Highly potent m-CENK cells and methods

Abstract

To provide a method for generating memory-like cytokine-enhanced natural killer (M-CENK) cells.SOLUTION: The method comprises obtaining a plurality of mononuclear cells and contacting said plurality of mononuclear cells with a corticosteroid and optionally a cytokine or cytokine analog; incubating said plurality of mononuclear cells in presence of said corticosteroid and said optional cytokine or cytokine analog, thereby enriching said mononuclear cells in NK cells; and inducing said enriched NK cells with TxM fusion proteins to generate said M-CENK cells, wherein said TxM fusion proteins comprise a proteinaceous moiety having IL-12 activity, a proteinaceous moiety having IL-15 activity, and a proteinaceous moiety having IL-18 activity.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] This application claims priority to the applicant's co-pending U.S. Provisional Patent Application No. 63 / 156,269, filed March 3, 2021, and U.S. Provisional Patent Application No. 63 / 217,097, filed June 30, 2021, each of which is incorporated herein by reference in its entirety.

[0002] The field of the invention is cell-based therapeutics and related methods, particularly as it relates to memory-like cytokine-enhanced NK cells (M-CENK) with improved cytotoxicity and expansion properties. [Background technology]

[0003] The background discussion includes information that may be useful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the invention(s) claimed herein, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference contradicts or is contrary to the definition of such term provided herein, the definition of such term provided herein shall apply and the definition of such term in the reference shall not apply.

[0005] Natural killer (NK) cells constitute a group of innate immune cells and are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cellular cytotoxicity by target-directed release of granulysin and perforin. Most NK cells possess a specific cell surface marker profile (e.g., CD3 ) in addition to a diverse collection of activating and inhibitory receptors. - , CD56 + , CD16 + , CD57 + , CD8+ Recently, NK cells have become an important component of certain cancer treatments, but the generation of large numbers of NK cells (and especially autologous NK cells) presents a significant obstacle due to the relatively low proportion of NK cells in whole blood.

[0006] To obtain therapeutically meaningful quantities of NK and NK-like cells, NK cells can be generated from various progenitor cells. For example, various stem cell factors (SCF), FLT3 ligands, interleukin (IL)-2, IL-7, and IL-15 have been reported to induce and expand cytokine-induced killer (CIK) cells from umbilical cord blood in various in vitro methods (Anticancer Research 30:3493-3500 (2010)). Similarly, as reported in U.S. Patent Application Publication No. 2018 / 0044636, CD34 + Hematopoietic cells can be exposed to IL-12 and other agents. In yet other approaches, human hemangioblasts were sequentially exposed to two different cytokine cocktails, as described in WO 2011 / 068896, and different cytokine cocktails were used with post-embryonic hematopoietic stem cells, as taught in WO 2012 / 128622. While at least some of these methods provide for a large n-fold expansion of NK cells, the methods and reagents for such expansion are both time- and financially demanding. Furthermore, it should be noted that many of the known methods require the culture of NK cells on a feeder cell layer, which is problematic from a number of technical and regulatory perspectives.

[0007] In a simpler method, acute myeloid leukemia (AML) cells can be exposed to a TpoR agonist, thereby inducing the AML cells to form NK cells. However, such an approach appears less viable as a source of therapeutic cells. An alternative method also relies on culturing peripheral blood cells in the presence of various interleukins, stem cell factors, and FLT3 ligand, as disclosed in International Publication No. WO 2011 / 103882. Yet another method, U.S. Patent Application Publication No. 2013 / 0295671, teaches stimulating pre-existing NK cells with anti-CD16 and anti-CD3 antibodies in addition to cytokines. While procedurally simpler, such methods still require careful manipulation of cells and the use of specialized reagents, significantly increasing costs.

[0008] In yet another known method, U.S. Patent No. 10,125,351 describes using umbilical cord blood or peripheral blood as a cell source, isolating nucleated cells by subjecting them to density gradient separation, and then culturing them in a medium containing interferon, interleukin, CD3 antibody, and human albumin. Most advantageously, this method is suitable for perfusion culture in a bioreactor, thereby significantly reducing operational problems. Unfortunately, the yield of NK cells is still relatively low.

[0009] Regardless of the specific production method, cultured NK cells typically do not exhibit memory-like properties that are particularly desirable for cancer immunotherapy. In at least some attempts to generate memory-like NK cells, selected NK cells have been exposed to IL-12, IL-15, and IL-18, and the exposed NK cells exhibit a memory-like phenotype, correlated with expression of CD94, NKG2A, NKG2C, and CD69, and loss of CD57 and KIR (see Blood (2012) Vol. 120, No. 24; 4751-4760). Similarly, as described in WO 2018 / 089476 and U.S. Pat. No. 10,300,089, memory-like NK cells have also been prepared by preactivating NK cells with various stimulatory cytokines and subsequently contacting the preactivated cells with PM21 particles, EX21 exosomes, or FC21 feeder cells. Yet another approach to generating memory-like NK cells involved exposing freshly isolated NK cells to an IL-18 / IL-12-TxM fusion protein complex, as described in WO 2018 / 165208. While such methods typically produced NK cells with at least some memory-like properties, the cytotoxicity of such activated NK cells against target cells of choice was still suboptimal, likely due to lack of or low expression of certain activating receptors and / or expression of certain inhibitory receptors.

[0010] Yet another known approach involves generating cytokine-induced memory-like NK cells (CIML NK) in the laboratory using patient blood. However, such methods are typically limited in their utility due to the relatively limited number of CIML NK cells that can be generated. Consequently, multiple samples must be taken from the patient at multiple times to generate a sufficient dose for an entire treatment regimen. Yet another approach to generating CIML NK cells, as described in WO 2021 / 006876, involves activating mononuclear cells from umbilical cord blood or whole blood with anti-CD16 antibodies and N-803, followed by expansion with a cytokine cocktail. While conceptually simple, various challenges remain, including the presence of CD3+ cells and suboptimal cytotoxicity against at least some target cells.

[0011] Alternatively, NK cells can be isolated from apheresis products using beads, and then these isolated cells are directly induced to produce the CIML phenotype. While this approach allows for the reduction of CD3+ cells in NK cell preparations, it also limits the expansion potential of the cells. Furthermore, it is unclear whether such cells can be frozen and later thawed back into a therapeutically effective cell preparation. Finally, such isolated and induced NK cells tend to have relatively low potential cytotoxicity. Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, while various systems and methods for targeted antiviral therapy and vaccines are known in the art, all or nearly all suffer from several drawbacks. Among other challenges, many CIML NK cell preparations yield only limited numbers of cells and may therefore not be therapeutically effective. Moreover, the cost and inconsistent activity of cytokine mixtures, particularly when the memory phenotype is induced with multiple, distinctively distinct cytokines, can hinder the reproducibility of therapeutic formulations with predictable activity. Thus, there remains a need for improved compositions and methods for NK cell-based therapies, particularly memory-like cytokine-enhanced NK cell-based compositions and methods. [Means for solving the problem]

[0013] The present invention relates to M-CENK cells, various compositions and methods for their generation and expansion, and various uses thereof. Remarkably, the M-CENK cells as presented herein possess excellent cytotoxicity, allowing for rapid and substantial expansion, and they also exhibit therapeutic activity after cryopreservation. Most advantageously, the M-CENK cells can be prepared in desired quantities from mononuclear-derived cytokine-enhanced NK cells in a simple and effective manner, and induction of the M-CENK phenotype is achieved with a single protein complex, preferably TxM, with IL-12 / IL-15 / IL-18 activity.

[0014] In one aspect of the inventive subject matter, the inventors contemplate a method of generating memory-like cytokine-enhanced natural killer (M-CENK) cells, the method comprising the steps of obtaining a plurality of mononuclear cells and another step of contacting the plurality of mononuclear cells with a corticosteroid and optionally a cytokine. In yet another step, the plurality of mononuclear cells is incubated in the presence of a corticosteroid and optional cytokine to enrich for the mononuclear cells in NK cells, and then the enriched NK cells are induced with a TxM fusion protein to generate M-CENK cells, wherein the TxM fusion protein comprises a protein portion having IL-12 activity, a protein portion having IL-15 activity, and a protein portion having IL-18 activity.

[0015] In some embodiments, the plurality of mononuclear cells are cryopreserved prior to the incubating step. In such embodiments, the cryopreserved mononuclear cells are preferably thawed and washed in a medium containing a corticosteroid and optional cytokines. It is further contemplated that the incubating step is carried out for 14 to 21 days and / or until NK cells are enriched to at least 65% of the total viable cells. Additionally, it is contemplated that the enriched NK cells are induced with TxM at a concentration of 1 to 25 μg / mL, typically for 12 to 16 hours.

[0016] Although not limiting the inventive subject matter, it is generally preferred that the corticosteroid be hydrocortisone and the optional cytokine be N-803. Moreover, it is also preferred that the optional cytokine be included in the incubating step. Contemplated methods will typically include the steps of harvesting M-CENK cells and formulating the harvested M-CENK cells for infusion. If desired, the harvested M-CENK cells are cryopreserved prior to infusion. In a further aspect of the inventive subject matter, the incubating step is performed in an automated bioreactor.

[0017] Consequently, the inventors also contemplate a method of producing memory-like cytokine-enhanced natural killer (M-CENK) cells, comprising the steps of obtaining a plurality of monocyte-derived cytokine-enhanced NK cells (CENK), and the further step of inducing the enriched NK cells with a TxM fusion protein such that M-CENK cells are generated, wherein the TxM fusion protein comprises a protein portion having IL-12 activity, a protein portion having IL-15 activity, and a protein portion having IL-18 activity.

[0018] Alternatively, the inventors also contemplate memory-like cytokine-enhanced natural killer (M-CENK) cells produced by the methods as provided herein. Most typically, the cells will be included in a pharmaceutical composition with a pharmaceutically acceptable carrier that, in some embodiments, is or comprises a cryopreservation medium. Furthermore, the pharmaceutically acceptable carrier will generally be formulated for injection and / or administered in amounts of 0.5-1.5 x 10 7 It is contemplated that the cell density may be in the range of 1000 to 10000 cells / mL.

[0019] In yet another embodiment, the inventors contemplate a method of treating an individual with cancer, comprising administering the cells and compositions provided herein. Accordingly, compositions for use in treating cancer are also contemplated. In one embodiment, the M-ceNK cells provided herein can be used to kill cancer stem cells and mesenchymal cells, and can be administered to a patient for this purpose.

[0020] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, as well as from the figures in the accompanying drawings, in which like numerals represent like elements. [Brief explanation of the drawings]

[0021] [Figure 1]CD56+CD3-M-CENK cell enrichment is depicted in a bivariate dot plot. Activation of apheresis material with N-803 and hydrocortisone resulted in significant enrichment of CD56+CD3-M-CENK cells. [Figure 2] 1 depicts M-CENK enrichment kinetics from the same apheresis product lot that are comparable when thawed for different days. [Figure 3] 1 shows exemplary results of phenotyping of M-CENK cells. [Figure 4] 1 depicts exemplary results of cell health markers for M-CENK cells upon harvesting. [Figure 5] Exemplary results are depicted that demonstrate potent cytotoxicity against tumor cells. The cytotoxicity of M-CENK cells against two target cells, K562 and MS-1 cells, was tested using a calcein AM-based cytotoxicity assay. M-CENK cells from different donors exhibited cytotoxicity in the range of 60-80% against the NK-resistant cell line MS-1 at an E:T ratio of 20:1. [Figure 6] Figure 1 shows comparable results for CD56 and IFN-gamma expression for M-CENK cells made from the same lot of apheresis material, but thawed at different times. [Figure 7] 1 depicts exemplary cytotoxic activity of M-CENK cells against a set of target tumor cell lines. [Figure 8] 1 depicts exemplary activity of M-CENK cells against a set of target tumor cell lines. [Figure 9] 1 depicts exemplary activity of M-CENK cells against a set of target tumor cell lines. [Figure 10] 1 depicts exemplary IFN-γ expression in M-CENK cells. [Figure 11] 1 depicts exemplary cell viability results for M-CENK cells according to the present subject matter. [Figure 12] 1 depicts further exemplary cell health results for M-CENK cells according to the present subject matter. [Figure 13]1 depicts exemplary cytotoxicity results for M-CENK cells against MS-1 cells according to the present subject matter. [Figure 14] 1 depicts exemplary cytotoxicity results for M-CENK cells against K562 cells according to the present subject matter. [Figure 15A] 1 illustrates a schematic depiction of an exemplary TxM. [Figure 15B] Draw the TxM sequence. [Figure 16] 1 is a graph depicting efficient IFN-gamma production by TxM-induced M-CENK cells. [Figure 17] 1 is a graph depicting potent killing of NK-resistant MS-1 cells by TxM-induced M-CENK cells. [Figure 18] 1 depicts the results of comparing cell killing of NK-resistant MS-1 cells by cytokine cocktail-induced M-CENK cells compared to TxM induction. [Figure 19] 1 depicts the results of comparing cell killing of K562 cells by cytokine cocktail-induced M-CENK cells compared to TxM induction. [Figure 20] 1 depicts the comparative results of IFN-gamma production in cytokine cocktail-induced M-CENK cells compared to TxM induction. [Figure 21] 1 depicts further comparison of NK-specific markers in cytokine cocktail-induced M-CENK cells compared to TxM induction. [Figure 22] Further comparison of memory cell phenotypes in cytokine cocktail-induced M-CENK cells compared with TxM induction is shown. [Figure 23] 1 illustrates the lysis of small cell lung cancer by NK cells. [Figure 24] 1 illustrates lysis of ovarian cancer by NK cells. [Figure 25] Illustrating NK cell lysis of breast cancer and NSCLC [Figure 26] Illustrates the CD56 / CD16 profile of healthy donor NK cells compared to ImmunityBio NK cells. [Figure 27]1 illustrates that ceNK and M-ceNK cells express the activating receptors NKp30, NKp44, and NKG2D to a greater extent. [Figure 28] NK activating receptor expression is illustrated. [Figure 29] 1 illustrates NK intracellular protein expression. [Figure 30] NK inhibitory receptor expression is illustrated. [Figure 31] 1 illustrates an outline of the M-CENK-DS manufacturing process using the NANT 001 bioreactor. [Figure 32] An example of the M-CENK production flow process is shown below. [Figure 33] The efficacy of M-CENK lots produced by the above process is illustrated. [Figure 34] 1 illustrates the M-CENK surface phenotype. [Figure 35] This demonstrates that M-CENK is a potent cancer cell killer. [Figure 36] Illustrates the stability of apheresis material intermediates in LN2. [Figure 37] 1 illustrates the stability of cryopreserved M-CENK cell preparations. [Figure 38] 1 illustrates a comparison of M-CENK production from healthy donors and patients. [Figure 39] Presenting the Phase 1 Protocol for Clinical Trial QUILT-3.076 (A Study of Autologous M-CENK in Subjects with Locally Advanced or Metastatic Solid Tumors) [Figure 40-1]This figure demonstrates that the novel fusion protein superkine (18 / 12 / TxM), which combines IL-15, IL-12, and IL-18, induces signaling through all target cytokine receptors. (A) Schematic diagram showing the structure of the 18 / 12 / TxM molecule. (B-D) Freshly isolated NK cells from three to five healthy donors were stimulated with IL-12 (10 ng / mL), IL-15 (50 ng / mL), or IL-18 (50 ng / mL) (IL12 / 15 / 18) or 18 / 12 / TxM (38.8 nM) and evaluated at various time intervals by gating on CD56bright and CD56dim NK cells. Summary data showing the fold change in phosphorylated (B) STAT5, AKT, and ERK, (C) STAT4, or (D) p65 after stimulation. Data shown are means ± SEM, and comparisons were performed using paired t-tests. N = 3-5 human donors. (E-G) Evaluation of individual cytokine activity was performed using reporter cell lines. (E) Proliferation of the IL-15-dependent 32β cell line was assessed 3 days after incubation with various concentrations of 18 / 12 / TxM or N-803. (F) Bioactivity was measured after incubation with various concentrations of IL-12 or 18 / 12 / TxM with HEK12 cells. (G) Bioactivity was measured after incubation with various concentrations of IL-18 or 18 / 12 / TxM with HEK18 cells. [Figure 40-2]This figure demonstrates that the novel fusion protein superkine (18 / 12 / TxM), which combines IL-15, IL-12, and IL-18, induces signaling through all target cytokine receptors. (A) Schematic diagram showing the structure of the 18 / 12 / TxM molecule. (B-D) Freshly isolated NK cells from three to five healthy donors were stimulated with IL-12 (10 ng / mL), IL-15 (50 ng / mL), or IL-18 (50 ng / mL) (IL12 / 15 / 18) or 18 / 12 / TxM (38.8 nM) and evaluated at various time intervals by gating on CD56bright and CD56dim NK cells. Summary data showing the fold change in phosphorylated (B) STAT5, AKT, and ERK, (C) STAT4, or (D) p65 after stimulation. Data shown are means ± SEM, and comparisons were performed using paired t-tests. N = 3-5 human donors. (E-G) Evaluation of individual cytokine activity was performed using reporter cell lines. (E) Proliferation of the IL-15-dependent 32β cell line was assessed 3 days after incubation with various concentrations of 18 / 12 / TxM or N-803. (F) Bioactivity was measured after incubation with various concentrations of IL-12 or 18 / 12 / TxM with HEK12 cells. (G) Bioactivity was measured after incubation with various concentrations of IL-18 or 18 / 12 / TxM with HEK18 cells. [Figure 41-1]Short-term activation with the 18 / 12 / TxM superkine activates NK cells, resulting in the induction of IFN-γ and CD25 expression and increased cytotoxicity. (A-G) Freshly isolated NK cells were activated with increasing concentrations of 18 / 12 / TxM or IL-12 (10 ng / mL) + IL-15 (50 ng / mL) + IL-18 (50 ng / mL) for 16 hours, and the expression of the indicated markers was assessed. (A) Representative flow plots showing IFN-γ and CD25 expression. (B) NK cells were incubated with various concentrations of 18 / 12 / TxM to identify the optimal concentration for maximal CD25 induction. (C, D) Summary data for NK cells stimulated with 38.8 nM 18 / 12 / TxM for 16 hours. (C) Percent CD25-positive NK cell rate and (D) CD25 expression shown as CD25 MFI. (E) NK cells were incubated with various concentrations of 18 / 12 / TxM to identify the optimal concentration for maximal IFN-γ induction. (F, G) Summary data for NK cells stimulated with 38.8 nM 18 / 12 / TxM for 16 hours. (F) Percent IFN-γ positive NK cell rate and (G) IFN-γ expression shown as IFN-γ mfi. Data were compared using RM one-way ANOVA (*p<0.05, ****p<0.0001). (n=6 donors, two independent experiments). [Figure 41-2]Short-term activation with the 18 / 12 / TxM superkine activates NK cells, resulting in the induction of IFN-γ and CD25 expression and increased cytotoxicity. (A-G) Freshly isolated NK cells were activated with increasing concentrations of 18 / 12 / TxM or IL-12 (10 ng / mL) + IL-15 (50 ng / mL) + IL-18 (50 ng / mL) for 16 hours, and the expression of the indicated markers was assessed. (A) Representative flow plots showing IFN-γ and CD25 expression. (B) NK cells were incubated with various concentrations of 18 / 12 / TxM to identify the optimal concentration for maximal CD25 induction. (C, D) Summary data for NK cells stimulated with 38.8 nM 18 / 12 / TxM for 16 hours. (C) Percent CD25-positive NK cell rate and (D) CD25 expression shown as CD25 MFI. (E) NK cells were incubated with various concentrations of 18 / 12 / TxM to identify the optimal concentration for maximal IFN-γ induction. (F, G) Summary data for NK cells stimulated with 38.8 nM 18 / 12 / TxM for 16 hours. (F) Percent IFN-γ positive NK cell rate and (G) IFN-γ expression shown as IFN-γ mfi. Data were compared using RM one-way ANOVA (*p<0.05, ****p<0.0001). (n=6 donors, two independent experiments). [Figure 42]These results demonstrate that activation with the 18 / 12 / TxM superkine stimulates NK cell proliferation, similar to IL12 / 15 / 18. To track cell division, purified NK cells were labeled with CFSE and activated for 16 hours with either LD IL15 (1 ng / mL IL-15), IL12 / 15 / 16 (10 ng / mL IL12 + 50 ng / mL IL-15 + 50 ng / mL IL-18), or 38.8 nM 18 / 12 / TxM. After incubation, cells were washed three times to remove the preactivation cytokines and cultured with LD IL15. After 7 days, cells were analyzed for CFSE dilution. (A) Representative bivariate plots of both CD56bright and CD56dim NK cells demonstrating cell division (CFSE dilution) by 18 / 12 / TxM and IL12 / 15 / 18. (B) Summary results showing enhanced proliferation of both CD56bright and CD56dim NK cells 7 days after activation with 18 / 12 / TxM or IL12 / 15 / 18 compared to low-dose IL-15 controls. Summary results are shown as mean ± SEM of percentage of cells per generation (n = 4 donors, 2 independent experiments). Comparisons were made between conditions separately using one-way repeated measures ANOVA. Purified NK cells were >95% CD56+CD3-, and <0.5% CD3+ T cells. *P < 0.05; **P < 0.01; **P < 0.001. [Figure 43]This figure illustrates the comparable multidimensional phenotypic changes in NK cells after activation with 18 / 12 / TxM and IL12 / 15 / 18. Mass cytometry analysis reveals similar changes in NK cell phenotype. Freshly isolated human NK cells were activated with 18 / 12 / TxM or IL12 / 15 / 18 for 16 hours, and the expression of 36 markers was assessed using mass cytometry at baseline or 1 or 6 days after activation. (A) Representative viSNE maps from one donor showing NK cell populations at baseline and 1 or 6 days after activation with IL12 / 15 / 18 or 18 / 12 / TxM. Overlay of these populations demonstrates that population-level changes are similar between activation conditions. (B-C) Data are reported as the mean log fold change in median expression over baseline at (B) 1 day or (C) 6 days after activation. (n = 2 donors, 1 independent experiment). R-squared values ​​were determined by simple linear regression. [Figure 44-1] This figure illustrates that 18 / 12 / TxM induces functional memory-like NK cells in vitro. (A) Functional assay scheme. Briefly, NK cells from healthy donors were isolated and activated with LD IL15, 18 / 12 / TxM, or IL12 / 15 / 18 for 16 hours, washed with 1 ng / mL IL-15, and incubated for 1 week. Functional assessment was performed by stimulating NK cells with K562 cells (5:1 E:T ratio) or IL-12 and IL-15, and the indicated markers were evaluated by flow cytometry (n = 9 donors, 3 independent experiments). (B) Representative flow plots showing IFN-γ induction in NK cells stimulated with K562 and IL12 + IL15. (C) Summary data showing the percent IFN-γ-positive NK cells stimulated with K562 or IL-12 / 15 as mean ± SEM. Analysis was performed using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). N=15 unique human donors, 6 independent experiments. (D) Percent specific killing measured by chromium release assay after incubation with K562 cells at various E:T ratios (n=4 donors, 2 independent experiments). Analysis was performed using two-way ANOVA. [Figure 44-2] This figure illustrates that 18 / 12 / TxM induces functional memory-like NK cells in vitro. (A) Functional assay scheme. Briefly, NK cells from healthy donors were isolated and activated with LD IL15, 18 / 12 / TxM, or IL12 / 15 / 18 for 16 hours, washed with 1 ng / mL IL-15, and incubated for 1 week. Functional assessment was performed by stimulating NK cells with K562 cells (5:1 E:T ratio) or IL-12 and IL-15, and the indicated markers were evaluated by flow cytometry (n = 9 donors, 3 independent experiments). (B) Representative flow plots showing IFN-γ induction in NK cells stimulated with K562 and IL12 + IL15. (C) Summary data showing the percent IFN-γ-positive NK cells stimulated with K562 or IL-12 / 15 as mean ± SEM. Analysis was performed using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). N=15 unique human donors, 6 independent experiments. (D) Percent specific killing measured by chromium release assay after incubation with K562 cells at various E:T ratios (n=4 donors, 2 independent experiments). Analysis was performed using two-way ANOVA. [Figure 45-1]Figure 1 illustrates the induction of ML NK cell molecular programs by 18 / 12 / TxM activation. (A-D) Day 1 and (E-F) Day 6 after 16 hours of activation with low-dose IL-15, IL12 / 15 / 18, or 18 / 12 / TxM. (A) Venn diagram demonstrating the number of genes with statistically significant differences in expression (p<0.05) shared (purple) and characteristically distinct genes between low-dose and 18 / 12 / TxM (red) and IL12 / 15 / 18 (blue) activated NK cells on day 1. (B) Scatter plot comparing the log2 (fold change) of genes induced after IL12 / 15 / 18 or 18 / 12 / TxM activation. (C-D) Volcano plots showing the number of genes with differential expression between low-dose IL15 and (C) 18 / 12 / TxM or (D) IL12 / 15 / 18 on the day after activation. (E) Scatter plot showing the log2 (fold change) of genes induced after IL12 / 15 / 18 or 18 / 12 / TxM activation, filtered to show genes with a log2 fold change greater than 1 or less than -1. (F) Scatter plot showing similar gene induction between conditions on day 6. RNA sequencing analysis was performed using Phantasus. Differential gene expression analysis was performed using the LIMMA package. N = 3 different donors per condition. Data shown are representative from two independent experiments with 3 donors each. [Figure 45-2]Figure 1 illustrates the induction of ML NK cell molecular programs by 18 / 12 / TxM activation. (A-D) Day 1 and (E-F) Day 6 after 16 hours of activation with low-dose IL-15, IL12 / 15 / 18, or 18 / 12 / TxM. (A) Venn diagram demonstrating the number of genes with statistically significant differences in expression (p<0.05) shared (purple) and characteristically distinct genes between low-dose and 18 / 12 / TxM (red) and IL12 / 15 / 18 (blue) activated NK cells on day 1. (B) Scatter plot comparing the log2 (fold change) of genes induced after IL12 / 15 / 18 or 18 / 12 / TxM activation. (C-D) Volcano plots showing the number of genes with differential expression between low-dose IL15 and (C) 18 / 12 / TxM or (D) IL12 / 15 / 18 on the day after activation. (E) Scatter plot showing the log2 (fold change) of genes induced after IL12 / 15 / 18 or 18 / 12 / TxM activation, filtered to show genes with a log2 fold change greater than 1 or less than -1. (F) Scatter plot showing similar gene induction between conditions on day 6. RNA sequencing analysis was performed using Phantasus. Differential gene expression analysis was performed using the LIMMA package. N = 3 different donors per condition. Data shown are representative from two independent experiments with 3 donors each. [Figure 46]This figure illustrates that 18 / 12 / TxM induces functional memory-like NK cells in vivo, with antitumor activity comparable to that of NK cells induced by IL12 / 15 / 18. Experimental design for (A), (B), and (C). NSG mice were intravenously injected with 1 × 10 K562-luciferase cells. Three days later, BLI was performed to confirm leukemia engraftment. On day 4, mice were administered retro-orbitally with either control (no NK cells) or 5 × 10 NK cells activated with low-dose IL-15, IL12 / 15 / 18, or 18 / 12 / TxM. Mice were treated with rhIL-2 every other day (every other data), and tumor burden (BLI) was monitored. (B) Representative BLI of recipient mice engrafted with K562-luc at the indicated days after tumor challenge. (C) Summary of BLI measurements over time showing tumor burden reduction in mice receiving NK cells activated with IL12 / 15 / 18 and 18 / 12 / TxM. Data are presented as mean ± SEM. Summary data are from two independent experiments with 9-10 mice per group. Differences were determined using two-way analysis of variance (2-way ANOVA). *P<0.05; **P<0.01; **P<0.001. [Figure 47] This figure illustrates that 77.6 nM of 18 / 12 / TxM induces signaling through all target receptors. (A-C) Freshly isolated NK cells from three to five healthy donors were stimulated with IL-12 (10 ng / mL), IL-15 (50 ng / mL), and IL-18 (50 ng / mL) or 18 / 12 / TxM (77.6 nM), and CD56 bright and CD56 dim NK cells were assessed at various time intervals. (A) Phosphorylation of signaling mediators downstream of IL-15 signaling: STAT5, pAKT, and pERK. (B) Phosphorylation of pSTAT4 downstream of IL-12 signaling. (C) Phosphorylation of p65 downstream of IL-18 signaling. Summary data were compared using paired t-tests. [Figure 48]This figure illustrates the lack of difference in NK cell viability between activation conditions. Freshly isolated human NK cells were activated with either LD IL-15 (1 ng / mL), IL12 / 15 / 18, or 18 / 12 / TxM for 16 hours and cultured in LD IL-15 for 7 days. Viability was assessed by measuring the percentage of Zombie-Green-negative NK cells by flow cytometry. N = 5 human donors, two independent experiments. Statistical analysis was performed using one-way ANOVA (*P < 0.05). [Figure 49-1] Illustrates phenotypic differences in NK cells at baseline, day 1, and day 6 after activation with IL-12 / 15 / 18 or 18 / 12 / TxM. (A) Summary data demonstrating median expression of the indicated markers from FIG. 43. [Figure 49-2] Illustrates phenotypic differences in NK cells at baseline, day 1, and day 6 after activation with IL-12 / 15 / 18 or 18 / 12 / TxM. (A) Summary data demonstrating median expression of the indicated markers from FIG. 43. [Figure 49-3] Illustrates phenotypic differences in NK cells at baseline, day 1, and day 6 after activation with IL-12 / 15 / 18 or 18 / 12 / TxM. (A) Summary data demonstrating median expression of the indicated markers from FIG. 43. [Figure 50-1]This figure illustrates the induction of functional memory-like cells in vitro by 18 / 12 / TxM. Functionality was assessed as described in Figure 5 and evaluated for (A-C) CD107a and (D-F) TNF expression. (A) Representative flow plots showing CD107a induction in NK cells stimulated with K562 and IL-12+IL-15. (B-C) Summary data showing the percent CD107a-positive NK cell rate after stimulation with (B) K562 or (C) IL-12+IL-15. (D) Representative flow plots showing TNF induction in NK cells stimulated with K562 and IL-12+IL-15. (D-F) Summary data showing the percent TNF-positive NK cell rate after stimulation with (E) K562 or (F) IL-12+IL-15. (n = 15 donors, 7 independent experiments). Analysis was performed using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 50-2] This figure illustrates the induction of functional memory-like cells in vitro by 18 / 12 / TxM. Functionality was assessed as described in Figure 5 and evaluated for (A-C) CD107a and (D-F) TNF expression. (A) Representative flow plots showing CD107a induction in NK cells stimulated with K562 and IL-12+IL-15. (B-C) Summary data showing the percent CD107a-positive NK cell rate after stimulation with (B) K562 or (C) IL-12+IL-15. (D) Representative flow plots showing TNF induction in NK cells stimulated with K562 and IL-12+IL-15. (D-F) Summary data showing the percent TNF-positive NK cell rate after stimulation with (E) K562 or (F) IL-12+IL-15. (n = 15 donors, 7 independent experiments). Analysis was performed using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 51]The NL call phenotypic mass cytometry panel is illustrated. The metal isotope, marker name, antibody clone, and supplier for this mass cytometry phenotypic panel are shown. The asterisk (*) after the supplier indicates that the antibody was custom conjugated using the Fluidigm antibody labeling kit according to the manufacturer's instructions. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present inventors have discovered that it is possible to generate M-CENK cells with superior cytotoxicity that can be expanded to desired quantities and cryopreserved and thawed without loss of functional properties. Notably, in contrast to protocols for generating CIML NK cells, this method does not require the use of anti-CD16 antibodies to activate NK cells in a mononuclear cell mixture. In contrast, the method presented herein uses hydrocortisone, preferably in combination with N-803 (or IL-15) and human AB serum. Furthermore, it was unexpectedly observed that enrichment and expansion can be performed from cryopreserved apheresis material, and that the M-CENK cells can also be cryopreserved and thawed without loss of cytotoxicity.

[0023] Accordingly, the present inventors contemplate M-CENK (memory-like cytokine-enhanced NK cells) and methods for their production, as well as cell-based therapeutics comprising such cells, particularly cryopreserved M-CENK suspensions for infusion. Viewed another way, it should be appreciated that selective enrichment and expansion of NK cells from (thawed) patient apheresis material can be achieved by including hydrocortisone (and typically N-803 or other cytokines or cytokine analogs with IL-15-like activity) in the growth medium, thereby producing high-quality NK cells from the apheresis product. The cells thus obtained are then activated to produce a memory phenotype. Consequently, it should be appreciated that multiple doses of high-quality M-CENK cells can be prepared from cryopreserved material without repeated blood draws from the donor. Indeed, M-CENK cells produced by the process presented herein can be cryopreserved in custom-made media for preservation, and the freeze-thaw procedures developed for cryopreservation ensure maintenance of cell characteristics and viability, as described in more detail below.

[0024] For example, in one step of a contemplated method, a cryopreserved apheresis material intermediate is prepared as follows: leukocyte apheresis product (MNC, apheresis) from a patient is processed and cryopreserved as apheresis material intermediate (AMI) to enable the production of M-CENK product. The cryopreservation medium is formulated to ensure high viability of the apheresis product after thawing. Most preferably, the cryopreservation medium contains PlasmaLyte A, 5% albumin (human) USP, and DMSO. The freshly prepared medium is filter-sterilized using a 0.2 μm PES filter unit. The cryopreservation medium is mixed with the MNC apheresis product in a 1:1 ratio (by volume), thus generating the AMI. The formulated product is loaded into separate cryobags. 10-20 bags can be produced from each apheresis product. The loaded cell bags are then stored frozen at or below -85°C using a controlled-rate freezer (e.g., a CryoMed freezer).

[0025] Enrichment and expansion of cytokine-enhanced NK cells can then be performed as follows: Cryopreserved apheresis material intermediate (AMI) is thawed in a 37°C water bath and used for expansion. The thawed cells are washed, for example, using a Sepax C-Pro device or a tabletop centrifuge, and resuspended in growth medium consisting of NK-MACS medium containing 50-100 ng / mL N-803, 0.2-2.0 μM hydrocortisone, and human AB serum. A strategy has been developed in which, by adding growth medium, NK cells can be successfully enriched and expanded for 20 days. For example, Figure 1 shows an exemplary CD56+CD3 - Cell enrichment is depicted in a bivariate dot plot. As can be easily seen from this plot, activation of the apheresis material with N-803 and hydrocortisone resulted in increased cell enrichment in CD56+CD3 - Significant enrichment of CENK cells was obtained. Figure 2 shows that virtually identical CENK enrichment kinetics can be achieved from the same apheresis product lot when thawed at different days (here: 15 days compared to 380 days).

[0026] Generation of M-CENK cells was then performed as follows: by day 20 of expansion, the cultures had a population of at least 1 × 10 6 When the culture reaches a density of at least 85% CD56-positive cells at 100-300 ng / mL, N-803 (100-300 ng / mL), IL-12 (1-100 ng / mL), and IL-18 (5-250 ng / mL) are added. The cells are stimulated with the cytokine cocktail for 14-16 hours, thereby inducing a memory-like phenotype in CD56-positive cells. After the M-CENK induction step is complete, the cells are washed using a Sepax C-Pro device. Most typically, a 5% albumin (human) solution is used as the wash and resuspension solution. For cryopreservation, M-CENK cells are formulated in medium containing 5% albumin (human) USP:CryoStor 10 (CS10) (1:1). The ability of M-CENK to kill cancer cell targets is enhanced through its increased IFN-γ production. In addition, these cells are phenotypically CD56+, CD25+, DNAM-1+, and NKP30+, NKG2D+, NKG2A+, and CD3-.

[0027] Figure 3 provides exemplary results of phenotyping of M-CENK cells generated in accordance with the present subject matter, where NK markers included DNAM-1, CD25, NKG2A, TIGIT, NKp30, CD16, and NKG2D. Furthermore, the cells so generated also had high viability / survival, as can be seen from the data in Figure 4. Similarly, freezing and thawing had no adverse effect on IFN-γ secretion, as depicted in Figure 6.

[0028] With regard to cytotoxicity, the inventors observed that the M-CENK cells possessed excellent cytotoxic activity against a variety of target cells, even against MS-1 cells (see Figure 5), which are known to be resistant to killing by NK cells, at favorable effector-to-target ratios. Figures 7-9 depict further examples of the cytotoxicity of M-CENK cells against a wide variety of cancer cells. It should be appreciated, therefore, that the improved NK cell-based therapeutic formulation (M-CENK™, a suspension for injection, cryopreserved) can be easily prepared and used, even after prolonged cryopreservation.

[0029] It should be understood, of course, that numerous alternative methods or formulations can be used to freeze the apheresis material, or the apheresis material can be unfrozen or fresh material can be combined with previously frozen material prior to the enrichment and expansion steps. Similarly, it is contemplated that NK cells can also be first purified from whole blood, umbilical cord blood, or apheresis material and then subjected to expansion. Such expanded cells can then be activated to achieve a memory phenotype. Similarly, while hydrocortisone is generally preferred for the enrichment and expansion steps, it should be understood that numerous hydrocortisone analogs and other corticosteroids (e.g., cortisol, corticosterone, cortisone, aldosterone, etc.) are also considered suitable for use herein.

[0030] In addition, it should be noted that M-CENK cells can be frozen using a variety of cryopreservation media, and any known cryopreservation media is deemed suitable for use herein. It is also noted that with respect to the cryopreservation process, enriched and expanded NK cells can be frozen and, upon thawing, subjected to activation for generation of a memory phenotype. [Example]

[0031] manufacturing process Overview of an Exemplary Manufacturing Process: The manufacturing process begins with receiving autologous leukapheresis product (MNC, apheresis), which is processed and cryopreserved as apheresis material intermediate (AMI) to enable on-demand production. After thawing, the AMI is processed for media exchange using Sepax C-Pro, first removing the frozen formulation medium with Plasmalyte A, and then eluting it into NK-GM containing 10% human AB serum, N-803, and hydrocortisone, which is then seeded into a NANT 001 bioreactor for expansion and enrichment into CENK. When the desired number and purity of CENK cells are generated, cytokine-induced memory-like (CIML)-NK cells are generated as M-CENK by treating the cells with a cytokine cocktail containing N-803, IL-12, and IL-18 cytokines. After induction, cells were harvested, concentrated, and washed with 5% albumin (human) using Sepax C Pro, eluted in 5% albumin (human), and then diluted to a total of approximately 0.25-0.75 x 10 in a 100 mL volume. 9 The desired VCD (0.25–0.75 × 10) for M-CENK cells / bag 7 Mix with CryoStor 10 (CS10) at a 1:1 ratio to give a total volume of 1000 cells / mL and freeze for storage.

[0032] Apheresis to obtain peripheral blood mononuclear cells: Autologous M-CENK cell manufacturing begins upon receipt of fresh apheresis material at the manufacturing facility. After completion of chain of custody documentation, samples are aseptically removed from the apheresis transport pack, allowing for determination of cell viability, total nucleated cell (TNC) count, and phenotypic characteristics.

[0033] Cryopreservation of Apheresis Material Intermediate (AMI): Cryopreservation and processing steps were involved from receipt of apheresis material at the time of manufacture to thawing of patient-specific mononuclear cells (MNCs) and apheresis material intermediate (AMI). The AMI cryopreservation process begins with determining the total nucleated cell (TNC) count and percent viability of the fresh MNC apheresis product.

[0034] The cryoformulation medium is freshly prepared, filter-sterilized using a 0.2 μm PES filter unit, and stored on ice until use. The cryoformulation medium is prepared using a mixture of PlasmaLyte A and 5% albumin (human) USP, and DMSO. The apheresis material is transferred to an Erlenmeyer flask and adjusted to the desired cell density. The cryoformulation medium is mixed with the MNC apheresis product in a 1:1 ratio, and the cells are formulated to produce apheresis material intermediate (AMI). The formulated product is then diluted to the desired cell count (2–10 × 10). 8 The apheresis material is then packed into separate cryobags to achieve a volume of 1000 cells. Several bags (10–20) are produced from each apheresis material. The packed cell bags are then frozen at or below −85°C using a controlled-rate freezer (CryoMed freezer) and then transferred to a vapor-phase liquid nitrogen (−120°C or below) freezer for long-term storage.

[0035] Composition of NK growth medium: The basal medium used for the production of M-CENK is the designated NK growth medium (NK-GM). The medium is prepared at the beginning of each study, then sterile-filtered using a 0.2 μm PES filter unit and stored on ice until use.

[0036] Throughout this process, various media supplements are added aseptically at specific steps. (1) At the time of inoculation into the NANT 001 bioreactor, thawed apheresis material intermediate (AMI) is suspended in NK-GM containing 50–100 ng / mL N-803 (0.8 nM) and 0.2–2.0 μM hydrocortisone. (2) During the subsequent media addition step, NK-GM containing 50–100 ng / mL N-803 is added to the bioreactor. (3) Stimulation of the memory phenotype is achieved by the addition of NK-GM containing a cytokine cocktail [N-803 (100–300 ng / mL), IL-12 (1–100 ng / mL), and IL-18 (5–250 ng / mL)]. Before each medium addition step, the desired amount of NK-GM is mixed with a fixed concentration of cytokines / supplements (N-803 + hydrocortisone, or N-803 alone, or N-803 + IL-12 + IL-18), and then filter-sterilized using a 0.2 μm PES filter unit. The prepared medium is aseptically transferred to NANT 001 for cell expansion / stimulation.

[0037] CENK Expansion Using the NANT 001 Platform: An exemplary overview of the M-CENK manufacturing process is provided below. The NANT 001 Bioreactor Platform System (ImmunityBio, Inc.) is a self-contained bioreactor that runs preprogrammed protocols that direct automated procedures and real-time monitoring throughout the harvest, concentration, expansion, and M-CENK derivation stages of the manufacturing process. Programmable process parameters include pH monitoring, cell imaging, temperature, and agitation parameters. The NANT 001 bioreactor includes a thermostatic chamber, a touchscreen user interface, a barcode reader, a pH estimation unit, an integrated imaging system, and gas flow control. The components are designed as a single-use, closed-system, easily loaded for safe, GMP-compliant cell processing. They include a waste bag (up to 4 L), sterile disconnects, a harvest bottle, two auxiliary bags (up to 100 mL each), sterile connections, a cell culture flask, a 636 cm2 flask, a medium bag (up to 3 L), and a buffer bag (up to 3 L). Exemplary systems suitable for use herein are described, for example, in U.S. Pat. No. 10,801,005 and U.S. Patent Application Publication No. 2017 / 0037357, which are incorporated herein by reference.

[0038] Thawing MNC, apheresis-cryopreserved material, medium exchange, and NANT 001 inoculation using Sepax C-Pro: First, start the Sepax C-Pro device using the CultureWash software program. After installing the single-use disposable kit, load 1 L of wash solution (PlasmaLyte A) and 100 mL of resuspension solution (NK-GM containing 50-100 ng / mL N-803 and 0.2-2.0 μM hydrocortisone) into the device as per the batch record. Remove the cryopreserved apheresis material intermediate (AMI) from cryostorage, inspect the cryobag for visible signs of damage, and immediately place in a 37°C water bath for rapid thawing.

[0039] After thawing, samples were aseptically removed to determine cell viability and TNC counts before connecting the thawed cryobag material to a Sepax C-Pro device. The MNC apheresis-cryopreserved material was then washed twice with washing solution (PlasmaLyte A) using a Sepax C-Pro device and transferred to a cell collection bag containing NK-GM containing 50-100 ng / mL N-803 and 0.2-2.0 μM hydrocortisone, with a total of 3.5 × 10 cells / ml. 6 Resuspend the cells at 1.0–5.0 × 10 cells / mL or higher. Then remove the cell collection bag from the Sepax C-Pro device. Take a sample to confirm cell viability and cell count, and if necessary, resuspend the cells at the desired inoculation cell density (1.0–5.0 × 10 cells / mL). 6 After adjusting the inoculum to 100 cells / mL, transfer 50 mL of the inoculum into a NANT 001 bioreactor containing 100 mL of pre-warmed NK-GM containing the same concentrations of N-803 (74 ng / mL) and hydrocortisone (1 µM) to begin the NK cell enrichment and expansion phase. The initial cell culture volume in the NANT 001 bioreactor is 150 mL, with a cell density range of 0.5-1.5 x 10. 6 cells / mL. If more than one NANT 001 bioreactor inoculation from the same patient is planned, thaw multiple AMI bags.

[0040] Cell harvesting, enrichment, and expansion using the NANT 001 bioreactor: 1.20–1.80 × 10 cells per NANT 001 bioreactor unit. 8 After inoculation of thawed AMI cells, the culture is monitored daily using microscopy, sampling at specific stages throughout the cell recovery, enrichment, and expansion process. In-process monitoring (IPM) is performed to determine cell viability, cell number, and phenotypic results (percent CD56-positive cells). This test is used to control the need for subsequent addition of fresh growth medium containing N-803 (without hydrocortisone). On day 20 of expansion, the percentage of CD56-positive cells in the bioreactor reached 85% or greater, and the total cell count reached 1 x 10 6Once the cell / mL is exceeded, the culture is transitioned to the cytokine stimulation phase of the M-CENK manufacturing process. Prior to the cytokine stimulation phase, a sample is taken for bioburden testing.

[0041] In-Process Monitoring (IPM): Visually inspect cultures for signs of turbidity or visible contamination during each medium and N-803 addition. Viable cell density, cell viability, and cell phenotype analyses are performed on designated days as per batch records to obtain NK enrichment profiles and ensure the production bioreactor is within the desired VCD specifications.

[0042] Stimulation of CENK with a cytokine cocktail (human IL-12, IL-18, and N-803): NANT 001 bioreactor cultures expanded to 1 x 10 by day 20. 6 When the culture reached a density of 85% or more CD56-positive cells at ≥ 100 cells / mL, fixed concentrations of N-803 (100–300 ng / mL), IL-12 (1–100 ng / mL), and IL-18 (5–250 ng / mL) were added to the culture in fresh NK-GM for a final total culture volume of up to approximately 650 mL. The cells were stimulated with the cytokine cocktail for 14–16 hours, thereby inducing CD56-positive cells with a memory-like phenotype (M-CENK) contained within the NANT 001 bioreactor. This cytokine cocktail stimulation phase in the bioreactor was then terminated by bulk cell harvest of the culture using the NANT 001 automated unloading function. Samples for mycoplasma testing were collected prior to culture harvest.

[0043] NANT 001 Bioreactor: Cell Culture Harvesting and Sepax Concentration, and Washing: Following completion of the M-CENK induction step, the NANT 001 is manually advanced to perform an automated unloading and harvesting protocol. The automated unloading and harvesting is performed using a closed system utilizing direct sterile welding between the NANT 001 bioreactor and the collection bag. After unloading of M-CENK, the NANT 001 bioreactor is flushed with NK-GM to capture any remaining cells.

[0044] After the automated unloading and collection step is complete, the intermediate collection bag containing the M-CENK cells is weighed and then directly welded to the Sepax C-Pro device for downstream processing and drug substance formulation. The Sepax C-Pro from GE utilizes single-use disposable technology, allowing for direct, sterile welding to the BCH intermediate bag. Using a single-use disposable kit and a designated wash and resuspension program, the Sepax C-Pro first performs a cell concentration step, followed by a two-chamber volume buffer exchange / wash step using 5% albumin (human) solution. Finally, the cells are concentrated and 0.1–2.5 × 10 cells are placed in an approximate 50 mL volume of 5% human albumin solution in the attached 300 mL cell collection bag. 7 The M-CENK is eluted at the expected cell density in cells / mL. The collection bag / vessel containing the M-CENK is then removed from the device and a QC sample is aliquoted to determine cell viability, cell density, and endotoxin. If multiple NANT 001 bioreactors were populated with MNCs from the same patient as inoculum for seeding, the M-CENK can be pooled.

[0045] Formulation of M-CENK drug product: M-CENK preparations generated from multiple NANT 001 bioreactors inoculated with the same patient's AMI are pooled at this stage and volume-corrected to 0.5–1.5 × 10 7 A VCD of 1000 cells / mL is achieved. The formulated drug formulation is then prepared by mixing M-CENK in 5% human albumin with an equal volume (1:1) of CryoStor® CS10 in a flask on ice. The formulated cells are transferred to a CellFreeze® infusion bag (CF-750) on ice to prepare multiple drug formulation bags and small QC bags. The filled infusion bags are then frozen to -85°C or below using a controlled rate freezer. The frozen drug formulation is then transferred to a vapor phase LN2 freezer (-120°C or below) for long-term storage. Upon thawing, QC and sterility testing is performed on the QC bags. The process flow for M-CENK cell expansion and harvest using the NANT 001 bioreactor is shown below.

[0046] An overview of the M-CENK-DS manufacturing process using the NANT 001 bioreactor is shown in Figure 31. An example of the M-CENK production flow process is shown in Figure 32. The potency of M-CENK lots produced by this process is shown in Figure 33.

[0047] IL-12 / IL-18 / N-803-induced M-CENK Viability and Viable Cell Density: One measure that reflects the structural integrity of M-CENK cells is percent viability. Viability is routinely used in process and final product release measurements and is an indicator of product quality. The following experiments describe exemplary tests to verify product characteristics and quality.

[0048] CD56 expression: Neural cell adhesion molecule (NCAM1), also known as CD56, is a member of the immunoglobulin superfamily. NK cells are characterized by the expression of CD56 and the absence of CD3. M-CENK cells derived from PB-NK retain CD56 expression.

[0049] IFN-γ Expression: NK cells are cytolytic, cytokine-producing effector cells of the innate immune system. NK cells are a major source of IFN-gamma (IFN-γ), which interferes with tumor activity. IFN-γ production was analyzed using a flow cytometry-based intracellular cytokine staining assay. As can be seen in Figure 10, high amounts of IFN-γ produced by the NANT 001 process were detected in M-CENK cells, and expression was highly uniform (99.6% of CD56+ cells stained positive for IFN-γ, with an MFI of 15759 (red) compared to 122 (blue) in the unstained sample).

[0050] Phenotypic analysis: (A) DNAM-1: A cell surface glycoprotein that functions as an adhesion molecule and synergizes with activating receptors to elicit NK cell-mediated cytotoxicity. DNAM-1+ve NK cells produce a higher level of IFNγ compared to their DNAM-1-ve counterparts after stimulation with IL-12 and IL-18. DNAM-1 is upregulated in M-CENK cells, as observed in the phenotyping assay described below. (B) TIGIT: A checkpoint receptor that can negatively affect the cytotoxic activity of NK cells. No significant changes in TIGIT expression were observed with M-CENK generation. TIGIT expression was analyzed in the phenotyping assay described below. (C) CD25: Natural killer cells express the IL-2Rα chain (p55), which is identified as CD25 for the formation of the high-affinity IL-2R. CD25 is upregulated in M-CENK cells. (D) CD16: Present on select CD56+ peripheral blood NK cells. Upon recognition of antibody-coated cells, this sends a potent signal to NK cells, leading to target elimination through direct killing and cytokine production.

[0051] GSH Cell Viability: One measure reflecting the health status of M-CENK cells is the intracellular reducing power available to the cells, or percent viability. Expression of intracellular reduced thiol (glutathione; GSH) can be analyzed by staining cell lines with a specific dye (VitaBright-48™, VB48), which reacts with thiols to form fluorescent products that, in combination with acridine orange (AO) and propidium iodide (PI), stain nucleated and dead cells, respectively. Stained samples are then analyzed using a NucleoCounter® NC3000™ imaging cytometer. As can be seen from the exemplary results in Figure 11, M-CENK cells from different culture batches expanded in the NANT 001 bioreactor exhibited characteristics of healthy cells with high viability (GSH+ve, PI-ve) and were comparable to each other.

[0052] Annexin V Cell Health: Another measure reflecting the health of M-CENK cells is the presence of apoptotic, pre-apoptotic, and necrotic cells in culture. The Annexin V assay can detect the translocation of phosphatidylserine to the outer layer of the cell membrane, an indicator of early apoptosis. Quantification of early apoptotic cells can be achieved by staining cells with Annexin V-AF488 conjugate, in addition to Hoechst 33342 and PI to stain nucleated and dead cells, respectively. Stained samples are then analyzed using a NucleoCounter® NC3000™ Imaging Cytometer. M-CENK cells from different culture batches expanded in the NANT 001 bioreactor displayed healthy cell characteristics (annexin negative, PI negative) and were comparable to each other. Exemplary results are shown in Figure 12.

[0053] M-CENK Cytotoxicity Against MS-1 Cells: A key functional assay used to measure the activity of M-CENK cells is to assess their cytotoxicity against the MS-1 target cell line, a cell line relatively resistant to the overall cytotoxicity of NK cells. As an example of scaled-up characterization, the graph in Figure 13 below shows the cytotoxicity results of M-CENK cells (red) plotted over a wide range of effector-to-target (E:T) cell ratios. Control CENK cells (blue) were also expanded in the NANT 001 bioreactor, but M-CENK production was not induced.

[0054] Cytotoxicity of M-CENK Cells Against K562 Cells: Examination of the natural cytotoxicity of M-CENK cells against the K562 cell line is part of an expanded characterization of M-CENK cells. The graph in Figure 14 presents the results of comparing the natural cytotoxicity of M-CENK cells (red) against K562 cells compared to control CENK cells (blue) that were expanded in a NANT 001 bioreactor but not stimulated with the cytokine cocktail.

[0055] TxM-induced M-CENK cells The TxM used in the following studies was obtained from ImmunityBio, Inc. and is a fusion protein containing N-803, IL-12, and IL-18 as shown in Figure 15A, and Figure 15B depicts the sequence used for the TxM. This superkine, in which IL-18 was fused to the IL-15 portion of N-803 and the IL-12 single-chain heterodimer of IL-18 was fused to the IL-15 receptor alpha portion of N-803, was evaluated for its ability to induce an NK memory phenotype as a surrogate for the cytokine N-803.

[0056] The production of CENK from mononuclear cells was described above, however, in this step, the induction / stimulation of CENK with the cytokine cocktail (human IL-12, IL-18, and N-803) was replaced with induction / stimulation with TxM as just described. In this regard, it should be noted that the TxM used herein had an equimolar ratio of 1:1:1 of IL-15 analog (N-803), IL-18, and IL-12 (single chain). Notably, the molar ratio of the components of TxM is substantially different from that of the cytokine cocktail.

[0057] Furthermore, it should be noted that TxM resulted in simultaneous activation due to the close association of all three cytokine functions, whereas induction / stimulation of CENK with a cytokine cocktail (human IL-12, IL-18, and N-803) would allow spatially and temporally separate activation events. Surprisingly, TxM enabled substantially identical, if not improved, formation of M-CENK compared to the use of a cytokine cocktail. Additionally, TxM is provided as a single protein complex and only needs to be added to CENK cells once (as opposed to three times), greatly reducing the risk of contamination. Furthermore, because TxM is provided as a single protein complex, the inconsistent potency of individual cytokine preparations (e.g., lot-to-lot variability) can be completely avoided.

[0058] For comparison with TxM-induced cells, N-803-enriched NK cells (CENK, cytokine-enhanced NK cells) were incubated with a cytokine cocktail containing a fixed concentration of N-803 (175 ng / mL), IL-12 (10 ng / mL), and IL-18 (50 ng / mL) or with TxM (9.8 μg / mL) alone. Cells were stimulated for 14–16 h, which induced CD56+ cells with a memory-like phenotype (M-CENK), as described in further detail below.

[0059] After recovery, M-CENK cells from both treatment experiments were evaluated for memory cell characteristics in various assays. Cytokine priming is generally required for NK cell proliferation and function. However, cytokines can also lead to dose-dependent death of NK cells. Therefore, the viability of N-803-expanded NK cells was evaluated before and after TxM stimulation. As can be seen in the table below, TxM-induced M-CENK cells exhibited high viability, comparable to that of CENK cells (>90%).

[0060] [Table 1]

[0061] M-CENK cells are the primary IFN-gamma-producing cells. To determine whether TxM-induced M-CENK cells developed the ability to produce IFN-gamma, a flow cytometry-based staining method was used. As can be seen in Figure 16, a significantly higher number of IFN-gamma-expressing cells was observed in M-CENK cells compared to CENK cells. This graph depicts exemplary results demonstrating that TxM-induced M-CENK cells are effective producers of IFN-gamma as a proinflammatory cytokine.

[0062] Regarding cytotoxicity, various experiments were performed to establish that TxM-induced M-CENKS cells have significant cytotoxicity against various cell lines.

[0063] In one set of experiments, the cytotoxicity of TxM-induced M-CENK cells was tested against MS-1 cells, a skin cancer cell line that is generally resistant to NK cell cytotoxicity. The cytotoxicity of M-CENK cells against the MS-1 cell line was measured in a cytotoxicity assay across a wide range of effector-to-target (E:T) cell ratios. Notably, M-CENK, but not CENK, induced significant lysis of MS-1 cells, suggesting that the resulting preparations acquired potent cytotoxic activity against resistant tumor cells. Specifically, Figure 17 demonstrates that TxM-induced M-CENK cells are potent killers of NK-resistant MS-1 cells, as can be readily seen from the graph.

[0064] Next, M-CENK cells from both treatments were compared in a cytotoxicity assay. Here, cells generated from either treatment induced similarly potent cytotoxicity against MS-1, suggesting that TxM can serve as an alternative to cytokine cocktails. Figure 18 depicts an exemplary comparison of cytokine cocktail-induced M-CENK cells compared to TxM-induced M-CENK cells for killing NK-resistant MS-1 cells.

[0065] To determine whether TxM-induced M-CENK cells retained the natural cytotoxic activity of NK cells, M-CENK cells were mixed with K562 target cells in a calcein-based cytotoxicity assay. Effector and target cells were mixed together at various ratios. M-CENK cells generated from both treatments induced potent and comparable cytotoxicity against K562 cells, suggesting that the activation step using TxM retained the natural cytotoxicity of NK cells. Figure 19 shows a typical comparison of the ability of cytokine cocktail-induced M-CENK cells to kill K562 cells compared to TxM-induced M-CENK cells.

[0066] In further experiments, M-CENK cells from both treatments (TxM-induced and cytokine cocktail-induced) were compared for their potential to produce IFN-gamma. Remarkably, M-CENK generated from either treatment induced potent IFN-gamma, as observed in a flow cytometry-based assay. Specifically, Figure 20 depicts exemplary comparative results of IFN-gamma production in TxM-induced and cytokine cocktail-induced M-CENK cells.

[0067] It is well known that activating receptors on NK cells play a key role in eliciting NK cell antitumor responses. Therefore, we investigated whether TxM treatment could affect the expression of one or more NK-specific receptors. Notably, the expression of NKG2D, NKp30, NKp44, NKG2A, and NKG2C was confirmed to be comparable in cells generated from either treatment, as shown in the exemplary results in Figure 21. Here, the graph shows a typical comparison of NK-specific markers on TxM-induced and cytokine cocktail (IL-12 + IL-18 + N-803)-induced M-CENK cells.

[0068] To establish that TxM-treated CENK cells would indeed differentiate into the M-CENK phenotype, we assayed the treated cells for the presence of specific markers associated with memory phenotypes. More specifically, differentiation of CENK into M-CENK is generally accompanied by changes in the expression of specific receptors, including DNAM-1 (DNAX accessory molecule-1), CD25, and CD16. Therefore, changes in the expression status of these receptors were detected using a flow cytometry-based assay. Again, M-CENK cells from both treatments showed comparable expression of these receptors. Figure 22 shows an exemplary comparison of the memory cell phenotypes of TxM-induced and cytokine cocktail-induced M-CENK cells.

[0069] Taken together, these experimental results clearly and unexpectedly demonstrate that TxM fusion proteins can act as a substitute for the IL-12 / IL-18 / N-803 cocktail for inducing the NK memory phenotype (M-CENK) in CENK cells. These cytokines can also act as a substitute for other NK cell-activating cytokines (e.g., IL-21), which often increase cytotoxicity but may induce apoptosis at high concentrations.

[0070] M-ceNK cells result in enhanced cytotoxic effects The present inventors also discovered that when various NK cells were treated with a cytokine cocktail containing N-803 (or TxM with an IL15 receptor scaffold as presented herein), such cells had excellent cytotoxicity against various cancer cells, and even against cancer cells that had undergone EMT (endothelial-mesenchymal transition). Notably, the cells treated in this way exhibited CD56 bright , CD16 low The treated cells exhibited a phenotype of NKp44 and TIGIT expression, and showed higher expression of NKp44 and lower expression of TIGIT compared to untreated cells. Figures 23 to 30 provide exemplary results of such enhanced cytotoxicity. Furthermore, given the above findings, it is also contemplated that T cell diversity can be achieved in a similar manner.

[0071] NK cell lysis assays included NK cell effectors such as ceNK, M-ceNK, healthy donor NK cells (2 donors), and healthy donor NK cells (2 donors) pretreated with N-803. Tumor cells included SCLC-H69 and H841, ovarian cancer-OVCAR3 and SK-OV-3, breast cancer-MDA-MB-231, and NSCLC-H441. NK cells and tumor cells were cocultured for 6 hours, and cell counts were collected using the Celigo system. E:T ratios of 20:1, 10:1, and 5:1 were evaluated. The results are shown in Figures 23-25, respectively. Figure 23 illustrates the lysis of small cell lung cancer by NK cells. These results demonstrate that ceNK and M-ceNK cells are highly effective in lysing SCLC tumors of epithelial and mesenchymal phenotypes. Figure 24 illustrates the lysis of ovarian cancer by NK cells. These results demonstrate that ceNK and M-ceNK cells produce similar lysis of epithelial targets as N803-pretreated NK cells in ovarian cancer cell lines, but produce higher lytic activity of mesenchymal target cells. Figure 25 illustrates lysis of breast cancer and NSCLC by NK cells. ceNK and M-ceNK cells are effective in lysing MDA-MB-231 TNBC tumor cells, producing the most effective NK lysis of H441 NSCLC cells. These results demonstrate that the M-ceNK cells disclosed herein can be used as a therapy for cancer stem cells and mesenchymal stem cells.

[0072] For a summary of NK receptors assessed by flow cytometry, see Chan, C et al., Cell Death & Difference, 2016, incorporated herein by reference in its entirety. NK Cell CD56 / CD16 Profile—The CD56 / CD16 profile of healthy donor NK cells differs significantly from that of ImmunityBio NK cells, the latter being highly CD56+, as shown in Figure 26. ceNK and M-ceNK cells express more highly the activating receptors NKp30, NKp44, and NKG2D, as shown in Figures 27-28. Figure 29 illustrates intracellular NK protein expression. ceNK and M-ceNK cells express perforin and granzymes at levels comparable to N-803-activated NK cells. IFN-γ is significantly higher in M-ceNK. Figure 30 illustrates NK inhibitory receptor expression. ceNK and M-ceNK cells express significantly lower levels of the inhibitory receptors TIM3, KLRG1, and TIGIT.

[0073] Figure 34 illustrates the M-CENK surface phenotype. As shown therein, positive expression was observed for CD56, CD25, NKp46, NKp44, NKp30, NKG2D, NKG2A, DNAM-1, and TIGIT. The effect of M-CENK on various cancer cells is shown in Figure 35, illustrating that M-CENK is a potent cancer cell killer.

[0074] The stability of apheresis material intermediates and cryopreserved M-CENK cell preparations under LN2 conditions is shown in Figures 36-37, respectively. A comparison of M-CENK production from healthy donors and patients (Figure 38) demonstrates that M-CENK can be obtained from both healthy donors and patients. Figure 39 shows the phase 1 protocol for clinical trial QUILT-3.076 (a study of autologous M-CENK in subjects with locally advanced or metastatic solid tumors).

[0075] Fusion protein scaffold 18 / 12 / TxM activates IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells In one embodiment, Fehniger and coworkers described the creation of a novel triple cytokine fusion molecule, 18 / 12 / TxM, containing an IL-15 superagonist scaffold (N-803) fused to IL-18 and IL-12. This trimeric molecule retained specific and unique IL-12, IL-15, and IL-18 activity and generated potent human memory-like natural killer cells in vitro and in vivo. See Cubitt CC, et al., "A novel fusion protein scaffold 18 / 12 / TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells," Molecular Therapy: Oncolytics (2022), which is incorporated by reference in its entirety.

[0076] Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as cellular immunotherapies for various malignancies. NK cells are particularly dependent on interleukin-15 (IL-15) for their survival, proliferation, and cytotoxic function. After brief activation by IL-12, IL-15, and IL-18, NK cells differentiate into memory-like cells with enhanced effector function. N-803 is an IL-15 superagonist that contains an IL-15 mutant (IL-15N72D) linked to the sushi domain of IL-15Rα fused to the Fc region of IgG1, resulting in physiological transpresentation of IL-15. Here, we describe the engineering of a novel triple cytokine fusion molecule, 18 / 12 / TxM, using an N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to heteromeric single-chain IL-12 p70 by the sushi domain of IL-15Rα. This molecule exhibits trispecific cytokine activity through its binding and signaling through distinct cytokine receptors. Compared with activation by individual cytokines, 18 / 12 / TxM induces similar short-term NK cell activation and memory-like differentiation at both the transcriptional and protein levels, as well as identical in vitro and in vivo antitumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cell therapy.

[0077] Natural killer (NK) cells are cytotoxic innate lymphoid cells that account for approximately 5–20% of circulating blood lymphocytes and are important for the elimination of virus-infected and malignantly transformed cells. NK cell function is tightly regulated by a balance of germline-encoded activating, costimulatory, and inhibitory receptors expressed on the cell surface. Through these receptors, NK cells can recognize and spontaneously kill cells either by the loss of self-identifying molecules, such as major histocompatibility complex (MHC) class I, which bind to inhibitory receptors on the NK cell (detecting "loss of self"), or by upregulating ligands recognized by activating receptors on the NK cell, which can overcome inhibitory signals. Human NK cells are identified by the surface expression of CD56 and the absence of CD3, and characteristically differ in CD56 expression based on their relative CD56 expression. bright and CD56 dim can be divided into CD56 dim NK cells typically express FcγRIII (CD16), whereas CD56 bright NK cells have low or no expression.

[0078] NK cells constitutively express several cytokine receptors and are particularly dependent on IL-15 for development, homeostasis, and function. IL-15 signaling is mediated by CD56 bright Promoting NK cell survival, proliferation, and priming (at high doses), and CD56 dim It has been shown to enhance the cytotoxicity of a subset of IL-15 receptors. There are three receptor subunits of the IL-15 receptor type: IL-15Rα (CD25), IL-15Rβ (CD122), and IL-15R (CD132). The signaling components of the IL-15 receptor are not private; its β subunit is shared with IL-2, and its γ subunit (common γ chain) is shared with IL-2, IL-4, IL-7, IL-9, and IL-21. Physiologically, IL-15 mediates its effects through transpresentation, which is expressed via IL-15Rβγ cIL-15Rα is expressed on the surface of accessory cells (such as dendritic cells and monocytes / macrophages) that present IL-15 to NK cells carrying IL-15. In addition to the effects mediated by IL-15, the cytokines IL-12 and IL-18 are also important for NK cell survival and function. The primary effect of IL-12 on NK cells occurs via STAT4-mediated signaling and includes interferon-γ (IFN-γ) and tumor necrosis factor (TNF) production. IL-18 has been described as a signal transduction pathway that leads to MAPK and NF-kB activation, functioning synergistically with IL-12 and IL-15 while also priming NK cells for IFN-γ production. Indeed, paradigm-shifting studies have demonstrated that combined activation by IL-12, IL-15, and IL-18 induces memory-like NK cells, defined by enhanced proliferation, expression of the high-affinity IL-2 receptor αβγ (IL-2Rαβγ), and increased IFN-γ production after cytokine-, tumor-, or activating receptor-mediated restimulation. These cytokine-induced memory-like (ML) NK cells represent a promising NK cell therapy and have shown promising results in first-in-human clinical trials in patients with relapsed / refractory AML.

[0079] N-803 is an IL-15 superagonist containing an IL-15 mutant (IL-15N72D) linked to the N-terminal sushi domain of IL-15Rα fused to the Fc region of IgG1. This results in accessory cell-independent IL-15 transpresentation, prolonged in vivo pharmacokinetics, increased in vivo biological activity, and increased effector function compared to IL-15. Given the potent functional effects induced by combined stimulation with IL-12, IL-15, and IL-18, we hypothesized that a single molecular construct capable of signaling through all three cytokine pathways would be beneficial for generating memory-like NK cells for both research and clinical applications. Therefore, we constructed the fusion protein 18 / 12 / TxM by linking IL-18 to the IL-15N72D domain using N-803 as a scaffold and heteromeric single-chain IL-12 p70 to the sushi domain of IL-15Rα, which was previously linked to the Fc domain of human IgG1. This noncovalently associated heterodimeric, homodimeric, and ternary cytokine fusion protein retained specific and unique IL-18, IL-12, and IL-15 activities in vitro and in vivo.

[0080] The stability of the N-803 structure offers a biochemical strategy for decorating this "scaffold" with additional components while maintaining IL-15-based signaling. Here, we investigated the ability of this novel 18 / 12 / TxM fusion protein to generate memory-like NK cells compared with the individual combinations of recombinant human IL-12, IL-15, and IL-18. This included in vitro testing of each cytokine receptor signaling activity, short-term effector function, and the ability to generate memory-like NK cells.

[0081] The fusion 18 / 12 / TxM superkine induces signaling through all target cytokine receptors We first sought to construct a fusion protein consisting of human IL-18, IL-12, and IL-15 to replace the use of individual recombinant cytokines. To this end, N-803 (an IL-15 superagonist, formerly known as ALT-803) was linked to IL-18 via the IL-15N72D domain and to the heteromeric single-chain IL-12 p70 by the sushi domain of IL-15Rα linked to the Fc domain of human IgG1 (Figure 40A). The ability of this triple cytokine fusion protein (hereafter referred to as 18 / 12 / TxM) to induce signaling through all target cytokine receptors was assessed. Freshly isolated and purified NK cells were stimulated with 18 / 12 / TxM (38.8 nM) or an optimal combination of recombinant human (rh) IL-12 (10 ng / mL), IL-18 (50 ng / mL), and IL-15 (50 ng / mL) (IL12 / 15 / 18) and evaluated at various time points for their ability to induce phosphorylation of key signaling intermediates. 18 / 12TxM induced IL-15 signaling through phosphorylation of STAT5, AKT, and ERK, with high efficiency at CD56. bright and CD56 dim At higher concentrations, IL12 / 15 / 18 stimulation stimulated CD56 NK cells in both NK cell subsets (Fig. 40B). dim Induce slightly higher levels of phosphorylated (p)ERK in cells and CD56 bright IL-12 signaling induced slightly lower pAKT expression in NK cells (Fig. 47A). STAT4 phosphorylation by IL-12 signaling was significantly suppressed by CD56 at a low TxM dose (38.8 nM). bright There was a modest but statistically significant difference in NK cells (p=0.005), and no significant difference at high TxM concentrations (77.6 nM) or CD56 dimNo differences were observed in NK cells (Figures 40C and 47B). IL-18 signaling-mediated p65 phosphorylation was similarly induced by 18 / 12 / TxM and IL-12 / 15 / 18 (Figures 40D and 47C). Next, we assessed the ability of 18 / 12 / TxM to activate individual cytokine bioassays. To elucidate IL-15 activity, we assessed the proliferation of a murine hematopoietic cell line, IL-2 / 15-dependent 32D-IL2 / 15Rβ (32Dβ) cells. Increasing concentrations of 18 / 12 / TxM or N-803 were added to 32Dβ cells, which were incubated at 37°C for 3 days, and proliferation was measured using PrestoBlue cell viability assay. Although the ability of 18 / 12 / TxM to promote cell proliferation was reduced compared to N-803 (EC 50 The IL-12 activity of 18 / 12 / TxM was 1.7 nM compared to 0.03 nM for N-803, which may be due to the linkage of IL-18 to the IL-15N72D domain (Figure 40E). To determine the IL-12 activity of 18 / 12 / TxM, activation of IL-12 reporter HEK-blue (HEK12) cells expressing the STAT4-inducible secreted embryonic alkaline phosphatase (SEAP) gene was assessed. Increasing concentrations of 18 / 12 / TxM or recombinant IL-12 were added to HEK12 cells for 20–22 h at 37°C. The activity of SEAP was measured using QUANTI-Blue (Invivogen), and the 50% effective concentration (EC) for IL-12 bioactivity was determined based on the relationship between absorbance and protein concentration. 50 ) was determined, and the bioactivity of recombinant IL-12 was used as a positive control. 50 The EC values ​​for rhIL-12 were 99.1 pM and 86.1 pM, respectively, demonstrating similar biological activity to recombinant IL-12 (Figure 40F). Finally, for IL-18, IL-18 reporter HEK-Blue (HEK18) cells expressing the NF-κB / AP-1 inducible SEAP gene were seeded with increasing concentrations of 18 / 12 / TxM. After 20-22 hours of incubation at 37°C, the activity of SEAP was measured as described above. The EC values ​​for 18 / 12 / TxM were 50 EC of 7.1 pM, and recombinant IL-18 (EC of 0.54 pM) 50), which may be due to the ligation of IL-18 to IL15N72D (Figure 40G). Collectively, these data support that appropriate concentrations of 18 / 12 / TxM stimulate signaling through the IL-12, IL-15, and IL-18 receptors.

[0082] 18 / 12 / Short-term activation by TxM superkines leads to NK cell activation Short-term activation of human NK cells with IL-12, IL-15, and IL-18 leads to increased expression of IL-2 receptor alpha (IL-2Rα, CD25) and enhanced IFN-γ production. To determine the optimal concentration for 18 / 12 / TxM activation, purified human NK cells were activated with increasing concentrations of 18 / 12 / TxM or IL12 / 15 / 18 for 16 hours. Induction of the activated phenotype was assessed as increased cell surface CD25 expression and intracellular IFN-γ compared to control resting NK cells, as determined by flow cytometry (Figure 41A). The optimal concentration for maximal induction of CD25 was reached at 38.8 nM 18 / 12 / TxM, with an EC50 of 2.095 nM (Figure 41B). Short-term activation of purified human NK cells with 38.8 nM of 18 / 12 / TxM or IL12 / 15 / 18 demonstrated similar CD25 induction compared to control NK cells (Figures 41C, 41D). Similarly, IFN-γ induction was near-maximal at 38.8 nM, at which time EC 50 The IL-12 / TxM fusion protein (IL-12 / TxM) was 2.64 nM (Figure 41E). Short-term activation demonstrated that the induction of intracellular IFN-γ by 18 / 12 / TxM was similar compared to IL12 / 15 / 18, although both were significantly higher than the low-dose IL-15 (1 ng / mL) control (Figures 41F, 41G). Collectively, these data indicate that the 18 / 12 / TxM fusion protein produces nearly identical short-term activation via the IL-12, 15, and 18 receptors, resulting in IFN-γ and CD25 expression, compared to the combination of rhIL-12, IL-15, and IL-18.

[0083] Activation with the 18 / 12 / TxM superkine stimulates NK cell proliferation Previous studies have demonstrated that activation with IL-12, IL-15, and IL-18 leads to a memory-like phenotype, including robust proliferation and expansion of NK cells. To address the ability of 18 / 12 / TxM to induce proliferation, purified human NK cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated with 18 / 12 / TxM (38.8 nM), IL12 / 15 / 18, or low-dose IL-15 (LD IL15) for 16 hours. After activation, NK cells (CD56 dim and CD56 bright NK cells (containing both IL-12 / 15 / 18 and IL-12 / TxM subsets) were washed and placed in LD IL15 for 6 days. Consistent with previous data, activation with IL-12 / 15 / 18 or 18 / 12 / TxM induced robust proliferation compared to activation with low-dose IL-15 (Figures 42A and 42B). Interestingly, in this set of experiments, activation with 18 / 12 / TxM resulted in enhanced proliferation and a higher proportion of NK cells expanding beyond three generations compared to IL12 / 15 / 18 (Figure 42B). This increase in cell cycle progression with 18 / 12 / TxM was not due to differences in survival between the activation conditions (Figure 48). This enhanced proliferation could be due to the N-803 scaffold inducing proliferation to a greater extent than IL-15 alone due to enhanced signaling from the IL-15Rα and IgG1-Fc components, or alternatively, it could involve simultaneous signaling through the IL-12, IL-15, and IL-18 receptors.

[0084] Multidimensional phenotypic changes are similar between IL12 / 15 / 18-induced and 18 / 12 / TxM-induced memory-like NK cells Memory-like NK cells undergo dramatic changes in multiple cell surface and intracellular markers both immediately after activation with IL12 / 15 / 18 and after 6 days of differentiation. We previously developed a custom mass cytometry panel containing markers related to NK cell lineage, maturation, and functional capacity (Figure 51) and identified a multidimensional ML NK cell phenotype. To compare the multidimensional phenotype, we used mass cytometry to profile human NK cells before activation (baseline), after 16 hours of incubation with IL-12 / 15 / 18 or 18 / 12 / TxM (D1), and after 6 days of ML differentiation following activation (D6). tSNE analysis using median marker expression revealed distinctively distinct NK cell populations at baseline, day 1, and day 6 post-activation. Notably, the same specific clustered NK cell subsets were identified after activation with either IL12 / 15 / 18 or 18 / 12 / TxM (Figure 43A). Furthermore, comparison of median expression changes after overnight activation for well-defined markers of acute NK cell activation, such as increases in CD25, CD69, and CD137 and decreases in CD56 and CD16, was identical between IL12 / 15 / 18- and 18 / 12 / TxM-activated NK cells (Figures 43B and 49). In accordance with previous studies of differentiated memory-like NK cells, both IL12 / 15 / 18- and 18 / 12 / TxM-activated NK cells demonstrated similar upregulation of NKG2A, CD69, Ki67, CD25, CD137, granzyme B, perforin, and the activating receptors NKp44, NKG2D, and CD94 on day 6. They also demonstrated similar downregulation of CD56, CD16, CD57, NKp30, and NKp80, as previously reported (Figure 43C). Using mass cytometry, we were able to determine that trimeric superkine 18 / 12 / TxM could induce a memory-like NK cell phenotype identical to that induced by the combination of individual recombinant cytokines. These data indicate that 18 / 12 / TxM activation produces short- and long-term changes in NK cells similar to those seen with IL-12, IL-15, and IL-18 activation.

[0085] 18 / 12 / TxM induces functional memory-like NK cells in vitro Previous studies have shown that ML NK cells can be induced after overnight stimulation of purified NK cells ex vivo with saturating doses of IL12 / 15 / 18. These cells exhibit ML characteristics, including 1) enhanced proliferation, 2) IL-2Rα expression, 3) increased IFN-γ production, and 4) increased perforin- and granzyme-mediated cytotoxicity. To demonstrate the generation of ML NK cells by 18 / 12 / TxM, primary human NK cells were activated with 18 / 12 / TxM (38.8 nM), IL12 / 15 / 18, or LD IL15 for 16 hours, washed, and supported with low-dose IL-15 for 6 days to allow memory-like differentiation (Figure 44A). IFN-γ production was assessed as a functional readout for ML NK cell generation after 6 h of restimulation with cytokines (IL-12 [10 ng / ml] and IL-15 [50 ng / mL]) or leukemia targets (K562 cells, effector:target ratio of 5:1) (Figure 44B). Activation with 18 / 12 / TxM induced a slightly greater degree of IFN-γ expression after K562 stimulation than with IL-12 / 15 / 18 (Figure 44C). IFN-γ expression after IL-12 + IL-15 stimulation was slightly higher in IL12 / 15 / 18-activated NK cells compared with 18 / 12 / TxM, but was robustly induced in both conditions compared with LD controls (Figure 44C). Induction of CD107a (a surrogate marker of degranulation) after K562 stimulation was similar among LD, IL12 / 15 / 18, and 18 / 12 / TxM NK cells, consistent with previous reports that degranulation is unaffected by memory-like differentiation (Figures 50A-C). Interestingly, activation with 18 / 12 / TxM elevated TNF expression even in the absence of stimulation, suggesting that 18 / 12 / TxM induces a higher baseline expression of this cytokine (Figures 50D-F). In addition to cytokine secretion, the ability of 18 / 12 / TxM to promote tumor killing was assessed in a standard 4-hour cytotoxicity assay using K562 target cells. Specific target cell killing was identical between IL12 / 15 / 18- and 18 / 12 / TxM-activated NK cells and was higher than that of LD NK cells at all E:T ratios assessed (Figure 4D).These data indicate that the 18 / 12 / TxM molecule is as capable as IL12 / 15 / 18 of inducing memory-like functions, including enhanced IFN-γ and cytotoxicity.

[0086] 18 / 12 / TxM activation induces a molecular program similar to that of IL-12, IL-15, and IL-18 To confirm the phenotypic and functional similarities induced by 18 / 12 / TxM and IL-12 / 15 / 18, we performed bulk RNA sequencing. Purified NK cells from three different donors were isolated, and RNA was isolated before activation with either 18 / 12 / TxM, IL-12 / 15 / 18, or IL-15 (baseline), after overnight activation (D1), and 6 days after activation with IL-15 (D6). Analysis of transcript counts revealed similar gene expression profiles at D1 postactivation between IL-12 / 15 / 18- and 18 / 12 / TxM-activated NK cells compared with LD IL-15. Analysis of genes with significant differences (p<0.05) in expression at D1 after IL12 / 15 / 18 or 18 / 12 / TxM stimulation demonstrated that the vast majority (5,812 genes) of changes were shared between treatments, with 808 unique genes expressed in the TxM condition and 332 unique genes expressed after IL12 / 15 / 18 treatment (Figure 45A). Indeed, direct comparison of genes expressed in NK cells activated with 18 / 12 / TxM or IL12 / 15 / 18 revealed nearly identical expression profiles (r 2=0.9679) (Figure 45B). Consistent with the phenotypic observations of D1, short-term activation with both 18 / 12 / TxM and IL12 / 15 / 18 led to dramatic increases in the expression of IL2Rα (CD25), IFN-γ, granzyme B, LTA, TNFSF4 (OX40L), NIFK, CCL3, and CSF2 (GM-CSF) (Figures 45C, 45D). Six days after activation, when differentiation was supported with LD IL-15, there were no statistically significant differences in gene expression between LD IL-15 and 18 / 12 / TxM- or IL12 / 15 / 18-activated NK cells. Although the vast majority of gene expression changes were minimal (logFC < 0.5 or > -0.5), direct comparison of genes induced in 18 / 12 / TxM- and IL12 / 15 / 18-activated NK cells at day 6 that showed differential trends (logFC > 1 or < -1) compared with LD revealed similar changes between these two treatment groups (Figure 45E). Despite the dramatic molecular activation profile the day after activation, no statistically significant genes were differentially expressed between LD IL-15- and IL12 / 15 / 18-activated NK cells by day 6. This is consistent with previous studies in our laboratory and may be due to the heterogeneity of memory-like differentiated NK cells at day 6 and the potential for the dominant IL-15-induced transcriptional profile to mask unique transcriptional profiles. For some differentially expressed genes, trends differed between LD IL-15- and 18 / 12 / TxM- or IL12 / 15 / 18-treated NK cells at day 6, including increased expression of CXCR6, CCR1, and granzyme K. Direct comparison of gene expression changes induced by 18 / 12 / TxM or IL12 / 15 / 18 revealed no statistically significant differences, suggesting that they induce similar transcriptional profiles by day 6 (Figure 45F). Accordingly, using bulk RNA sequencing on enriched NK cells, 18 / 12 / TxM and IL12 / 15 / 18 induced nearly identical transcriptional changes at 24 h, both of which were distinct from control NK cells. However, this analytical approach did not identify significant differences between day 6 ML NK cells induced by either initial activation.Based on the subsets of ML NK cells with enhanced function, we expect that only a small proportion of cells at day 6 represent functional ML NK cells with unique transcriptional signatures. In this setting, single-cell RNA-seq approaches may be required to identify subset-based transcriptome changes.

[0087] 18 / 12 / TxM induces memory-like NK cell antitumor activity in vivo To confirm that the molecular and phenotypic changes induced by 18 / 12 / TxM in vitro also translate into enhanced in vivo function, we performed leukemia-engrafted NOD-SCID-IL2Rg - / - The antitumor activity was compared in nonsteroidal anti-cancer (NSG) mice. Briefly, NSG mice were inoculated with luciferase-expressing K562 tumor cells (0.5 × 10 6 NK cells (3-5 × 10 cells / mouse) were engrafted, and 4 days later, NK cells (3-5 × 10 cells / mouse) preactivated with 18 / 12 / TxM, IL-2 / 15 / 18, or LD IL-15 were added. 6 IL12 / 15 / 18 or 18 / 12 / TxM (cells / mouse) were injected into the tumors (Figure 46A). Tumor growth was assessed using whole-body bioluminescence imaging (BLI) on days 3, 11, and 17 (Figure 46B). ML NK cells induced with either IL12 / 15 / 18 or 18 / 12 / TxM demonstrated enhanced tumor control on day 17 (Figure 46C). These data demonstrate that activating NK cells with 18 / 12 / TxM can induce a ML NK cell phenotype similar to that induced by IL12 / 15 / 18, which exerts enhanced control of tumor targets in vivo.

[0088] Using N-803 as a scaffold, we constructed a novel fusion protein, 18 / 12 / TxM, by linking IL-18 to the IL-15N72D domain and heteromeric single-chain IL-12 p70 to the sushi domain of IL-15Rα, which was previously linked to the Fc domain of human IgG1. This noncovalently associated heterodimeric homodimeric triple cytokine fusion protein retained specific and unique IL-18, IL-12, and IL-15 activities in vitro, as measured by activation, proliferation, and signaling through their cognate receptors. Furthermore, when used at appropriate concentrations, 18 / 12 / TxM exhibited functional equivalents to the individual cytokine combinations after overnight ex vivo stimulation of primary NK cells and after 6 days of in vitro differentiation into memory-like NK cells, as well as in vivo in NSG mice. The ability of 18 / 12 / TxM to induce this memory-like phenotype was confirmed at the protein and transcriptional levels using high-dimensional methods, including mass cytometry phenotyping and bulk RNA sequencing. These phenotypic changes translated into equivalent cytotoxic effector functions in vitro and similar tumor control in vivo. Thus, 18 / 12 / TxM is an alternative to IL-12, IL-15, and IL-18 in generating memory-like NK cells.

[0089] Interestingly, activation with 18 / 12 / TxM resulted in higher levels of proliferation of purified NK cells than with the combination of individual cytokines. This finding suggests that IL-12, IL-15, and IL-18 receptors on the same or different cells are not activated sequentially, but rather that simultaneous engagement of these receptors on the same cell may result in distinctly different biological outcomes. Another possibility is that spatial linkage of cytokines on the scaffold enhances membrane clustering of cytokine receptors and signaling molecules as a result of binding from trimeric molecules. It is also possible that the Fc portion of 18 / 12 / TxM activates NK cells through downstream signaling events following engagement with the FcγRIII receptor (CD16). However, CD56 dim(CD16+) and CD56 bright Given the observation that increased proliferation occurred in both (CD16-) and (CD16-) NK cells, it is also possible that Fc-FcR interactions allow facile transpresentation of 18 / 12 / TxM by CD16+ NK cells to nearby NK cells. Further investigations using FcR-null mutants of 18 / 12 / TxM will be required to clarify this possible contribution.

[0090] Minimal differences in gene expression were observed between LD IL-15-activated and IL-12 / 15 / 18-activated NK cells on day 6. This is consistent with previous observations that only a small proportion of NK cells are capable of undergoing full memory-like differentiation, making it difficult to identify their gene signatures using bulk RNA sequencing methods and after extensive IL-15-supported culture in vitro. Further studies using deeper sequencing methods, such as single-cell RNA sequencing, will be essential to characterize the unique transcriptional changes in memory-like differentiated NK cells. It is also possible that memory-like differentiation is primarily orchestrated by epigenetic changes, which can be revealed by methods such as ATAC-seq.

[0091] IL-15 has been used as an ideal candidate for clinical immunotherapy combinations due to its ability to stimulate NK cell (and CD8+ T cell) activation. However, physiological activation by IL-15 requires binding to the IL-15Rα chain prior to target cell activation, limiting the research and clinical role of free IL-15. N-803 consists of two IL-15Rα subunits fused to a human IgG1 Fc, linked to an IL-15 superagonist (N72D mutation, which enhances biological activity), resulting in higher biological activity and a longer serum half-life compared to free IL-15. Previous studies have demonstrated that preactivation of NK cells with IL12 / 15 / 18 leads to ML NK cell differentiation, representing a promising approach to enhance adoptive allogeneic NK cell therapy. However, the use of these individual cytokines, either alone or in combination, for research and clinical purposes can be fraught with production challenges and lot-to-lot variability. Additionally, this superkine represents a promising platform for replacing various NK cell-activating cytokines (IL-2, IL-21) or tumor-targeting molecules (e.g., CD20, EGFR, HER2, or CD34) to direct activated NK cells to kill tumor cells. Indeed, N-803 has been used as a functional scaffold fused to a CD20-targeting antibody component and demonstrated superior antitumor activity compared to the individual components alone. Other studies have demonstrated enhanced antitumor activity when N-803 was used in combination with either tumor-targeting or checkpoint inhibitor antibodies, representing a promising avenue for the development of further fusion proteins.

[0092] This disclosure demonstrates that fusion of three distinct cytokines via human IgG1 Fc binding induced activity equivalent to that of the individual cytokine combinations in vitro and in vivo. While these studies preactivated NK cells in vitro using 18 / 12 / TxM prior to infusion, the Fc backbone confers additional in vivo half-life, which may support its in vivo use in other contexts. Additionally, the use of the N-803 protein scaffold linked to three distinct cytokine targets represents a novel method for expanding and stimulating NK cells for adoptive cell therapy.

[0093] Materials and Methods Recombinant proteins: hIL18 / IL12 / TxM protein, lot number 305-86(1) and N-803 protein, lot number 01062016, were manufactured and purified by Altor BioScience, Miramar, FL. Endotoxin-free recombinant human (rh) IL-12 (Biolegend), IL-15 (Miltenyi), IL-18 (Invivogen), and IL-2 (R&D Systems, Minneapolis, MN) were used in these studies.

[0094] Flow cytometry antibodies: The following Beckman Coulter antibodies were used: CD3 (clone UCHT1), CD45 (clone A96416), CD56 (clone N901), NKG2A (clone Z199.1), NKp46 (clone BAB281). The following BD antibodies were used: CD16 (clone 3G8), IFN-γ (clone B27), CD107a (clone H4A3), CD57 (NK-1). CD69 (FN50), CD137 (clone 4-1BB), perforin (clone dG9), Ki67 (clone B56), ERK1 / 2 (pT202, pY204), AKT (pS473), STAT4 (38 / p-Stat4), STAT5 (47 / Stat5, pY694), p38 (pT290 / pY182), and p65 (pS529). The following Biolegend antibodies were used: NKG2D (clone 1D11), NKp30 (clone P30-15), NKp44 (clone P44-8), and IgG1 control (clone MG1-45). The following eBioscience antibodies were used: granzyme B (GB12), and TNF (clone Mab11).

[0095] Cell Line: K562 cells (ATCC, CCL-243) were obtained from ATCC in 2008, cryopreserved viably, thawed for use in these studies, and maintained in continuous culture as described for no longer than 2 months. Prior to our studies, K562 cells were authenticated in 2014 and 2015 by confirming their cell growth morphology (lymphoblastoid), growth characteristics, and functionality as NK cell-sensitive targets. Cells were cultured in RPMI 1640 medium (Hyclone / GE Healthcare, Logan, UT) supplemented with L-glutamine, HEPES, NEAA, sodium pyruvate, and Pen / Strep / glutamine containing 10% FBS.

[0096] HEK-Blue IL-18 cells (interleukin-18 sensor cells) and HEK-Blue IL-12 cells (interleukin-12 sensor cells) from Invivogen (San Diego, CA) were cultured in complete HEK-Blue medium (I10 medium) consisting of IMDM, 10% FBS (HyClone / GE Healthcare, Logan, UT); 1x penicillin-streptomycin-glutamine (Thermo Fisher Scientific, Dallas, TX); 100 μg / ml normacin, and 1x HEK-Blue selection (InvivoGen, San Diego, CA). 32D-IL2 / 15Rβ (32Dβ) cells were constructed at Altor BioScience (Miramar, FL) and cultured at 37°C and 5% CO2 at a density of 1.5 x 10 cells / ml in complete 32Dβ medium containing IMDM-10 medium + 25 ng / ml rhIL-2. 4 ~2×10 6 The cell density was maintained at 1000 cells / ml.

[0097] NK cell purification and cell culture: Human platelet apheresis donor PBMCs were obtained by Ficoll centrifugation. RosetteSep (StemCell Technologies, ≥95% CD56 + CD3 - ) and used for selection experiments. 3–5 × 10 cells were used. 6 Cells were seeded at 1000 cells / mL and pre-activated for 16 hours with 38.8 nM 18 / 12 / TxM (9.5 μg / mL), rhIL-12 (10 ng / mL) + rhIL-18 (50 ng / mL) + rhIL-15 (50 ng / mL), or control condition (rhIL-15, 1 ng / mL). Cells were washed three times to remove cytokines and cultured in complete HAB10 medium containing RPMI 1640 medium supplemented with rhIL-15 (1 ng / mL) + 10% human AB serum (Sigma-Aldrich, St. Louis, MO) for 6 days, replacing 50% of the medium with fresh rhIL-15 every 2–3 days.

[0098] Assessment of IL-18 and IL-12 activity: HEK-Blue IL-18 and HEK-Blue IL-12 cells were maintained in complete HEK-Blue medium at 37°C and 5% CO2. Following the manufacturer's cell handling recommendations, HEK-Blue Selection was added to this growth medium after two passages. Growth medium was refreshed twice weekly, and cells were passaged when they reached 70-80% confluency. To measure IL-18 or IL-12 activity, the respective sensor cells were dissociated in PBS and resuspended at 280,000 cells / ml in complete HEK-Blue assay medium. Twenty microliters of semi-logarithmic serial dilutions (concentration ranges listed below) of cytokine control and hIL18 / IL12 / TxM were added to a flat-bottom 96-well plate, followed by 180 μL of cells for a final cell count of approximately 50,000 cells in 200 μL. The plate was incubated at 37°C and 5% CO2 for approximately 20 hours. To assess IL-18 or IL-12 activity, the resulting secreted alkaline phosphatase was quantified using QUANTI-Blue detection reagent (Invivogen, San Diego, CA). QUANTI-Blue reagent was prepared according to the manufacturer's instructions. After warming to room temperature, 180 μL was added to 20 μL of culture supernatant in a 96-well flat-bottom plate and incubated at 37°C and 5% CO2 for 18 hours. Absorbance was then measured at 650 nm to determine cell activation based on the reduction of QUANTI-Blue by secreted alkaline phosphatase. EC values ​​for IL-18 or IL-12 bioactivity of 18 / 12 / TxM were calculated from dose-response curves generated using nonlinear regression variable slope curve fitting in GraphPad Prism 7. 50 It was decided that:

[0099] Concentration range: For detection of IL-18 activity, semi-log serial dilutions were performed ranging from 10 ng / ml (556 pM) to 0.05 pg / ml (0.0028 pM) for IL-18 and from 3350 ng / ml (13673 pM) to 0.0167 ng / ml (0.0683 pM) for 18 / 12 / TxM, corresponding to final pM concentrations of 56 pM to 0.00028 pM for IL-18 cytokine and 1367 pM to 0.00683 pM for 18 / 12 / TxM. For detection of IL-12 activity, semi-log serial dilutions ranging from 1000 ng / ml (17483 pM) to 5 pg / ml (0.0875 pM) for IL-12 and from 85.7 ug / ml (349,796 pM) to 0.428 ng / ml (1.748 pM) for 18 / 12 / TxM were performed, corresponding to final pM concentrations of 1748.3 to 0.00875 pM for IL-12 cytokine and 34,979.6 to 0.1748 pM for 18 / 12 / TxM molecule.

[0100] Assessment of IL-15 activity: To measure IL-15 activity, assay plates were prepared as follows: 100 μL of IMDM-10 medium was added to each well of a 96-well flat-bottom plate. Next, 100 μL of 4x concentrated N-803 (225 ng / ml; approximately 2400 pM) or IL18 / IL12 / TxM (18,000 ng / ml; approximately 73468 pM) was added to column 1. Drugs were serially diluted 2-fold through column 10, leaving 100 μL in each well. Cells were washed three times with IMDM-10 medium and resuspended in IMDM-10 medium at a density of 1×10 5Cells were resuspended at 0.1 cells / ml and 100 μL of cells were added to columns 1-11 of the assay plate for a total assay volume of 200 μL. 100 μL of IMDM-10 was added to column 12. The assay plate was placed at 37°C and 5% CO2 for approximately 72 hours. To assess IL-15 activity, 20 μL of 10× PrestoBlue cell viability reagent was added directly to the assay plate after approximately 72 hours, and the plate was placed at 37°C and 5% CO2 for an additional approximately 4 hours. Absorbance was measured at 570 nm and 600 nM for normalization. The EC values ​​for IL-15 bioactivity of hIL18 / IL12 / TxM were calculated from dose-response curves generated using nonlinear regression variable slope curve fitting in GraphPad Prism 7, using column 11 (cells without drug) as a negative control. 50 It was decided that:

[0101] Phosphorylation assay: Freshly isolated human NK cells were incubated in cytokine-free HAB10 medium for 30 minutes at 37°C. Individual cytokines (IL-12, IL-15, or IL-18) were added to the wells at the indicated concentrations for various time intervals (2 hours of stimulation for STAT4, 1 hour for Akt and ERK, and 15 minutes for NF-κB-p65, STAT5, and p38 detection). After incubation, cells were fixed with 4% paraformaldehyde (PFA) and incubated at room temperature for 10 minutes. Cells were then pelleted, resuspended in cold 100% methanol, and incubated at 4°C for 30 minutes. Cells were washed three times with FACS buffer (PBS, 0.5% BSA, 2 mM EDTA). After washing, cells were suspended in a surface antibody master mix (CD3, CD16, CD56, CD45) and the appropriate phospho-flow antibodies and stained overnight at 4°C. The following morning, cells were washed twice, and samples were acquired on a BeckmanCoulter Gallios flow cytometer and analyzed using FlowJo Version 9.3.2 (TreeStar) software.

[0102] Assessment of cytokine production by functional assays: After a 6-day resting period to allow memory-like NK cell differentiation, control and memory-like NK cells were harvested and then restimulated in standard functional assays. Briefly, cells were incubated with K562 leukemia targets at a 5:1 effector-to-target (E:T) ratio, unless otherwise noted, in the presence of CD107a for 6 hours. After 1 hour of stimulation, brefeldin A and monensin (GolgiStop / GolgiPlug, BD) were added, and 5 hours later, cells were stained for CD45, CD3, CD56, and CD25. Cells were fixed (Cytofix / Cytoperm, BD) and permeabilized (Perm / Wash, BD) before staining for intracellular IFN-γ and TNF. Cells were acquired on a Gallios 3 flow cytometer and analyzed using FlowJo Version 9.3.2 (TreeStar) software.

[0103] Assessment of specific killing: On day 6 or 7 post-activation, control or ML-NK cells were resuspended in 1 ng / mL IL-15 and challenged with K562 targets at various E:T ratios in a standard 4-hour 51Cr release assay. 51Cr release was detected in a Wallac Microbeta Tri-lux scintillation counter. Percent specific lysis was calculated as follows: [(cpm exp -cpm spontaneous ) / cpm max -cpm spontaneous )]×100.

[0104] Flow cytometry analysis: Cell staining was performed as previously described, and data were acquired on a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN) and analyzed using Kaluza Version 1.2 (Beckman Coulter) or FlowJo Version 9.3.2 (TreeStar) software. Statistical analysis was performed using GraphPad Version 7.0 software.

[0105] RNA Sequencing: One million purified NK cells were frozen at -80°C in Trizol until RNA isolation using the Direct-zol RNA MicroPrep Kit (Zymo Research). Next-generation RNA sequencing was performed using an Illumina HiSeq 2500 sequencer. RNASeq reads were aligned to the Ensembl release 76 top-level assembly in STAR version 2.0.4b. Gene counts were derived from the number of uniquely aligned, unambiguous reads using Subread:featureCount version 1.4.5. Sequencing data were analyzed using Phantasus, a browser-based gene expression analysis software. Differences between conditions were analyzed using differential expression analysis using the LIMMA package, and results were filtered to only include genes with a false positive rate-corrected p-value of 0.05 or less.

[0106] Mass cytometry: All mass cytometry data were collected on a CyTOF2 mass cytometer (Fluidigm) and analyzed using Cytobank. Mass cytometry data were analyzed using previously described methods, and sample staining and data collection were performed as previously described.

[0107] NSG xenograft model and BLI imaging: On day 0, K562 luciferase-expressing tumor cells (1 × 106) were inoculated into 8- to 12-week-old male and female NOD-SCID-IL2Rγ xenografts. - / - NSG mice (The Jackson Laboratory, Bar Harbor, ME) were intravenously injected via the tail vein. All mice were irradiated with 2.5 cGy two days before tumor injection. On day 3, BLI was performed to confirm leukemia cell engraftment. On day 4, 5 × 10 6Control (NK cells in 1 ng / mL IL-15), NK cells activated with IL-12 / 15 / 18 (10 ng / mL IL-12, 50 ng / mL IL-15, 50 ng / mL IL-18), or 18 / 12 / TxM (38 nM) were administered retroorbitally to mice (9–10 mice per group from two independent experiments). Mice were treated with rhIL-2 (50,000 IU per mouse) every other day (every other data), and tumor burden (BLI) was monitored weekly.

[0108] In vivo BLI imaging was performed with an IVIS 50 (1–60 s exposure, bin 8, FOV 12 cm, open filter) (Xenogen, Alameda, CA). Mice were injected intraperitoneally with D-luciferin (150 mg / kg in PBS, Gold Biotechnology, St. Louis, MO) and anesthetized with isoflurane (2% vaporized in O) before imaging. Total photon flux (photons / second) was measured from a fixed region of interest across the mouse using the Living Image 2.6 software program.

[0109] In some embodiments, numbers expressing properties such as quantities of ingredients, concentrations, reaction conditions, and the like, used to describe and claim particular embodiments of the present invention should be understood to be modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the description and appended claims are approximations that may vary depending on the desired properties sought to be achieved by the particular embodiment. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein by reference as if it were individually set forth herein.

[0110] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct administration and indirect administration of the pharmaceutical composition or drug, where direct administration of a pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and where indirect administration includes providing or making the pharmaceutical composition or drug available for direct administration by a medical professional (e.g., by injection, infusion, oral delivery, topical delivery, etc.). Furthermore, it should be noted that the terms "prognosing" or "predicting" a condition, susceptibility to disease development, or response to an intended treatment is meant to encompass the act of predicting or the prediction (but not the treatment or diagnosis) of the condition, susceptibility, and / or response, including the rate of progression, reversal, and / or duration of the condition in a subject.

[0111] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples provided in connection with specific embodiments herein, or the use of exemplary language (e.g., "etc."), are intended merely to better illustrate the invention and do not impose limitations on the scope of the invention as originally claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0112] As used herein and throughout the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, as used herein, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise. Also, as used herein, and unless the context clearly dictates otherwise, the term "coupled to" is intended to include both direct coupling (where the two elements coupled to each other touch each other) and indirect coupling (where at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used synonymously.

[0113] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the present subject matter should not be limited except as indicated by the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, that is, indicating that a referenced element, component, or step may be present with, utilized in, or combined with other elements, components, or steps not expressly mentioned. When the specification or claims refer to at least one of something selected from the group consisting of A, B, C, ..., and N, the statement should be interpreted as requiring only one element from that group, and not A+N, or B+N, etc.

Claims

1. Cryopreserved memory-like cytokine-enhanced natural killer (M-CENK) cells, the M-CENK cells comprising: a. Thawing cryopreserved mononuclear cells in medium containing hydrocortisone (HC) and a fusion protein of IL-15 and IL-15Rα, which results in prolonged in vivo pharmacokinetics; b. Incubating the monocytes in the presence of the HC and the IL-15 and IL-15Rα fusion protein for 14 to 21 days, thereby enriching the monocytes for cytokine-enhanced NK cells (CENK); c. inducing the mononuclear cells enriched for CENK with a cytokine composition to generate M-CENK cells, wherein the cytokine composition comprises a second fusion protein comprising a portion having IL-12 activity, a portion having IL-15 activity, and a portion having IL-18 activity; and d. harvesting, formulating for injection, and cryopreserving the M-CENK cells; A cell produced by a method comprising:

2. The cells of claim 1, wherein the cryopreserved mononuclear cells are thawed, washed, and resuspended in a medium containing the HC and N-803.

3. The cells of claim 1 or 2, wherein the incubating step is carried out until the CENK cells are enriched to at least 65% of all live cells.

4. The cells of any one of claims 1 to 3, wherein the mononuclear cells enriched for CENK are induced for a period of 12 to 16 hours.

5. A composition comprising the M-CENK cells of claim 1 in combination with a pharmaceutically acceptable carrier.

6. The composition of claim 5 , wherein the pharmaceutically acceptable carrier comprises a cryopreservation medium.

7. 0.5 to 1.5 x 10 ７ 7. The composition of claim 5 or 6, having a cell density of 100 cells / mL.

8. 8. A pharmaceutical composition for use in treating an individual with cancer, comprising the composition of any one of claims 5 to 7, said pharmaceutical composition being formulated for administration by infusion.

9. 9. The pharmaceutical composition of claim 8, wherein the composition of any one of claims 5 to 7 treats cancer in the individual by killing cancer stem cells and mesenchymal cells.

10. A composition according to any one of claims 5 to 7 for use in the treatment of cancer.

11. 8. A composition according to any one of claims 5 to 7 for use in treating cancer in an individual by killing cancer stem cells and mesenchymal cells.

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