Methods and materials for expanding NK cells in vitro using feeder cell-free systems

Abstract

Methods and materials for expanding natural killer (NK) cells in vitro, without the use of feeder cells, are provided herein. For example, provides methods and materials for culturing NK cells in vitro in the presence of factors (e.g., IL-2, an activator of protein kinase C, and an activator of nuclear factor of activated T-cells) that can promote NK cell expansion without the use of feeder cells.

Description

[0001] METHODS AND MATERIALS FOR EXPANDING NK CELLS IN VITRO USING FEEDER CELL-FREE SYSTEMS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application Serial No. 63 / 678,817, filed August 2, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under CBET-1845366 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials for expanding natural killer (NK) cells in vitro, without the use of feeder cells. For example, this document provides methods and materials for culturing NK cells in vitro in the presence of factors (e.g., IL-2, an activator of protein kinase C and an activator of nuclear factor of activated T-cells) that can promote NK cell expansion in the absence of feeder cells.

[0008] BACKGROUND

[0009] Natural killer (NK) cells are cytotoxic lymphocytes in the innate immune system, making up 5-20% of all circulating lymphocytes in humans (Arachchige et al., Innate Immunity, 27(3):212-229, 2021), and are important for host immune surveillance against tumor cells and pathogen-infected cells. NK cells mediate anti-viral and anti-cancer immunity through cytokine and chemokine secretion, and through the release of cytotoxic granules (see, e.g., Vivier et al., Science, 331(6013):44-49, 2011; Caligiuri, Blood, 112(3):461-469, 2008; and Roda et al., Cancer Res., 66(1):517-526, 2006). Upon recognizing transformed, infected, or stressed target cells, NK cells establish immunological synapses on contact points with the target cell in the absence of antibodies and major histocompatibility complex (MHC), and execute their rapid cytotoxic response by releasing cytolytic granules and inflammatory cytokines.

[0010] NK cells have emerged as a cell type that can be used in cell-based immunotherapies, as they present a low risk of inducing cytokine release syndrome, neurotoxicity, and graft- versus-host disease. For clinical applications, however, NK cells need to be expanded tens of thousands-fold in population in order to meet the demand and to reduce therapy cost by economy of scale. Traditionally, NK cells are activated and switched to a proliferative state by exposing them to feeder cells derived from various cancers. The process can be costly, cumbersome, and inefficient in scaling up.

[0011] SUMMARY

[0012] NK cell expansion involves several processes, including proliferation, activation, and differentiation. This complex process is influenced by cytokines (e.g., interleukin-2 (IL-2), interleukin- 15 (IL-15), and interleukin- 12 (IL-12)), interactions with other immune cells, and environmental signals. In vivo and in vitro expansion strategies can harness these factors to enhance NK cell numbers and functionality for immune responses and therapeutic applications. Traditionally, expansion protocols have used feeder cells, which generally include irradiated peripheral blood mononuclear cells or cancer cell lines (e.g., K562 cells genetically modified to express one or more cytokines such as IL-21 and 4-1BBL). While feeder cells are effective for the ex vivo expansion of NK cells, there are drawbacks. For example, the use of K562 variant cell lines carries the risk of injecting tumor-derived cells or their genetic material into patients. Additionally, co-culture systems are inherently complex and variable, leading to inconsistency in the culture process. The use of feeder cells in NK cell manufacturing also requires cell line development, expansion, banking, quality control, and large-scale irradiation, significantly increasing manufacturing costs. The methods provided herein avoid these issues, and unlike other attempts at feeder-free approaches, can successfully achieve levels of NK cell expansion obtained when using feeder cells.

[0013] This document provides methods and materials for expanding NK cells in feeder-free systems. As demonstrated herein, mechanisms of NK cell activation by feeder cells were elucidated by RNA-seq and ATAC-seq analyses at different co-culture time points. The principal transcription factors at the start of co-culture, as well as those that become activated at later stages during the co-culture period, were identified. Soluble factors that can activate these transcription factors were then used in feeder cell-free cultures to expand NK cells. These factors included two small molecules, two cytokines, and a monoclonal antibody, which were aimed at replicating the behaviors of the identified transcription factors and signaling pathways. As demonstrated herein, the concentration and exposure time of these soluble factors were evaluated, resulting in successful and substantial NK cell expansion - comparable to that achieved with feeder cell expansion systems. Functional studies demonstrated that the feeder cell-free expanded NK cells were cytotoxic toward tumor cells.

[0014] In a first aspect, this document features a method for generating an expanded population of NK cells in vitro. The method can include, consist of, or consist essentially of: administering, to a starting population of NK cells, IL-2, an activator of protein kinase C (PKC) and an activator of nuclear factor of activated T-cells (NF AT); culturing the population of NK cells in the presence of the activator of PKC and the activator of NF AT for about 3 hours to about 24 hours; and subsequently culturing the population of NK cells without the activator of PKC and the activator of NF AT but in the presence of interleukin-2 (IL-2) and, optionally, interleukin-21 (IL-21), a 4-1BB agonist, or both IL-21 and a 4-1BB agonist, for a period of time sufficient to expand the population of NK cells by at least 10- fold as compared to the starting population of NK cells. The PKC activator can include one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB. The method can include administering the PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL. The PKC activator can be PMA, and the method can include administering the PMA at a concentration of about 1 ng / mL to about 100 ng / mL. The PKC activator can be PEP005, and the method can include administering the PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL. The NF AT activator can include a molecule that can increase intracellular calcium levels. The molecule can be a calcium ionophore, a Piezo- 1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor. The NF AT activator can include a calcium ionophore, and the calcium ionophore can include one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I). The NF AT activator can be ionomycin, and the method can include administering the ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL. The NF AT activator can include a Piezo-1 activator, and the Piezo-1 activator can include one or more of Yodal, Yoda2, Jedi 1, and Jedi2. The NF AT activator can include a SERCA inhibitor, and the SERCA inhibitor can include one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ). The 4-1BB agonist can be an anti -4- IBB antibody. The method can include culturing the population of NK cells for a period of time sufficient to expand the population of NK cells by at least 100- fold. The method can include culturing the population of NK cells for a period of time sufficient to expand the population of NK cells by at least 1000-fold. The method can include culturing the population of NK cells in the presence of the activator of PKC and the activator of NF AT for about 6 hours. The method can include culturing the population of NK cells in the presence of the activator of PKC and the activator of NF AT for about 12 hours. The period of time can be from about 3 days to about 10 days. The period of time can be from about 4 days to about 8 days. The method may not include the use of feeder cells.

[0015] In another aspect, this document features a method for generating an expanded population of NK cells in vitro, where the method includes, consists of, or consists essentially of: (a) administering, to a starting population of NK cells, a first activator of PKC and a first activator of NF AT; (b) culturing the population of NK cells in the presence of the first activator of PKC and the first activator of NF AT for about 3 hours to about 24 hours; (c) subsequently culturing the population of NK cells without the activator of PKC and the activator of NF AT but in the presence of IL-2 and, optionally, IL-21, a 4- IBB agonist, or both IL-21 and a 4-1BB agonist, for about 3 days to about 10 days; (d) administering, to the cultured population of NK cells of step (c), a second activator of PKC, a second activator of NFAT, and a Fas ligand (FasL) antagonist; (e) further culturing the population of NK cells in the presence of the second activator of PKC, the second activator of NFAT, and the FasL antagonist for about 3 hours to about 24 hours; (f) subsequently further culturing the population of NK cells without the second activator of PKC and the second activator of NFAT but in the presence of the FasL antagonist, IL-2, and optionally IL-21, the 4-1BB agonist, or both IL -21 and the 4- IBB agonist, for about 3 days to about 10 days to further expand the population of NK cells; and optionally (g) repeating steps (d) to (f), thereby expanding the population of NK cells to a desired number. The first PKC activator and the second PKC activator can be the same. The first PKC activator and the second PKC activator can be different. The first PKC activator and the second PKC activator can include one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, PDBu, and TPPB. The method can include administering the first PKC activator and the second PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL. The first PKC activator can be PMA or the second PKC activator can be PMA, or the first PKC activator and the PKC activator can be PMA, and the method can include administering the PMA at a concentration of about 1 ng / mL to about 100 ng / mL. The first PKC activator can be PEP005 or the second PKC activator can be PEP005, or the first PKC activator and the second PKC activator both can be PEP005, and wherein the method can include administering the PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL. The first NF AT activator and the second NF AT activator can be the same. The first NF AT activator and the second NF AT activator can be different. The first NF AT activator or the second NF AT activator, or both the first NF AT activator and the second NF AT activator, can include a molecule that can increase intracellular calcium levels. The molecule can be a calcium ionophore, a Piezo- 1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor. The first NF AT activator or the second NF AT activator, or both the first NF AT activator and the second NF AT activator, can include a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I). The first NF AT activator or the second NF AT activator, or both the first NF AT activator and the second NF AT activator can be ionomycin, and the method can include administering the ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL. The first NF AT activator or the second NF AT activator, or both the first NF AT activator and the second NF AT activator, and include a Piezo- 1 activator, and the Piezo- 1 activator can include one or more of Yodal, Yoda2, Jedi 1 , and Jedi2. The first NF AT activator or the second NF AT activator, or both the first NF AT activator and the second NF AT activator, and include a SERCA inhibitor, and the SERCA inhibitor can include one or more of thapsigargin, CPA, and BHQ. The 4-1BB agonist can be an anti-4-lBB antibody. The FasL antagonist can be selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti- FasL antibodies. The FasL antagonist can be an anti-FasL antibody. The method can include expanding the population of NK cells to a number that is at least 100- fold the number of NK cells in the starting population. The method can include expanding the population of NK cells to a number that is at least 1000-fold the number of NK cells in the starting population. The method may not comprise the use of feeder cells.

[0016] In another aspect, this document features a method for selectively generating an expanded population of NK cells in vitro. The method can include, consist of, or consist essentially of: (a) seeding a starting population of cells onto a solid surface in a culture vessel, where the starting population of cells includes NK cells, and where the solid surface has an anti-4-lBB antibody, a B7-H6 polypeptide, or both an anti-4-lBB antibody and a B7- H6 polypeptide attached thereto, and in the presence of IL-2 and IL-21; (b) culturing NK cells from the starting population in the culture vessel for about 5 days to about 10 days, thereby yielding a selected population of NK cells; (c) administering, to the selected population ofNK cells, an activator of PKC, an activator of NF AT, IL-2, and optionally IL- 21, a 4-1BB agonist, or both IL-21 and a 4-1BB agonist; (d) culturing the selected population of NK cells in the presence of the activator of PKC, the activator of NF AT, the IL-2, and the optional IL-21 and / or 4-1BB agonist, for about 7 days to about 12 days; and optionally (e) repeating steps (c) and (d) until cell growth stops, thereby selectively generating an expanded population of NK cells. The starting population of cells can be an enriched NK cell population. The starting population of cells can be a mixed cell population. The PKC activator can include one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, PDBu, and TPPB. The method can include administering the PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL. The PKC activator can be PMA, and the method can include administering the PMA at a concentration of about 1 ng / mL to about 100 ng / mL. The PKC activator can be PEP005, and the method can include administering the PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL. The NF AT activator can include a molecule that can increase intracellular calcium levels. The molecule can be a calcium ionophore, a Piezo-1 activator, or a SERCA inhibitor. The NF AT activator can include a calcium ionophore, and the calcium ionophore can include one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I). The NF AT activator can be ionomycin, and the method can include administering the ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL. The NF AT activator can include a Piezo-1 activator, and the Piezo-1 activator can include one or more of Yodal, Yoda2, Jedil, and Jedi2. The NF AT activator can include a SERCA inhibitor, and the SERCA inhibitor can include one or more of thapsigargin, CPA, and BHQ. The 4-1BB agonist can be an anti-4- 1BB antibody. The method can include culturing the selected population of NK cells for a period of time sufficient to expand the population of NK cells by at least 100-fold. The method can include culturing the selected population of NK cells for a period of time sufficient to expand the population of NK cells by at least 1000-fold. Step (c) can include culturing the selected population of NK cells in the presence of the activator of PKC, the activator of NF AT, IL-2, IL-21, and the 4- IBB agonist for about 10 days. The method may not include the use of feeder cells.

[0017] In another aspect, this document features an article of manufacture. The article of manufacture can include, consist of, or consist essentially of: a vessel containing an activator of PKC, a vessel containing an activator of NF AT, and optionally, one or more of: a vessel containing IL-2, a vessel containing IL-21, a vessel containing a 4-1BB agonist, a vessel containing a FasL agonist, and a solid substrate to which an anti-4-lBB antibody, B7-H6, or another NK cell binding agent is attached. The activator of PKC and the activator of NF AT can be contained in the same vessel. The activator of PKC and the activator of NF AT can be contained in separate vessels. The activator of PKC can include one or more of PMA, PEP005, prostratin, bryostatin 3, PDBu, and TPPB. The activator of NF AT can include a molecule that can increase intracellular calcium levels. The molecule can be a calcium ionophore, a Piezo-1 activator, or a SERCA inhibitor. The activator of NF AT can include a calcium ionophore, and the calcium ionophore can include one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I). The activator of NFAT can include a Piezo-1 activator, and the Piezo-1 activator can include one or more of Yodal, Yoda2, Jedi 1 , and Jedi2. The activator of NFAT can include a SERCA inhibitor, and the SERCA inhibitor can include one or more of thapsigargin, CPA, and BHQ. The 4- IBB agonist can be an anti-4-lBB antibody. The FasL antagonist can be selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti-FasL antibodies.

[0018] This document also features NK cells expanded using a method provided herein, as well as compositions containing NK cells that were expanded using a method provided herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0019] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0020] DESCRIPTION OF DRAWINGS

[0021] FIG. 1 is a schematic illustrating a representative experimental design for characterizing NK cell activation with feeder cells. Donor-derived NK cells were enriched from peripheral blood mononuclear cells (PBMCs) that were isolated from leukocyte reduction system chambers (LRSCs). NK cells were rested in IL-2 containing medium overnight and co-cultured with membrane-stained, irradiated feeder cells. At different time points, NK cells were sorted and prepared for RNA sequencing (RNA-seq) and AT AC sequencing (ATAC-seq), which were used to determine the dynamics in signaling pathways before and during NK cell activation.

[0022] FIG. 2 shows the output of chromVAR analysis of ATAC-seq data using the Homer database for transcription factor binding motifs (TFBMs). The top 50 most variable TFBMs were selected and clustered based on their Z-score dynamics. The names of the TFBMs are shown on the right-hand side, and the TF family is indicated in parentheses.

[0023] FIG. 3 is a schematic illustrating the signaling pathways and their downstream transcription factors involved in activation of NK cells by feeder cells. The arrows indicate how small molecules including protein kinase C (PKC) activators (e.g., phorbol myristate acetate (PMA) and PEP005) and Ca2+ionophores (e.g., ionomycin) can activate signaling molecules that result in the activation of the same downstream transcription factors. FIGS. 4A-4D include schematics and graphs illustrating the design and results of time course experiments conducted to determine the concentration and exposure duration of small molecules (PKC activators and calcium ionophores) for NK cell expansion. FIG. 4A illustrates the concentration profde of different factors added to the culture for the 6h exposure samples. FIG. 4B is a graph plotting the fold expansion of NK cells 7 days after adding PMA / ionomycin to the culture for samples that were exposed to small molecules for 6 hours. FIG. 4C illustrates the concentration profile of different factors added to the culture for the 24h exposure samples. FIG. 4D is a graph plotting the fold expansion of NK cells 7 days after adding PMA / ionomycin to the culture for samples that were exposed to small molecules for 24 hours.

[0024] FIGS. 5A-5C show that restimulation with PMA and ionomycin led to significant cell death. FIG. 5A is a schematic illustrating a culture protocol for stimulation and restimulation of NK cells with PMA and ionomycin. FIG. 5B is a graph plotting the percentage reduction in viable cell number within 1 day after stimulation (Day 0 - Day 1) and 1 day after restimulation (Day 7 - Day 8). FIG. 5C is a graph plotting the fold expansion of NK cells 7 days after stimulation (week 1) and 7 days after restimulation (week 2), compared to the initial cell numbers at the time of stimulation and re stimulation.

[0025] FIGS. 6A and 6B include NK cell growth curves after stimulation with PMA and ionomycin. NK cells were stimulated with PMA and ionomycin on day 0, and viable cell density and culture volume were measured to plot the growth curve (FIG. 6A). Viability of the NK cells was also measured daily using acridine orange / propidium iodide (AO / PI) staining (FIG. 6B).

[0026] FIGS. 7A-7B show that NK cell activation resulted in increased Fas expression. NK cells at different time points during activation with PMA and ionomycin were evaluated for Fas expression using flow cytometry. The histograms in FIG. 7A show the distribution of Fas fluorescence intensity for each sample. FIG. 7B is a graph plotting the geometric mean fluorescence intensity of Fas expression in NK cells from two different donors at the indicated time points, stained with an APC-anti Fas antibody.

[0027] FIGS. 8A-8C show that use of a FasL inhibitory antibody improved NK cell fold expansion during restimulation. Different factors were added to NK cell cultures during stimulation (FIG. 8A) and restimulation (FIG. 8B), with FasL Ab being the main difference between the stimulation and restimulation protocols. FIG. 8C is a graph plotting the fold expansion at day 20 for NK cells that were stimulated using the two protocols. **P<0.01.

[0028] FIG. 9 is a graph plotting the fold expansion of NK cells at Day 20 compared to Day 0, where the NK cells were grown on feeder cells or were stimulated and restimulated with PMA + FasL Ab or with PEP005 + FasL Ab.

[0029] FIGS. 10A-10B show that expanded cells express 'NK cell identity markers. FIG. 10A is a representative flow cytometry plot for expanded NK cells, showing a high percentage (96.3%) of CD56+CD3" cells. FIG. 10B is a graph plotting the percentage of CD56+CD3‘ cells among the population of expanded cells using different methods, including treatment with PMA / ionomycin, PMA / ionomycin with FasL mAb, PEP005 / ionomycin, PEP005 / ionomycin with FasL mAb, or feeder cells.

[0030] FIGS. 11A-11B show the cytotoxicity of expanded NK cells toward K562 and HL60 cells. NK cells that were expanded using different methods for 20 days were used in a cytotoxicity assay toward K562 cells (FIG. 11 A) and HL60 cells (FIG. 11B). Different ratios of NK cells to target cells were used, as indicated above the groups of bars in each figure.

[0031] FIGS. 12A-12B show cytokine production of expanded NK cells challenged with K562 and HL60 cells. NK cells that were expanded using different methods for 20 days were used in a cytokine production assay. NK cells were cocultured with K562 cells and HL60 cells, and increases in the percentage of IFN-y cells (FIG. 12A) and TNF-a+cells (FIG. 12B) were calculated.

[0032] FIGS. 13A-13D show the expansion of T cells from PBMCs stimulated with PEP005 and ionomycin. FIG. 13A is a graph plotting the number of total viable T cells and the total cell number after 10 days, for Donor 75. FIG. 13B is a graph plotting the number of total viable T cells and the total cell number after 10 days, for Donor 76. FIG. 13C is a graph plotting the calculated fold expansion of viable T cells 10 days after stimulation for both Donor 75 and Donor 76. FIG. 13D is a graph plotting viable T cell numbers at day 0 and day 10, where the composition of cells was measured using the flow cytometry that was used to calculate viable T cell numbers in FIGS. 13A and 13B.

[0033] FIGS. 14A and 14B show that a small molecule system can be used in combination with other expansion systems. FIG. 14A is a schematic illustrating a plate-bound expansion system, showing NK cell receptors along with added soluble and plate-bound proteins that can engage these receptors. FIG. 14B is a schematic illustrating a stimulation schedule for a combined plate- and small molecule-based expansion system, as well as small molecule- alone and plate-bound alone controls.

[0034] FIG. 15 is a graph plotting growth curves for NK cells expanded with a plate-bound system followed by a small molecule-based system. Cumulative cell numbers for Donor 71 (squares), Donor 72 (circles), Donor 75 (triangles), and Donor 76 (diamonds) are shown. Plate bound-alone groups (dotted lines) and plate-bound to small molecule groups (solid lines) were included for all donors. Small molecule-alone groups (dashed lines) were included for two donors (Donor 75 and Donor 76).

[0035] FIG. 16A is a schematic showing an experimental design to expand NK cells using immobilized factors in a plate-bound system (IF), small molecules (also referred to herein as soluble factors) in a SF system, or a combination of the two (Combined IF and SF system). FIG. 16B is a graph plotting expansion of NK cells from a first donor (Donor 1) using a SF system, a feeder cell system, an IF system, and a combined IF and SF system. FIG. 16C includes representative flow cytometry plots for NK cells from Donor 1, expanded using an SF system (left panel), a combined system (center panel), or a feeder cell system (right panel) FIG. 16D is a graph plotting K562 cell killing by NK cells expanded using the indicated systems. FIG. 16E includes graphs plotting the percent increase in IFNy (top panel) and TNFa (bottom panel) secretion by NK cells expanded using the indicated systems, as determined by flow cytometry after blocking secretion for 4 hours and then co-culturing with K562 cells, relative to cells without co-culture.

[0036] FIG. 17A is a graph plotting expansion of NK cells from a second donor (Donor 2) using a SF system, a feeder cell system, an IF system, and a combined IF and SF system. FIG. 17B includes representative flow cytometry plots for NK cells from Donor 2, expanded using an SF system (left panel), a combined system (center panel), or a feeder cell system (right panel). FIG. 17C is a graph plotting K562 cell killing by NK cells expanded using the indicated systems. FIG. 17D includes graphs plotting the percent increase in IFNY (toP panel) and TNFa (bottom panel) secretion by NK cells expanded using the indicated systems, as determined by flow cytometry after blocking secretion for 4 hours and then co-culturing with K562 cells, relative to cells without co-culture. FIGS. 18A-18B include graphs plotting expansion of NK cells from a third donor (Donor 3; left panel of FIG. 18A) and a fourth donor (Donor 4; left panel of FIG. 18B) using a SF system, a feeder cell system, an IF system, and a combined IF and SF system. FIGS. 18A and 18B also include representative flow cytometry plots for NK cells from Donors 3 and 4, expanded using an SF system (center panels) or a combined system (right panels).

[0037] FIG. 19 is a plot showing transcriptome- wide comparison of feeder and small molecule expanded cells. The log fold change of transcript expression at day 38 relative to day 18 for the SF system is plotted on the y-axis and the log fold change of transcript expression at day 38 relative to day 18 for a traditional feeder system is plotted on the x-axis. Regression is shown with a dashed line. Transcripts were grouped based on whether the absolute value of both the feeder system expression and the soluble factor system expression was < 1, or if the absolute value of either the feeder system or the soluble factor system was < 1, or if the absolute value in both systems was > 1.

[0038] DETAILED DESCRIPTION

[0039] Provided herein are materials and methods that can be used to generate expanded populations of NK cells in vitro, in the absence of feeder cells. The methods provided herein can include, for example, contacting a population of NK cells in culture with one or more agents that can lead to activation of transcription factors identified, as described in the Examples herein, as being involved in activation and expansion of NK cells cultured on feeder cells. For example, the methods provided herein can include culturing NK cells in the presence of one or more agents (e.g., one or more soluble small molecules) that can activate or lead to activation of AP-1 (e.g., one or more activators of PKC), one or more agents (e.g., one or more soluble small molecules) that can activate or lead to activation of NF-KB (e.g., one or more activators of PKC), and one or more factors (e g., one or more soluble small molecules) that can activate or lead to activation of NF AT. In some cases, a method provided herein also can include culturing NK cells in the presence of one or more FasL antagonists. The methods provided herein can include culturing the NK cells for a period of time sufficient to expand the population of NK cells by at least 10-fold (e.g., at least 100-fold, at least 1000-fold, or at least 10,000-fold). The methods provided herein do not use feeder cells. The feeder cell-free and fully soluble processes provided herein can decrease culture complexity and reduce regulatory concerns that would otherwise be associated with using feeder cells - typically cancer cell lines - or beads in the process. Further, the methods disclosed herein present scalable ways to expand NK, cells with much higher expansion potential compared to previously developed methods.

[0040] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0041] As used herein, the term “about,” when used herein in reference to a value, refers to a value that is ± 10% of the referenced value.

[0042] Methods for expanding NK cells

[0043] In some embodiments, this document provides methods for generating expanded populations of NK cells in vitro, without the use of feeder cells.

[0044] The methods provided herein can include administering, to a population of NK cells in culture, one or more agents that can activate, or lead to activation of, AP-1. For example, the methods provided herein can include contacting a population of NK cells with one or more agents that lead to activation of AP-1. As described in Example 1 and illustrated in FIG. 3 herein, PKC is an upstream activator of AP-1. Thus, in some cases, the methods provided herein can include contacting a population of NK cells with one or more activators of PKC.

[0045] The methods provided herein also can include administering, to a population of NK cells in culture, one or more agents that can activate, or lead to activation of, NF-KB. AS described in Example 1 and illustrated in FIG. 3 herein, PKC is an upstream activator of NF- KB. Thus, in some cases, the methods provided herein can include contacting a population of NK cells with one or more activators of PKC.

[0046] In addition, the methods provided herein can include administering, to a population of NK cells in culture, one or more agents that can activate, or lead to activation of, NF AT. As described in Example 1 and illustrated in FIG. 3 herein, NF AT can be activated by increased intracellular Ca2+levels. Intracellular Ca2+levels can be increased by calcium ionophores. Thus, in some cases, the methods provided herein can include contacting a population of NK cells with one or more calcium ionophores that can activate or lead to activation of NF AT.

[0047] In some cases, a method provided herein can include contacting a population of NK cells with (a) one or more activators of PKC and (b) one or more activators of NF AT, and culturing the population of NK cells for a period of time sufficient to expand the population of NK cells such that the number of NK cells in the population is increased by at least 10- fold (e.g., at least 100-fold, at least 1000-fold, or at least 10,000-fold) as compared to the number of NK cells in the starting population of NK cells.

[0048] NK cells for use in the methods provided herein can be sourced from any appropriate origin, including, without limitation, peripheral blood, umbilical cord blood, and pluripotent stem cells. In some cases, a starting population of NK cells can be included within a mixed cell population (e.g., PBMCs). In some cases, a starting population of NK cells can be relatively pure (e.g., an enriched population of NK cells isolated from peripheral blood or umbilical cord blood, or a population of NK cells derived from induced pluripotent stem cells; iPSC). Isolation of NK cells from peripheral or umbilical cord blood typically yields limited numbers of NK cells, but those numbers can be increased using in vitro methods for expansion of NK cells as described herein, which can provide larger doses of NK cells for administration to patients. In some cases, NK cells from an expanded population generated according to the methods provided herein can be cryopreserved for future use (e.g., in allogeneic cell therapy).

[0049] In some cases, the methods provided herein can include contacting a population of NK cells in vitro with one or more PKC activators. Any appropriate activator of PKC can be used. Examples of suitable PKC activators include, without limitation, PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), TPPB, DCP-LA, R59949, decursin, (-)-indolactam V, prostratin, ingenol, l-oleoyl-2-acetyl-sn-glycerol, daphnoretin, l-stearoyl-2-arachidoyl-sn-glycerol, R 59-022, pinoresinol dimethyl ether, C8- ceramide, SC- 10, and decursinol angelate. In some cases, methods provided herein can include contacting NK cells with PMA or PEP005.

[0050] In some cases, the methods provided herein can include contacting a population of NK cells in vitro with one or more NF AT activators. Any appropriate activator of NF AT can be used. Examples of suitable NF AT activators include, without limitation, molecules that can increase intracellular calcium levels (e.g., calcium ionophores, Piezo-1 activators, sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitors, or any combination thereof). In some cases, a method provided herein can included contacting NK cells in vitro with one or more calcium ionophores (e.g., ionomycin, A23187 (Calcimycin), 4-Br-A23187, ETH 1001 (Calcium ionophore I), or any combination thereof). In some cases, a method provided herein can include contacting NK cells in vitro with one or more Piezo-1 activators (e.g., Yodal, Yoda2, Jedi 1 , Jedi2, or any combination thereof). In some cases, a method provided herein can include contacting NK cells in vitro with one or more SERCA inhibitors (e.g., thapsigargin, cyclopiazonic acid (CPA), 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ), or any combination thereof).

[0051] In some cases, a method provided herein can include contacting NK cells with one or more activators of PKC (e.g., PMA and / or PEP005) and one or more activators of NFAT (e.g., a calcium ionophore such as ionomycin), where the NK cells are incubated with the PKC activator(s) and the NFAT activator(s) for about 24 hours or less. For example, NK cells can be cultured in the presence of a PKC activator (e.g., PMA and / or PEP005) and an NFAT activator (e.g., ionomycin) for a length of time ranging from about 1 hour to about 24 hours (e g., from about 1 hour to about 12 hours, from about 3 hours to about 24 hours, from about 2 hours to about 6 hours, from about 4 hours to about 8 hours, from about 5 hours to about 7 hours, from about 6 hours to about 10 hours, from about 8 hours to about 12 hours, from about 10 hours to about 14 hours, from about 12 hours to about 16 hours, from about 16 hours to about 20 hours, from about 20 hours to about 24 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours). In some cases, a method provided herein can include culturing NK cells in the presence of a PKC activator (e.g., PMA and / or PEP005) and an NFAT activator (e.g., ionomycin) for less than 12 hours (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 11 hours). For example, a PKC activator and an NFAT activator can be present in an NK cell culture for about 4 hours to about 8 hours (e.g., about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours). Any appropriate concentration of a PKC activator can be used in the methods provided herein. For example, a method provided herein can include culturing a population of NK cells with a PKC activator (e.g., PMA and / or PEP005) at a concentration of about 20 pg / mL to about 1 pg / mL (e.g., about 20 pg / mL to about 100 pg / mL, about 100 pg / mL to about 500 pg / mL, about 500 pg / mL to about 1 ng / mL, about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 20 pg / mL, about 100 pg / mL, about 200 pg / ml, about 400 pg / mL, about 600 pg / mL, about 800 pg / mL, about 1 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 100 ng / mL, about 200 ng / mL, about 400 ng / mL, about 600 ng / mL, about 800 ng / mL, or about 1 pg / mL) of culture medium. In some cases, a method provided herein can include culturing NK cells with PMA, where the method includes contacting a population of NK cells in culture with PMA at a concentration of about 100 pg / mL to about 1 pg / mL (e.g., about 100 pg / mL to about 1 ng / mL, about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 100 pg / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 500 ng / mL, or about 1 pg / mL). In some cases, a method provided herein can include culturing NK cells with PEP005, where the method includes contacting a population of NK cell in culture with PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL (e.g., about 0.2 ng / mL to about 0.5 ng / mL, about 0.5 ng / mL to about 1 ng / mL, about 1 ng / mL to about 2 ng / mL, about 2 ng / mL to about 3 ng / mL, about 3 ng / mL to about 5 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 15 ng / mL, about 15 ng / mL to about 20 ng / mL, about 0.2 ng / mL, about 1.0 ng / mL, about 2 ng / mL, about 3 ng / mL, about 3.5 ng / mL, about 4 ng / mL, about 4.5 ng / mL, about 5 ng / mL, about 5.5 ng / mL, about 6 ng / mL, about 6.5 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, or about 20 ng / mL) of culture medium.

[0052] Any appropriate concentration of an NF AT activator can be used in the methods provided herein. For example, a method provided herein can include culturing a population of NK cells with an NF AT activator (e.g., ionomycin) at a concentration of about 10 ng / mL to about 1 mg / mL (e.g., about 10 ng / mL to about 50 ng / mL, about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 150 ng / mL, about 150 ng / mL to about 200 ng / mL, about 200 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 pg / mL to about 5 pg / mL, about 5 pg / mL to about 10 pg / mL, about 10 pg / mL to about 100 pg / mL, about 100 pg / mL to about 500 pg / mL, about 500 pg / mL to about 1 mg / mL, about 10 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 mg / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, or 1 mg / mL). In some cases, a method provided herein can include contacting a population of NK cells in culture with an NF AT activator that is a Piezo-1 activator (e.g., Yodal, Yoda2, Jedi 1 , Jedi2, or any combination thereof) at a concentration of about 10 ng / mL to about 1 mg / mL. In some cases, a method provided herein can include contacting a population of NK cells with an NF AT activator that is a SERCA inhibitor (e.g., thapsigargin, CPA, BHQ, or any combination thereof) at a concentration of about 10 ng / mL to about 1 mg / mL.

[0053] In some cases, a method provided herein can include culturing a population of NK cells in the presence of one or more PKC activators and one or more NF AT activators for about 1 to about 24 hours, and then culturing the population of NK cells for an appropriate length of time without the one or more PKC activators and the one or more NF AT activators. For example, a method provided herein can include culturing a population of NK cells in the presence of one or more PKC activators and one or more NF AT activators for about 1 to about 24 hours, and then culturing the population of NK cells without the one or more PKC activators and the one or more NF AT activators for a period of time from about 2 days to about 20 days (e.g., from about 2 days to about 6 days, from about 4 days to about 8 days, from about 6 days to about 10 days, from about 8 days to about 12 days, from about 10 days to about 14 days, from about 12 days to about 16 days, from about 14 days to about 18 days, from about 16 days to about 20 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, or about 20 days).

[0054] In some cases, a method provided herein can include culturing NK cells in the presence of a FasL antagonist in addition to one or more PKC activators (e.g., PMA or PEP005) and / or one or more NF AT activators (e.g., ionomycin). Without being bound by a particular mechanism of action, inclusion of a FasL antagonist can enhance and / or facilitate restimulation of NK cell expansion (e.g., by reducing restimulation-induced NK cell death). In some cases, a method provided herein can include stimulating NK cell expansion by culturing a population of NK cells with one or more PKC activators and / or one or more NFAT activators for a suitable period of time (e.g., about 3 to about 24 hours), followed by incubation of the NK cells without any PKC activators or NFAT activators for a suitable period of time (e.g., about 7 to about 12 days), and then restimulating expansion of the NK cells by incubating the NK cells with one or more PKC activators and / or one or more NFAT activators, along with one or more FasL antagonists.

[0055] Any appropriate FasL antagonist can be used in the methods provided herein. Examples of suitable FasL antagonists include, without limitation, APG101, RG7826, apocept, Kp7-6, soluble Fas, decoy receptors, and anti-FasL antibodies. A FasL antagonist can be included in NK cell culture media at any appropriate concentration. For example, a method provided herein can include culturing a population of NK cells in media that contains a FasL antagonist (e.g., an anti- FasL antibody) at a concentration of about 5 ng / mL to about 50 pg / mL (e.g., about 5 ng / mL to about 50 ng / mL, about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 250 ng / mL, about 250 ng / mL to about 500 ng / mL, about 250 ng / mL to about 750 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 pg / mL to about 10 pg / mL, about 10 pg / mL to about 50 pg / mL, about 5 ng / mL, about 10 ng / mL, about 50 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, about 1 pg / mL, about 10 pg / mL, about 20 pg / mL, about 30 pg / mL, about 40 pg / mL, or about 50 pg / mL) of culture medium.

[0056] In some cases, one or more FasL antagonists (e.g., an anti- FasL antibody) in addition to one or more PKC activators (e.g., PMA and / or PEP005) and / or one or more NFAT activators (e.g., ionomycin) can be administered to NK cells in culture about 8 days to about 15 days (e g., about 8 days to about 10 days, about 9 days to about 11 days, about 10 days to about 12 days, about 11 days to about 14 days, about 12 days to about 15 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days) after initial stimulation of the NK cells with one or more PKC activators (e.g., PMA and / or PEP005) and / or one or more NFAT activators (e.g., ionomycin). In some cases, administration of one or more FasL antagonists in addition to one or more PKC activators (e.g., PMA and / or PEP005) and / or one or more NFAT activators (e.g., ionomycin) can be repeated about every 8 days to about every 15 days (e.g., about every 8 to 10 days, about every 9 to 11 days, about every 10 to 12 days, about every 11 to 14 days, about every 12 to 15 days, about every 8 days, about every 9 days, about every 10 days, about every 11 days, about every 12 days, about every 13 days, about every 14 days, or about every 15 days). For example, administration of a FasL antagonist in addition to a PKC activator (e.g., PMA or PEP005) and / or an NF AT activator (e.g., ionomycin) can be repeated every 10 days until expansion of the NK cells stops.

[0057] In some cases, one or more FasL antagonists can be present in NK cell culture media for about 4 hours to about 10 days (e.g., about 4 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 16 hours, about 16 hours to about 24 hours, about 1 day to about 2 days, about 2 days to about 4 days, about 4 days to about 6 days, about 6 days to about 8 days, about 8 days to about 10 days, about 4 hours, about 6 hours, about 6 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 2 days, about 4 days, about 6 days, about 8 days, or about 10 days). For example, a FasL antagonist (e.g., an anti- FasL antibody) together with a PKC activator (e.g., PMA or PEP005) and / or an NF AT activator (e.g., ionomycin) can be present in NK cell culture for about 6 hours. For example, a FasL antagonist (e g., an anti- FasL antibody) together with a PKC activator (e.g., PMA or PEP005) and / or an NF AT activator (e.g., ionomycin) can be present in NK cell culture for about 4 days.

[0058] In any of the methods provided herein, a medium in which NK cells are cultured also can contain one or more cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21) and / or a 4- 1BB agonist. For example, one or more cytokines (e.g., IL-2 and / or IL-21) and / or a 4-1BB agonist (e.g., an anti-4-lBB antibody, such as Urelumab, Utomilumab, and / or ADG106, a 4-1BBL polypeptide, and / or a 4-1BB antibody clone, such as the 4b4-l clone from Biolegend, Cat. #309806) can be included in NK cell culture media along with one or more PKC activators and / or one or more NF AT activators and / or one or more FasL antagonists. Alternatively, or in addition, one or more cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21) and / or a 4-1BB agonist (e.g., an anti-4-lBB antibody) can be included in NK cell culture media without any PKC activators, NF AT activators, or FasL antagonists. For example, a method provided herein can include incubating NK cells in a culture medium that contains IL-2, IL-21, and a 4- IBB agonist (e.g., Urelumab). Any appropriate concentration of a cytokine can be used in the methods provided herein. In some cases, a method provided herein can include culturing a population of NK cells in media that contains IL-2 at a concentration of about 1 lU / mL to about 10,000 lU / mL (e.g., about 1 lU / mL to about 10 lU / mL, about 10 lU / mL to about 50 lU / mL, about 50 lU / mL to about 100 lU / mL, about 100 lU / mL to about 500 lU / mL, about 500 lU / mL to about 1000 lU / mL, about 1000 lU / mL to about 5000 lU / mL, about 5000 lU / mL to about 10,000 lU / mL, about 1 lU / mL, about 10 lU / mL, about 50 lU / mL, about 100 lU / mL, about 150 lU / mL, about 200 lU / mL, about 500 lU / mL, about 1000 lU / mL, about 5000 lU / mL, or about 10,000 lU / mL) of culture medium.

[0059] In some cases, a method provided herein can include culturing a population of NK cells in media that contains IL-21 at a concentration of about 500 pg / mL to about 5 pg / mL (e.g., about 500 pg / mL to about 1 ng / mL, about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 25 ng / mL, about 25 ng / mL to about 50 ng / mL, about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 pg / mL to about 5 pg / mL, about 500 pg / mL, about 1 ng / mL, about 10 ng / mL, about 25 ng / mL, about 50 ng / mL, about 75 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 500 ng / mL, about 1 pg / mL, or about 5 pg / mL) of culture medium.

[0060] In some cases, a method provided herein can include culturing a population of NK cells with in media that contains a 4-1BB agonist (e.g., Urelumab) at a concentration of about 10 ng / mL to about 100 pg / mL (e.g., about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 pg / mL to about 2 pg / mL, about 2 pg / mL to about 5 pg / mL, about 5 pg / mL to about 10 pg / mL, about 10 pg / mL to about 100 pg / mL, about 10 ng / mL, about 100 ng / mL, about 500 ng / mL, about 1 pg / mL, about 1.25 pg / mL, about 1.5 pg / mL, about 2 pg / mL, about 5 pg / mL, about 10 pg / mL, about 50 pg / mL, or about 100 pg / mL) of culture medium.

[0061] NK cells can be cultured in the presence of one or more cytokines (e.g., IL -2 and / or IL-21) and / or one or more 4-1BB agonists (e.g., Urelumab) for any appropriate length of time. For example, NK cells can be cultured in the presence of IL-2 and / or IL-21 and / or a 4- 1BB agonist for about 4 days to about 10 days (e.g., about 4 days to about 6 days, about 5 days to about 7 days, about 6 days to about 8 days, about 7 days to about 9 days, about 8 days to about 10 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days). In some cases, a method provided herein can include culturing NK cells in the presence of one or more activators of PKC (e.g., PMA and / or PEP005) and / or one or more activators of NFAT (e.g., ionomycin), optionally in the presence of one or more cytokines (e.g., IL-2 and / or IL-21) and / or one or more 4-1BB agonists (e.g., Urelumab) for about 1 hour to about 24 hours, and then incubating the NK cells without the one or more PKC activators and the one or more 'NFAT activators, but in the presence of one or more cytokines (e.g., IL-2 and / or IL-21) and / or one or more 4-1BB agonists (e.g., Urelumab) for about 4 days to about 10 days.

[0062] In some cases, a method provided herein for expanding populations of NK cells can include contacting a starting population of NK cells with IL-2, one or more activators of PKC (e.g., PMA and / or PEP005) and / or one or more activators of NFAT (e.g., ionomycin), culturing the NK cells in the presence of the activator(s) of PKC and / or NFAT for about 3 hours to about 24 hours, and subsequently culturing the NK cells without the activator(s) of PKC and / or NFAT but in the presence of IL-2 and / or interleukin-21 (IL-21) and / or a 4-1BB agonist, for a period of time (e.g., 4 to 10 days) sufficient to expand the population of NK cells by at least 10-fold (e.g., at least 100-fold, or at least 1000-fold) as compared to the starting population of NK cells.

[0063] In some cases, a method provided herein for expanding populations of NK cells can include stimulating a starting population of NK cells by adding, to the culture medium, one or more activators of PKC (e.g., PMA and / or PEP005) and / or one or more activators of NFAT (e.g., ionomycin), culturing the population of NK cells in the presence of the activator(s) of PKC and / or the activator(s) of NFAT for about 3 hours to about 24 hours, subsequently culturing the NK cells without the activator(s) of PKC and / or NFAT but in the presence of IL-2 and / or IL-21 and / or and a 4-1BB agonist for about 3 days to about 10 days, and then restimulating the NK cells by adding, to the culture medium, one or more FasL antagonists along with one or more activators of PKC and / or one or more activators of NFAT, further culturing the NK cells in the presence of the FasL antagonist(s) and the activator(s) of PKC and / or NFAT for about 3 hours to about 24 hours, and then culturing the NK cells without the activator(s) of PKC and / or NFAT but in the presence of the FasL antagonist(s) along with IL-2 and / or IL-21 and / or one or more 4-1BB agonists for about 3 days to about 10 days. In some cases, one or more steps of this method can be repeated to further expand the population of NK cells. For example, the restimulation step and the subsequent steps of this method can be repeated (e.g., until cell expansion stops or reaches a desired point).

[0064] In some cases, such as when the starting population of cells is a mixed population that includes NK cells along with other cell types (e g., other types of cells within a sample of peripheral blood), NK cells can be enriched or isolated from the mixed population and then expanded. Enrichment or isolation of NK cells from a mixed population can be achieved by, for example, placing the mixed population of cells in a vessel (e.g., a culture plate, flask, or dish) having a surface to which NK cell-engaging molecules are attached. In some cases, the NK cell-engaging molecules can stimulate NK cells but not T cells. For example, a biological sample (e.g., a blood sample or a PBMC sample) containing NK cells in combination with one or more other cell types (e.g., T cells) can be placed into a culture vessel having a surface with an anti-4-lBB antibody attached thereto. As another example, a biological sample (e.g., a blood sample or a PBMC sample) containing NK cells in combination with one or more other cell types can be placed into a culture vessel having a surface with a B7-H6 polypeptide attached thereto in the presence of IL-2 and IL-21. In some cases, a biological sample containing NK cells in combination with one or more other cell types can be placed into a culture vessel having a surface with an anti -4- IBB antibody and a B7-H6 polypeptide attached thereto in the presence of IL-2 and IL-21. The anti-4-lBB antibody and / or the B7- H6 polypeptide can be attached to the surface of the culture vessel via any appropriate means (e g., via a biotin-streptavidin interaction or via a non-covalent interaction). Any appropriate anti-4-lBB antibody can be used. Examples of suitable anti-4-lBB antibodies include, without limitation, Urelumab, Utomilumab, and ADG106. Any appropriate B7-H6 polypeptide can be used. Examples of suitable B7-H6 polypeptides include, without limitation, a human B7-H6 polypeptide having the amino acid sequence set forth in NCBI Reference Sequence NP_001189368 (e.g., version NP_001189368.1), and fragments thereof to which NKp30 can interact (e.g., the mature polypeptide sequence set forth in amino acid residues 24-454 of the amino acid sequence set forth in NCBI Reference Sequence NP_001124587.1.

[0065] For example, a method provided herein can be used to selectively generate an expanded population of NK cells. The method can include, for example, seeding a mixed population of cells into a culture vessel, where the mixed population of cells includes NK cells and where the culture vessel has a solid surface to which an anti-4-lBB antibody and / or a B7-H6 polypeptide are attached in the presence of IL-2 and IL-21, such that NK cells within the mixed population interact with and / or become attached to the anti -4- IBB antibody and / or the B7-H6 polypeptide. The method can further include culturing the NK cells in the culture vessel for about 5 days to about 10 days (e.g., about one week), optionally in the presence of one or more cytokines and / or one or more 4-1BB agonists as described herein, and then stimulating the enriched population of NK cells to expand by adding one or more activators of PKC (e.g., PMA and / or PEP005) and / or one or more activators of NF AT (e.g., ionomycin), and optionally one or more cytokines and / or one or more 4- IBB agonists as described herein, and culturing the enriched population of NK cells in the presence of the activator(s) of PKC and / or NF AT for about 7 days to about 12 days, optionally with the one or more cytokines and / or the one or more 4-1BB agonists. In some cases, the NK cells can be released from the solid surface by pipetting up and down before culturing them (optionally in the presence of one or more cytokines and / or one or more 4-1BB agonists as described herein) and stimulating them to expand using one or more activators of PKC (e.g., PMA and / or PEP005) and / or one or more activators of NF AT (e g., ionomycin), and optionally one or more cytokines and / or one or more 4-1BB agonists as described herein. In some cases, one or more steps of this method can be repeated to further expand the population of NK cells. For example, the stimulation step and the subsequent culturing step of this method can be repeated (e.g., until cell expansion stops or reaches a desired point).

[0066] In some cases, NK cells can be expanded as described herein and, during the expansion process, contacted with one or more nucleic acid modifying agents in order to generate engineered NK cells. For example, NK cells undergoing expansion in a feeder cell- free system (e g., NK cells that have begun diving) provided herein can be contacted with a nucleic acid modifying agent. Examples of suitable nucleic acid modifying agents include, without limitation, rare-cutting endonucleases and base editors. In some cases, the methods provided herein can include expanding the population of engineered NK cells generated as described herein (e.g., NK cells undergoing expansion in a feeder cell-free system that have been contacted with a nucleic acid modifying agent). For example, a nucleic acid modifying agent can be selected to introduce a break (e g., a single-strand break or a double-strand break) into NK cell DNA(e.g., genomic DNA). In general, a double-strand break at a target sequence to be modified can be repaired by one of two primary pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). In NHEJ, the ends of the broken chromosome are rejoined, sometimes imprecisely, which can introduce small insertions or deletions (indels) at the break site (Gorbunova and Levy, Nucleic Acids Res 1997, 25:4650-4657). When indels occur in coding sequences, they may create frame shift mutations that disrupt gene function. In HR, or gene targeting (GT), the DNA break is repaired using a template with homology to the break site. The repair template can be the sister chromatid, a homologous chromosome, or an exogenous template containing one or more specific sequence modifications to be incorporated into the break site.

[0067] Any appropriate nucleic acid modifying agent can be used to modify NK cell nucleic acids. Examples of nucleic acid modifying agents include, without limitation, targeted rare- cutting endonucleases such as meganucleases (Puchta et al., Nucleic Acids Res 1993, 21 :5034-5040; Salomon and Puchta, EMBO J 1998, 17:6086-6095; and Jacoby et al., Nucl. Acids Res. 10.1093 / nar / gkrl303, 2012), zinc-finger nucleases (ZFNs) (Kim et al., Proc Natl Acad Sci USA 1996, 93:1156-1160; Townsend et al., Nature 2009, 459:442-445; and Sander et al., Nature Methods, 8:67-69, 2011), transcription activator-like effector (TALE) endonucleases (Christian et al., Genetics 2010, 186:757-761; Bogdanove and Voytas, Science 2011, 333: 1843-1846; and U.S. Publication No. 2011 / 0145940), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, such as a CRISPR / Cas9 system (Hwang et al., Nat Biotechnol 2013, 31 :227-229; Shan et al., Nat Biotechnol 2013 , 31 :686- 688; Cong et al., Science 339:819-823, 2013; and Mali et al., Science 339:823-826, 2013).

[0068] CRISPR / Cas systems use RNA base pairing to direct DNA or RNA cleavage by a Cas endonuclease. CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) sequences direct the Cas enzyme to a specific target DNA sequence (Makarova et al., Nat Rev Microbiol, 9(6):467-477, 2011). The modification of a single targeting RNA can be sufficient to alter the nucleotide target of a Cas protein. In some cases, crRNA and tracrRNA can be engineered as a single cr / tracrRNA hybrid to direct Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012). Any appropriate Cas endonuclease can be used. In some cases, the Cas endonuclease can be a Cas9 endonuclease (e.g., a Streptococcus pyogenes Cas9 endonuclease). Other suitable Cas polypeptides include, without limitation, Casl2j

[0069] (also referred to as CasO).

[0070] Other examples of nucleic acid modifying agents that can be used to generate engineered NK cells include, without limitation, TnpB polypeptides, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editing agents. TnpBs are Cas-related polypeptides that work with guide RNAs to modify DNA. TnpBs are relatively small in size - often less than about 500 amino acids. Base editing and prime editing technology can avoid the creation of double-stranded DNA breaks, and may provide enhanced editing efficiency and product purity. These technologies are described elsewhere (see, e.g., Kaya (2024), “Base Editing and Prime Editing” in: A Roadmap for Plant Genome Editing. Ricroch, Eriksson, Miladinovic, Sweet, Van Laere, and Wozniak-Gientka (eds), Springer, Cham.; https : / / doi . org / 10.1007 / 978 -3 -031 -46150-7_2) .

[0071] In some cases, a nucleic acid modifying agent used to generate engineered NK cells can be a TALE endonuclease. TAL effectors of plant pathogenic bacteria in the genus Xanthomonas play important roles in disease and trigger defense by binding to host DNA and activating effector-specific host genes (see, e.g., Gu et al., Nature 435: 1122, 2005; Yang et al., Proc Natl Acad Sci USA 103:10503, 2006; Kay et al., Science 318:648, 2007; Sugio et al., Proc Natl Acad Sci USA 104: 10720, 2007; and Romer et al., Science 318:645, 2007). Specificity depends on an effector-variable number of imperfect, typically 34 amino acid repeats (Schomack et al., J Plant Physiol 163:256, 2006). Polymorphisms are present primarily at repeat positions 12 and 13, which are referred to herein as the repeat variable- diresidue (RVD). TALE nucleases contain (1) a DNA binding domain derived from a TAL effector, where the domain can be engineered to bind to a specific sequence based on the RVDs included in the repeats, and (2) an endonuclease domain, typically from a type II restriction endonuclease such as Fold (Kim et al., Proc Natl Acad Sci USA 93: 1156-1160, 1996). Other useful endonucleases include, for example, Hhal, Hind ll, Noil, BbvCI, EcoRI, Bgll, and A / w . The fact that some endonucleases (e.g., FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TALE nuclease. For example, in some cases each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created. Thus, TALE nucleases can function as heterodimers, where each monomer of the pair is targeted to a selected target sequence, and when the monomers are bound to their targets, the nuclease dimerizes and cleaves the DNA at the target sequence between the monomer binding sites. See, e.g., U.S. Patent No. 8,586,363.

[0072] Nucleic acid modifying agents can be delivered to NK cells using any appropriate method. In some cases, a nucleic acid modifying agent can be delivered to NK cells via a viral vector (e.g., a lentivirus vector, an adenovirus vector, or an adeno-associated virus vector).

[0073] Methods of Treatment

[0074] NK cells that are expanded using the methods provided herein can be used, for example, as cell therapies to treat mammals (e.g., humans, non-human primates, dogs, cats, rabbits, mice, rats, horses, cows, sheep, or pigs) who have, or are at risk of developing, diseases such as cancer, infections, autoimmune disorders, or stress-related disorders. In some cases, a method provided herein can include generating an expanded population of NK cells and subsequently administering at least a portion of the expanded NK cells to a mammal in need thereof. In some cases, the starting population of NK cells can have been obtained from the mammal to be treated (e.g., a mammal that has, or is at risk of developing, a disease such as cancer, an infection, or a stress-related disorder). In some cases, however, the starting population of NK cells can have been obtained from a different mammal than the mammal that will eventually be treated with the expanded NK cells. In such cases, the donor mammal from which the starting population of NK cells is obtained can be a mammal of the same species as the mammal to which the expanded NK cells will be administered. In some cases, the donor mammal and the recipient mammal can be immunologically matched. In some cases, after their in vitro culture and expansion, NK cells obtained according to the methods provided herein can be cryopreserved (e.g., in multiple vials), and / or can be prepared for transplantation to a mammal.

[0075] NK cell populations that have been expanded according to the methods provided herein can be administered to a recipient mammal using any appropriate methods (e.g., by intravenous infusion or intratumoral injection). Single or multiple (e.g., two, three, four, or more than four) administrations of expanded NK cells can be administered to a mammal (e.g., a human) depending on, for example, the dosage and frequency as required and tolerated by the patient. Each administration can provide a sufficient quantity of expanded NK cells (e.g., about 106to about 109NK cells per kilogram of patient weight) to effectively treat or ameliorate a condition, disease, or symptom in a mammal in need thereof.

[0076] Compositions and Articles of Manufacture

[0077] This document also provides compositions containing a populations of NK cells that have been expanded using the methods described herein. The NK cells can be provided in any pharmaceutically acceptable composition. The pharmaceutically acceptable compositions can include a population of NK cells, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The term “pharmaceutically-acceptable carrier” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material.

[0078] Any appropriate method can be used to formulate suitable pharmaceutical compositions. See, e.g., Remington, The Science and Practice of Pharmacy, 21sted., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs, Ed. Hickey (CRC Press, Boca Raton, FL). Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers can include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The compositions provided herein typically are sterile and fluid to the extent that easy syringability exists. The compositions also typically are stable under the conditions of manufacture and storage, and may be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0079] This document also provides articles of manufacture that include agents for use in the methods provided herein. For example, this document provides articles of manufacture that include a vessel containing an activator of PKC and / or a vessel containing an activator of 'NF AT. In some cases, an article of manufacture provided herein can include a vessel containing an activator of PKC and / or a vessel containing an activator of NF AT, along with a vessel containing one or more cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21), and / or a vessel containing one or more 4-1BB agonists. In some cases, an article of manufacture provided herein can include a vessel containing an activator of PKC and / or a vessel containing an activator of NF AT, along with a vessel containing a FasL agonist, and optionally a vessel containing one or more cytokines (e.g., IL-2, IL-12, IL-15, IL-18, and / or IL-21) and / or a vessel containing one or more 4-1BB agonists. It is to be noted that in some cases, any combination of the aforementioned activators and other agents can be included in the same vessel. In other cases, each of the aforementioned activators and other agents can be included within their own, separate vessels. Further, in some cases, an article of manufacture can include a solid substrate to which an anti-4-lBB antibody and / or a B7-H6 polypeptide and / or another NK cell binding agent is attached, along with one or more vessels containing any of the aforementioned activators and agents.

[0080] Exemplary Embodiments

[0081] Embodiment 1 is a method for generating an expanded population of NK cells in vitro, wherein the method comprises, consists of, or consists essentially of: administering, to a starting population of NK cells, interleukin-2 (IL-2), an activator of protein kinase C (PKC) and an activator of nuclear factor of activated T-cells (NF AT); culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 3 hours to about 24 hours; and subsequently culturing said population of NK cells without said activator of PKC and said activator of NF AT but in the presence of IL-2 and, optionally, interleukin-21 (IL-21), a 4-1BB agonist, or both IL-21 and a 4-1BB agonist, for a period of time sufficient to expand said population of NK cells by at least 10-fold as compared to said starting population of NK cells. Embodiment 2 is the method of embodiment 1, wherein said PKC activator comprises one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

[0082] Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein said method comprises administering said PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

[0083] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein said PKC activator is PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

[0084] Embodiment 5 is the method of any one of embodiments 1 to 3, wherein said PKC activator is PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

[0085] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein said NF AT activator comprises a molecule that can increase intracellular calcium levels.

[0086] Embodiment 7 is the method of embodiment 6, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor.

[0087] Embodiment 8 is the method of embodiment 6, wherein said NF AT activator comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I), or wherein said NF AT activator comprises a Piezo- 1 activator, and wherein said Piezo- 1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2, or wherein said NFAT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

[0088] Embodiment 9 is the method of embodiment 8, wherein said NFAT activator is ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

[0089] Embodiment 10 is the method of embodiment 6, wherein said NFAT activator comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1, and Jedi2. Embodiment 11 is the method of embodiment 6, wherein said NF AT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

[0090] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein said 4- 1BB agonist is an anti -4- IBB antibody.

[0091] Embodiment 13 is the method of any one of embodiments 1 to 12, comprising culturing said population of NK cells for a period of time sufficient to expand said population of NK cells by at least 100-fold.

[0092] Embodiment 14 is the method of any one of embodiments 1 to 12, comprising culturing said population of NK cells for a period of time sufficient to expand said population of NK cells by at least 1000-fold.

[0093] Embodiment 15 is the method of any one of embodiments 1 to 14, comprising culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 6 hours.

[0094] Embodiment 16 is the method of any one of embodiments 1 to 14, culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 12 hours.

[0095] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein said period of time is from about 3 days to about 10 days.

[0096] Embodiment 18 is the method of any one of embodiments 1 to 16, wherein said period of time is from about 4 days to about 8 days.

[0097] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein said method does not comprise the use of feeder cells.

[0098] Embodiment 20 is a method for generating an expanded population of NK cells in vitro, wherein said method comprises, consists of, or consists essentially of: (a) administering, to a starting population of NK cells, a first activator of protein kinase C (PKC) and a first activator of nuclear factor of activated T-cells (NF AT); (b) culturing said population of NK cells in the presence of said first activator of PKC and said first activator of NF AT for about 3 hours to about 24 hours; (c) subsequently culturing said population of NK cells without said activator of PKC and said activator of NF AT but in the presence of interleukin-2 (IL-2) and, optionally, interleukin-21 (IL-21), a 4-1BB agonist, or both IL-21 and a 4-1BB agonist, for about 3 days to about 10 days; (d) administering, to said cultured population of NK cells of step (c), a second activator of PKC, a second activator of NF AT, and a Fas ligand (FasL) antagonist; (e) further culturing said population of NK cells in the presence of said second activator of PKC, said second activator of NF AT, and said FasL antagonist for about 3 hours to about 24 hours; (f) subsequently further culturing said population of NK cells without said second activator of PKC and said second activator of NF AT but in the presence of said FasL antagonist, said IL-2, and optionally said IL-21, said 4-1BB agonist, or both said IL-21 and said 4-1BB agonist, for about 3 days to about 10 days to further expand said population of NK cells; and optionally (g) repeating steps (d) to (f), thereby expanding said population of NK cells to a desired number.

[0099] Embodiment 21 is the method of embodiment 20, wherein said first PKC activator and said second PKC activator are the same.

[0100] Embodiment 22 is the method of embodiment 20, wherein said first PKC activator and said second PKC activator are different.

[0101] Embodiment 23 is the method of any one of embodiments 20 to 22, wherein said first PKC activator and said second PKC activator comprise one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

[0102] Embodiment 24 is the method of any one of embodiments 20 to 23, wherein said method comprises administering said first PKC activator and said second PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

[0103] Embodiment 25 is the method of any one of embodiments 20 to 24, wherein said first PKC activator is PMA, said second PKC activator is PMA, or both said first PKC activator and said second PKC activator are PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

[0104] Embodiment 26 is the method of any one of embodiments 20 to 24, wherein said first PKC activator is PEP005, said second PKC activator is PEP005, or both said first PKC activator and said second PKC activator are PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

[0105] Embodiment 27 is the method of any one of embodiments 20 to 26, wherein said first

[0106] NF AT activator and said second NF AT activator are the same. Embodiment 28 is the method of any one of embodiments 20 to 26, wherein said first NF AT activator and said second NF AT activator are different.

[0107] Embodiment 29 is the method of any one of embodiments 20 to 26, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a molecule that can increase intracellular calcium levels.

[0108] Embodiment 30 is the method of embodiment 29, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor.

[0109] Embodiment 31 is the method of embodiment 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

[0110] Embodiment 32 is the method of embodiment 31, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator are ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

[0111] Embodiment 33 is the method of embodiment 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedil, and Jedi2.

[0112] Embodiment 34 is the method of embodiment 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

[0113] Embodiment 35 is the method of any one of embodiments 20 to 34, wherein said 4- 1BB agonist is an anti-4-lBB antibody.

[0114] Embodiment 36 is the method of any one of embodiments 20 to 35, wherein said FasL antagonist is selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti- FasL antibodies. Embodiment 37 is the method of any one of embodiments 20 to 35, wherein said FasL antagonist is an anti-FasL antibody.

[0115] Embodiment 38 is the method of any one of embodiments 20 to 37, comprising expanding said population of NK cells to a number that is at least 100-fold the number of NK cells in said starting population.

[0116] Embodiment 39 is the method of any one of embodiments 20 to 37, comprising expanding said population of NK cells to a number that is at least 1000-fold the number of NK cells in said starting population.

[0117] Embodiment 40 is the method of any one of embodiments 20 to 39, wherein said method does not comprise the use of feeder cells.

[0118] Embodiment 41 is a method for selectively generating an expanded population of NK cells in vitro, wherein said method comprises, consists of, or consists essentially of: (a) seeding a starting population of cells onto a solid surface in a culture vessel in the presence of interleukin-2 (IL-2) and interleukin-21 (IL-21), wherein said starting population of cells comprises NK cells, and wherein said solid surface has an anti-4- IBB antibody, a B7-H6 polypeptide, or both said anti -4- IBB antibody and said B7-H6 polypeptide attached thereto; (b) culturing NK cells from said starting population in said culture vessel for about 5 days to about 10 days, thereby yielding a selected population of NK cells; (c) administering, to said selected population of NK cells, an activator of protein kinase C (PKC), an activator of nuclear factor of activated T-cells (NF AT), IL-2, and optionally IL-21, a 4- IBB agonist, or both IL-21 and a 4-1BB agonist; (d) culturing said selected population of NK cells in the presence of said activator of PKC, said activator of NFAT, said IL-2, and optionally said IL- 21, said 4-1BB agonist, or both IL-21 and said 4-1BB agonist, for about 7 days to about 12 days; and optionally (e) repeating steps (c) and (d) until cell growth stops, thereby selectively generating an expanded population of NK cells.

[0119] Embodiment 42 is the method of embodiment 41, wherein said starting population of cells is an enriched NK cell population.

[0120] Embodiment 43 is the method of embodiment 41, wherein said starting population of cells is a mixed cell population. Embodiment 44 is the method of any one of embodiments 41 to 43, wherein said PKC activator comprises one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

[0121] Embodiment 45 is the method of any one of embodiments 41 to 44, wherein said method comprises administering said PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

[0122] Embodiment 46 is the method of any one of embodiments 41 to 45, wherein said PKC activator is PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

[0123] Embodiment 47 is the method of any one of embodiments 41 to 45, wherein said PKC activator is PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

[0124] Embodiment 48 is the method of any one of embodiments 41 to 47, wherein said NF AT activator comprises a molecule that can increase intracellular calcium levels.

[0125] Embodiment 49 is the method of embodiment 48, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor .

[0126] Embodiment 50 is the method of embodiment 48, wherein said NF AT activator comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

[0127] Embodiment 51 is the method of embodiment 50, wherein said NF AT activator is ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

[0128] Embodiment 52 is the method of embodiment 48, wherein said NF AT activator comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1, and Jedi2.

[0129] Embodiment 53 is the method of embodiment 48, wherein said NF AT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

[0130] Embodiment 54 is the method of any one of embodiments 41 to 53, wherein said 4- 1BB agonist is an anti -4- IBB antibody. Embodiment 55 is the method of any one of embodiments 41 to 54, comprising culturing said selected population of NK cells for a period of time sufficient to expand said population of NK cells by at least 100-fold.

[0131] Embodiment 56 is the method of any one of embodiments 41 to 54, comprising culturing said selected population of NK cells for a period of time sufficient to expand said population of NK cells by at least 1000-fold.

[0132] Embodiment 57 is the method of any one of embodiments 41 to 56, wherein step (c) comprises culturing said selected population of NK cells in the presence of said activator of PKC, said activator of NF AT, said IL-2, said IL-21, and said 4-1BB agonist for about 10 days.

[0133] Embodiment 58 is the method of any one of embodiments 41 to 57, wherein said method does not comprise the use of feeder cells.

[0134] Embodiment 59 is an article of manufacture comprising, consisting of, or consisting essentially of a vessel containing an activator of PKC, a vessel containing an activator of NF AT, and optionally, one or more of: a vessel containing IL-2, a vessel containing IL-21, a vessel containing a 4-1BB agonist, a vessel containing a FasL agonist, and a solid substrate to which an anti-4-lBB antibody, B7-H6, or another NK cell binding agent is attached.

[0135] Embodiment 60 is the article of manufacture of embodiment 59, wherein said activator of PKC and said activator of NF AT are contained in the same vessel.

[0136] Embodiment 61 is the article of manufacture of embodiment 59, wherein said activator of PKC and said activator of NF AT are contained in separate vessels.

[0137] Embodiment 62 is the article of manufacture of any one of embodiments 59 to 61, wherein said activator of PKC comprises one or more of PMA, PEP005, prostratin, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

[0138] Embodiment 63 is the article of manufacture of any one of embodiments 59 to 62, wherein said activator of NF AT comprises a molecule that can increase intracellular calcium levels.

[0139] Embodiment 64 is the article of manufacture of embodiment 63, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor. Embodiment 65 is the article of manufacture of embodiment 63, wherein said activator of NF AT comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

[0140] Embodiment 66 is the article of manufacture of embodiment 63, wherein said activator of NF AT comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2.

[0141] Embodiment 67 is the article of manufacture of embodiment 63, wherein said activator of NF AT comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4- benzohydroquinone (BHQ).

[0142] Embodiment 68 is the article of manufacture of any one of embodiments 59 to 67, wherein said 4-1BB agonist is an anti-4-lBB antibody.

[0143] Embodiment 69 is the article of manufacture of any one of embodiments 59 to 68, wherein said FasL antagonist is selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti-FasL antibodies.

[0144] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0145] EXAMPLES

[0146] Example 1 - Scalable Expansion of NK Cells Using Soluble Molecules Materials and Methods

[0147] Isolation and enrichment ofNK cells: Leukocyte reduction system chambers (LRSCs) were obtained from Innovative Blood Resources (St. Paul, MN). Peripheral blood mononuclear cells (PBMCs) were isolated from the LRSCs using density gradient centrifugation with Ficoll-Paque Premium medium (Cytiva) and SEPMATE™-50 tubes (STEMCELL Technologies). NK cells were then enriched from the PBMCs using the EASYSEP™ Human NK Cell Enrichment Kit (STEMCELL Technologies). The NK cells were rested overnight in B0 medium supplemented with 100 lU / mL IL-2 (R&D Systems, Inc.; Minneapolis, MN). B0 medium consists of a 2: 1 (vokvol) blend of DMEM containing 4.5 g / L glucose, L-glutamine, and sodium pyruvate (Gibco), and Ham’s F12 Medium (Corning). It was supplemented with 20 pM 2-mercaptoethanol (Gibco), 50 pM ethanolamine (Sigma), 10 pg / mL ascorbic acid (Sigma), 1.6 ng / mL sodium selenite (Sigma), 100 lU / mL penicillin / streptomycin (Gibco), and 10% heat-inactivated human AB serum (Valley Biomedical).

[0148] NK cell co-culture with feeder cells for multi-omics analyses: Feeder cells, specifically K562 cells genetically engineered to express membrane-bound (mb)-IL-21 and mb-4-lBBL, were irradiated and stained with PKH26 dye (Sigma Aldrich). NK cells were co-cultured with these feeder cells in 24-well plates at concentrations of 5*105cells / mL and l * 106cells / mL, respectively. Before and after co-culture initiation, cells in culture were collected and incubated with SYTOX™ Green Dead Cell Stain (Invitrogen) in PBS containing 2% human AB serum. The cells were sorted to collect viable NK cells (negative for PKH26 and SYTOX™ Green) using a BD FACSARIA™ II (BD Biosciences; San Jose, CA) at various time points: before co-culture initiation, and at 6 hours, 1 day, 2 days, 3 days, and 7 days after initiation.

[0149] Sample preparation for RNA-Seq and ATAC-Seq: The collected cells were divided into different populations to prepare samples for RNA-seq and ATAC-seq. For ATAC sequencing, cells collected after 3 days were not used. Total RNA was extracted from samples using a Quick-RN A Microprep Kit from Zymo Research (Irvine, CA), followed by isolation of poly-A containing mRNA molecules and generation of unique dual-indexed (UDI) TruSeq stranded mRNA libraries. These libraries were pooled and sequenced on a NovaSeq 6000 platform (Illumina, Inc.; San Diego, CA) at the University of Minnesota Genomics Center.

[0150] ATAC-seq samples were cryopreserved in B0 media with 10% DMSO. A total of 50,000 cells per sample were processed using the OMNI-ATAC protocol. Afterward, the transposed DNA was purified with a MINELUTE™ PCR cleanup kit (Qiagen; Venlo, Netherlands). Dual -indexed ATAC-seq libraries were generated from transposed DNA using Nextera reagents (Illumina, Inc.). Libraries were pooled and sequenced in a single lane of a NovaSeq SI 2x50-bp run (Illumina, Inc.).

[0151] ATAC-seq data analysis: Trimmomatic was utilized to trim adapter sequences and remove low-quality bases from Fastq files. For mapping the reads to the human reference genome (hg38), Bowtie2 was employed with parameters set to -p 2 -X 2000 - very-sensitive, excluding mitochondrial reads and removing duplicates using MarkDuplicates. Peak calling was performed with MACS2 using parameters -p 0.01 - nomodel -shift -75 -extsize 150 -bdg, and IDR was applied to generate a reproducible peak list across all conditions. Consensus peaks were identified by taking the union of reproducible peaks from all conditions. The raw peak count matrix, representing the number of reads in each peak, was obtained using featureCounts, with EDASeq correcting for GC content bias.

[0152] To evaluate the dynamics of transcription factor binding motif (TFBM) accessibility in samples, the chromVAR package in R with TFBM data from HOMER and Jaspar was used. ChromVAR calculated a “raw accessibility deviation” for each TFBM, representing the difference between the mapped reads to the TFBM-encompassing peaks and the expected reads based on the average of all samples. ChromVAR also created a “background” peak set with matching GC content and calculated both the average accessibility and the raw background accessibility deviation. These deviations were then used to determine a Z-score for each TFBM and sample, indicating the relative gain or loss of accessibility compared to the average sample profile.

[0153] NK cell stimulation with small molecules: Unless otherwise specified in the results, small molecule-mediated expansion of NK cells was performed using a combination of PMA and ionomycin (both from Sigma Aldrich; Burlington, MA) or using a combination of PEP005 (Tocris Bioscience; Minneapolis, MN) and ionomycin. For PMA / ionomycin expansion, NK cells were cultured in 96-well plates at 200,000 cells / mL (50,000 cells / well) in B0 medium supplemented with rhLL-2 (100 lU / mL) (R&D Systems; Minneapolis, MN), PMA (10 ng / mL), and ionomycin (1 pg / mL) for 6 hours. After this period, the cell culture medium was replaced with B0 containing IL-2 (100 lU / mL), IL-21 (50 ng / mL, R&D Systems), and Urelumab (1.25 pg / mL, ThermoFisher Scientific or MedChemExpress), and the NK cells were maintained in this medium until day 4. On day 4, the NK cells were transferred to 24-well culture plates, and B0 with IL-2 (100 lU / mL) was added. Cells were cultured under these conditions from day 4 to day 7. On day 7, the number of viable cells was determined, and the fold expansion of NK cells was calculated. ForNK cells cultured for 10 days, B0 with IL-2 (100 lU / mL) was added on day 7 to support their growth until day 10. The same protocol was used for NK cells expanded with PEP005 / ionomycin, with PMA replaced by PEP005 (5 nM) during the initial 6 hours.

[0154] Restimulation ofNK cells: NK cells were expanded through a 6-hour exposure to small molecules, as described in the previous section. Following this expansion, the NK cells were counted and restimulated using the same method as on day 0. For samples designated as containing FasL antibody, 500 ng / mL of FasL Ab (R&D Systems) was added during the 6- hour exposure to either PMA / ionomycin or PEP005 / ionomycin, and also from the 6 hour timepoint to day 4 during restimulation. NK cells that underwent restimulation on day 10 were cultured for an additional 10 days, and their fold expansion was evaluated at that time.

[0155] Feeder cell-mediated NK cell expansion: Feeder cell-mediated NK cell expansion involved co-culturing feeder cells with NK cells at a ratio of 2: 1 on day 0 and 1 : 1 on day 10 in 96-well plates in B0 supplemented with IL-2 (100 lU / mL), starting with an NK cell density of 200,000 cells / mL (50,000 cells / well). Medium was added to the culture on days 4 and 7 to support growth. NK cell counts were performed on day 20, and fold expansion was determined.

[0156] NK cell marker expression: Expanded NK cells were washed in flow buffer (1% AB serum and 0.5 mM EDTA in PBS) and incubated with antibodies against CD56 and CD3 that were diluted in flow buffer containing a fixable viability dye (eFluor 780, Invitrogen), for 30 minutes at 4°C. After incubation, the cells were centrifuged, washed twice, and fixed in 2% paraformaldehyde. The samples were then washed again and resuspended in flow buffer until flow cytometry analysis was performed.

[0157] Cytokine production assay: Enriched NK cells were co-cultured with target cells (K562 or HL60) at a 2: 1 ratio (NK celktarget cell) in a 96-well plate. After 1 hour, GolgiStop (BD Biosciences) and GolgiPlug (BD Biosciences) were added to inhibit Golgi transport, allowing cytokines to accumulate within the cells. Four hours after co-culture initiation, cells were collected and stained for NK cell surface markers. Subsequently, intracellular staining was performed to detect IFN' and TNFa using specific conjugated antibodies. The cells were analyzed by flow cytometry to assess cytokine production at the single-cell level.

[0158] Cytotoxicity assay: Expanded NK cells were counted and prepared for a cytotoxicity assay. Target cells (K562 or HL60 cell lines) were stained with a cell tracker dye (CELLTRACKER™ Green CMFDADye, Invitrogen) and co-cultured with NK cells at the ratios indicated in the figures. After 4 hours, cells were collected from the culture plates and stained with a fixable viability dye. Cells were analyzed using flow cytometry to determine the percentage of non-viable target cells.

[0159] Plate-bound expansion system: Molecules were non-covalently bound to the surface to simulate molecular presentation on the cell surface. Tissue culture-treated 96-well plates were incubated with 100 pL of protein solution at 5 pg / ml. overnight in PBS, then washed twice with PBS before use. The proteins were present in equal mass concentration. Reagent grade, biotinylated, agonist antibody (clone 4B4-1, isotype mouse IgGlK, Biolegend, Cat# 309806) was used to provide binding to 4-1BB, while the recombinant B7-H6, ligand of NKp30, purchased as an oligosaccharide-biotinylated IgGl Fc-chimera (R&D systems, Cat# BT7144), was used to bind NKp30. Soluble IL-21 was added at 50 ng / mL. NK cells rested overnight in 100 lU / mL IL-2 were seeded at 50,000 cells / mL in 200 pL in flat-bottomed 96- well plates. Samples from the culture well were taken on days 4 and 7 for counting. Media changes were performed as needed.

[0160] Results

[0161] Experimental design for multi-omics analysis of NK cells cocultured with feeder cells: To elucidate the mechanism of feeder cell-mediated NK cell activation, an experiment was designed to analyze NK cells at different time points following their coculture with feeder cells using RNA-seq and ATAC-seq techniques, as shown in FIG. 1. First, PBMCs were isolated from LRCSs via density gradient centrifugation, and were subsequently enriched for NK cells using immunomagnetic negative selection. The enriched NK cells were cultured overnight in B0 media supplemented with 100 HJ / mL of IL-2. Before the start of coculture, these cells were sorted using fluorescence-activated cell sorting (FACS) to account for any changes that could arise from the sorting process (referred to as Oh samples). Membrane-stained, irradiated feeder cells were co-cultured with NK cells at a 2: 1 ratio, and samples were taken at 6 hours, 1 day, 2 days, 3 days, and 7 days (hereafter referred to as 6h, Id, 2d, 3d, and 7d) after initiation of co-culture for FACS. Sorted NK cells were prepared for downstream RNA-seq and ATAC-seq analysis. Donors 1, 2, and 3 were used for RNA-seq analysis experiments, while Donors 1 and 2 at Oh, 6h, Id, 2d, and 7d from the same samples were utilized for ATAC-seq sample preparations. ATAC-seq identified transcription factors involved in NK cell activation: To understand which TFs are activated following NK cell coculture with feeder cells, chromVAR analysis was applied to ATAC-seq data and the dynamics of TFBM accessibility were evaluated. The results are summarized in FIG. 2, in which samples are represented in columns, TFBMs are represented in rows, and the TFBM Z-scores, which are measures of TF binding activity, are shown by heatmap colors. Cluster 1 and Cluster 2 showed substantially higher Z-scores right after coculture initiation. TFs such as NF AT and the NFAT-AP-1 dimer are located in Cluster 1. These TFs showed a transient increase in Z- score, followed by a decline starting 24 hours after coculture. NF AT and its dimer with AP-1 have been reported to be involved in T cell activation through T cell receptor (TCR) signaling (see, Gaud et al., Nat Rev Immunol., 18:485-497, 2018).

[0162] Cluster 2 TFs exhibited heightened TFBM Z-scores at the 6-hour and 1-day time points, followed by a reduction at the 2-day and 7-day time points. This cluster consisted of the NF-KB and AP-1 families of TFs. NF-KB TFS are involved in regulating immune response, inflammation, cell proliferation, and survival. The AP-1 (Activator Protein- 1) family is a group of TFs that regulate gene expression in response to various stimuli in immune cells, including cytokines and infections. The AP-1 and NF-KB TFS are downstream of NK cell activating receptors, and are involved in immune response and proliferation. Given the apparent importance of transcription factors activated immediately after coculture initiation, and the results of studies described elsewhere demonstrating the roles of NF AT, NF-KB, and AP-1 in T cell activation, it was determined that these transcription factors are likely to be involved in the activation and effective expansion of NK cells.

[0163] Identifying small molecules capable of activating the transcription factors: NF-KB, AP-1, and NF AT are downstream TFs of TCR signaling (Gaud et al., supra). Similarly, activation of NK cells through their activating receptors involves comparable signaling pathways. One group of activating receptors, which contain immunoreceptor tyrosine-based activation motifs (IT AMs) in their intracellular domains (such as NKp30, NKp44, NKp46, and CD16a), signals through ZAP-70, closely resembling TCR signaling. Additionally, NKG2D and 2B4 primarily signal through Vavl. PKC, which is downstream of both Vavl and ZAP-70, plays an important role in this signaling cascade. As illustrated in FIG. 3, activation of PKC can result in the activation of both NF-KB and AP-1 transcription factors. Moreover, NK cell activating receptor signaling leads to an increase in intracellular calcium levels, which in turn activates NF AT transcription factors. Small molecule PKC activators such as Phorbol 12-myristate 13-acetate (PMA) and Ingenol 3-angelate (PEP005) can effectively activate AP-1 and NF-KB (FIG. 3). Additionally, ionomycin, a calcium ionophore, can increase intracellular Ca2+levels and activate NF AT (FIG. 3). Thus, the work described herein indicated that when combined, a PKC activator and a calcium ionophore can mimic the activation of NK cells by feeder cells.

[0164] Concentration and exposure time of small molecules for NK cell expansion: To determine the optimal concentrations and exposure durations of PMA and ionomycin for maximizing NK cell expansion over 7 days, an experiment was designed with varying levels of each factor. As shown in FIGS. 4A and 4C, NK cells were cultured in 96-well plates in BO medium supplemented with IL-2 (100 ZU / mL), PMA, and ionomycin for either 6 hours or 24 hours. After this initial period, the cell culture medium was replaced with B0 containing IL-2 (100 lU / mL), IL-21 (50 ng / mL), and Urelumab (1.25 pg / mL), and the NK cells were maintained in this medium until day 4. On day 4, NK cells were transferred to 24-well culture plates, and B0 with IL-2 (100 lU / mL) was added to the wells. Cells were cultured under these conditions from day 4 to day 7. At day 7, the number of viable cells was determined, and the fold-expansion of NK cells was calculated.

[0165] Various conditions based on the concentration of PMA and ionomycin at the start of culture were tested, and the final fold expansions were plotted. For the 6-hour exposure conditions, the highest fold expansion was achieved with 10 ng / mL of PMA and 1 pg / mL of ionomycin (FIG. 4B). For the 24-hour exposure conditions, the highest fold expansion was observed with 1 ng / mL of PMA and 1 pg / mL of ionomycin (FIG. 4D). Of note, prolonged exposure to PMA and ionomycin (more than 2 days) led to significant NK cell death. These results suggested that by adjusting the exposure time and concentration of PMA and ionomycin, NK cells can be effectively expanded.

[0166] NK cell restimulation results in cell death: While NK cells expanded effectively with PMA and ionomycin following initial stimulation, producing enough NK cells for several doses may require restimulation to increase their expansion potential. To determine the feasibility of restimulating NK cells, the protocol of exposing NK cells to PMA and ionomycin for 6 hours was repeated, as depicted in FIG. 5A. The results plotted in FIG. 5B illustrate the percent reduction differences between initial stimulation (day 0 to day 1) and restimulation (day 7 to day 8) of NK cells with PMA and ionomycin. Upon restimulation on day 7 with PMA and ionomycin, NK cell numbers were significantly decreased compared to the first stimulation. Consistent with the observed reduction in cell numbers after restimulation, the fold expansion of NK cells as also significantly decreased with restimulation as compared to the initial stimulation (FIG. 5C). These results indicated that substantial cell death following restimulation was responsible for the observed reduction in fold expansion.

[0167] Growth curve ofNK cells after first stimulation with PMA and ionomycin: To understand the dynamics of viable cell number and viability of NK cells after activation by PMA and ionomycin, these parameters were measured over time following the 6h exposure protocol described above. Viable cell numbers were measured daily forNK cells stimulated with PMA and ionomycin on day 0 and then cultured for 11 days without restimulation. Stimulation initially caused cell death, as evidenced by the reduction in viability and viable cell number (FIGS. 6A-6B). However, the viable cell number then increased until day 10, suggesting that it was beneficial to keep the cells in culture as long as they were actively proliferating. Based on these observations, it appeared that stimulating NK cells every 10 days could enhance their expansion by reducing the number of required restimulations and overall cell death. Consequently, the NK cell expansion protocol was modified to include restimulation every 10 days instead of every 7 days.

[0168] FAS expression increases following NK cell activation: Reactivation-induced cell death (RICD) in T cells is a phenomenon characterized by T cell apoptosis upon repeated antigenic stimulation, and is crucial for immune response regulation and homeostasis (Lee et al., Front Cell Death., 2, 2023). The Fas-Fas ligand (Fas-FasL) pathway plays a central role in orchestrating the apoptosis of restimulated T cells. Fas, a member of the death receptor group, is essential in lymphocyte immune responses (Lee et al., supra). Upon stimulation, FasL translocates to lymphocyte membranes, inducing apoptosis in Fas-expressing cells (Snow et al., Immunol Rev. 236:68-82, 2010).

[0169] Studies were conducted to examine whether NK cells also upregulate Fas expression upon stimulation with PMA and ionomycin. Using flow cytometry, the surface expression level of Fas on NK cells was measured before (day 0) and 3 days, 7 days, and 10 days after stimulation with PMA and ionomycin. The results showed that Fas expression was significantly increased in activated NK cells, and remained high for at least 10 days following initial stimulation (FIGS. 7A-7B).

[0170] Inhibiting Fas, FasL interaction could reduce restimulation-induced cell death: NK cells upregulated Fas expression upon stimulation, becoming susceptible to FasL-Fas- induced cell death through fratricide, where activated NK cells induce apoptosis in each other. To investigate whether blocking the interaction of Fas / FasL could reduce restimulation-induced cell death, cell culture was supplemented with an inhibitory FasL monoclonal antibody during the early phases of restimulation with PMA / ionomycin (FIGS. 8A-8B). These studies demonstrated that early supplementation of a FasL antagonist during NK cell restimulation enhanced overall NK cell expansion, potentially by reducing RICD. By day 20, NK cells exhibited over a 20,000-fold expansion (FIG. 8C).

[0171] PEP005 shows superior expansion potential compared to PMA: Both PMA and PEP005 are activators of PKC. To assess the expansion potential of PEP005, PMA was replaced with PEP005 at a concentration of 5 nM (2.15 ng / mL), while maintaining all other conditions constant. Cells were stimulated every 10 days with ionomycin and either PMA or PEP005, as described above. For restimulation, FasL Ab was added to enhance expansion, and the overall fold expansion after 20 days was calculated.

[0172] The expansion results for NK cells obtained from multiple donors, stimulated with PMA / ionomycin, PEP005 / ionomycin, or feeder cells, are shown in FIG. 9. PEP005 / ionomycin demonstrated superior expansion potential compared to PMA / ionomycin, and there was no statistically significant difference observed between the PEP005 / ionomycin group and the feeder cell expansion group.

[0173] Expanded cells express NK cell identity markers: NK cells were expanded for 20 days using PMA / ionomycin, PEP005 / ionomycin, and feeder cells, as described above. Following expansion, NK cells were stained with anti-CD56 and anti-CD3 antibodies and analyzed by flow cytometry (FIG. 10A). NK cells were identified as CD56+CD3" lymphocytes. The cells expanded with PMA / ionomycin, PEP005 / ionomycin, and feeder cells for 20 days showed a high percentage of NK cell marker expression (FIG. 10B), demonstrating that treatment of NK cells with small molecules effectively led to expansion. NK cells expanded with small molecules exhibit cytotoxicity towards tumor cells: NK cells perform two key functions: cytotoxicity and cytokine production. These functions can be evaluated using flow cytometry-based assays that measure responses at the single-cell level when NK cells encounter tumor cells. To assess the functionality of expanded NK cells, their cytotoxicity toward K562 and HL60 tumor cell lines was evaluated. NK cells were expanded for 20 days using PMA / ionomycin, PMA / ionomycin with FasL Ab, PEP005 / ionomycin, PEP005 / ionomycin and FasL Ab, or feeder cells, and were then cocultured with target cells (K562 or HL60 cell lines) for 4 hours. The percentage of non- viable target cells was determined using flow cytometry. These studies demonstrated that NK cells expanded using different expansion systems for 20 days exhibited comparable cytotoxicity toward both K562 (FIG. 11 A) and HL60 (FIG. 11B) tumor cell lines. In addition, the overall cytotoxicity increased as the ratio of NK cells to target cells increased. Thus, the expanded NK cells maintained their cytotoxicity toward tumor cell lines.

[0174] NK cells expanded with small molecules produce cytokines after encountering tumor cells: After encountering and activation with target cells, NK cells upregulate the production of cytokines such as IFN-y and TNF-a. To evaluate the ability of expanded NK cells to produce cytokines, expanded NK cells were cocultured with target (K562 or HL60) cells at a 2: 1 ratio (NK cell: target cell). After 1 hour, Golgi transport was inhibited, allowing cytokines to accumulate within the NK cells. Four hours after coculture initiation, NK cells were collected and stained for IFN-y and TNF-a, and the expression levels of these cytokines were determined using flow cytometry. The percentage increase in IFN-y and TNF-a positive NK cells is plotted in FIG. 12A and FIG. 12B, respectively. These results showed that NK cells expanded with different methods exhibited similar behavior in terms of cytokine production after being challenged with K562 and HL60 cells, indicating that the NK cells retained their cytokine production function after expansion with PMA / ionomycin and PEP005 / ionomycin. Overall, the functional assays performed with expanded NK cells indicated that the cells stimulated and expanded with small molecules remained cytotoxic and were capable of producing cytokines when challenged with tumor cells.

[0175] Small molecules are capable of expanding T cells: In order to determine if small molecules can expand T cells, PBMCs were stimulated with Pep005 / ionomycin. The composition of the starting population was determined by evaluating surface expression of CD56 (a NK cell marker) and CD3 (a T cell marker). After 10 days, total cell numbers were evaluated and CD56 / CD3 expression was again determined. To determine T cell numbers at day 0 and day 10, the percentage of cells that were CD567CD3+was multiplied by total cell number. For donor 75, total T cell number increased from 4.7>< 104cells at day 0 to 6.9* 106cells at day 10 (FIG. 13A), which was about 148-fold (FIG. 13C), while the total PBMC number increased from 2.4* 1 O’ to 1 .04* 107, or 44-fold. For donor 76, the total T cell number increased from 1.2* 105at day 0 to 1.28* 107at day 10 (FIG. 13B), which was about 105-fold (FIG. 13C), while the total PBMC number increased from 2.3* 105to 1.62x 107, about 69- fold. While T cells comprised 20% and 52% of starting PBMCs for donor 75 and donor 76, respectively, their final composition was 66% and 79% (FIG. 13D). This suggested that not only did T cells expand in response to stimulation by PEP005 / ionomycin, but they also constituted a majority of the population by the end of culture. Thus, the stimulation methods described herein have utility for both NK cells and T cells. This dual utility, however, may present a concern if the objective is obtaining pure NK cells, particularly if the starting population is not pure.

[0176] Plate-bound system followed by small molecules for NK cell expansion: While small molecules can expand NK cells significantly, there may be advantages to combining this strategy with other expansion strategies. The plate-bound strategy discussed herein can expand cells up to day 21 but not beyond. Although the plate-bound strategy may enable up to 104-fold expansion by day 21, availability of plate-bound protein to cells is restricted to exposed surface area and thus is not necessarily scalable. On the other hand, the small molecule strategy described herein is scalable, although the small molecule strategy also can be used to expand T cells, suggesting that target proteins of these small molecules are shared by both cell types. If not starting with a highly pure population of NK cells, the small molecule strategy may not be reliable for generating pure NK cells by the end of the expansion process, as there may be T cell impurities. Studies were conducted to investigate the ability of a combined plate-bound and small molecule system to overcome these issues, first by evaluating if plate-bound NK cells are capable of expansion.

[0177] Together, NKp30 agonism via B7-H6, 4-lBB-agonism, IL -21, and IL-2 were combined to create a feeder-free system for NK cell expansion (FIG. 14A). Tissue culture- treated plates were coated overnight with B7-H6 and anti-4- IBB agonist via noncovalent interactions, while IL-21 and IL-2 were delivered in soluble form. This system is referred to herein as “plate-bound,” and cells expanded using this system are referred to as “plate- expanded” or “plate-bound.” As indicated in FIG. 14B, at day 7, cells were restimulated by reseeding at 50,000 cells / mL on plates freshly coated with protein, and again restimulated at day 14. Plate-expanded cells were cultured for 21 days total and are referred herein to as the “plate-bound-alone” group. Another group of rested cells, which are referred to as the “small-molecule alone” group, was stimulated with small molecules at 50,000 cells / mL as described above for PEP005 and ionomycin. These cells were restimulated with small molecules every 10 days. At day 7, a subset of plate-expanded cells was restimulated with small molecules using the same conditions, and were restimulated with small molecule every 10 days. This group is referred to herein as the “plate-bound to small molecule group.” Expansion of cells from four separate donors was performed for the plate-bound to small molecule group and the plate-bound alone group, while cells from two donors were expanded in the small molecule-alone group.

[0178] As shown in FIG. 15, plate bound-alone cells produced 9* 107-4*108total cells, expanding the original population about 1000- to 3000-fold by the end of the culture period (14 days for donors 71 and 72, and 21 days for donors 75 and 76). Cells grew slightly slower after day 14 and stopped growing completely after day 21. The small molecules-alone group produced 6.6x l013-2.5>< 1014cells, expanding around 1.16* 108to 4.5>< 108-fold after 38 days. The plate-bound to small molecule group produced 3x l0n-8.3*1013cells, expanding around 107to 5.8* 108-fold, reaching similar levels of magnitude as the small molecule-alone group (in two biological replicates). Thus, restimulation with small molecules is capable of growing plate-expanded cells beyond 21 days, and the overall fold expansion appears comparable to small-molecule alone groups.

[0179] Example 2 - A combined system for feeder cell-free NK cell expansion with high purity The immobilized factor (IF) system presents an advantage over the soluble factor (SF) feeder-free system provided herein system in selectively expanding NK cells over T cells, while the SF system has the advantage of a high level of population expansion in feeder cell-free conditions. Studies were conducted to explore the feasibility of first expanding NK cells preferentially and reducing T cell proliferation, and then switching to the SF system for further expansion. While the IF system is not ideal for large-scale operations, the cell population in the early stages of NK expansion is at small laboratory scale. Donor cells were expanded in the IF-system and then were restimulated with soluble factors via the SF system on days 7 and 18 (FIG. 16A, bottom). For comparison, cells were expanded with IF and SF systems in parallel (FIG. 16A, top and middle).

[0180] For the IF experiments, molecules were non-covalently bound to the surface of plates to simulate molecular presentation on a cell surface. Tissue culture-treated 96-well plates were incubated with polypeptide solutions, each at 5 pg / mL overnight in PBS, then washed twice with PBS before use. The polypeptides were present in equal mass concentration. Reagent grade, biotinylated, agonist antibody (clone 4B4-1, isotype mouse IgGlK, Biolegend, Catalog no. 309806) was used to provide binding to 41BB, along with recombinant B7H6, ligand of NKp30, purchased as an oligosaccharide-biotinylated IgGl Fc- chimera (R&D systems, Catalog no. BT7144). Soluble IL-21 was added at a concentration of 50 ng / mL, and IL-2 at 100 lU / mL. NK cells were stimulated with small molecules at 5 x 104cells / well (~1.7 x 105 / mL) as described above (5 ng / mL (5 nM) Pep005, 0.75 pg / mL (1.3 pM) ionomycin (Millipore Sigma), and 100 lU / mL IL2, which were diluted after 4 hours with B0 media containing 50 ng / mL (3.3 nM) IL21 (R&D Systems), 100 lU / mL IL2, and 1.25 pg / mL (8.6 nM) Urelumab (ThermoFisher Scientifc or MedChemExpress)). With subsequent stimulations (day 18 and on), anti-TNFR (R&D Systems MAB225-SP) and anti- FasL (R&D Systems Cat# MAB126-100) were also added, each at 500 ng / mL. Media changes were performed as necessary and cells were moved to 24-well plates and then 6-well plates upon reaching confluency.

[0181] Results obtained using starting populations of NK cells from four representative donors are shown in FIGS. 16B-16E, 17A-17D, and 18A-18B. For Donor 1, enriched NK cells expanded comparably in the combined system and the SF system, reaching about a 105- fold increase in population by day 28 (FIG. 16B). For Donor 2, the fold expansion in the combined system was somewhat lower than in the SF system (FIG. 17A). The lower extent of expansion in the combined system was largely attributed to the lower expansion initially (day 7) in the IF system. Feeder expansion with Donor 1 reached only 6 x 104-fold expansion, while the IF system reached 1560-fold expansion after 21 days. The purity of the expanded cells was evaluated at day 28. About 3% of cells from the SF system were T cells (CD3+) (FIG. 16C, left panel) while only 0.43% of the total population were T cells in the combined system (FIG. 16C, center panel). In the expanded cells from the SF system, 4.5% of cells were CD56+ / CD3+ (FIG. 16C, left panel), while 0.56% were CD56+ / CD3+ in the combined system (FIG. 16C, center panel). Feeder-expanded cells contained 0.27% T cells, and 0.38% were CD56+ / CD3+ (FIG. 16C, right panel). Thus, expanding enriched NK cells with the IF system for 7 days before expanding with the SF system reduced T cell contamination. Donors 2-4 also showed reduction in T cell expansion with the combined system at day 18 (FIGS. 17B, 18A, and 18B, respectively). These studies demonstrated that while the SF system can be used as a complete system by itself (e.g., for IPSC-derived NK, for which T cell expansion is not a concern), or the SF system can also be used in this combined system in which the IF system is used before the SF system to reduce T cell expansion.

[0182] The expanded cells were evaluated for cytotoxicity using K562 as target cells at varying ratios. For Donor 1, cells expanded in all three systems were capable of killing the target K562 cells at day 28 (FIG. 16D). The results were very similar for Donor 2 (FIG. 17C). The NK cells expanded in all three systems were active in IFNy and TNFa production as evaluated by flow cytometry after blocking secretion for 4 hours and then co-culturing with K562 cells, relative to cells without co-culture (FIGS. 16E and 17D) Overall, the data showed that cells expanded using the combined system exhibited less T cell expansion and were cytotoxic and capable of enhanced IFNy+ and TNFa+ production in response to tumor cell challenge.

[0183] Example 3 - Comparing the transcriptomes of SF and feeder-expanded NK cells To compare the transcriptomes of NK cells expanded using the SF (feeder-cell-free) and feeder-expanded systems, sequence data in Fastq format were aligned to the human genome (hg38) using the STAR aligner. Raw counts were obtained using HT-seq. Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were computed using Cufflinks. These values were then normalized to transcripts per million (TPM) values in R. Genes that were expressed in at least three samples with counts per million (CPM) > 1 were included for subsequent analysis, resulting in 15,295 transcripts. Transcriptomes of SF- and feeder-expanded cells from two donors at day 18 and day 38 allowed the comparison of transcript dynamics for these two methods of NK expansion. The graph shown in FIG. 19 presents the log2(TPM at day 38 / TPM at day 18) for the feeder system on the x-axis and the SF system on the y-axis. Transcripts were categorized based on whether they had at least a 2-fold change in TPM in both comparisons or if they had a change of 2-fold or higher in only the SF system or only the feeder system. The trendline for all plotted transcripts had a slope of 0.8, indicating that corresponding transcripts largely changed similarly from day 18 to day 38 in the two systems. Only 33 genes changed their expression levels in opposite directions. The majority of transcripts with expression levels changed more than 2-fold were downregulated. The observation that the transcriptome changes between days 18 and 38 were similar for the SF system and the feeder system suggested that the two systems also elicit similar cellular responses.

[0184] Example 4 - In vivo studies

[0185] To evaluate the function of NK cells (e.g., NK cells from mammalian blood, engineered NK cells, cord blood-derived, or iPSC-derived NK cells) expanded using an SF system provided herein in vivo, the NK cells are administered to a mouse cancer model (e.g., a mouse leukemia model, such as NOD scid gamma (NSG) mice intravenously xenografted with 5xlO5-lxlO6luciferase-expressing HL60 promyelocytic leukemia cells, or a mouse myeloma model, such as NSG mice intraperitoneally injected with luciferase-expressing MM. IS cells; or an ovarian cancer model, such as NSG mice intraperitoneally injected with 2xl04luciferase-expressing OVCAR8 tumor cells). NK cells are administered via any appropriate route of administration (e.g., intravenous or intraperitoneal administration in PBS) and at any appropriate dose (e.g., lxl06-lxl07cells / dose, with or without supplemental cytokine delivery, such as IL-15 or IL-2). Control animals are administered NK cells expanded using a feeder cell system. To demonstrate that the cells expanded using the SF system provided herein are as effective as NK cells expanded using a traditional feeder cell expansion system, the persistence of each type of administered NK cells and the extent of tumor regression in the mice are assessed via bioluminescent imaging and blood sampling. In some cases, an in vitro assay employing cytokine starvation of expanded NK cells that mimics in vivo conditions is performed.

[0186] OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for generating an expanded population of NK cells in vitro, wherein said method comprises: administering, to a starting population of NK cells, interleukin-2 (IL-2), an activator of protein kinase C (PKC) and an activator of nuclear factor of activated T-cells (NF AT); culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 3 hours to about 24 hours; and subsequently culturing said population of NK cells without said activator of PKC and said activator of NF AT but in the presence of IL-2 and, optionally, interleukin-21 (IL-21), a 4-1BB agonist, or both IL-21 and a 4-1BB agonist, for a period of time sufficient to expand said population of NK cells by at least 10-fold as compared to said starting population of NK cells.

2. The method of claim 1, wherein said PKC activator comprises one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

3. The method of claim 1, wherein said method comprises administering said PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

4. The method of claim 1, wherein said PKC activator is PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

5. The method of claim 1, wherein said PKC activator is PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

6. The method of claim 1, wherein said NF AT activator comprises a molecule that can increase intracellular calcium levels.

7. The method of claim 6, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor.

8. The method of claim 6, wherein said NF AT activator comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I), or wherein said NF AT activator comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1, and Jedi2, or wherein said NF AT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5- di -t-buty 1 -1,4 -benzohydroquinone (BHQ) .

9. The method of claim 8, wherein said NF AT activator is ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

10. The method of claim 6, wherein said NF AT activator comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2.

11. The method of claim 6, wherein said NF AT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

12. The method of claim 1, wherein said 4-1BB agonist is an anti-4-lBB antibody.

13. The method of claim 1, comprising culturing said population of NK cells for a period of time sufficient to expand said population of NK cells by at least 100-fold.

14. The method of claim 1, comprising culturing said population of NK cells for a period of time sufficient to expand said population of NK cells by at least 1000-fold.

15. The method of claim 1, comprising culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 6 hours.

16. The method of claim 1, culturing said population of NK cells in the presence of said activator of PKC and said activator of NF AT for about 12 hours.

17. The method of claim 1, wherein said period of time is from about 3 days to about 10 days.

18. The method of claim 1, wherein said period of time is from about 4 days to about 8 days.

19. The method of claim 1, wherein said method does not comprise the use of feeder cells.

20. A method for generating an expanded population of NK cells in vitro, wherein said method comprises:(a) administering, to a starting population of NK cells, a first activator of protein kinase C (PKC) and a first activator of nuclear factor of activated T-cells (NF AT);(b) culturing said population of NK cells in the presence of said first activator of PKC and said first activator of NF AT for about 3 hours to about 24 hours;(c) subsequently culturing said population of NK cells without said activator of PKC and said activator of NF AT but in the presence of interleukin-2 (IL-2) and, optionally, interleukin-21 (IL-21), a 4-1BB agonist, or both IL-21 and a 4-1BB agonist, for about 3 days to about 10 days;(d) administering, to said cultured population of NK cells of step (c), a second activator of PKC, a second activator of NF AT, and a Fas ligand (FasL) antagonist;(e) further culturing said population of NK cells in the presence of said second activator of PKC, said second activator of NF AT, and said FasL antagonist for about 3 hours to about 24 hours;(f) subsequently further culturing said population of NK cells without said second activator of PKC and said second activator of NF AT but in the presence of said FasL antagonist, said IL-2, and optionally said IL-21, said 4- IBB agonist, or both said IL-21 and said 4-1BB agonist, for about 3 days to about 10 days to further expand said population of NK cells; and optionally(g) repeating steps (d) to (f), thereby expanding said population of NK cells to a desired number.

21. The method of claim 20, wherein said first PKC activator and said second PKC activator are the same.

22. The method of claim 20, wherein said first PKC activator and said second PKC activator are different.

23. The method of claim 20, wherein said first PKC activator and said second PKC activator comprise one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

24. The method of claim 20, wherein said method comprises administering said first PKC activator and said second PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

25. The method of claim 20, wherein said first PKC activator is PMA, said second PKC activator is PMA, or both said first PKC activator and said second PKC activator are PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

26. The method of claim 20, wherein said first PKC activator is PEP005, said second PKC activator is PEP005, or both said first PKC activator and said second PKC activator are PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

27. The method of claim 20, wherein said first NF AT activator and said second NF AT activator are the same.

28. The method of claim 20, wherein said first NF AT activator and said second NF AT activator are different.

29. The method of claim 20, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a molecule that can increase intracellular calcium levels.

30. The method of claim 29, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor.

31. The method of claim 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

32. The method of claim 31, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator are ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

33. The method of claim 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2.

34. The method of claim 29, wherein said first NF AT activator, said second NF AT activator, or both said first NF AT activator and said second NF AT activator comprise a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

35. The method of claim 20, wherein said 4- IBB agonist is an anti -4- IBB antibody.

36. The method of claim 20, wherein said FasL antagonist is selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti- FasL antibodies.

37. The method of claim 20, wherein said FasL antagonist is an anti-FasL antibody.

38. The method of claim 20, comprising expanding said population of NK cells to a number that is at least 100-fold the number of NK cells in said starting population.

39. The method of claim 20, comprising expanding said population of NK cells to a number that is at least 1000-fold the number of NK cells in said starting population.

40. The method of claim 20, wherein said method does not comprise the use of feeder cells.

41. A method for selectively generating an expanded population of NK cells in vitro, wherein said method comprises:(a) seeding a starting population of cells onto a solid surface in a culture vessel in the presence of interleukin-2 (IL-2) and interleukin-21 (IL-21), wherein said starting population of cells comprises NK cells, and wherein said solid surface has an anti -4- IBB antibody, a B7-H6 polypeptide, or both said anti-4-lBB antibody and said B7-H6 polypeptide attached thereto;(b) culturing NK cells from said starting population in said culture vessel for about 5 days to about 10 days, thereby yielding a selected population of NK cells;(c) administering, to said selected population of NK cells, an activator of protein kinase C (PKC), an activator of nuclear factor of activated T-cells (NF AT), IL-2, and optionally IL-21, a 4-1BB agonist, or both IL-21 and a 4-1BB agonist;(d) culturing said selected population of NK cells in the presence of said activator of PKC, said activator of NF AT, said IL-2, and optionally said IL-21, said 4-1BB agonist, or both IL-21 and said 4-1BB agonist, for about 7 days to about 12 days; and optionally(e) repeating steps (c) and (d) until cell growth stops, thereby selectively generating an expanded population of NK cells.

42. The method of claim 41, wherein said starting population of cells is an enriched NK cell population.

43. The method of claim 41, wherein said starting population of cells is a mixed cell population.

44. The method of claim 41, wherein said PKC activator comprises one or more of PMA, PEP005, prostratin, bryostatin 1, bryostatin 3, phorbol 12,13-dibutyrate (PDBu), and TPPB.

45. The method of claim 41, wherein said method comprises administering said PKC activator at a concentration of about 20 pg / mL to about 1 pg / mL.

46. The method of claim 41, wherein said PKC activator is PMA, and wherein said method comprises administering said PMA at a concentration of about 1 ng / mL to about 100 ng / mL.

47. The method of claim 41, wherein said PKC activator is PEP005, and wherein said method comprises administering said PEP005 at a concentration of about 0.2 ng / mL to about 20 ng / mL.

48. The method of claim 41, wherein said NF AT activator comprises a molecule that can increase intracellular calcium levels.

49. The method of claim 48, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor .

50. The method of claim 48, wherein said NF AT activator comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

51. The method of claim 50, wherein said NF AT activator is ionomycin, and wherein said method comprises administering said ionomycin at a concentration of about 10 ng / mL to about 1 mg / mL.

52. The method of claim 48, wherein said NF AT activator comprises a Piezo- 1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2.

53. The method of claim 48, wherein said NF AT activator comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

54. The method of claim 41, wherein said 4-1BB agonist is an anti-4-lBB antibody.

55. The method of claim 41, comprising culturing said selected population of NK cells for a period of time sufficient to expand said population of NK cells by at least 100-fold.

56. The method of claim 41, comprising culturing said selected population of NK cells for a period of time sufficient to expand said population of NK cells by at least 1000-fold.

57. The method of claim 41, wherein step (c) comprises culturing said selected population of NK cells in the presence of said activator of PKC, said activator of NF AT, said IL-2, said IL-21, and said 4-1BB agonist for about 10 days.

58. The method of claim 41, wherein said method does not comprise the use of feeder cells.

59. An article of manufacture comprising: a vessel containing an activator of PKC, a vessel containing an activator of NF AT, and optionally, one or more of: a vessel containing IL-2, a vessel containing IL-21, a vessel containing a 4- IBB agonist, a vessel containing a FasL agonist, and a solid substrate to which an anti-4-lBB antibody, B7-H6, or another NK cell binding agent is attached.

60. The article of manufacture of claim 59, wherein said activator of PKC and said activator of NF AT are contained in the same vessel.

61. The article of manufacture of claim 59, wherein said activator of PKC and said activator of NF AT are contained in separate vessels.

62. The article of manufacture of claim 59, wherein said activator of PKC comprises one or more of PMA, PEP005, prostratin, bryostatin 3, phorbol 12, 13 -dibutyrate (PDBu), and TPPB.

63. The article of manufacture of claim 59, wherein said activator of NF AT comprises a molecule that can increase intracellular calcium levels.

64. The article of manufacture of claim 63, wherein said molecule is a calcium ionophore, a Piezo-1 activator, or a sarco / endoplasmic reticulum Ca ATPase (SERCA) inhibitor.

65. The article of manufacture of claim 63, wherein said activator of NF AT comprises a calcium ionophore, and wherein said calcium ionophore comprises one or more of ionomycin, A23187 (Calcimycin), 4-Br-A23187, and ETH 1001 (Calcium ionophore I).

66. The article of manufacture of claim 63, wherein said activator of NF AT comprises a Piezo-1 activator, and wherein said Piezo-1 activator comprises one or more of Yodal, Yoda2, Jedi 1 , and Jedi2.

67. The article of manufacture of claim 63, wherein said activator of NF AT comprises a SERCA inhibitor, and wherein said SERCA inhibitor comprises one or more of thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-t-butyl-l,4-benzohydroquinone (BHQ).

68. The article of manufacture of claim 59, wherein said 4-1BB agonist is an anti-4-lBB antibody.

69. The article of manufacture of claim 59, wherein said FasL antagonist is selected from the group consisting of APG101, RG7826, apocept, Kp7-6, soluble Fas polypeptides, decoy receptors, and anti-FasL antibodies.

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