were left otherwise undisturbed. On Day 8, NK cells were enriched for, and were then treated with Dasatinib (1 pM / ml; in the presence of the noted cytokine culture conditions) for 24 hours and then frozen on Day 9. NK cells were thawed and cocultured with GSC267 spheroids at a 3:1 E:T ratio. The data showed a readily observable decrease in GCS267 spheroid size in the wells treated with NT NK cells expanded in IL2+IL12+IL18+TGFb culture conditions relative to P+E NK cells and tumor controls. FIG. 10B provides quantification of the total integrated red intensity over time for the test groups as represented in FIG. 10A. The data showed a significant decrease in total integrated red intensity when GCS267 spheroids were exposed to NT NK cells expanded with IL2+IL12+IL18+TGFb, relative to controls.

[0412] FIGs. 11A-11C show how in orthotopic PDAC mouse models, IL2+IL12+IL18+TGFb expanded TROP2 CAR NK Cells (iC9 / TROP2CAR / IL15) prepared as described in FIG. 8 exhibited higher in vivo antitumor activity against PATC148 (PDAC) cancer cells, and displayed higher engraftment rates relative to controls. NK cells were prepared as described in FIG. 8 (e.g., P+E or IL2+IL12+IL18+TGFb expansion conditions), and on Day 8 the NK cells were treated with Dasatinib (1 pM / ml) for 24 hours and then frozen on Day 9. 8-week-old NSG mice were engrafted with 0.3 M firefly luciferase-labeled PATC148 (PDAC) cells. Six days after tumor engraftment, the mice were irradiated. Seven days after tumor engraftment, NK cells were thawed and infused into mice via intraperitoneal (IP) injection (Day 0). FIG. 11A shows bioluminescence imaging (BLI) of the tumor, the data showed that TROP2 CAR NK cells expanded with IL2+IL12+IL18+TGFb were superior at reducing the tumor burden when compared to controls at Day 7 and Day 13 post treatment. FIG. 11B shows a graph plotting the average radiance from the different groups of mice BLI data displayed in FIG. 11 A. Mice treated with IL2+IL12+IL18+TGFb expanded NK cells showed significantly improved tumor control relative to P+E expanded TROP2 CAR NK cells & tumor controls. FIG. 11C is a graph showing NK cell (hCD45+CD56+CD16+) engraftment as measured by presence in blood of mice 10 days after receiving NK cells or sham injection. Compared to the P+E expanded TROP2 CAR NK cells, the IL2+IL12+IL18+TGFb expanded NK cells showed significantly improved NK cell engraftment. These results suggest that expanding NK cells with IL-2, IL- 12, IL- 18, and TGF-P enhanced their engraftment, in vivo expansion, and tumor control, making it a promising approach for improving NK cell-based therapies.

[0413] FIGs. 12A-12C show how in orthotopic GIST mouse models, IL2+IL12+IL18+TGFb expanded TROP2 CAR NK Cells (iC9 / TROP2CAR / IL15) prepared as described in FIG. 8 exhibited significantly superior in vivo antitumor activity against OVCAR5 (GIST) cancer cells, and displayed significantly increased engraftment rates relative to controls. NK cells were prepared as described in FIG. 8 (e.g., P+E or IL2+IL12+IL18+TGFb expansion conditions), and on Day 8 the NK cells were treated with Dasatinib (1 pM / ml) for 24 hours and then frozen on Day 9. 8-week-old NSG mice were engrafted with 0.3 M firefly luciferase-labeled OVCAR5 (GIST) intraperitoneally (IP). Three days after tumor engraftment, NK cells were thawed and infused into mice via intraperitoneal (IP) (Day 0). FIG. 12A shows bioluminescence imaging (BLI) of the tumor, the data showed that TROP2 CAR NK cells expanded with IL2+IL12+IL18+TGFb were superior at reducing GIST tumor burden when compared to controls at Day 7 and Day 13 post treatment. FIG. 12B shows a graph plotting the average radiance from the different groups of mice BLI data displayed in FIG. 12A. Mice treated with IL2+IL12+IL18+TGFb expanded NK cells showed significantly improved tumor control relative to P+E expanded TROP2 CAR NK cells & tumor controls. FIG. 12C is a graph showing NK cell (hCD45+CD56+CD16+) engraftment as measured by presence in the blood of mice 10 days after receiving NK cells or sham injection. Compared to the P+E expanded TROP2 CAR NK cells, the IL2+IL12+IL18+TGFb expanded NK cells showed significantly improved NK cell engraftment. These results suggest that expanding NK cells with IL-2, IL- 12, IL- 18, and TGF-P enhanced their engraftment, in vivo expansion, and tumor control, making it a promising approach for improving NK cell-based therapies.

[0414] FIGs. 13A-13B provide schematic diagrams detailing exemplary methodologies and combinations for expansion and preparation of NK cells. FIG. 13A provides an exemplary workflow for expansion and preparation methods provided herein; NK cells are isolated and resuspend. The isolated NK cells are pre-activated using cytokines IL- 15, IL- 18, and IL- 12 overnight, for example 16 hours. The NK cells are then washed twice with PBS and stimulated with APCs, Activating Beads, and / or Feeder cells, with the following expansion conditions: “P+E”: IL-2; “KS” (“K&S” or “K+S”): IL-2, IL12, IL18, and TGFb; and KS + mTOR inhibitor (e.g., “KSmT”): IL-2, IL12, IL18, TGFb, and mTOR inhibitor. Five days following expansion start, the NK cells are optionally enriched for and / or optionally engineered (e.g., transduced or electroporated, etc.) in media comprising the cytokine and / or small molecule expansion conditions. Following optional enrichment and / or optional engineering, for P+E, continue with IL-2; for KS, cells can be split into two or more cultures, and either cultured with IL-2, TGFb, IL 12, and IL18, or that combination with the addition of an mTOR inhibitor added to the culturefor the last 3 days (e.g., “KS-mT” (last 3 days)), or the KS combination with the addition of a PI3K inhibitor added to the culture for the last 3 days (e.g., “KS-P” (last 3 days)); and for KS+ mTOR inhibitor, cells can be split into two or more cultures, and either continued in culture with IL-2, TGFb, IL12, IL18, and mTOR inhibitor (e.g., “KSmT”), or that combination with the addition of a PI3K inhibitor for the last 3 days (e.g., “KSmT-P”), or that combination with the addition of an IDH1 / 2 inhibitor for the last 3 days (e.g., “KSmT-ID”). By Day 8 the cells have been prepared, and can optionally be deactivated through the addition of an RTK inhibitor, such as Dasatinib, for 24 hours to temporarily deactivate the prepared NK cells prior to freezing on Day 9. FIG. 13B provides an exemplary workflow for expansion and preparation methods provided herein; NK cells were isolated from CB, and resuspend to about 1 x 10A6 / ml. The isolated NK cells were pre-activated using cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) overnight (e.g., 16 hours). After pre-activation, the NK cells were washed twice with PBS and stimulated with uAPCs (e.g., as described in US 2020 / 0390815 Al, which is incorporated herein by reference in its entirety for the purposes described herein), with the following expansion conditions: “P+E”: IL-2 (200 U / ml); “KS” (“K&S” or “K+S”): IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), and TGFb (0.3 ng / ml); and KS + Rapamycin 1 nM (“KSR”): IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), TGFb (0.3 ng / ml), and Rapamycin (1 nM). Media was replaced every 2-3 days and supplemented with the noted cytokines and / or small molecules. On Day 5 after expansion start, the NK cells were enriched for and resuspended in media without the uAPCs, and optionally engineered (e.g., transduced or electroporated, etc.) in media comprising the aforementioned cytokine cocktails and / or small molecules. 16-24 hours after enrichment and optional engineering, expansion was continued in the absence of uAPCs, for P+E, continue with IL-2 (200 U / ml); for KS, cells were split into two or more populations and either cultured with IL-2 (200 U / ml), TGFb (0.3 ng / ml), IL12 (10 ng / ml), and IL18 (20 ng / ml), or that combination with the addition of Rapamycin (10 nM) added to the culture for the last 3 days (“KS-R” (last 3 days)), or that combination with the addition of a PI3K inhibitor for the last 3 days (e.g., “KS-I”); and for KS+ Rapamycin 1 nM conditions (KSRs), cells were split into two or more populations and either continued in culture with IL-2 (200 U / ml), TGFb (0.3 ng / ml), IL12 (10 ng / ml), and IL18 (20 ng / ml), and Rapamycin (1 nM) (“KSR”), or the KSR combination with the addition of Idelalisib (10 nM) for the last 3 days (“KSR-I”), or the KSR combination with addition of 2-Deoxy-d-Glucose (2-DG) (2 mM) for the last 3 days (“KSR-2DG”; not shown in figure), or the KSR combination with addition of Enasidenib (1 pM) for the last 3 days (“KSR-E”) On Day 8, the prepared NK cellswere either utilized, or optionally the prepared NK cells were deactivated through addition of Dasatinib (1 pM) for 24 hours, followed by cry opreservation on Day 9.

[0415] FIGs. 14A-14B NK cells prepared and expanded with IL-2, IL- 12, IL- 18, and TGF-P (“KS”), or KS combined with Rapamycin (“KSR”), or KSR with Idelalisib (“KSR-I”) (as outlined in FIG. 13B), exhibited significantly higher transduction efficiency and sustained proliferation rates compared to IL-2-expanded NK cells (“P+E”). The populations of prepared NK cells as presented and described in FIG. 13B (e.g., KS-R, KS, KS-I, KSR, KSR-I, KSR- 2DG, and control P+E) were prepared, with the cells being enriched for and transduced on Day 5. On Day 8, Dasatinib (1 pM) was added for 24 hours and deactivated prepared NK cells were frozen on Day 9. Prior to cry opreservation, NK cell levels were measured, and NK cells were stained for heterologous proteins to determine transduction efficiency. FIG. 14A is a graphical representation of the transduction efficiencies as measured using flow cytometry, the results showed increased levels of transduction efficiency in at least KS, KS-R, KS-I, KSR, and KSR- I, expanded NK cells relative to P+E expanded NK cells. FIG. 14B is a graphical representation of the average NK cell population fold increase prior to cryopreservation, the results showed that the KS, KS-R, KS-I, KSR, KSR-I, and KSR-2DG did not hinder the proliferative capacity of NK cells relative to P+E expanded NK cells.

[0416] FIGs. 15A-15C Prepared NK cells expanded with IL2+IL12+IL18+TGFb (“KS”), or KS with Rapamycin (“KSR”), or KSR with Idelalisib (KSR-I), or KS with Rapamycin last 3 days only (KS-R), or KS with Idelalisib last 3 days only (KS-I) (as described in FIG. 13B), displayed distinct and improved chemokine profiles when compared to P+E expanded NK cells. The populations of prepared NK cells as presented and described in FIG. 13B (e.g., KS- R (“KS+Rapa (10nM)”), KS, KS-I (“KS+Ida(10nM)”), KSR (“KS+Rapa(lnM)”), KSR-I (“KS+Rapa(lnM)+Ida(10nM)”), and control P+E) were prepared, with the cells being enriched for on Day 5. On Day 8, prior to deactivation, NK cells were labeled to facilitate detection of different chemokines (e.g., CD62L, CXCR4, and CCR7), and analyzed using flow cytometry. The data showed that, compared to P+E expanded NK cell populations, NK cells expanded with KS, KSR, KS-R, KS-I, or KSR-I conditions exhibited an enhanced chemokine profile with increased percentages of the NK cells being positive for CD62L (FIG. 15A), CXCR4 (FIG. 15B), and / or CCR7 (FIG. 15C).

[0417] FIGs. 16A-16B Prepared engineered NK cells expanded with IL2+IL12+IL18+TGFb (“KS”), or KS with Rapamycin (“KSR”), or KSR with Idelalisib (KSR-I), or KS with Rapamycin last 3 days only (KS-R), or KS with Idelalisib last 3 days only (KS-I) (as described in FIG. 13B), exhibited higher antitumor activity against CCRF-Cem (T-ALL) cancer cellswhen compared to P+E engineered NK cells in a long-term IncuCyte® cytotoxicity assay. FIG. 16A shows representative images of CCRF-CEM (tumor cells transduced with mCherry) over time (Days 0, 0.5, 1.5, 2.5, or 3.5) after the tumor cells were treated with populations of prepared NK cells as presented and described in FIG. 13B (e.g., prepared using culture conditions: KS, KS-R (“KS+Rapamycin lOnM (last 3 days)”), KS-I (“KS+Idelalisib lOnM (last 3 days)”), KSR (“KS+Rapamycin InM”), KSR-2DG (“KS+Ramaycin 1 nM 2DG (last 3 days)”), KSR-I (“KS+Rapamycin 1 nM + Idelalisib lOnM (last 3 days)”), or control (“P+E”), with the NK cells being enriched for on Day 5, transduced with anti-CD5 CAR and IL-15 secreting constructs (“CAR5 / IL-15”; e.g., as described in PCT publication WO 2022 / 221548 Al published October 20, 2022, which is incorporated herein by reference in its entirety for any purpose), subjected to a second expansion step, deactivated on Day 8, frozen on Day 9, and subsequently thawed and utilized for the assay. The data showed a significant decrease in tumor cell count in the wells treated with KS, KSR, KS-R, KS-I, KSR-2DG, or KSR-I prepared NK cells when compared to NK cells prepared using P+E. The results were particularly strikingly robust for NK cells prepared using KSR-I conditions. FIG. 16B provides quantification of the total count of tumor cells (measured as total orange object count per image) over time from the experiment described in FIG. 16A. The results showed that NK cells expanded using the KS comprising protocols (IL-2, IL- 12, IL- 18, TGF-P) significantly reduced tumor cell numbers compared to NK cells expanded with the P+E protocol. This indicates that the KS comprising protocols enhanced NK cell anti-tumor efficacy, improving their ability to target and eliminate tumor cells.

[0418] FIGs. 17A-17D Prepared NK cells expanded with KS, KSR, KSR-I, KS-R, KSR-2DG, or KS-I (largely as described in FIG. 13B, with the exception that in FIG. 17C, “KS” does not include TGFB unless noted as such) and transduced with anti-CD5 CAR and IL- 15 secreting constructs (“CAR5 / IL-15”; e.g., as described in PCT publication WO 2022 / 221548 Al published October 20, 2022, which is incorporated herein by reference in its entirety for any purpose) exhibited higher in vivo antitumor activity against CCRF-CEM (T-ALL) cancer cell lines with improved immune cell engraftment when compared to P+E prepared NK cell controls. Female mice, aged 10 weeks, were exposed to sublethal irradiation (300cGy) on day -4. On day -3, they were intravenously injected with 100,000 CCRF-CEM cells that expressed the Firefly luciferase (FFluc) gene, through the tail vein. A baseline measurement of bioluminescence imaging (BLI) was taken after injecting the tumor (day -2). The mice were then divided into various groups to ensure that each group had a similar average BLI signal at baseline. Each group consisted of at least 4 or 5 mice (n= 4 or 5 mice per group). On day 0, themice were injected with 3 x 10A6 NK cells that had been prepared using the noted culture conditions, and subsequently thawed following freezing. The mice were thereafter followed with weekly bioluminescence imaging (BLI) to evaluate the extent of tumor growth, in addition to weight measures and toxicity scoring conducted three times per week. Blood was collected from the mice on day 20 to monitor NK cell engraftment by flow cytometry. FIG. 17A shows BLI imaging depicting tumor burden (CCRF-CEM-FFluc bioluminescence) among the different groups of mice at baseline and then at multiple time points following treatment. FIG. 17B is a graph showing average radiance of BLI quantification among the various groups of mice, with each individual mouse represented by a single line. FIG. 17C is a bar graph showing absolute NK cell numbers in blood collected from the mice at day 20. Results are also shown for additional control NK cells prepared using “P+E+Rapa” (at 1 nM concentration for full expansion period; third column) and KS without TGFb but including rapamycin (“KS+Rapa”; at 1 nM concentration for full expansion period; fourth column). FIG. 17D provides results of 2-way ANOVA statistical analysis, with P values representing non-significant (ns) = >0.05, * = <0.5, ** = <0.01, *** = <0.001, and **** = <0.0001, for comparison of BLI across the test and control groups depicted in in FIG. 17B. These findings demonstrate that animals treated with the engineered NK cells exhibited robust tumor suppression and successful NK cell engraftment. Specifically, NK cells prepared under the KS ("KS+TGFP"), KSR ("KS+TGFP+Rapa"), KS-I ("KS-TGFP-Ide"), KS-R ("KS-TGFP-Rapa(lOnM)"), KSR-2DG ("KS-(TGFP-Rapa)+2DG"), and KSR-I ("KS(TGFP-Rapa)+Ide") conditions showed enhanced tumor control and engraftment compared to the P+E conditions.

[0419] FIGs. 18A-18B Prepared NK cells expanded with KS, KSR, or KSR-I (as described in FIG. 13B) displayed robust Crispr-Cas9 knockout efficacy that was not diminished relative to P+E prepared control NK cells. Six days following expansion start (e.g., day 5), NK cells were enriched for and electroporated with Crispr-Cas9 ribonucleoprotein complexes targeting Glucocorticoid Receptor (GR; N3CR1) and Transforming Growth Factor Beta Receptor 2 (TGFBR2) (as described in PCT application publication WO 2018 / 195339 Al published October 25, 2018, and PCT application publication WO 2018 / 195339 A2 published June 4, 2020, each of which are incorporated herein by reference in their entirety). FIG. 18A shows a representative gel showing knockout efficacy as measured by PCR amplification of the targeted GR loci. FIG. 18B is a graph displaying knockout efficacy of GR relative to P+E WT NK cells. Together, these results showed that prepared NK cells cultured with KS, KSR, or KSR-I conditions had excellent Crispr-Cas9 knockout efficacy on par or better than NK cells prepared using P+E culture methods.

[0420] FIGs. 19A-19H Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and engineered to have double knockout (DKO) of TGFBR2 and GR genes showed improved antitumor activity against Glioblastoma (GBM) cell line spheroids (GCS272, GCS267, GCS20, and GCS231) when compared to P+E prepared DKO NK cells. Glioblastoma cell line spheroids were seeded at 10,000 cells and allowed to grow for 48 hours before challenge with DKO NK cells at a 3:1 E:T ratio. FIG. 19A are representative images of GCS272 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded using P+E, KS, KSR, or KSR-I culture conditions. The data showed a readily appreciable decrease in GCS272 spheroid size in the wells treated with prepared NK cells expanded with KS, KSR, or KSR-I culture conditions relative to NK cells prepared with P+E culture conditions. FIG. 19B provides quantification of the total integrated red intensity over time as pictured in FIG. 19A, the results demonstrated a significant decrease in total integrated red intensity when GCS272 spheroids were treated with prepared NK cells expanded with KS, KSR, or KS-I conditions relative to tumor only or P+E prepared NK cells. FIG. 19C are representative images of GCS267 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded using P+E, KS, KSR, or KSR-I culture conditions. The data showed a readily appreciable decrease in GCS267 spheroid size in the wells treated with prepared NK cells expanded with KS, KSR, or KSR-I culture conditions relative to NK cells prepared with P+E culture conditions. FIG. 19D provides quantification of the total integrated red intensity over time as pictured in FIG. 19C, the results demonstrated a significant decrease in total integrated red intensity when GCS267 spheroids were treated with prepared NK cells expanded with KS, KSR, or KS-I conditions relative to tumor only or P+E prepared NK cells. FIG. 19E are representative images of GCS20 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded using P+E, KS, KSR, or KSR-I culture conditions. The data showed a readily appreciable decrease in GCS20 spheroid size in the wells treated with prepared NK cells expanded with KSR or KSR-I culture conditions relative to NK cells prepared with P+E culture conditions. FIG. 19F provides quantification of the total integrated red intensity over time as pictured in FIG. 19E, the results demonstrated a significant decrease in total integrated red intensity when GCS20 spheroids were treated with prepared NK cells expanded with KSR or KS-I conditions relative to tumor only or P+E prepared NK cells. FIG. 19G are representative images of GCS231 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded using P+E, KS, KSR, or KSR-I culture conditions. The data showed a readily appreciable decrease inspheroid size in the wells treated with prepared NK cells expanded with KSR or KSR-I culture conditions relative to NK cells prepared with P+E culture conditions. FIG. 19H provides quantification of the total integrated red intensity over time as pictured in FIG. 19G, the results demonstrated a significant decrease in total integrated red intensity when GCS231 spheroids were treated with prepared NK cells expanded with KSR or KS-I conditions relative to tumor only or P+E prepared NK cells. For FIGs. 19B, 19D, 19F, and 19H, 2-way ANOVA statistical analysis was performed, with P values representing non-significant (ns) = >0.05, * = <0.5, ** = <0.01, *** = <0.001, and **** = <0.0001. These results indicate that NK cells expanded with KS, KSR, or KSR-I exhibit a substantial improvement in antitumor activity, demonstrating enhanced efficacy across multiple GCS models compared to NK cells expanded with P+E.

[0421] FIGs. 20A-20B Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and engineered to have double knockout (DKO) of TGFBR2 and GR genes showed improved antitumor activity against WM266-4 (Melanoma) cell line spheroids when compared to P+E prepared DKO NK cells. Melanoma cell line spheroids were seeded at 10,000 cells and allowed to grow for 48 hours before challenge with DKO NK cells at a 3: 1 E:T ratio. FIG.20A are representative images of WM266-4 (melanoma) spheroids (tumor cells transduced with GFP) left alone or treated with prepared NK cells expanded using P+E, KS, KSR, or KSR-I culture conditions. The data showed a readily appreciable decrease in WM266-4 spheroid size in the wells treated with prepared NK cells expanded with KS, KSR, or KSR-I culture conditions relative to NK cells prepared with P+E culture conditions. FIG. 20B provides quantification of the total integrated green intensity over time as pictured in FIG. 20A, the results demonstrated a significant decrease in total integrated green intensity when WM266-4 spheroids were treated with prepared NK cells expanded with KSR or KS-I conditions relative to tumor only or P+E prepared NK cells. 2-way ANOVA statistical analysis was performed, with P values representing non-significant (ns) = >0.05, * = <0.5, ** = <0.01, *** = <0.001, and **** = <0.0001. These results indicate that NK cells expanded with KS, KSR, or KSR-I exhibited a substantial improvement in antitumor activity compared to NK cells expanded with P+E.

[0422] FIGs. 21A-21B Prepared NK cells expanded with KS, KS without TGFb (e.g., IL2, IL12, and IL18 only), KSR, KSR-I, KSR-2DG, and KS-R, culture conditions (as described in FIG. 13B) displayed distinct phenotypes when compared to prepared NK cells cultured with P+E conditions, or P+E with added rapamycin (“P+E-Rapa”; with 1 nM rapamycin from Day 0 onwards). Prepared NK cells were expanded using the noted conditions and then stained with50-color mass cytometry and analyzed using Cytobank. FIG. 21A shows a uMAP representation of how the prepared NK cells cultured comprising KS conditions (with or without TGFb) had distinct clustering relative to prepared NK cells cultured comprising P+E conditions (with or without rapamycin). FIG. 21B provides a heatmap showing marker expression levels for the various test groups. The results showed that prepared NK cells cultured comprising KS conditions had distinct phenotypes relative to P+E cultured NK cells, for example, NK cell populations expanded in KS comprising culture conditions had an increase in CCR7, CCR4, CLA, CD127, CD16, DNAM, TRAIL, CD2, TIM3, CXCR2, CD25, CD39, LAG3, PDL1, CCR9, and PD1 expression which are closely associated with improved trafficking, greater tumor infiltration, and enhanced cytotoxicity; and a decrease in KI67, NKp46, NKP44, NKG2D, CD8, Perforin, granzyme B, CD56, CXCR6, CCR5, CCR6, NKp30, TIGIT, CXCR3, CX3CR1, and KLRG1 expression relative to P+E cultured NK cells which had relatively downregulated markers associated with strong activation and potentially linked to activation-induced cell death.

[0423] FIGs. 22A-22D Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and engineered to have double knockout (DKO) of TGFBR2 and GR genes showed improved antitumor activity against Glioblastoma (GBM) cell line spheroids (GCS272, GCS267, GCS20, and GCS231) when compared to P+E prepared DKO NK cells in long term IncuCyte® assays when prepared using three different CB donors. Glioblastoma cell line spheroids were seeded at 10,000 cells and allowed to grow for 48 hours before challenge with DKO NK cells at a 3:1 E:T ratio. The representative images were taken at completion of the experiments on the seventh day. Experiments are related to those depicted and quantified in FIGs. 19A-19H. FIG. 22A provides representative images of GCS272 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded with the noted culture conditions. The data showed a readily appreciable and significant decrease in BLI intensity in the wells treated with prepared NK cells cultured with KS, KSR, and KSR-I conditions relative to P+E prepared NK cells or tumor only conditions. FIG. 22B provides representative images of GCS267 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded with the noted culture conditions. The data showed a readily appreciable and significant decrease in BLI intensity in the wells treated with prepared NK cells cultured with KS, KSR, and KSR-I conditions relative to P+E prepared NK cells or tumor only conditions. FIG. 22C provides representative images of GCS20 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded with the noted culture conditions. The datashowed a readily appreciable and significant decrease in BLI intensity in the wells treated with prepared NK cells cultured with KSR and KSR-I conditions relative to P+E prepared NK cells or tumor only conditions. FIG. 22D provides representative images of GCS231 (GBM) spheroids (tumor cells transduced with mCherry) left alone or treated with prepared NK cells expanded with the noted culture conditions. The data showed a readily appreciable and significant decrease in BLI intensity in the wells treated with prepared NK cells cultured with KS, KSR, and KSR-I conditions relative to P+E prepared NK cells or tumor only conditions.

[0424] FIG. 23 Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and engineered to have double knockout (DKO) of TGFBR2 and GR genes showed improved antitumor activity against WM266-4 (Melanoma) cell line spheroids when compared to P+E prepared DKO NK cells in long-term IncuCyte® spheroid assays when prepared using three different CB donors. Melanoma cell line spheroids were seeded at 10,000 cells and allowed to grow for 48 hours before challenge with DKO NK cells at a 3:1 E:T ratio. The representative images were taken at completion of the experiments on the seventh day. Experiments are related to those depicted and quantified in FIGs. 20A-20B. Provided are representative images of WM266-4 (Melanoma) spheroids (tumor cells transduced with GFP) left alone or treated with prepared NK cells expanded with the noted culture conditions. The data showed a readily appreciable and significant decrease in BLI intensity in the wells treated with prepared NK cells cultured with KSR and KSR-I conditions relative to P+E prepared NK cells or tumor only conditions.

[0425] FIG. 24 Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and either non-transduced (NT) or engineered to express uTNK15 and TCR / FCR #2 (“TCR / FCR”; see PCT application PCT / US2023 / 085140 filed December 20, 2023, and PCT application PCT / US2024 / 027601 filed May 3, 2024, each of which are incorporated herein by reference in their entirety for any purpose described herein) and left unloaded or loaded with the antibody Avelumab (anti-PDLl ; at 300 pg / ml) displayed improved antitumor activity against Karpas (T-NHL) tumor cells relative to P+E NK cells (engineered or not engineered, combined with Avelumab or not combined), tumor only controls (“Karpas”), or antibody controls (“Karpas+ Avelumab”; at 300 pg / ml) in long term IncuCyte® assays. Provided are representative images of Karpas (tumor cells transduced with mCherry) cells left alone or treated with the noted prepared NK cells either engineered or left NT, and combined with antibody or not. The tumor cells and NK cells were cultured at an E:T ratio of 1:1. The data demonstrated a clear and significant reduction in tumor cell count over time in wells treated with both engineered and non-engineered NK cells expanded under KS cultureconditions compared to P+E. This effect was further enhanced when combined with the antibody Avelumab, leading to improved antitumor activity.

[0426] FIG. 25 Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and either non-transduced (NT) or engineered to express uTNK15 and TCR / FCR #2 (“TCR / FCR”; see PCT application PCT / US2023 / 085140 filed December 20, 2023, which is incorporated herein by reference in its entirety for any purpose described herein) and left unloaded or loaded with the antibody Glofitamab (bispecific anti-CD20 and anti-CD3; 20 pg / ml) displayed improved antitumor activity against Raji (B-ALL) tumor cells relative to P+E NK cells (engineered or not engineered, combined with Glofitamab or not combined), tumor only controls (“Raji”), or antibody controls (“Raji+ Glofitamab”; at 20 pg / ml) in long term IncuCyte® assays. Provided are representative images of Raji (tumor cells transduced with mCherry) cells left alone or treated with the noted prepared NK cells either engineered or left NT, and combined with antibody or not. The tumor cells and NK cells were cultured at an E:T ratio of 1:1. The data demonstrated a clear and significant reduction in tumor cell count over time in wells treated with both engineered and non-engineered NK cells expanded under KS culture conditions compared to P+E. This effect was further enhanced when combined with the antibody Glofitamab-T Cell engager, leading to improved antitumor activity.

[0427] FIG. 26 Prepared NK cells expanded with KS, KSR, and KSR-I culture conditions (as described in FIG. 13B) and either non-transduced (NT) or engineered to express anti-TROP2 CAR (“TROP2”; e.g., as described in PCT publication WO 2023 / 283644 A2 published January 12, 2023, which is incorporated herein by reference in its entirety for any purpose) showed improved antitumor activity against WiDR (CRC) tumor cell spheroids compared to P+E NK cells in a long-term IncuCyte® spheroid assay. Provided are representative images of WiDR spheroids (CRC, tumor cells transduced with GFP) left alone or treated with the noted prepared NK cells either engineered or left NT. The tumor cells and NK cells were cultured at an E:T ratio of 1 : 1. The data demonstrated a clear and significant reduction in tumor size over time in wells treated with TROP2 CAR / IL15 NK cells expanded under KS culture conditions compared to P+E.

[0428] FIGs. 27A-27B Prepared NK cells expanded with KSR culture conditions (as described in FIG. 13B) engineered to express uTNK15 and TCR / FCR #2 (“TCR / FCR”; see PCT application PCT / US2023 / 085140 filed December 20, 2023, which is incorporated herein by reference in its entirety for any purpose described herein) and loaded with the antibody Elranatamab (bispecific anti-BCMA and anti-CD3) displayed improved in vivo antitumor activity against multiple myeloma mouse models (MM Is tumor cells) relative to P+Eengineered NK cells. Female mice, aged 10 weeks, were exposed to sublethal irradiation (300cGy) on day -8. On day -7, they were intravenously injected (through the tail vein) with 250,000 MM Is cells that express the Firefly luciferase (FFluc) gene. Baseline measurement of bioluminescence imaging (BLI) was taken after injecting the tumor (day -7). The mice were then divided into various groups to ensure that each group had a similar average BLI signal at baseline. Each group consisted of 3 or 4 mice. On day 0, the mice were injected with combination Elranatamab (5 mg / kg per mouse) and 1.5 x 10A6 of PluReceptor NK cells that were pre-activated with IL-12 (10 ng / ml), IL18 (50 ng / ml) and IL15 (50 ng / ml) for 16 hours, and then expanded with either KSR culture conditions (e.g., IL2 (200 lU / ml) + IL12 (10 ng / ml) + IL 18 (20 ng / ml) + TGFb (0.3 ng / ml) + Rapamycin (1 nM) + uAPC) or P+E (IL2 (200 lU / ml) + uAPC). The mice were thereafter followed with weekly bioluminescence imaging (BLI) to evaluate the extent of tumor growth FIG. 27A provides BLI imaging showing tumor burden (MMls-FFluc bioluminescence) among the different groups of mice at baseline and then at multiple time points following treatment. FIG. 27B is a graph showing average radiance of BLI quantification among the various groups of mice. These results showed that TCR / FCR engineered NK cells prepared using KS culture conditions and armed (e.g., with Elranatamab) displayed improved in vivo tumor control relative to armed P+E prepared TCR / FCR engineered NK cells prepared using P+E culture conditions.

[0429] FIGs. 28A-28B Prepared NK cells expanded with low doses of exogenously provided TGFb in combination with IL2, IL 12, and IL 18 (KS conditions) had improved antitumor activity against SKOV3 (Ovarian Carcinoma) cancer cell line spheroids when compared to NK cells expanded with IL2, IL12, IL18 and higher doses of TGFb, or P+E expanded NK cells, when measured using long-term IncuCyte spheroid and xCELLigence killing assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KS (IL2, IL12, IL18, and TGFb) with IL-2 (200 U / ml), IL12 (10 ng / ml) and IL18 (20 ng / ml), and varying concentrations of TGFb (0.3 ng / ml, 1 ng / ml, or 10 ng / ml, as noted). FIG. 28A provides representative images of SKOV3 spheroids (tumor cells transduced with GFP) over time when left alone or treated at a 1 : 1 E:T ratio with prepared NK cells expanded as described above. The data showed a significant decrease in SKOV3 spheroid size in the wells treated with NK cells expanded with KS conditions comprising low dose exogenously provided TGFb (e.g., 0.3 ng / ml) compared to KS conditions comprising higher doses of TGFb, or NK cells expanded with P+E conditions. FIG. 28B providesxCELLigence assay data (at a 1:1 E:T ratio) showing both faster and improved antitumor activity in prepared NK cells expanded with KS conditions comprising low dose TGFb (e.g., 0.3 ng / ml) when compared to prepared NK cells expanded with KS conditions comprising higher doses of TGFb (e.g., >1 ng / ml), or prepared NK cells expanded with P+E conditions. The results indicate that, even at higher doses, TGFP facilitated more effective tumor control than P+E-expanded NK cells. However, this effect was comparatively slower and less pronounced than the tumor control observed with a lower dose of TGFp.

[0430] FIG. 29 Prepared NK cells expanded with low doses of exogenously provided TGFb in combination with IL2, IL12, and IL18 (KS conditions) had improved antitumor activity against PATC148 (PDAC) cancer cell line spheroids when compared to NK cells expanded with IL2, IL12, IL18 and higher doses of TGFb, or P+E expanded NK cells, when measured using long-term IncuCyte spheroid killing assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL- 12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KS (IL2, IL12, IL18, and TGFb) with IL-2 (200 U / ml), IL12 (10 ng / ml) and IL18 (20 ng / ml), and varying concentrations of TGFb (0.3 ng / ml, 1 ng / ml, or 10 ng / ml). FIG. 29 provides representative images of SKOV3 spheroids (tumor cells transduced with GFP) over time when left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described above. The data revealed a notable reduction in the size of PATC148 spheroids in wells treated with NK cells expanded under KS conditions with a low dose of exogenously added TGFb (e.g., 0.3 ng / ml), compared to NK cells expanded under KS conditions with higher doses of TGFb or NK cells prepared using P+E conditions. Additionally, the addition of medium or high doses of TGFb resulted in better tumor control compared to P+E conditions.

[0431] FIG. 30 Prepared NK cells expanded with low doses of exogenously provided TGFb in combination with IL2, IL 12, and IL 18 (KS conditions) had improved antitumor activity against WiDR (CRC) cancer cell line spheroids when compared to NK cells expanded IL2, IL12, IL18 and higher doses of TGFb, or P+E expanded NK cells, when measured using longterm IncuCyte spheroid killing assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KS (IL2, IL12, IL18, and TGFb) with IL-2 (200 U / ml), IL12 (10 ng / ml) and IL18 (20 ng / ml), and varying concentrations of TGFb (0.3 ng / ml, 1 ng / ml, or 10 ng / ml). FIG. 30 providesrepresentative images of WiDR spheroids (tumor cells transduced with GFP) over time when left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described above. The data showed a significant decrease in WiDR spheroid size in the wells treated with NK cells expanded with KS conditions comprising low dose exogenously provided TGFb (e.g., 0.3 ng / ml) compared to KS conditions comprising higher doses of TGFb, or prepared NK cells expanded with P+E conditions. The data revealed a notable reduction in the size of WiDR spheroids in wells treated with NK cells expanded under KS conditions with a low dose of exogenously added TGFb (e.g., 0.3 ng / ml), compared to NK cells expanded under KS conditions with higher doses of TGFb or NK cells prepared using P+E conditions. Additionally, the addition of medium or high doses of TGFb resulted in better tumor control compared to P+E conditions.

[0432] FIG. 31 Prepared NK cells expanded with KS conditions (IL2, IL12, IL18, and TGFB) exhibited a slightly higher rate of cell proliferation relative to NK cells expanded with the same conditions plus added rapamycin. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); KS (IL2, IL12, IL18, TGFb) with IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), or KS with varying concentrations of rapamycin (KSR, 0.5 nM, 1.0 nM, 10 nM, 100 nM, or 200 nM). The results showed that NK cells expanded with KSR conditions comprising lower doses of rapamycin had expansion / proliferation rates comparable to NK cells expanded with KS conditions, while higher concentrations of rapamycin inhibited cell proliferation.

[0433] FIGs. 32A-32D show how preparing NK cells with culture media comprising low doses of rapamycin resulted in improved antitumor activity relative to NK cells prepared with higher doses of rapamycin. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KS without TGFb (IL2, IL 12, IL18, and rapamycin) with IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), and varying concentrations of rapamycin (0.5 nM, 1.0 nM, 10 nM, 100 nM, or 200 nM). NK cells were left non-transduced and otherwise undisturbed, with media being refreshed every 2-3 days. Prepared NK cells were co-cultured with tumor cells at a 1:1 effector-to-target ratio. The addition of low-dose rapamycin significantly improved NK cell antitumor activity against OVCAR5 (GIST tumors) as shown in FIG. 32A and FIG. 32B with two independent repeatsof Xcelligence tumor cell killing assays. The addition of low-dose rapamycin significantly improved NK cell antitumor activity against WiDR (CRC) as shown in FIG. 32C and FIG. 32D with two independent repeats of Xcelligence tumor cell killing assays. The data demonstrate a dose-dependent enhancement of NK cell antitumor activity by rapamycin across multiple tumor models, with the most pronounced improvements observed at concentrations of 0.5 and 1 nM. These findings suggest that rapamycin at these doses may effectively amplify the antitumor efficacy of NK cells in various tumor settings.

[0434] FIGs. 33A-33B Preparing NK cells with KSR culture media comprising Enasidenib (“KSR-E”, see FIG. 13B) improved NK cells’ antitumor activity against PATC148 (PDAC) cells and spheroids relative to KSR or P+E NK cells in long-term IncuCyte spheroid and xCELLigence assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KSR (IL2, IL12, IL18, TGFb, and rapamycin) with IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), TGFb (0.3 ng / ml), and rapamycin (InM). Five days following expansion, NK cells were enriched for and transduced with a construct comprising a CAR targeting TROP2 and secreted IL- 15 (e.g., iC9 / TROP2CAR / IL-15) in the presence of the noted cytokine cocktail. NK cells were maintained under the following conditions: for P+E, continue with IL-2 (200 U / ml); for KSR, conditions, the cells were split to either continue with KSR including IL-2 (200 U / ml), TGFb (0.3 ng / ml), IL12 (10 ng / ml), and IL18 (20 ng / ml), and Rapamycin (1 nM); or KSR with added Enasidenib (1 pM) (“KSR-E”) until Day 8 of expansion. Prepared NK cells were then challenged against PATCI 48 (PDAC) cells or spheroids (tumor cells transduced with GFP). FIG. 33A provides representative IncuCyte assay images of PATC148 (PDAC) spheroids left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data showed a significant decrease in spheroid size in the wells treated with prepared NK cells cultured with KSR-E relative to NK cells prepared with P+E or KSR alone. FIG. 33B provides results of xCELLigence assay of PATC148 (PDAC) cells left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data showed KSR-E prepared NK cells had increased and swifter antitumor activity against PATC148 cells relative to KSR or P+E prepared NK cells. The data demonstrate that treating NK cells with Enasidenib for the final 3- 4 days before freezing significantly enhanced their antitumor activity across multiple tumor models. This suggests that Enasidenib treatment effectively augment the therapeutic potential of NK cells in targeting and combating tumor growth.

[0435] FIGs. 34A-34B Preparing NK cells with KSR culture media comprising Enasidenib (“KSR-E”, see FIG. 13B) improved NK cells’ antitumor activity against SKOV3 (ovarian cancer) cells and spheroids relative to KSR or P+E NK cells in long-term IncuCyte spheroid and xCELLigence assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KSR (IL2, IL12, IL18, TGFb, and rapamycin) with IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), TGFb (0.3 ng / ml), and rapamycin (InM). Five days following expansion, NK cells were enriched for and transduced with a construct comprising a CAR targeting TROP2 and secreted IL- 15 (e.g., iC9 / TROP2CAR / IL-15) in the presence of the noted cytokine cocktail. NK cells were maintained the following conditions: for P+E, continue with IL-2 (200 U / ml); for KSR, conditions, the cells were split to either continue with KSR including IL-2 (200 U / ml), TGFb (0.3 ng / ml), IL12 (10 ng / ml), and IL18 (20 ng / ml), and Rapamycin (1 nM); or KSR with added Enasidenib (1 pM) (“KSR-E”) until Day 8 of expansion. Prepared NK cells were then challenged against SKOV3 (ovarian cancer) cells and spheroids (tumor cells transduced with GFP). FIG. 34A provides representative IncuCyte assay images of SKOV3 spheroids left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data showed a significant decrease in spheroid size in the wells treated with prepared NK cells cultured with KSR-E relative to NK cells prepared with P+E or KSR alone. FIG. 34B provides results of xCELLigence assay of SKOV3 cells left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data showed KSR-E prepared NK cells had increased and swifter antitumor activity against SKOV3 cells relative to KSR or P+E prepared NK cells. The data demonstrate that treating NK cells with Enasidenib for the final 3-4 days before freezing significantly enhanced their antitumor activity across multiple tumor models. This suggests that Enasidenib treatment can effectively augment the therapeutic potential of NK cells in targeting and combating tumor growth.

[0436] FIGs. 35A-35B Preparing NK cells with KSR culture media comprising Enasidenib (“KSR-E”, see FIG. 13B) improved NK cells’ antitumor activity against WiDR (CRC) cells and spheroids relative to KSR or P+E NK cells in long-term IncuCyte spheroid and xCELLigence assays. NK cells were isolated from CB, resuspend at 1 x 10A6 cells / ml, and then pre-activated with cytokines IL-15 (50 ng / ml), IL-18 (50 ng / ml), and IL-12 (10 ng / ml) for 16 hours. The NK cells were then washed twice with PBS and expansion was started (Day 0) using uAPCs under the following conditions: P+E: IL-2 (200 U / ml); or KSR (IL2, IL12, IL18, TGFb,and rapamycin) with IL-2 (200 U / ml), IL12 (10 ng / ml), IL18 (20 ng / ml), TGFb (0.3 ng / ml), and rapamycin (InM). Five days following expansion, NK cells were enriched for and transduced with a construct comprising a CAR targeting TROP2 and secreted IL- 15 (e.g., iC9 / TROP2CAR / IL-15) in the presence of the noted cytokine cocktail. NK cells were maintained the following conditions: for P+E, continue with IL-2 (200 U / ml); for KSR, conditions, the cells were split to either continue with KSR including IL-2 (200 U / ml), TGFb (0.3 ng / ml), IL12 (10 ng / ml), and IL18 (20 ng / ml), and Rapamycin (1 nM); or KSR with added Enasidenib (1 pM) (“KSR-E”) until Day 8 of expansion. Prepared NK cells were then challenged against WiDR (CRC) cells and spheroids (tumor cells transduced with GFP). FIG. 35A provides representative IncuCyte assay images of WiDR spheroids left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data showed a significant decrease in spheroid size in the wells treated with prepared NK cells cultured with KSR-E relative to NK cells prepared with P+E or KSR alone. FIG. 35B provides results of xCELLigence assay of WiDR cells left alone or treated at a 1:1 E:T ratio with prepared NK cells expanded as described. The data demonstrated that treating NK cells with Enasidenib for the final 3-4 days before freezing significantly enhanced their antitumor activity across multiple tumor models. This suggests that Enasidenib treatment can effectively augment the therapeutic potential of NK cells in targeting and combating tumor growth.

[0437] FIGs. 36A-36B NK cells cultured in KS Media (IL-2, IL- 12, IL- 18, TGFb) showed superior proliferation and antitumor activity against WiDR spheroids (CRC; colorectal tumor cells transduced with GFP) compared to NK cells prepared using P+E or P+E+TGFb. NK cells were cultured in P+E (IL-2), P+E+TGFb (IL-2 with supplemented TGFb), or KS (IL-2, IL- 12, IL-18, and TGFb) for 14 days. To test the anti-tumor cytotoxicity of NK cells cultured in the different culture media, NK cells were challenged against tumor spheroids. For functional analysis, GFP-labeled WiDR(CRC) tumor cell lines were plated to form spheroids and monitored using IncuCyte. After 24 hours, different NK cell populations were added to the spheroids at a 1:1 ratio. Quantification of total integrated green intensity over time showed a notable decrease when NK cells were expanded with IL-2, IL-12, IL-18, and TGF-P (KS), indicating improved NK cell cytotoxic potential. FIG. 36A provides representative IncuCyte assay images of the spheroids left alone or treated with NK cells expanded (cultured) as described. FIG. 36B provides results of xCELLigence assay of tumor spheroids left alone or treated with NK cells expanded as described. The data showed a substantial decrease in spheroid size in the wells treated with prepared NK cells cultured with IL-2, IL- 12, IL- 18, and TGFb relative to NK cells cultured with IL-2 (P+E) or IL-2 and TGFb (P+E+TGFb). Theseantitumor activity data demonstrate synergistic impacts of cytokines, as described herein, and TGF-P, relative to the efficacy of either factor (e.g., cytokine) alone. This integrated approach demonstrates a more effective inhibition of tumor progression and improved therapeutic outcomes.

[0438] FIGs. 37A-37B NK cells cultured in KS Media (IL-2, IL- 12, IL- 18 and TGFb) showed superior proliferation and antitumor activity against SKOV3 (Ovarian Carcinoma) compared to NK cells prepared using P+E or P+E+TGFb culture media. NK cells were cultured in media supplemented with IL-2 (i.e., P+E), or IL-2 and TGFb (i.e., P+E+TGFb), or IL-2, IL-12, IL-18 and TGFb (i.e., KS) for 14 days. To test the anti-tumor cytotoxicity of NK cells cultured in the different culture media, NK cells were challenged against tumor spheroids. GFP-labeled SKOV3 tumor cell lines were plated to form spheroids and monitored using IncuCyte. After 24 hours, different NK cell populations were added to the spheroids at a 1:1 ratio. Quantification of total integrated green intensity over time revealed a significant reduction in total integrated green intensity when spheroids were treated with NK cells expanded in IL-2, IL- 12, IL- 18, and TGF-P, suggesting enhanced cytotoxicity. FIG. 37A provides representative IncuCyte assay images of the spheroids left alone or treated with NK cells expanded (i.e., “cultured”) as described. FIG. 37B provides results of xCELLigence assay of tumor spheroids left alone or treated with NK cells expanded as described. The data showed a substantial decrease in spheroid size in the wells treated with prepared NK cells cultured with IL-2, IL- 12, IL- 18, and TGFb relative to NK cells cultured with IL-2 (P+E) or IL-2 and TGFb (P+E+TGFb). These antitumor activity data demonstrate synergistic impacts of cytokines, as described herein, and TGF-P, relative to the efficacy of either factor (e.g., cytokine) alone. This integrated approach demonstrates a more effective inhibition of tumor progression and improved therapeutic outcomes.

[0439] FIGs. 38A-38E NK cells cultured in KS Media (IL-2, IL- 12, IL- 18, and TGF-P demonstrated superior proliferation and antitumor activity across multiple tumor models compared to the combination of IL-2, IL- 12, and IL- 18 (without TGF-P). NK cells derived from cord blood were pre-stimulated with IL-12, IL-18, and IL-15 for 16-24 hours. After preactivation, they were washed to eliminate residual cytokines and then cultured under two conditions: IL-2, IL- 12, IL- 18, and uAPC, or IL-2, IL- 12, IL- 18, TGF-P, and uAPC. FIG. 37A shows cell proliferation and expansion as monitored by assessing cell counts at regular three- day intervals. After 14 days of expansion, NK cells were co-cultured with mCherry-expressing K562 cells at a 1:2 effector-to-target (E:T) ratio (FIGs. 38B-38C), or NK cells were co-cultured with mCherry-expressing Raji cells at a 1:2 E:T ratio (FIGs. 38D-38E). Real-time monitoringusing the IncuCyte system revealed that NK cells cultured with IL-2, IL- 12, IL- 18, TGF-P, and uAPC demonstrated significantly enhanced antitumor activity against cancer cells, as shown in the figures. The results showed that culturing NK cells with IL-2, IL- 12, IL18 was less effective than those cultured with KS media. These antitumor activity data demonstrate synergistic impacts of cytokines, as described herein, and TGF-P, relative to the efficacy of either factor (e.g., cytokine) alone. This integrated approach demonstrates a more effective inhibition of tumor progression and improved therapeutic outcomes, as well as improved NK cell proliferation.

[0440] FIGs. 39A-39C Show how NK cells cultured with KS Media (IL-2, IL- 12, IL- 18, TGF- P) or modified versions thereof exhibited distinct phenotypic characteristics at both the protein and mRNA levels relative to NK cells prepared using P+E or without TGFb. NK cells were isolated from cord blood and pre- stimulated with IL- 12, IL- 15, and IL- 18 for 16-24 hours. Following pre-stimulation, they were washed twice with PBS to remove residual cytokines before being co-cultured with uAPC under various conditions: IL-2 alone (P+E), or IL-2 with Rapamycin (P+E+Rapa), or IL-2, IL- 12, IL- 18, and TGF-P (KS), or KS with Rapamycin during the second half of culture (a second step) only (KS - Rappa), or IL-2, IL- 12, IL- 18, TGF-P, and Rapamycin (KSR), or IL-2, IL- 12, IL- 18, TGF-P, Rapamycin, and Idelalisib (KSR-I), or IL-2, IL- 12, IL- 18, TGF-P, Rapamycin, and 2-DG (KSR-2DG), or IL-2, IL- 12, and IL- 18 (KS no TGFb) for 14 days. After the 14-day expansion, samples were stained for 50-color mass cytometry and analyzed using Cytobank software. FIG. 39A Provides a UMAP analysis and FIG. 39B provides a heat map analyses of the NK cell population markers following expansion. The results demonstrated that the P+E and KS conditions resulted in NK cells that exhibited distinct phenotypic profiles. NK cells expanded under any KS condition showed higher expression of TRAIL, DNAM-1, CD25, CLA, CD16, and multiple chemokines, which are important for NK cell function and trafficking. FIG. 39C Provides results of RNA sequencing from the aforementioned NK cell populations. As shown in the heatmap, NK cells expanded under KS conditions exhibited higher expression of DNAM and TRAIL, similar or lower levels of SMAD2 / 3, reduced TIGIT expression, comparable CD38 levels, and decreased perforin and granzyme B, likely as a mechanism to prevent activation-induced cell death. Together, these results showed that SMAD3, CD38, perforin, granzyme, and FasL mRNA / protein levels were altered (reduced or increased, as the data shows) during NK cell activation and expansion by KS / KSR / KSRI, etc., while TRAIL mRNA / protein levels were increased during NK cell activation and expansion by KS / KSR / KSRI, etc.

[0441] FIGs. 40A-40B Show how increasing the amount of TGF-P during KS culture conditions resulted in increased baseline SMAD2 / 3 phosphorylation in NT NK (40A) and CAR NK cells (40B). NK cells were isolated from cord blood and pre-stimulated with IL-12, IL-15, and IL- 18 for 16-24 hours. After pre-stimulation, they were washed twice with PBS to remove residual cytokines before being co-cultured with uAPC, IL-2, IL- 12, IL- 18, and varying concentrations of TGF-P (0.3, 1, and 10 ng / ml). Following the 14-day expansion, SMAD2 / 3 phosphorylation was assessed using flow cytometry. As shown in the figure, higher concentrations of TGF-P resulted in increased baseline SMAD2 / 3 phosphorylation.DETAILED DESCRIPTIONI. Examples of Definitions

[0442] It is to be understood that the present disclosure is not limited solely to the particular aspects described herein, as such may, of course, vary, while still being encompassed by the teachings provided herein. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0443] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, certain embodiments of methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0444] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijned. (2005) Oligonucleotide Synthesis: Methods and Applications; Hanies and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition; all of which are incorporated herein by reference.

[0445] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0446] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0447] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0448] The term “antigen presenting cells (APCs)” refers to a class of cells capable of presenting one or more antigens in the form of a peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. The term “APC” encompasses intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells, or molecules, naturally occurring or synthetic capable of presenting antigen, such as purified MHC Class I molecules complexed to y2-microglobulin.

[0449] An “autoimmune disease” refers to a disease in which the immune system produces an immune response (for example, a B cell or a T cell response) against an antigen that is part of the normal host (that is, an autoantigen), with consequent injury to tissues. An autoantigen may be derived from a host cell, or may be derived from a commensal organism such as the microorganisms (known as commensal organisms) that normally colonize mucosal surfaces.

[0450] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0451] ‘ ‘Culture condition” as used herein refers to the conditions under which any cell described herein is cultured and includes media composition (e.g., cytokines, growth factors, antigen presenting cells), time (e.g., length of time in a given culture condition), and environmental conditions such as the temperature, humidity, and CO2 levels.

[0452] As used herein, “derived” refers to a segment, domain, or portion of an amino acid sequence that is obtained from a larger protein, such as but not limited to a wild-type protein. This term encompasses any fragment, subsequence, or isolated part of the original protein which may retain specific functional or structural characteristics. The derived segment may be obtained through various methods, including but not limited to, enzymatic digestion, chemical cleavage, or genetic manipulation. It is contemplated that a derived segment can maintain portions of at least one biological activity of the original protein while also possibly exhibiting enhanced or modified properties.

[0453] As used herein, a “disruption” of a gene refers to the elimination or reduction of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the disruption. Exemplary gene products include mRNA and protein products encoded by the gene. Disruption in some cases is transient or reversible and in other cases is permanent. Disruption in some cases is of a functional or full length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, gene activity or function, as opposed to expression, is disrupted. Gene disruption is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and / or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene silencing, knockdown, knockout, and / or gene disruption techniques, such as gene editing. Examples include antisense technology, such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in transient reduction ofexpression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and / or homologous recombination. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and / or complete absence of expression of a normal or “wild type” product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and mis sense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

[0454] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, construct, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific embodiments, a vector and / or construct is engineered through recombinant nucleic acid technologies, and a cell is engineered, e.g., through transfection or transduction of an engineered vector (also referred to as an engineered construct, which can comprise or consist of one or more polynucleotides). Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0455] The term “exogenous” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to external factors that originate outside of a biological specimen (e.g., a cell, population of cells, etc.). As used herein, exogenous components, reagents, and / or conditions, are components, reagents, and / or conditions that are added to compositions or methods described herein, although this does not necessarily preclude the possibility of the same components, reagents, and / or conditions also being present through a function endogenous to a biological specimen present in said composition and / or method.

[0456] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition istherefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0457] The term “heterologous” when used in reference to a gene refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise gene sequences that comprise cDNA forms of a gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or antisense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally found and / or expressed).

[0458] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0459] An “immune disorder,” “immune-related disorder,” or “immune-mediated disorder” refers to a disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft versus host disease and inflammatory and allergic conditions.

[0460] An “immune response” is a response of a cell of the immune system, such as a B cell, or a T cell, or innate immune cell to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen- specific response”).

[0461] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal e.g., human) administration, it will be understood that preparations should meet sterility,pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0462] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0463] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human. The term “individual” is interchangeable with “subject” and “patient”.

[0464] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0465] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.II. Methods of Making

[0466] Embodiments of the disclosure include methods of producing NK cells. The methods encompass 1, 2, 3, 4, 5, or more steps, one or some of which have a specific duration in time. The NK cells may be of any kind, including non-transduced or manipulated to express one or more heterologous genes. The various steps may or may not be in a particular order. In particular embodiments, the production methods are in vitro. One or some of the method steps may or may not be optional. In certain embodiments, culture conditions are replenished through media change, media addition, and / or cell splitting at a certain frequency, such as every 1 day, 2 days, 3 days, 4 days, or 5 days. In certain embodiments, culture conditions are replenished upon cell confluency reaching a certain level, such as 1 x 10A6 cells per ml. In certain embodiments, culture conditions are replenished every 2 or 3 days, without disturbing the cells.

[0467] In specific embodiments, there may be a pre-activating step. In some embodiments, the pre-activation step comprises exposure of NK cells to one or more cytokines. Any cytokine(s) used in the pre-activation and / or expansion steps may be recombinant human cytokines. In certain aspects, the cytokines are IL-12, IL-15, and / or IL-18. In specific embodiments, the duration of the pre-activating step may be in a particular range, such as between 12 hours and 20 hours, or 14 hours and 18 hours, or about 16 hours. In some embodiments, the duration of the pre-activating step is about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or any range or value derivable therein. In some embodiments, the duration of the pre-activating step is about 12-20 hours, 13-19 hours, 14-18 hours, 15-17 hours, 16 hours, or 16-24 hours.

[0468] Embodiments of the disclosure include at least one expansion step and in particular embodiments, the duration of time of the expansion step is particular. In specific embodiments, the duration in time for the expansion step is about 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-14, 3- 13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-14, 9-13, 9-12, 9- 11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, or 13-14 days. The duration of the expansion step may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. The duration of the expansion step may comprise one, or two, or more than two different culturing steps, each of which may be of a particular duration. In some embodiments, the duration of a first culturing step during expansion may be about 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4,2-3, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9,4-8, 4-7, 4-6, 4-5, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-14, 9- 13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, or 13-14 days. In some embodiments, the duration of a second culturing step during expansion may be about 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4,4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7,5-6, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8- 13, 8-12, 8-11, 8-10, 8-9, 9-14, 9-13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, or 13-14 days. In some embodiments, the duration of an additional culturing step beyond the first and / or second culturing step may be about 1-14, 1-13, 1-12, 1- 11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-14, 6-13, 6-12,6-11, 6-10, 6-9, 6-8, 6-7, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8-13, 8-12, 8-11, 8-10, 8- 9, 9-14, 9-13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, or 13-14 days. In some embodiments, during the expansion step the NK cells may be exposed to one or more particular agents, such as IL-2, antigen presenting cells (APCs), one or more of IL-12, IL-15, and IL-18, activating beads, TGFb, an mTOR inhibitor, a PI3K inhibitor, an AKT inhibitor, an IDH2 inhibitor, or any combination thereof.

[0469] In some embodiments, NK cells are expanded for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 days. In some embodiments, NK cells are subject to a second round of aAPC (e.g., uAPC) stimulation during a second culturing step. In some embodiments, one or more boluses of aAPCs are added to a culture comprising NK cells on Day 8 or Day 9 of expansion. In some embodiments, during a second culturing step, one or more boluses of aAPCs are added to a culture comprising engineered NK cells 1, 2, 3, 4, or 5 days following engineering (e.g., transduction, transfection, etc.) that was performed in the absence of aAPCs. In some embodiments, NK cells are collected (e.g., enriched for, selected, aspirated, etc.) prior to and / or following engineering, and then are subject to a second culturing step comprising aAPCs. In some embodiments, a second culturing step comprising aAPCs is for 1, 2, 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, a second culturing step comprising aAPCs is for 5 days or 6 days, and NK cells are harvested on Day 13 or Day 14 of expansion.

[0470] In some embodiments, NK cell populations are enriched for through any means known in the art. In some embodiments, NK cell populations are collected through selection such as via cell surface marker based selection. In some embodiments, NK cell populations are collected via centrifugation, aspiration, and / or decanting.

[0471] In some embodiments, NK cells are pre-activated. In some embodiments, pre-activation can occur as described in international PCT application publication WO 2019 / 165121 Al, published August 29, 2019, which is incorporated herein by reference in its entirety for the purposes described herein.

[0472] In some embodiments, NK cells are expanded in a culture media comprising exogenously provided IL-2, IL-12, IL-18, and TGFb (e.g., “KS” culture conditions). In some embodiments, NK cells are expanded in a KS culture condition for a first culturing step, and a different or the same culturing conditions for a second culturing step. In some embodiments, NK cells are expanded in a culture media comprising exogenously provided IL-2, IL- 12, IL- 18, TGFb, and rapamycin (e.g., “KSR” culture conditions). In some embodiments, NK cells are expanded in a KSR culture condition for a first culturing step, and a different or the same culturing conditions for a second culturing step. In some embodiments, NK cells are expanded in a culture media comprising exogenously provided IL-2, IL- 12, IL- 18, TGFb, and rapamycin (e.g., “KSR” culture conditions) for a first culturing step, and have a second culturing step that includes KSR conditions plus one or more additional agents, such as an IDH2 inhibitor (“KSR- ID”, e.g., “KSR-E”), a PI3K inhibitor (“KSR-P”, e.g., “KSR-I”), an AKT inhibitor, and / or a particular sugar (metabolizable or not; e.g., “KSR-2DG”). In some embodiments, NK cells are expanded in a culture media comprising feeder cells, and / or aAPCs (such as uAPCs, see e.g., international PCT application publication WO 2019 / 165097 Al, published August 29, 2019, and Enli Liu et al., “GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells”, Front Immunol., 2021; each of which re incorporated by reference herein in their entirety for the purposes described herein.

[0473] The culturing step may occur in a particular vessel, including a flask, a plate, a bioreactor, a hollow fiber bioreactor, and so forth.

[0474] In some embodiments, during methods of production of functional prepared NK cells as described herein, such methods can comprise pre-activating, expanding with one or more culture steps, optionally transducing and / or transforming, and optionally deactivating and freezing. At any one or more points during methodologies described herein, cells and media culture may be tested to determine characteristics of the media, cells, etc. In someembodiments, tests may include, for example but not limited to: cell counting / cell viability (e.g., TB / Sytox Green), sterility tests (e.g., BD BACTEC™), immunophenotyping, PCR, qPCR, MycoPCR, endotoxin tests (e.g., LAL), mycoplasma tests, cytokine measurements, visual inspections, vector copy number analysis (e.g., by qPCR), RCR (e.g., by qPCR), human pathogen virus tests, in-vitro adventitious virus testing, and / or HLA typing. Tests may be conducted at certain time points.

[0475] In some embodiments, NK cells are engineered, such as through transduction with a viral vector, and the engineering step may or may not occur during an NK cell expansion step of a method. The timing of the engineering step may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days following the onset of the expansion step. In some embodiments, the engineering step occurs on Day 4, Day 5, or Day 6 following onset of the expansion step. In some embodiments, the engineering step occurs on Day 4 following onset of the expansion step. In some embodiments, the engineering step occurs on Day 5 following onset of the expansion step. In some embodiments, the engineering step occurs on Day 6 following onset of the expansion step. In some embodiments, NK cells or their precursors may be engineered prior to expansion. In some embodiments, prepared NK cells may be engineered after expansion.

[0476] In some embodiments, an expansion step comprises at least two culturing steps that are separated by an NK cell enrichment step. In specific embodiments, the NK cells express one or more heterologous genes, such as a cytokine, an engineered antigen receptor (e.g., a chimeric antigen receptor (CAR), and / or a T cell receptor (TCR), etc.), and / or a suicide gene. In specific cases, the engineered antigen receptor may be a CAR and / or TCR, including one that targets a cancer antigen of any kind, including an antigen associated with a hematological cancer or a solid tumor.

[0477] In some embodiments, there is a culturing step in which the NK cells are exposed to an effective amount of one or more cytokines. In specific aspects, there may or may not be aAPCs in the culture. In specific embodiments, the one or more cytokines comprises IL-2. In some embodiments, the one or more cytokines comprises IL- 12. In some embodiments, the one or more cytokines comprises IL- 18. In some embodiments, the one or more cytokines comprises TGEb. In some embodiments, the one or more cytokines comprises IL-2, IL- 12, IL- 18, and TGEb. In some embodiments, during a culturing step the NK cells are exposed to an effective amount of one or more small molecules, such as but not limited to mTOR inhibitors (e.g., rapamycin, rapalogs, etc.), phosphoinositide 3-kinase (PI3K) inhibitors (e.g., Idelalisib), mTOR and PI3K dual action inhibitors, d-glucose mimetics (e.g., 2-deoxy-d-glucose (2-DG)),protein kinase B (PKB, AKT) inhibitors, isocitrate dehydrogenase 2 (IDH2) inhibitors, and / or metabolizable sugars.

[0478] In certain embodiments, the NK cells are cryopreserved following production. In some embodiments, prior to cryopreservation the NK cells are exposed to one or more deactivating agents, including a small molecule. The deactivating agent(s) may be exposed to the NK cells for a specific duration in time, such as from about 1-48 hours, including 12-48 hours, 18-48 hours, 24-48 hours, 24-36 hours, 12-24 hours, and so forth. The exposure to one or more deactivating agents may be about 12, 16, 18, 20, 24, 28, 36, 40, or 48 hours, in specific embodiments. In particular embodiments, the NK cells are washed following exposure to the deactivating agent(s), including prior to cryopreservation, in certain embodiments.

[0479] In particular embodiments, the NK cells are modified, such as including knockout or knockdown of 1, 2, 3, 4, or more endogenous genes of the cell. In specific embodiments, the NK cells are double knockout CRISPR gene-edited NK cells. In certain embodiments, the NK cells comprise engineered mutations in endogenous Glucocorticoid Receptor (GR; N3CR1) and / or Transforming Growth Factor Beta Receptor 2 (TGFBR2) genes.

[0480] Some embodiments of the present disclosure concern expansion of NK cells and may including the isolation, activation, and / or expansion of NK cells, such as for cancer immunotherapy and / or auto immune disorder immunotherapy.

[0481] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. Specifically, the NK cells may be isolated from cord blood (CB), peripheral blood (PB), bone marrow, or stem cells. In particular embodiments, the NK cells are isolated from pooled CB. The CB may be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells may be autologous or allogeneic. The isolated NK cells may be haplotype matched for the subject to be administered the cell therapy. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans.

[0482] In certain aspects, the NK cells are isolated by the previously described method of ex vivo expansion of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by ficoll density gradient centrifugation. The cell culture may be depleted of any cells expressing CD3 and may be characterized to determine the percentage of CD56+ / CD3‘ cells or NK cells. In other methods, umbilical CB is used to derive NK cells by the isolation of CD34+cells.

[0483] Pre-activation of the NK cells may comprise culturing the isolated NK cells in the presence of one or more cytokines. The NK cells may or may not be stimulated with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). In particular embodiments, the pre-activation cytokines may be IL-12, IL-15, and / or IL- 18 and may include all of IL- 12, IL- 15, and IL- 18. One or more additional cytokines may be used for the pre-activation step. The pre-activation may be for a short period of time such as 5-72 hours, such as 10-50 hours, particularly 10-20 hours, such as 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, specifically about 16 hours. The pre-activation culture may comprise IL-18 and / or IL- 15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, particular 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, specifically about 50 ng / mL. The pre-activation culture may comprise IL-12 at a concentration of 0.1-150 ng / mL, such as 0.5-50 ng / mL, particularly 1-20 ng / mL, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, specifically about 10 ng / mL.

[0484] The pre-activated NK cells may then be expanded in the presence of artificial antigen presenting cells (aAPCs). The pre-activated NK cells may be washed prior to expansion, such as 2, 3, 4, or 5 times, specifically 3 times. The aAPCs may be engineered to express CD137 ligand and / or a membrane-bound cytokine. The membrane-bound cytokine may be membranebound IL-21 (mIL-21) or membrane-bound IL- 15 (mIL-15). In particular embodiments, the aAPCs are engineered to express CD 137 ligand and mIL-21. The aAPCs may be derived from cancer cells, such as leukemia cells. The aAPCs may not express endogenous HLA class I, II, or CD Id molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, the aAPCs may be K562 cells, such as K562 cells engineered to express CD137 ligand and mlL- 21. The aAPCs may be irradiated. The engineering may be by any method known in the art, such as retroviral transduction. The expansion may be for about 2-30 days, such as 3-20 days, particularly 12-16 days, such as 12, 13, 14, 15, 16, 17, 18, or 19 days, specifically about 14 days. The pre-activated NK cells and aAPCs may be present at a ratio of about 3: 1-1:3, such as 2:1, 1:1, 1:2, specifically about 1:2. The expansion culture may further comprise cytokines to promote expansion, such as IL-2. The IL-2 may be present at a concentration of about 10- 500 U / mL, such as 100-300 U / mL, particularly about 200 U / mL. The IL-2 may be replenished in the expansion culture, such as every 2-3 days. The aAPCs may be added to the culture at least a second time, such as at about 7 days of expansion.

[0485] The cytokine used in the pre-activation and / or expansion steps may be recombinant human cytokines.

[0486] Following expansion, the NK cells may be immediately infused or may be stored, such as by cryopreservation. In certain aspects, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, or 5 days.

[0487] Activated and / or expanded NK cells can secrete type I cytokines, such as interferon-y, tumor necrosis factor-a and granulocyte-macrophage colony- stimulating factor (GM-CSF), which activate both innate and adaptive immune cells as well as other cytokines and chemokines. The measurement of these cytokines can be used to determine the activation status of NK cells. In addition, other methods known in the art for determination of NK cell activation may be used for characterization of the NK cells of the present disclosure.A. Chimeric Antigen Receptors

[0488] In certain embodiments, the present NK cells are genetically modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.

[0489] A CAR recognizes cell-surface tumor-associated antigen independent of human leukocyte antigen (HLA) and employs one or more signaling molecules to activate genetically modified NK cells for killing, proliferation, and cytokine production (Jena et al., 2010). In certain embodiments, the platform technologies disclosed herein to genetically modify NK cells comprise (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28 / CD3-^, CD137 / CD3-^, Dapl0 / CD3-^, DAP12 / CD3-^, NKG2D / CD3-^, CD40 / CD3-( 2B4 / CD3-< 41BB / CD3-<; or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the antigen-recognition domain to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPC) derived from K562 to be able to robustly and numerically expand CAR+NK cells (Singh et al., 2008; Singh et al., 2011).

[0490] Embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding an antigen- specific chimeric antigen receptor (CAR) polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof.In another embodiment, that specificity is derived from a peptide e.g., cytokine) that binds to a receptor.

[0491] It is contemplated that the human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure provides a full-length CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen- specific antibody. In a more specific embodiment, the fragment is an antigen- specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.

[0492] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. One of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin may be used. The hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization may be used. In other aspects, just the hinge portion of an immunoglobulin or portions of CD8a may be used.

[0493] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 2B4, NKG2D, CD40 and 4-1BB (CD137). In addition to a primary signal initiated by CD3^, an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for full activation of NK cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy .

[0494] The intracellular signaling domain of a chimeric antigen receptor is responsible for activation of at least one of the normal effector functions of the immune cell in which the chimeric antigen receptor has been placed. The term “effector function” refers to a specialized function of a differentiated cell, such as a NK cell. In specific embodiments, intracellularreceptor signaling domains in the CAR include those of the T-cell antigen receptor complex, such as the zeta chain of CD3, also Fc y RIII costimulatory signaling domains, CD28, CD27, DAP10, CD137, 0X40, CD2, alone or in a series with CD3zeta, for example. In specific embodiments, the intracellular domain (which may be referred to as the cytoplasmic domain) comprises part or all of one or more of TCR zeta chain, CD28, CD27, OX40 / CD134, 4- 1BB / CD137, Fc e RI y , ICOS / CD278, IL-2Rbeta / CD122, IL-2Ralpha / CD132, DAP10, DAP12, and CD40. In some embodiments, one employs any part of the endogenous T-cell receptor complex in the intracellular domain. One or multiple cytoplasmic domains may be employed, as so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effect, for example.

[0495] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a tumor associated antigen or a pathogen-specific antigen binding domain. Antigens include carbohydrate antigens recognized by pattern-recognition receptors, such as Dectin- 1. A tumor associated antigen may be of any kind so long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor associated antigens include CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c- Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, and mutated ras. Additional exemplary antigens include CD99, CLL-1, CD47, CD33, CS1, B7- H3, GD2, MUC1, GPC3, GPR5CD, CD38, CD70, TROP2, CD5, and BCMA.

[0496] In certain embodiments, the CAR may be co-expressed with a cytokine to improve persistence when there is a low amount of tumor-associated antigen. For example, CAR may be co-expressed with IL- 15 and / or IL-21. For example, CAR may be co-expressed with IL- 15, IL-4, IL-7, IL-9, IL- 10, IL-27, and / or IL-21.

[0497] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0498] It is contemplated that the chimeric construct can be introduced into NK cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers tothe DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0499] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into NK cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the NK cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0500] The CAR encoding construct may also comprise a sequence encoding a suicide gene, such as CD20, CD52, EGFRv3, or inducible caspase 9. An engineered NK cell provided herein may comprise a heterologous sequence encoding a suicide gene, such as CD20, CD52, EGFRv3, or inducible caspase 9.

[0501] The CAR may comprise a tumor antigen-binding domain. Any suitable antigen may be targeted in the present method. The antigen may be associated with certain cancer cells but not associated with non-cancerous cells, in some cases. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto- / self-antigens, tumor- / cancer- associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular aspects, the antigens include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL- HRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, -catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, Tumor- associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases e.g.,Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor- Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP- 4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRN1.B. Antigen Presenting Cells

[0502] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular major histocompatibility complex (MHC) molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The MHC is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans, the MHC is referred to as the HLA complex and in mice the H-2 complex.

[0503] In some cases, aAPCs are useful in preparing therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. W02007 / 103009.

[0504] In certain embodiments, methods of the disclosure comprise expansion of NK cells in the presence of aAPCs for at least a first culture condition. In certain embodiments, methodsof the disclosure comprise expansion of NK cells in the presence of aAPCs for a first culture condition and a second culture condition. In some embodiments, expansion of NK cells with a culture condition that does not comprise aAPCs is for less than about 4 or 5 days. In certain embodiments, if a culture condition will last for longer than 4 or 5 days, aAPCs are utilized to promote NK cell viability.

[0505] aAPC systems may comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co- stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcyRI), 41BB ligand (CD137 ligand), and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell- to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0506] In particular embodiments, the aAPCs have been engineered to express CD 137 ligand, such as by retroviral vectors. The aAPCs may further express membrane-bound cytokines, such as membrane -bound IL-21 (mIL-21) or membrane-bound IL- 15 (mIL-15). In particular aspects, the aAPCs express CD137 ligand and mIL-21. The aAPCs may be K562 leukemia cells engineered to express CD137 and mIL-21. The aAPCs may be developed to express a desired antigen, such as CD 19. As needed, additional stimulation cycles can be undertaken to generate larger numbers of NK cells.C. Gene Editing

[0507] In specific embodiments, the NK cells are modified by editing of one or more of their endogenous genes. In particular embodiments, one or more of the following genes are modified in the NK cells, such as by knockdown or knockout: NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1 (GR), PD1, PDL- 1, PDL-2, CD47, SIRPA, SHIP1, ADAM 17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and / or CD7.

[0508] In specific embodiments, an engineered mutation in an endogenous gene can be a mutation in GR, TGFBR2, CISH, and / or CD38. In specific embodiments, an NK cell is engineered to comprise a mutation in the endogenous genes GR and TGFBR2.

[0509] In certain embodiments, an engineered mutation is in an endogenous gene mutation as described in international PCT patent application publication WO 2020 / 113029 A2, published June 4, 2020; international PCT patent application publication WO 2021 / 146719 Al, published July 22, 2021; international PCT patent application publication WO 2021 / 108671 Al, published June 3, 2021; and / or international PCT patent application publication WO 2023 / 245041 A2, published December 21, 2023; each of which are hereby incorporated in their entirety for any purpose described herein.D. CD3 Chains, Invariant NK TCRs, and Cytokines

[0510] In certain embodiments, engineered NK cells are modified to express, a) part or all of a single chain or any combination of CD38, CD3e, CD3y, or CD3^; b) part or all of a single chain or any combination of invariant NK T cell receptor (iTCR) alpha (iTCRoc) chain or beta (iTCRP) chain; and c) a cytokine selected from the group consisting of IL-15, IL-12, IL-2, IL- 18, IL-21, IL-23, IL-7, GMCSF, and a combination thereof. In certain embodiments, the NK cells are modified to express part or all of one CD38, two of CD3e, one CD3y, and / or one CD3^. In certain embodiments, any one or more of CD38, CD3e, CD3y, and / or CD3^ are heterologously linked to one or more intracellular signaling domains. In certain embodiments an intracellular signaling domain is selected from the group consisting of CD 16, NKG2D, DAP 10, DAP 12, 2B4, 4- IBB, CD2, CD28, and a combination thereof. In certain embodiments, an intracellular signaling domain comprises a DAP10 intracellular signaling domain.

[0511] In certain embodiments, NK cells are complexed to one or more antibodies. In certain embodiments, the one or more antibodies are one or more bispecific or multi- specific antibodies, wherein at least one of the bispecific or multi- specific antibodies comprises an anti- CD3 antibody linked. In certain embodiments, the antibody is Blinatumomab, Tebentafusp, Mosunetuzumab, Teclistamab, Glofitamab, Epcoritamab, Flotetuzumab, APV0436, and / or TNB383B. In certain embodiments, the antibody is Blinatumomab. In certain embodiments, the antibody comprises, consists essentially of, or consists of Elranatamab, Glofitamab, Tafasitamab, Cetuximab, Imgatuzumab, Margetuximab, Amivantamab, Blinatumomab, Obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), Teclistamab, Talquetamab, Pertuzumab, Trastuzumab, Brentuximab, Mosunetuzumab, Epcoritamab, GEN3017, Loncastuximab tesirine, Belimumab, and / or Rituximab. In certain embodiments, the NK cellexpresses the antibody. In certain embodiments, the NK cells are complexed to one or more antibodies ex vivo and / or in vivo.E. Culture length

[0512] In some embodiments, methods described herein comprise expanding immune cells (e.g., NK cells) in one or more distinct culture media (e.g., pre-activation media, expansion culture media) for a defined length of time. The terms expansion media, expansion culture, and expansion culture media are used interchangeably herein. The terms pre-activation media, preactivation culture, pre-activation culture media, are used interchangeably herein. In some embodiments, methods described herein comprise a pre-activating step comprising culturing the immune cells in a pre-activation media. In some embodiments, methods described herein comprise an expanding step comprising culturing immune cells (e.g., NK cells) in an expansion culture media (e.g., a first expansion culture media). In some embodiments, methods described herein comprise an expanding step comprising culturing immune cells (e.g., NK cells) in a first expansion culture media and a second or subsequent expansion culture media (e.g., a second expansion culture media, a third expansion culture media, and / or a fourth expansion culture media, etc.). In some embodiments, methods described herein comprise a deactivating step comprising culturing, contacting, or exposing immune cells (e.g., NK cells) with one or more deactivating agent.

[0513] In some embodiments, methods described herein comprise, consist essentially of, of consist of (i) a pre-activation step comprising culturing immune cells (e.g., NK cells) in a pre- activation media, (ii) an expanding step comprising culturing immune cells (e.g., NK cells) in expansion culture media. In some embodiments, methods described herein comprise, consist essentially of, of consist of (i) a pre-activation step comprising culturing immune cells (e.g., NK cells) in a pre-activation media, (ii) an expanding step comprising (iia) culturing immune cells (e.g., NK cells) in expansion culture media and (iib) culturing immune cells (e.g., NK cells) in a second expansion culture media. In some embodiments, methods described herein comprise, consist essentially of, of consist of (i) a pre-activation step comprising culturing immune cells (e.g., NK cells) in a pre-activation media, (ii) an expanding step comprising (iia) culturing immune cells (e.g., NK cells) in expansion culture media and (iib) culturing immune cells (e.g., NK cells) in a second expansion culture media, and / or (iic) culturing immune cells (e.g., NK cells in a third, fourth, or fifth expansion culture media. In some embodiments, methods described herein comprise, consist essentially of, of consist of an expanding step comprising culturing immune cells (e.g., NK cells) in expansion culture media.1. Pre-activation step

[0514] In some embodiments, methods described herein comprise culturing immune cells (e.g., NK cells) in a pre-activation step. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for less than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or any range or value derivable therein. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for less than or about 12 hours. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for less than or about 16 hours. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for about 12 to 18 hours. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for less than or about 24 hours. In some embodiments, a pre- activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre- activation media for no more than 18, 19, 20, 21, 22, 23, or 24 hours, or any range or value derivable therein. In some embodiments, a pre-activation step as described herein comprises culturing immune cells (e.g., NK cells) in pre-activation media for no more than 12, 13, 14, 15, or 16 hours, or any range or value derivable therein.2. Expanding step

[0515] In some embodiments, methods described herein comprise an expanding step comprising culturing immune cells (e.g., NK cells) in expansion culture media, a second expansion culture media, and / or a subsequent expansion culture media. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or any range or value derivable therein. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for about 1 day. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 2 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 3 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 4 days. In some embodiments, an expansion step as described herein comprisesculturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 5 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 6 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 7 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in median expansion culture media for less than or about 8 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 9 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 10 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 11 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in expansion culture media for less than or about 12 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 13 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 14 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 15 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 16 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 17 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 18 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 19 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 20 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 21 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 22 days. In some embodiments, an expansion step as described herein comprises culturingimmune cells (e.g., NK cells) in an expansion culture media for less than or about 23 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 24 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 25 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 26 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 27 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for less than or about 28 days.

[0516] In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for or for no more than 7-9 days, or 13-14 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for 2-7 days, 3-6 days, or 4-5 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in an expansion culture media for no more than 3 days, no more than 4 days, no more than 5 days, no more than 6 days, no more than 7 days, no more than 8 days, or no more than 9 days.

[0517] In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in a first expansion culture media for or for no more than 7-9 days, or 13-14 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in a first expansion culture media for or for no more than 2-7 days, 3-6 days, or 4-5 days. In some embodiments, an expansion step as described herein comprises culturing immune cells (e.g., NK cells) in a first expansion culture media for no more than 3 days, no more than 4 days, no more than 5 days, no more than 6 days, no more than 7 days, no more than 8 days, or no more than 9 days.3. Second expansion culture media

[0518] In some embodiments, methods described herein comprise an expanding step comprising culturing immune cells (e.g., NK cells) in expansion culture media, a second expansion culture media, and / or a subsequent expansion culture media. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second (or subsequent) expansion culture media for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 days, or any range or value derivable therein. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 1 day. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 2 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 3 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 4 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 5 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 6 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 7 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 8 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion culture media for less than or about 9 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g.,NK cells) in second expansion culture media for less than or about 10 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g.,NK cells) in second expansion culture media for less than or about 11 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g.,NK cells) in second expansion culture media for less than or about 12 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g.,NK cells) in second expansion culture media for less than or about 13 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g.,NK cells) in second expansion culture media for less than or about 14 days.

[0519] In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion media for 2-7 days, 3-6 days, or 4-5 days. In some embodiments, an expanding step as described herein comprises culturing immune cells (e.g., NK cells) in second expansion media for no more than 3 days, 4 days, 5 days, 6 days, or 7 days.F. Expansion culture

[0520] The present disclosure provides, among other things, media (e.g., pre-activation media, expansion culture media, second expansion culture media, etc.) comprising large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., pre-activation media, expansion culture media, second expansion culture media) comprise exogenously provided large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., pre-activation media, expansion culture media, second expansion culture media) comprise exogenous or exogenous and endogenously provided large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., pre-activation media, expansion culture media, second expansion culture media) comprise heterologous large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., pre-activation media, expansion culture media, second expansion culture media) comprise synthetic (i.e., artificial, made by the hand of man) large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., pre-activation media, expansion culture media, second expansion culture media) comprise exogenously provided large molecules (e.g., IL-2, IL- 12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., smallmolecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof. In some embodiments, media as described herein (e.g., preactivation media, expansion culture media, second expansion culture media) comprise recombinant large molecules (e.g., IL-2, IL-12, IL-15, IL-18, TGFb, or any other large molecule described herein), small molecules (e.g., small molecule inhibitors of mTOR, PI3K, IDH1, IDH2, sugars or sugar analogs, or any other small molecule described herein), cells (e.g., aAPCs, uAPCs, APCs, feeder cells), activating beads, and / or combinations thereof.G. Expansion culture cytokines

[0521] In certain embodiments, provided herein are technologies (e.g., methods, compositions, kits, etc.) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10) exogenously provided cytokines and / or growth factors, such as but not limited to, IL-1, IL-2, IL-3, IL-4, IL- 6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or any combination thereof. In some embodiments, technologies provided herein comprise use of IL- 2, IL- 12, IL- 18, and TGFb for expansion of NK cells in one or more culture conditions. In some embodiments, NK cells are pre-activated prior to expansion. In some embodiments, NK cells are preactivated using a combination of IL-12, IL-15, and IL-18, prior to NK cell expansion. In some embodiments, preactivation does not comprise use of exogenous TGFb.

[0522] In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL-2. In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL-2 in at least a first culture condition, and in at least a second culture condition. In certain embodiments, IL-2 is a recombinant human IL-2. In certain embodiments, the recombinant human IL-2 is FDA approved. In certain embodiments, exogenously provided IL-2 is included in a composition or method of the disclosure at a concentration of about 10- 1000 U / mL, such as 100-800 U / mL, or 200 U / mL, or 600 U / mL. Inc certain embodiments, IL- 2 is included in a composition or method of the disclosure at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 U / mL, or any range or value derivable therein. In certain embodiments, exogenouslyprovided IL-2 is included in a composition or method of the disclosure at a concentration of about 200 U / mL. In certain embodiments, exogenously provided IL-2 is included in a composition or method of the disclosure at a concentration of about 600 U / mL.

[0523] In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL- 12. In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL- 12 in at least a first culture condition, and in at least a second culture condition. In certain embodiments, provision of exogenously provided IL- 12 in at least a first culture condition improves NK cell engineering (e.g., transduction and / or transfection, etc.) rates relative to culture conditions that do not comprise exogenous IL- 12. In certain embodiments, IL-12 is a recombinant human IL-12. In certain embodiments, the recombinant human IL- 12 is FDA approved. In certain embodiments, exogenously provided IL- 12 is included in a composition or method of the disclosure at a concentration of about 0.1 to 1000 ng / ml, or about 1 to 100 ng / ml, or about 5 to 15 ng / ml, or about 10 ng / ml. In certain embodiments, exogenously provided IL- 12 is included in a composition or method of the disclosure at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng / ml, or any range or value derivable therein. In certain embodiments, exogenously provided IL- 12 is included in a composition or method of the disclosure at a concentration of about 10 ng / ml.

[0524] In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL- 18. In certain embodiments, expansion of NK cells comprises addition of exogenously provided IL- 18 in at least a first culture condition, and in at least a second culture condition. In certain embodiments, provision of exogenously provided IL- 18 in at least a first culture condition improves NK cell engineering (e.g., transduction and / or transfection, etc.) rates relative to culture conditions that do not comprise exogenous IL- 18. In certain embodiments, IL- 18 is a recombinant human IL- 18. In certain embodiments, the recombinant human IL- 18 is FDA approved. In certain embodiments, exogenously provided IL- 18 is included in a composition or method of the disclosure at a concentration of about 0.1 to 1000 ng / ml, or about 1 to 100 ng / ml, or about 10 to 30 ng / ml, or about 15 to 25 ng / ml or about 20 ng / ml. In certain embodiments, exogenously provided IL- 18 is included in a composition or method of the disclosure at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng / ml, or any range or value derivable therein. In certain embodiments, exogenously provided IL- 18 is included in a composition or method of the disclosure at a concentration of about 20 ng / ml. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of one or more cytokines described herein. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of one or more of TGFb, IL-2, IL-12, IL-15, and / or IL-18. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of IL- 2. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of IL- 12. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of IL- 15. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of IL- 18.

[0525] TGFb is known to alter NK cell function in multi-faceted ways. It has been reported that TGFb can modulate development of NK cells and subsequently negatively affect their function upon reaching maturation. It has been reported that TGFb can promote an immature NK cell lineage, by preventing the progression of NK cells into CD16+ NK cells. It has been reported that TGFb can also induce formerly CD16+ NK cells to become CD16-. Keskin et al., Proc Natl Acad Sci US A, 104: 3378-3383 (2007). In addition, it has been reported that exposure of mature NK cells can inhibit anti-tumor activity through multiple mechanisms. For example, it has been reported that TGFb decreases IL-2 and IL- 15 induced NK cell proliferation (Wilson et al., PloS one 6: e22842 (2011)), and IL- 15 induced mTOR activation. Viel et al., Sci Signal., 16;9(415):ral9 (2016). TGFb has also been reported to inhibit IFNy secretion, which is important for stimulating the adaptive immune system and can sensitive tumors to NK cell lysis. Furthermore, TGFb has also been reported to inhibit TNFa and GM- CSF secretion, and modulates chemokine receptor expression. There have been several approaches towards generating NK and T-cells resistant to TGFb. These include dominant negative TGFBRII expression, knock out of TGFBRII, and / or treatment with TGFb small molecule inhibitors.

[0526] The TGFb superfamily is a large group of structurally related cell regulatory proteins. TGFb is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGFb-1 is a peptide of 112 amino acid residues derived by proteolytic cleavage from the C-terminal of a precursor protein. These proteins interact with a conserved family of cell surface serine / threonine-specific protein kinase receptors, andgenerate intracellular signals using a conserved family of proteins called SMADs. The major subfamilies of the TGFb superfamily include the TGFb subfamily (including the TGFb-1 to 4 isoforms), the decapentaplegic Vg-related (DVR) related proteins (e.g., bone morphogenic protein), growth differentiation factors (e.g., GDF-1 through GDF-15), and the activin and inhibin subfamily.

[0527] In certain embodiments, expansion of NK cells comprises addition of exogenously provided TGFb. In certain embodiments, TGFb comprises, consists essentially of, or consists of TGFbl, TGFb2, and / or TGFb3. In certain embodiments, TGFb comprises, consists essentially of, or consists of TGFbl. In certain embodiments, expansion of NK cells comprises addition of exogenously provided TGFb in at least a first culture condition, and in at least a second culture condition. In certain embodiments, provision of exogenously provided TGFb in at least a first culture condition improves NK cell survivability and / or proliferation rates relative to culture conditions that do not comprise exogenous TGFb. In certain embodiments, TGFb is a recombinant human TGFb. In certain embodiments, the recombinant human TGFb is FDA approved. In certain embodiments, exogenously provided TGFb is included in a composition or method of the disclosure at a concentration of about 0.001 to 1000 ng / ml, or about 0.01 to 100 ng / ml, or about 0.05 to 20 ng / ml, or about 0.1 to 10 ng / ml, or about 0.3 to 5 ng / ml, or about 0.3 to 1 ng / ml, or about 1 ng / ml, or about 0.3 ng / ml. In certain embodiments, exogenously provided TGFb is included in a composition or method of the disclosure at a concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 ng / ml, or any range or value derivable therein. In certain embodiments, exogenously provided TGFb is included in a composition or method of the disclosure at a concentration of about 0.3 ng / ml. In certain embodiments, exogenously provided TGFb is included in a composition or method of the disclosure at a concentration of below about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 ng / ml, or any range or value derivable therein. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of TGFb. In some embodiments, pre-activation and / or culturing of NK cells comprise culturing NK cells in the absence of TGFb. In some embodiments, expansion comprises culturing of NK cells in less than or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 ng / ml exogenously provided TGFb, or any range or value derivable therein.H. Expansion culture small molecules

[0528] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) modulators of NK cell metabolism. In some embodiments, a modulator of NK cell metabolism is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, a modulator of NK cell metabolism is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. In some embodiments, a modulator of NK cell metabolism is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. In some embodiments, a modulator of NK cell metabolism is utilized during the expansion process to improve anti-tumor efficacy and / or NK cell persistence in vivo. In some embodiments, a modulator of NK cell metabolism comprises, consists essentially of, or consists of modulators of mTOR, PI3K, AKT, IDH1 and / or IDH2. In some embodiments, a modulator of NK cell metabolism comprises, consists essentially of, or consists of inhibitors of mTOR, PI3K, AKT, IDH1 and / or IDH2. In some embodiments, a modulator of NK cell metabolism comprises, consists essentially of, or consists of a non-metabolizable or metabolizable sugar. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of one or more small molecules described herein. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of one or more of mTOR, PI3K, AKT, IDH1 and / or IDH2. In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of mTOR (e.g., rapamycin). In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of PI3K (e.g., idelalisib). In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of AKT (e.g., capivasertib). In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of IDH1 (Ivosidenib). In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of IDH2 (Enasidenib). In some embodiments, expansion of NK cells comprise culturing NK cells in the absence of a modulator of IDH1 and IDH2 (Vasandani).1. Inhibitors of mammalian target of rapamycin (mTOR)

[0529] The mammalian target of rapamycin (mTOR) is a serine / threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K) -related kinase (PIKK) family. The kinase exists in the forms of two complexes, mTORCl and mT0RC2, and it participates in cell growth, proliferation, metabolism, and survival. The kinase activity is closely related to the occurrenceand development of multiple human diseases. Inhibitors of mTOR block critical pathways to produce antiviral, anti-inflammatory, antiproliferative and other effects, and they have been applied to research in cancer, inflammation, central nervous system diseases and viral infections. Existing mTOR inhibitors are commonly divided into mTOR allosteric inhibitors, ATP-competitive inhibitors and dual binding site inhibitors, according to their sites of action. In addition, there exist several dual-target mTOR inhibitors that also target PI3K (described below in PI3K sub-header).

[0530] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) inhibitors of mTOR. In some embodiments, a mTOR inhibitor is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, a mTOR inhibitor is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. In some embodiments, a mTOR inhibitor is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. In some embodiments, a mTOR inhibitor is utilized during the expansion process to improve anti-tumor efficacy and / or NK cell persistence in vivo.

[0531] In certain embodiments, one or more mTOR inhibitor comprises, consists essentially of, or consists of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI- 027, OXA-01, XL388, CC214-1, CC-223, CC-115, and / or DHM25. In certain embodiments, a mTOR inhibitor comprises, consists essentially of, or consists more of rapamycin.

[0532] In certain embodiments, one or mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of about 0.001 nM to 1000 nM, or about 0.01 nM to 100 nM, or about 0.1 nM to 20 nM, or about 0.1 nM to 10 nM, or about 0.1 nM to 5 nM, or about 0.1 nM to 3 nM, or about 10 nM, or about 1 nM or any range or value possible in the aforementioned. In certain embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of less than or about 10 nM. In certain embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of less than or equal to about 10 nM. In certain embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of about 1 to 10 nM. In certain embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of lessthan or equal to about 1 nM. In some embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, at a concentration of less than or about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 nM, or any range or value derivable therein.

[0533] In certain embodiments, one or more mTOR inhibitor is present in a composition, or is provided during a method, wherein the composition and / or method is associated with a first and / or a second culturing step during an expansion process. In some embodiments, NK cells are exposed to one or more mTOR inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any range or value derivable therein. In some embodiments, NK cells are exposed to one or more mTOR inhibitor for 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, NK cells are exposed to one or more mTOR inhibitor for 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, NK cells are only exposed to one or more mTOR inhibitor for 4 or 5 days. In some embodiments, NK cells are not exposed to one or more mTOR inhibitor. In some embodiments, NK cells are exposed to one or more mTOR inhibitor during a first culture condition of an expansion process. In some embodiments, NK cells are exposed to one or more mTOR inhibitor during both a first culture condition and a second culture condition of an expansion process.2. Inhibitors of phosphoinositide 3-kinase (PI3K)

[0534] Phosphoinositide-3- kinase (PI3K) signaling regulates cellular proliferation and growth, survival, and metabolism. Activation of PI3K is a frequent hallmark of cancer, highlighted by the prevalence of somatic mutations in genes encoding key components of this pathway. Over the last two decades, the clinical development of PI3K inhibitors has evolved considerably; numerous medicines have now been approved by several regulatory agencies for the treatment of different types of cancer such as leukemia and breast cancer. Recent PI3K inhibitors have shown impressive levels of potency, selectivity, and pharmacological profiles, proving to be relatively safe for administration to patients as monotherapies or combination therapies.

[0535] PI3K is evolutionary conserved across metazoans and, in humans, it has undergone successive gene duplication events giving rise to different isoforms. There are four catalytic subunits encoded in the human genomes: pl 10a and pl 10[3 (encoded by PIK3CA and PIK3CB, respectively), which are ubiquitously expressed; and pl 105 and pl lOy (encoded by PIK3CD and PIK3CG, respectively), which are restricted to immune lineages. PI3K enzymatic activity atalyzes the synthesis of the second messenger phosphatidylinositol (3,4,5)-trisphosphate(PIP3) by phosphorylating phosphatidylinositol 4,5-bisphosphate (PIP2), an abundant lipid found at the plasma membrane. This reaction is mediated by a catalytic subunit, pl 10, which forms a heterodimer with a regulatory subunit, generally p85. Detailed structural studies have been undertaken to reveal the complex network of interactions between catalytic and regulatory subunits required for PI3K activation. The RAS GTPases directly interact with the pl 10 subunit to regulate PI3K activity. PI3K is commonly activated by receptor tyrosine kinase (RTK) stimulation. Some RTKs are particularly efficient at activating this enzyme, including IR, PDGFR, and HER2. Increased concentration of PIP3 at the plasma membrane triggers the recruitment of proteins containing PIP3-binding pleckstrin homology (PH) domains and activation of downstream pathway2. Among the proteins that contain PH domains, PDK1 and AKT kinases are the key canonical downstream effectors of PI3K activation. Activated AKT phosphorylates an array of effector proteins that control fundamental cellular processes, including mTORCl, an important downstream effector complex that regulates cell growth, translation, and metabolic fitness. AKT activates mTORCl by phosphorylating and inhibiting TSC2 and PRAS40, two negative regulators of mTORCl.

[0536] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) inhibitors of PI3K. In some embodiments, a PI3K inhibitor is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, a PI3K inhibitor is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. In some embodiments, a PI3K inhibitor is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. In some embodiments, a PI3K inhibitor is utilized during the expansion process to improve anti-tumor efficacy and / or NK cell persistence in vivo.

[0537] In certain embodiments, one or more PI3K inhibitor comprises, consists essentially of, or consists more of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, and / or umbrasilib. In certain embodiments, a PI3K inhibitor comprises, consists essentially of, or consists more of idelalisib.

[0538] In some embodiments, one or more PI3K and / or mTOR inhibitors is a dual action mTOR and PI3K inhibitor. In some embodiments, one or more PI3K and / or mTOR inhibitor comprises, consists essentially of, or consists of dactolisib (NVP-BEZ235), gedatolisib (PKI- 587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), and / or voxtalisib (XL765).

[0539] In certain embodiments, one or PI3K inhibitor is present in a composition, or is provided during a method, at a concentration of about 0.001 nM to 1000 nM, or about 0.01 nM to 100 nM, or about 0.1 nM to 20 nM, or about 5 nM to 15 nM, or about 7.5 nM to 12. 5 nM, or about 10 nM, or any range or value possible in the aforementioned. In certain embodiments, one or more PI3K inhibitor is present in a composition, or is provided during a method, at a concentration of about 1 to 10 nM. In certain embodiments, one or more PI3K inhibitor is present in a composition, or is provided during a method, at a concentration of less than or equal to about 10 nM. In some embodiments, one or more PI3K inhibitor is present in a composition, or is provided during a method, at a concentration of less than or about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 nM, or any range or value derivable therein.

[0540] In certain embodiments, one or more PI3K inhibitor is present in a composition, or is provided during a method, wherein the composition and / or method is associated with a second culturing step during an expansion process. In some embodiments, NK cells are exposed to one or more PI3K inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, NK cells are exposed to one or more PI3K inhibitor for 3, 4, or 5 days. In some embodiments, NK cells are only exposed to one or more PI3K inhibitor for 4 or 5 days. In some embodiments, NK cells are not exposed to one or more PI3K inhibitor. In some embodiments, NK cells are not exposed to one or more PI3K inhibitor during the first culture condition of an expansion process.3. Inhibitors of isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), and dual IDH1 and IDH2 (IDH1 / 2) inhibitors

[0541] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) inhibitors of isocitrate dehydrogenase 1 (IDH1). In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) inhibitors of isocitrate dehydrogenase 2 (IDH2). In some embodiments, an IDH2 inhibitor is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, an IDH2 inhibitor is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. In some embodiments, an IDH2 inhibitor is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. In some embodiments, an IDH2 inhibitor is utilized during the expansion process to improve anti-tumor efficacy and / or NK cell persistence in vivo.

[0542] In certain embodiments, one or more IDH2 inhibitor comprises, consists essentially of, or consists of Enasidenib and / or Ivosidenib. In certain embodiments, an IDH2 inhibitor comprises, consists essentially of, or consists of Enasidenib. In certain embodiments, one or more IDH1 inhibitor comprises Ivosidenib. In certain embodiments, one or more dual IDH1 and IDH2 inhibitor (i.e., IDH1 / 2 inhibitor) comprises Vorasidenib.

[0543] In certain embodiments, one or more IDH2 inhibitor is present in a composition, or is provided during a method, at a concentration of about 0.001 nM to 1000 pM, or about 0.01 nM to 100 pM, or about 0.1 nM to 10 pM, or about 1-5 pM, or about 2 pM, or any range or value possible in the aforementioned. In certain embodiments, one or more IDH2 inhibitor is present in a composition, or is provided during a method, at a concentration of about 1-10 pM, or about 2 pM. In certain embodiments, one or more IDH2 inhibitor is present in a composition, or is provided during a method, at a concentration of less than or equal to about 10 pM. In some embodiments, one or more IDH2 inhibitor is present in a composition, or is provided during a method, at a concentration of less than or about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 pM, or any range or value derivable therein.

[0544] In certain embodiments, one or more IDH2 inhibitor is present in a composition, or is provided during a method, wherein the composition and / or method is associated with a second culturing step during an expansion process. In some embodiments, NK cells are exposed to one or more IDH2 inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, NK cells are exposed to one or more IDH2 inhibitor for 3, 4, or 5 days. In some embodiments, NK cells are only exposed to one or more IDH2 inhibitor for 4 or 5 days. In some embodiments, NK cells are not exposed to one or more IDH2 inhibitor. In some embodiments, NK cells are not exposed to one or more IDH2 inhibitor during the first culture condition of an expansion process.4. Inhibitors of protein kinase B (PKB, AKT)

[0545] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) inhibitors of protein kinase B (PKB, AKT). In some embodiments, an AKT inhibitor is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, AKT inhibitor is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. In some embodiments, an AKT inhibitor is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. Insome embodiments, an AKT inhibitor is utilized during the expansion process to improve antitumor efficacy and / or NK cell persistence in vivo.

[0546] In certain embodiments, one or more AKT inhibitor comprises, consists essentially of, or consists of capivasertib (AZD-5363), ipatasertib, miltefosine, uprosertib (GSK2141795), miransertib (ARQ 092), afuresertib (GSK2110183), palomid 529 (P529), and / or perifosine (KRX-0401).

[0547] In certain embodiments, one or more AKT inhibitor is present in a composition, or is provided during a method, at a concentration of about 0.001 nM to 1000 nM, or about 0.01 nM to 100 nM, or about 0.1 nM to 10 nM, or about 1-5 nM, or about 2 nM, or any range or value possible in the aforementioned. In certain embodiments, one or more AKT inhibitor is present in a composition, or is provided during a method, at a concentration of about 1 to 10 nM, or about 2 nM. In certain embodiments, one or more AKT inhibitor is present in a composition, or is provided during a method, at a concentration of less than or equal to about 10 nM. In some embodiments, one or more AKT inhibitor is present in a composition, or is provided during a method, at a concentration of less than or about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 nM, or any range or value derivable therein.

[0548] In certain embodiments, one or more AKT inhibitor is present in a composition, or is provided during a method, wherein the composition and / or method is associated with a second culturing step during an expansion process. In some embodiments, NK cells are exposed to one or more AKT inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or any range or value derivable therein. In some embodiments, NK cells are exposed to one or more AKT inhibitor for 3, 4, or 5 days. In some embodiments, NK cells are only exposed to one or more AKT inhibitor for 4 or 5 days. In some embodiments, NK cells are not exposed to one or more AKT inhibitor. In some embodiments, NK cells are not exposed to one or more AKT inhibitor during the first culture condition of an expansion process.5. Metabolizable or non-metabolizable sugars

[0549] In some embodiments, technologies (e.g., methods, compositions, kits, etc.) provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 7, or more, etc.) metabolizable and / or non- metabolizable sugars. In some embodiments, a metabolizable and / or non-metabolizable sugar is utilized to improve metabolic fitness of NK cells during the expansion process and / or once prepared. In some embodiments, a metabolizable and / or non-metabolizable sugar is utilized to increase levels of oxidative phosphorylation and / or reduce levels of glycolysis in NK cells. Insome embodiments, metabolizable and / or non-metabolizable sugar is utilized during the expansion process to reduce the occurrence / rate of exhaustion of prepared NK cells in vivo. In some embodiments, metabolizable and / or non-metabolizable sugar is utilized during the expansion process to improve anti-tumor efficacy and / or NK cell persistence in vivo.

[0550] In certain embodiments, one or more metabolizable and / or non-metabolizable sugar comprises, consists essentially of, or consists of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D- Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), 6-Deoxy-D-Glucose (6DG), sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum lycopersici (E-FOL). In certain embodiments, one or more non-metabolizable sugar comprises, consists essentially of, or consists of a non-metabolizable glucose analog. In certain embodiments, one or more non- metabolizable glucose analog comprises, consists essentially of, or consists of 2-Deoxy-d- Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O-Methyl-D-Glucose (20MG), and / or 6- Deoxy-D-Glucose (6DG). In certain embodiments, one or more non-metabolizable glucose analogs comprises, consists essentially of, or consists of 2-Deoxy-d-Glucose (2-DG). In certain embodiments, one or more metabolizable sugar comprises, consists essentially of, or consists of sucrose, palatinose, turanose, and / or elicitor preparation of F. oxysporum lycopersici (E- FOL).

[0551] In certain embodiments, one or more metabolizable and / or non-metabolizable sugar is present in a composition, or is provided during a method, at a concentration of about 0.001 mM to 1000 mM, or about 0.01 mM to 100 mM, or about 0.1 mM to 10 mM, or about 1-5 mM, or about 2 mM, or any range or value possible in the aforementioned. In certain embodiments, one or more metabolizable and / or non-metabolizable sugar is present in a composition, or is provided during a method, at a concentration of less than or about 1 to 10 nM, or about 2 nM. In certain embodiments, one or more metabolizable and / or non-metabolizable sugar is present in a composition, or is provided during a method, at a concentration of less than or equal to about 10 nM. In some embodiments, one or more metabolizable and / or non-metabolizable sugar is present in a composition, or is provided during a method, at a concentration of less than or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 mM, or any range or value derivable therein.

[0552] In certain embodiments, one or more metabolizable and / or non-metabolizable sugar is present in a composition, or is provided during a method, wherein the composition and / or method is associated with a second culturing step during an expansion process. In some embodiments, NK cells are exposed to one or more metabolizable and / or non-metabolizablesugar for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, NK cells are exposed to one or more metabolizable and / or non-metabolizable sugar for 3, 4, or 5 days. In some embodiments, NK cells are not exposed to one or more metabolizable and / or non- metabolizable sugars. In some embodiments, NK cells are not exposed to one or more metabolizable and / or non-metabolizable sugars during the first culture condition of an expansion process.I. Cryopreservation

[0553] In certain embodiments, cells are cryopreserved as described in the inventors publication WO 2023 / 220632 Al (PCT / US2023 / 066825) filed May 10, 2023, and published November 16, 2023, which is incorporated herein by reference in its entirety.

[0554] In particular cases, prepared NK cells of the disclosure are preserved in a cryopreservation medium composition comprising at least one cryoprotectant, a serum (human or animal serum) or a non- serum alternative to serum (not human serum or animal serum), and optionally one or more cytokine and / or growth factor. In some cases, the cryoprotectant is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, or a combination thereof. The non-serum alternative may be of any kind, including at least platelet lysate and / or a blood product lysate (for example, human serum albumin). In embodiments of the composition wherein one or more (including two or more) cytokines are utilized, the cytokine may be a natural or a recombinant or a synthetic protein. At least one of the cytokines may be an Food and Drug Administration (FDA)-approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL- 18, IL-21, IL-22, interferon, tumor necrosis factor, stem cell factor, ELT3-ligand, APRIL, thrombopoietin, erythropoietin, or a combination thereof. Lor serum embodiments, the serum may be an animal-derived serum, such as human serum (including human AB serum) or bovine serum. DMSO and other cryoprotectants, when utilized may comprise 4-10%, 4-6%, 4-8%, 5- 10%, 5-8%, 6-10%, 6-8%, 8-10%, and so forth, of the composition. Lor embodiments wherein serum is employed, the serum may comprise 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5- 70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5- 10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%. 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 30-99%, 30-95%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99% of the composition. The composition may comprise at least or no more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of serum. In specific embodiments, the composition comprises platelet lysate that may be at any concentration in the composition, but in certain embodiments the platelet lysate comprises 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5- 50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10- 90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, ...

Claims

WHAT IS CLAIMED IS:

1. An in vitro method comprising: an expanding step comprising: a culturing step comprising culturing NK cells for about 1-14 days in an expansion culture media comprising exogenous:(i) IL-2, IL- 12, IL- 18, and TGFb; and(ii) antigen presenting cells (APCs), artificial antigen presenting cells (aAPCs), universal antigen presenting cells (uAPCs), activating beads, and / or feeder cells.

2. The method of claim 1, wherein the expanding step comprises a second culturing step comprising: culturing the NK cells for 1-14 days in a second expansion culture media comprising:(i) exogenous IL-2, IL-12, IL-18, and TGFb, and(ii) one or more mammalian target of rapamycin (mTOR) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, dual mTOR and PI3K inhibitor, isocitrate dehydrogenase 2 (IDH2) inhibitor, isocitrate dehydrogenase 1 (IDH2) inhibitor, dual IDH1 and IDH2 (IDH1 / 2) inhibitor, protein kinase B (PKB, AKT) inhibitor, additional cytokines, non-metabolizable glucose analogs, metabolizable sugars, APCs, aAPCs, uAPCs, activating beads, or a combination thereof.

3. The method of claim 1 or 2, further comprising a pre-activation step comprising culturing the NK cells in pre-activation media comprising IL-12, IL-15, and IL-18 prior to the expanding step.

4. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprises an mTOR inhibitor.

5. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprise exogenous IL-2 at a concentration of about 100 to 300 U / mL, IL- 12 at a concentration of about 1-100 ng / mL, IL- 18 at a concentration of about 1-200 ng / mL, and TGFb at a concentration of about 0.01 to 1 ng / mL.

6. The method of claim 1 or 2, wherein the expanding step comprises culturing NK cells for 1-9 days.

7. The method of claim 2, wherein the expanding step comprises culturing NK cells for about 1-4 days in the expansion culture media.

8. The method of claim 7, wherein the second culturing step comprises culturing NK cells for about 4 days in the expansion culture media.

9. The method of claim 2, wherein the second culturing step comprises culturing NK cells for 1-9 days in the second expansion culture media.

10. The method of claim 2, wherein the second culturing step comprises culturing NK cells for about 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 4 days, or 3 days in the second expansion culture media.

11. The method of claim 2, wherein the expanding step comprises culturing NK cells for or for less than 1, 2, 3, 4, 5, 6, or 7 days in the expansion culture media, and comprises culturing NK cells for or for less than 1, 2, 3, 4, 5, 6, or 7 days in the second expansion culture media.

12. The method of claim 11, wherein the expanding step comprises culturing NK cells for 3-6 days in the expansion culture media.

13. The method of claim 12, wherein the expanding step comprises or consists of culturing the NK cells for 3-5 or 4 days in the expansion culture media and culturing the NK cells for 2- 5 or 3 days in the second expansion culture media14. The method of claim 3, wherein the pre-activation step comprises culturing the NK cells for 12-24 hours, and the expanding step comprises culturing the NK cells for 3-5 or 4 days in the expansion culture media, and the second culturing step comprises culturing the NK cells for 2-5 or 3 days in the second expansion culture media.

15. The method of claim 2, wherein APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are provided in the second culturing step.

16. The method of claim 2, wherein no APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are provided in the second culturing step.

17. The method of claim 2, further comprising removing the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells from the expansion culture media before the second culturing step.

18. The method of claim 2, wherein the first culturing step and second culturing step are separated by an NK cell enrichment step.

19. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are removed from the expansion culture media or second expansion culture media by about 5, 6, 7, 8, or 9 days from the beginning of the expanding step.

20. The method of claim 19, wherein the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells are removed from the expansion culture media by about 5, 6 or 7 days from the beginning of the expanding step.

21. The method of claim 2, wherein the second culturing step comprises culturing the NK cells in the absence of APCs, aAPCs, uAPCs, activating beads, and / or feeder cells.

22. The method of claim 14, further comprising an enrichment step comprising removing the APCs, aAPCs, uAPCs, activating beads, and / or feeder cells from the expansion culture media immediately before the second culturing step.

23. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprises: an exogenous IL-2 concentration equal to or about 100 to 600 U / mL, IL- 12 concentration equal to or about 1-100 ng / mL, and IL- 18 concentration equal to or about 1-200 ng / mL.

24. The method of claim 23, wherein the expansion culture media and / or second expansion culture media comprises an exogenous TGFb concentration of less than 10 ng / mL, less than or about 5 ng / mL, or about 0.01 to 1 ng / mL, or about 0.1 to 0.5 ng / mL, or about 0.3 ng / mL.

25. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprises: an exogenous TGFb concentration of less than 10 ng / mL, less than or about 5 ng / mL, or about 0.01 to 1 ng / mL, or about 0.1 to 0.5 ng / mL, or about 0.3 ng / mL.

26. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprises a TGFb concentration of less than about 1 ng / mL or about 0.3 ng / mL.

27. The method of claim 4, wherein the expansion culture media and / or second expansion culture media comprises an mTOR inhibitor selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, WAY-001, WAY-600, WYE-687, WYE- 354, GDC-0349, GNE-555, PF-05139962, Ku-0063794, AZD8055, AZD2014, 4- morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4- hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSL027, OXA-01, XL388, CC214-1, CC-223, CC-115, DHM25, or combinations thereof.

28. The method of claim 27, wherein the expansion culture media comprises rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM.

29. The method of claim 28, wherein the first culturing step comprises rapamycin at a concentration of less than or equal to 10 nM, particularly about 1 nM.

30. The method of claim 26, wherein the expansion culture media and / or second expansion culture media comprises the mTOR inhibitor rapamycin at a concentration of about 0.1 nM to 200 nM particularly about 0.5 nM, 1 nM, or 10 nM 1 nM.

31. The method of claim 30, wherein the expansion culture media and the second expansion culture media comprise rapamycin.

32. The method of claim 17, wherein the method includes transforming and / or transducing the cells with an engineered construct.

33. The method of claim 32, wherein the transforming comprises transient introduction of one or more ribonucleoproteins, polynucleotides, and / or polypeptides.

34. The method of claim 32, wherein the transducing comprises stable transgene introduction into the genome of the NK cell.

35. The method of claim 32, wherein the transducing comprises contacting the NK cells with one or more RetroNectin bound retroviral constructs.

36. The method of claim 2, wherein the PI3K inhibitor is selected from the group consisting of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, umbrasilib, or combinations thereof.

37. The method of claim 36, wherein the PI3K inhibitor is Idelalisib.

38. The method of claim 37, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM, or about 10 nM, or about 0.1 nM to 10 pM.

39. The method of claim 37, wherein the second expansion culture media comprises Idelalisib at about 10 nM or at about 1 pM concentration.

40. The method of claim 2, wherein the dual mTOR and PI3K inhibitor is selected from the group consisting of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), voxtalisib (XL765), or combinations thereof41. The method of claim 40, wherein the dual mTOR and PI3K inhibitor is at a concentration of about 0.1 nM to 100 nM, or about 10 nM.

42. The method of claim 2, wherein the AKT inhibitor is selected from the group consisting of capivasertib (AZD-5363), ipatasertib, miltefosine, uprosertib (GSK2141795), miransertib(ARQ 092), afuresertib (GSK2110183), palomid 529 (P529), perifosine (KRX-0401), or combinations thereof.

43. The method of claim 42, wherein the second expansion culture media comprises the AKT inhibitor capivasertib.

44. The method of claim 2, wherein the non-metabolizable glucose analog is selected from the group consisting of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-0- Methyl-D-Glucose (20MG), 6-Deoxy-D-Glucose (6DG), or combinations thereof.

45. The method of claim 44, wherein the non-metabolizable glucose analog comprises consists of 2-DG at a concentration of about 0.1 mM to 20 mM or about 2 mM.

46. The method of claim 45, wherein the second expansion culture media comprises the non-metabolizable glucose analog 2-DG at a concentration of about 2 mM.

47. The method of claim 2, wherein the metabolizable sugar is selected from the group consisting of sucrose, palatinose, turanose, elicitor preparation of E oxysporum lycopersici ETOL), or combinations thereof.

48. The method of claim 47, wherein the second expansion culture media comprises the metabolizable sugar sucrose.

49. The method of claim 2, wherein the IDH2 inhibitor is selected from the group consisting of Enasidenib, Ivosidenib, or combinations thereof.

50. The method of claim 49, wherein the concentration of IDH2 inhibitor is about 1 nM to 100 pM, or 100 nM to 10 pM, or about 1 pM.

51. The method of claim 1 or 2, wherein the expansion culture media and / or second expansion culture media comprises about 200, 100, 150, 175, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 U / mL, of IL-2.

52. The method of claim 51, wherein the expansion culture media and second expansion culture media comprises about 200, 100, 150, 175, 225, 250, 275, 300, 325, 350, 375, or 400 U / mL, of IL-2.

53. The method of claim 32, wherein the transforming and / or transducing takes place in the presence of about 200 U / mL or 600 U / mL of IL-2.

54. The method of claim 53, wherein the transforming and / or transducing takes place in the presence of IL-2, IL-12, IL-18, and TGFb.

55. The method of claim 54, wherein the transforming and / or transducing takes place in the presence of IL-2, IL-12, IL-18, TGFb, and rapamycin.

56. The method of claim 55, wherein the transforming and / or transducing takes place in the presence of IL-2, IL-12, IL-18, TGFb, rapamycin, and idelalisib.

57. The method of claim 56, wherein the transforming and / or transducing takes place in the presence of IL-2, IL-12, IL-18, TGFb, rapamycin, and 2-DG.

58. The method of claim 56, wherein the transforming and / or transducing takes place in the presence of IL-2, IL-12, IL-18, TGFb, rapamycin, and Enasidenib.

59. The method of claim 1 or 2, further comprising a deactivating step comprising exposing the NK cells to an effective amount of one or more deactivating agents after the expanding step.

60. The method of claim 59, wherein the deactivating agent is Dasatinib.

61. The method of claim 60, wherein the Dasatinib concentration is about 1 pM.

62. The method of claim 59, wherein the deactivating step further comprises exposing theNK cells to IL-2.

63. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, and TGFb.

64. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, and rapamycin.

65. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and idelalisib.

66. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and 2-DG.

67. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to IL-2, IL- 12, IL- 18, TGFb, rapamycin, and Enasidenib.

68. The method of claim 59, wherein the deactivating step further comprises exposing the NK cells to one or more mTOR inhibitors, PI3K inhibitors, metabolizable sugars, and / or non- metabolizable glucose analogs.

69. The method of claim 59, wherein the deactivating step occurs for about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

70. The method of claim 69, wherein the deactivating step occurs for about 24 hours.

71. The method of claim 59, further comprising cryopreserving the prepared NK cells.

72. The method of claim 71, further comprising suspending the NK cells in a pharmaceutically acceptable media.

73. The method of claim 71, further comprising suspending the NK cells in PlasmaLyte-A containing 0.5% HSA.

74. The method of claim 71, further comprising thawing the cryopreserved NK cells prior to administering the NK cells to an individual in need thereof.

75. The method of claim 32, wherein the engineered construct comprises a nucleic acid sequence encoding an engineered antigen receptor.

76. The method of claim 75, wherein the engineered antigen receptor is a chimeric antigen receptor, a T cell receptor, or both.

77. The method of claim 75, wherein the engineered antigen receptor targets one or more antigenic peptide derived from and / or antigen selected from the group consisting of: CD 19, CD5, CD20, CD30, CD70, TROP2, PRAME, KRAS, IL13, EGVRv3, BCMA, GPRC5D, HER2, c-MET, NYESO1, HPV, CMV, or combinations thereof.

78. The method of claim 32, wherein the transfection and / or transduction step comprises introduction of an engineered mutation in one or more of endogenous NK cell genes.

79. The method of claim 78, wherein the engineered mutation is in one or more of GR, TGFBR2, CISH, and / or CD38 genes.

80. The method of claim 32, wherein the engineered construct comprises a nucleic acid sequence encoding one or more cytokines.

81. The method of claim 80, wherein the nucleic acid sequence encodes IL- 15, IL-21, or both IL- 15 and IL-21.

82. The method of claim 80, wherein the nucleic acid sequence encodes one or more autonomous secretory signal.

83. The method of claim 1 or 2, wherein the NK cells are obtained from cord blood (CB), peripheral blood (PB), stem cells, or bone marrow.

84. The method of claim 83, wherein the NK cells is obtained from CB.

85. The method of claim 84, wherein the CB is pooled from 2, 3, 4, 5, 6, 7, or 8 or more individual cord blood units.

86. The method of claim 1 or 2, wherein the NK cells are CD56+NK cells, CD16+ NK cells, or CD56+CD16+ NK cells.

87. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells express a membrane -bound cytokine.

88. The method of claim 87, wherein the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15).

89. The method of 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells have essentially no expression of endogenous HLA class I, II, or CD Id molecules.

90. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells express ICAM-1 (CD54) and LFA-3 (CD58).

91. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells are derived from leukemia cells.

92. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells are engineered to express CD137 ligand, CD48, CS1, and / or mIL-21.

93. The method of claim 1 or 2, wherein the APCs, aAPCs, uAPCs, and / or feeder cells have been engineered by retroviral transduction, are irradiated, or both.

94. The method of claim 3, wherein the pre-activation step is for about 10-20 hours.

95. The method of claim 3, wherein the pre-activation step is for 14-18 hours.

96. The method of claim 3, wherein the pre-activation step is for 16-24 hours.

97. The method of claim 3, wherein the pre-activation media comprises IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, 40-60 ng / mL, or 50 ng / mL.

98. The method of claim 3, wherein the pre-activation media comprises IL- 12 at a concentration of 0.1-150 ng / mL, 1-20 ng / mL, or 10 ng / mL.

99. The method of claim 1 or 2, wherein the NK cells and APCs, aAPCs, uAPCs, and / or feeder cells are present in the expansion culture media and / or second expansion culture media at a ratio of 3:1 to 1:3, or 1:2.

100. The method of claim 1 or 2, wherein the IL-2 in the expansion culture media and / or second expansion culture media is present at a concentration of 10-1000 U / mL, 100-300 U / mL, or 200 U / mL.

101. NK cells produced according to the method of claim 1 or 2.

102. NK cells produced according to the method of any one of claims 1-100.

103. The NK cells of claim 101, wherein the NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared utilizing other methods.

104. The NK cells of claim 101, wherein the NK cells have increased in vitro and / or in vivo cytotoxicity, tumor control, engraftment, and / or persistence relative to NK cells that are expanded and / or prepared in the absence of exogenous TGFb.

105. The NK cells of claim 101, wherein the NK cells have a shifted chemokine profile compared to NK cells prepared utilizing other methods.

106. The NK cells of claim 101, wherein the NK cells exhibit increased expression levels of CD62L, CXCR4, and / or CCR7 relative to NK cells prepared utilizing other methods.

107. The NK cells of claim 101, wherein at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the NK cells are positive for CD62L.

108. The NK cells of claim 101, wherein at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, of the NK cells are positive for CXCR4.

109. The NK cells of claim 101, wherein at least about 20%, 25%, 30%, 35%, 40%, or 45% of the NK cells are positive for CCR7.

110. The NK cells of claim 101, wherein the NK cells exhibit an increased level of transduction and / or transformation relative to NK cells prepared utilizing other methods.

111. The NK cells of claim 101, wherein the NK cells exhibit a transduction efficiency level of at least 50%, 55%, or 60%.

112. The NK cells of claim 101, wherein the NK cells exhibit a transfection efficiency level of at least 80%, 85%, 90%, or 95%.

113. The NK cells of claim 101, wherein the NK cells have a phenotype as described in FIGs. 21B, 39A, 39B, and / or 39C.

114. The NK cells of claim 101, wherein the NK cells have a reduced baseline SMAD2 / 3 phosphorylation level relative to NK cells prepared using concentrations of TGFb greater than or equal to 5, 6, 7, 8, 9, or 10 ng / ml.

115. The NK cells of claim 101, wherein the NK cells are characterized by higher expression of TRAIL, DNAM-1, CD25, CLA, and / or CD 16 relative to NK cells prepared using other methods.

116. The NK cells of claim 101, wherein the NK cells can be characterized by higher expression of DNAM and TRAIL, similar or lower levels of SMAD2 / 3, reduced TIGIT expression, comparable CD38 levels, and / or decreased perforin and granzyme B, relative to NK cells prepared using other methods.

117. The NK cells of claim 101, wherein the NK cells can be characterized by altered SMAD3, CD38, perforin, granzyme, and FasL mRNA / protein levels, and increased TRAIL mRNA / protein levels relative to NK cells prepared using other methods.

118. A composition comprising the NK cells of claim 101 and one or more antibodies.

119. The composition of claim 118, wherein the one or more antibodies are complexed to the NK cells.

120. A pharmaceutical composition comprising NK cells according prepared according to the method of claim 1 or 2, and a pharmaceutically acceptable carrier.

121. The pharmaceutical composition of claim 120 for use in the treatment of a disease or disorder in a subject.

122. The pharmaceutical composition of claim 121, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

123. The pharmaceutical composition of claim 122, wherein the cancer is a leukemia.

124. The pharmaceutical composition of claim 123, wherein the leukemia is an acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), or a chronic myelogenous leukemia (CML).

125. The pharmaceutical composition of claim 122, wherein the cancer is a solid tumor.

126. The pharmaceutical composition of claim 122, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

127. The pharmaceutical composition of claim 122, wherein the disorder is graft versus host disease (GVHD).

128. The pharmaceutical composition of claim 122, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

129. A method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of the prepared NK cells of claim 101 to the subject.

130. The method of claim 129, wherein the disease or disorder is cancer, inflammation, graft versus host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B cell malignancy, or an infection.

131. The method of claim 130, wherein the cancer is a leukemia.

132. The method of claim 130, wherein the cancer is a solid tumor.

133. The method of claim 130, wherein the cancer is glioblastoma, melanoma, gastrointestinal stromal tumor, pancreatic cancer, and / or ovarian cancer.

134. The method of claim 130, wherein the disorder is graft versus host disease (GVHD).

135. The method of claim 130, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren’s syndrome.

136. An in vitro composition for expansion of NK cells comprising, IL-2, IL-12, IL-18, and TGFb.

137. The composition of claim 136, further comprising one or more mTOR inhibitor, PI3K inhibitor, dual action mTOR and PI3K inhibitor, non-metabolizable glucose analogs, metabolizable sugars, IDH1 inhibitors, IDH2 inhibitors, IDH1 / 2 inhibitors, APCs, aAPCs, uAPCs, activating beads, and / or feeder cells, or a combination thereof.

138. The composition of claim 136, comprising a concentration of IL-2 of or of about 100 to 300 U / mL, a concentration of IL- 12 of or of about 1-100 ng / mL, a concentration of IL- 18 of or about 1-200 ng / mL, and the concentration of TGFb of or of about 0.01 to 5 ng / mL or 0.1 to 1 ng / mL.

139. The composition of claim 138, wherein the TGFb is TGFbl.

140. The composition of claim 137 or 138, wherein the composition comprises an mTOR inhibitor, and the mTOR inhibitor is selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, non-rapalog allosteric inhibitor R1-R5,WAY-001, WAY-600, WYE-687, WYE-354, GDC-0349, GNE-555, PF-05139962, Ku- 0063794, AZD8055, AZD2014, 4-morpholinopyrido [2,3-d]pyrimidine, 4-(N-phenyl-N'- substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines, Torinl, Torin2, PP242, MLN0128, OSI-027, OXA-Ol, XL388, CC214-1, CC-223, CC-115, DHM25, or combinations thereof.

141. The composition of claim 140, wherein the mTOR inhibitor is rapamycin.

142. The composition of claim 141, wherein the rapamycin is at a concentration of about 0.1 nM to 100 nM.

143. The composition of claim 137 or 138, wherein the composition comprises a PI3K inhibitor, and the PI3K inhibitor is selected from the group consisting of idelalisib, wortmannin, LY294002, buparlisib, copanlisib, alpelisib, taselisib, GDC-0077, duvelisib, inavolisib, umbrasilib, or combinations thereof.

144. The composition of claim 143, wherein the PI3K inhibitor is Idelalisib.

145. The composition of claim 144, wherein the Idelalisib is at a concentration of about 0.1 nM to 100 nM, or about 0.1 nM to 10 pM.

146. The composition of claim 137 or 138, wherein the composition comprises a dual action mTOR and PI3K inhibitor, and the dual action mTOR and PI3K inhibitor is selected from the group consisting of dactolisib (NVP-BEZ235), gedatolisib (PKI-587), omipalisib (GSK2126458), apitolisib (GDC-0980), bimiralisib (PQR309), voxtalisib (XL765), or combinations thereof.

147. The composition of claim 137 or 138, wherein the composition comprises a non- metabolizable glucose analog, and the non-metabolizable glucose analog is selected from the group consisting of 2-Deoxy-d-Glucose (2-DG), 3-O-Methyl-D-Glucose (30MG), 2-O- Methyl-D-Glucose (20MG), 6-Deoxy-D-Glucose (6DG), or combinations thereof.

148. The composition of claim 147, wherein the non-metabolizable glucose analog is 2-DG at a concentration of about 0.1 mM to 20 mM.

149. The composition of claim 137 or 138, wherein the composition comprises one or more metabolizable sugar, and the metabolizable sugar is selected from the group consisting of sucrose, palatinose, turanose, elicitor preparation of E oxysporum lycopersici (E-FOL), or combinations thereof.

150. The composition of claim 137 or 138, wherein the isocitrate dehydrogenase 2 (IDH2) inhibitor is Enasidenib and / or Ivosidenib.

151. The composition of claim 150, wherein the concentration of IDH2 inhibitor is about 1 nM to 100 pM, or about 100 nM to 10 pM, or about 1 pM.

152. The composition of claim 137 or 138, further comprising NK cells.

153. The composition of claim 152, wherein the NK cells are NK cells pre-activated by exposure to IL12, IL15, and IL18.

154. The composition of claim 152, further comprising one or more retroviral particles and / or polynucleotide constructs.

155. The composition of claim 152, further comprising one or more deactivating agents.

156. The composition claim 152, further comprising APCs, aAPCs, uAPCs, or feeder cells, optionally wherein the APCs, aAPCs, uAPCs, or feeder cells have been stably engineered to transgenically express CD137 ligand, CS1, CD48, and / or mIL21; optionally wherein the APCs, aAPCs, uAPCs, or feeder cells have been irradiated.

157. An in vitro method for activating and expanding NK cells comprising:(i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL- 12, IL- 15, and IL- 18; and(ii) an expansion step comprising a) a first culturing step of the NK cells in a first expansion culture media, wherein the first culturing is for about 2-8 days,wherein the first expansion culture media comprises:IL-2, IL- 12, IL- 18, TGFb; andAPCs, aAPCs, uAPCs, feeder cells, or activating beads; and b) a second culturing step of the NK cells in a second expansion culture media, wherein the second culturing is for about 2-8 days, wherein the second expansion media comprises:IL-2, IL- 12, IL- 18, TGFb; and wherein the second expansion media may further comprise one or more of: APCs, aAPCs, uAPCs, feeder cells or activating beads, an mTOR inhibitor, a PI3K inhibitor, a dual mTOR and PI3K inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a dual IDH1 and IDH2 inhibitor, an additional cytokine, a non-metabolizable glucose analog, a metabolizable sugar, or a combination thereof.

158. The method of claim 157, wherein the first and / or second expansion culture media comprises a TGFb concentration of about 0.3-10 ng / ml.

159. The method of claim 157, wherein the first and / or second expansion culture media comprises a TGFb concentration of about 0.3-5.0 ng / ml.

160. The method of claim 157, wherein the first and / or second expansion culture media comprises an mTOR inhibitor.

161. The method of claim 158, wherein the first and / or second expansion culture media comprises the mTOR inhibitor rapamycin.

162. The method of claim 157, wherein the second expansion culture media comprises the PI3K inhibitor Idelalisib.

163. The method of claim 157 or 160, wherein the first expansion culture media and / or the second expansion culture media comprises an exogenous IL-2 concentration of about 100-600 lU / ml, an IL- 12 concentration of about 5-25 ng / ml, an IL- 18 concentration of about 5-40 ng / ml, and a TGFb concentration of about 0.3-5.0 ng / ml.

164. An in vitro method comprising:(i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step is or comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL-18,(ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-8 days, and wherein the first expansion media comprises(a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin; and(b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-3 days, and wherein the second expansion culture media comprises:IL-2, IL- 12, IL- 18, TGFb, and rapamycin, and(v) optionally deactivating and cryopreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

165. An in vitro method comprising:(i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step is or comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL-18,(ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-8 days, and wherein the first expansion media comprises(a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin;(b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-3 days, and wherein the second expansion culture media comprises:IL-2, IL- 12, IL- 18, TGFb, rapamycin and Idelalisib, and(v) optionally deactivating and cryopreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

166. An in vitro method comprising:(i) a pre-activation step comprising culturing NK cells in pre-activation media, wherein the pre-activation step comprises about 12 to about 24 hours, and wherein the pre-activation media comprises IL-12, IL-15, and IL-18,(ii) an expansion step comprising culturing the NK cells in a first expansion culture media, wherein the culturing in a first expansion culture media is 5-13 days, and wherein the first expansion media comprises(a) IL-2, IL- 12, IL- 18, TGFb, and rapamycin;(b) APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iii) optionally transducing or electroporating the NK cells on about day 5 of the first culturing step in the absence of APCs, aAPCs, uAPCs, feeder cells, or activating beads; and(iv) a second culturing step comprising culturing the NK cells in a second expansion culture media, wherein the culturing in a second expansion culture media is 1-5 days, and wherein the second expansion culture media comprises:IL-2, IL- 12, IL- 18, TGFb, and rapamycin, and(v) optionally deactivating and cryopreserving the NK cells, wherein deactivating comprises contacting the NK cells with Dasatinib.

167. The method of claim 166, wherein the second expansion culture media comprises Idelalisib.

168. The method of any one of claims 164-167, wherein the pre-activation media comprises about 10 ng / ml IL-12, about 50 ng / ml IL15, and about 50 ng / ml IL- 18; wherein the first expansion media comprises about 200 lU / ml of IL-2, about 10 ng / ml IL- 12, about 20 ng. ml IL- 18, about 0.3 ng / ml TGFb, about 1 nM rapamycin, and uAPCs; wherein the second expansion culture media comprises about 200 lU / ml of IL-2, 10 ng / ml IL- 12, about 20 ng / ml IL- 18, about 0.3 ng / ml TGFb, and about 1 nM rapamycin; and / or wherein the Dasatinib concentration is about 1 pM.

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