Engineered natural killer cells and related methods

Abstract

The present disclosure provides a population of engineered natural killer cells, wherein the NK cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E, human leukocyte antigen F, and human leukocyte antigen G. The NK cells comprise a genetically engineered disruption of one or more copies of endogenous beta-2 microglobulin, as well as a genetically engineered disruption of one or more copies of a human leukocyte antigen class II-associated gene selected from the group consisting of regulatory factor X-related ankyrin-containing protein, regulatory factor 5, regulatory factor X-related protein, and class II transactivator. Also provided are methods for producing engineered natural killer cells from human pluripotent stem cells under feeder-free conditions, and methods for treating a disease or disorder in a subject by administering the NK cells to the subject.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to engineered natural killer (NK) cells and methods for preparing populations of engineered NK cells.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 477,785, filed December 29, 2022, the entire contents of which are incorporated herein by reference. Incorporation by Reference of Electronically Submitted Materials

[0003]

[0003] The computer-readable nucleotide / amino acid sequence listing, filed concurrently with this application and identified as follows, is incorporated by reference in its entirety: 58,824 byte file "57819P_Seqlisting.XML"; created on December 18, 2022. [Background technology]

[0004]

[0004] Engineering patient-derived immune cells to express chimeric antigen receptors (CARs) has transformed the landscape of adoptive cell therapy, providing scientists and clinicians the ability to harness the potent cytolytic potential of immune cells and direct them to specific antigen-expressing targets in an MHC-independent manner. Natural killer (NK) cells, which arise from the lymphoid system, are part of the innate immune system and have demonstrated the ability to recognize and kill specific tumor cells, making them attractive candidates for adoptive cell therapy. Typical sources of NK cells include peripheral blood NK cells (PB-NK), umbilical cord blood NK cells, and NK cell lines such as NK-92. While these sources are promising, they suffer from challenges such as genetic instability, inconsistent cytotoxicity, growth limitations, product heterogeneity, and / or host immunogenicity. There is a need in the art for improved NK cell-based therapies to overcome the limitations of current treatments. Summary of the Invention

[0005]

[0005] The present disclosure provides a population of natural killer (NK) cells derived from pluripotent stem cells. The present disclosure further provides a population of engineered NK cells, in which the NK cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The NK cells also comprise a genetically engineered disruption of one or more copies (e.g., all copies) of endogenous beta-2 microglobulin (B2M) and / or one or more copies (e.g., all copies) of a human leukocyte antigen (HLA) class II-associated gene selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), and class II transactivator (CIITA). For example, the NK cells comprise genetically engineered disruptions of endogenous B2M and RFXANK and express HLA-E. In another example, the NK cells comprise genetically engineered disruptions of all copies of endogenous B2M and all copies of RFXANK and express HLA-E. In various embodiments, the NK cells further comprise a nucleic acid molecule encoding a suicide gene product (e.g., a herpes simplex virus thymidine kinase (TK) suicide gene). The NK cells are optionally derived from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) and optionally express one or more cell surface markers selected from the group consisting of CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and NKp44. Optionally, the NK cells further express a chimeric antigen receptor (CAR); an Fc receptor (FcR); or a non-native NKG2D receptor (see, e.g., U.S. Pat. No. 10,259,858 and U.S. Patent Application Publication No. 2020 / 0138866) that in some aspects has the ability to bind to an antibody-based bispecific molecule (e.g., a MicAbody); or other types of receptors.

[0006]

[0006] The present disclosure further provides a method for producing NK cells from pluripotent stem cells under feeder-free, optionally serum-free, conditions. The method includes the steps of: (a) culturing pluripotent stem cells (PSCs) that have been genetically engineered to optionally disrupt one or more copies (e.g., all copies) of B2M and / or one or more copies (e.g., all copies) of an HLA class II-associated gene selected from the group consisting of RFXANK, RFX5, RFXAP, and CIITA, and optionally express an HLA class I protein, e.g., HLA-E; (b) differentiating the pluripotent stem cells (e.g., using embryoid bodies) into progenitor cells that can differentiate into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; and (c) differentiating the progenitor cells into NK cells under feeder-free, optionally serum-free, conditions in a culture medium containing one or more of SCF, IL-7, IL-15, and Flt3L. Optionally, the culture medium of step (c) further comprises a pyrimidol[4,5-b]indole derivative, such as (1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimidol[4,5-b]indol-4-yl)cyclohexane-1,4-diamine dihydrobromide dihydrate ("UM171"). In various embodiments, the PSCs are human PSCs. In various other embodiments, the method further comprises (d) culturing the NK cells in a culture medium comprising IL-15 and IL-18 (and optionally IL-21) and in the presence of an antibody or ligand that binds to a receptor on NK cells that promotes NK cell activation and proliferation. In one embodiment, the antibody or ligand binds to a receptor selected from the group consisting of DNAM-1, OX40, NKG2D, 2B4, NKp30, and NKp46. A combination of antibodies or ligands that bind to different targets selected from DNAM-1, OX40, NKG2D, 2B4, NKp30, and NKp46 can be included in step (d). For example, step (d) can include culturing NK cells in the presence of an antibody that binds to NKp30 and / or an antibody that binds to DNAM-1.

[0007]

[0007] The present disclosure also provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of NK cells, thereby treating the disease in the subject. In some aspects of the present disclosure, the disease is cancer. Optionally, one or more cytokines, e.g., IL-15 and / or IL-2, are administered to the subject before, during, or after administering the population of NK cells. Optionally, one or more antibody constructs are also administered to the subject before, during, or after administering the population of NK cells.

[0008]

[0008] Although various embodiments are presented herein using the term "comprising," under various circumstances, related embodiments may also be described using the terms "consisting of" or "consisting essentially of." The present disclosure contemplates that embodiments described with the term "comprising" a feature include embodiments "consisting of" or "consisting essentially of" the feature. The terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to include items alternatively or together, unless the context clearly requires otherwise.

[0009]

[0009] The recitation of ranges of values ​​herein, unless otherwise specifically stated herein, is intended as a shorthand method of referring individually to each individual value and each endpoint within the range, and each individual value and endpoint is incorporated into the specification as if it were individually stated herein. However, the specification also contemplates the same range excluding the lower and / or upper endpoint. When the term "about" is used in connection with a numerical value or general value, it means ±1%, ±2%, ±3%, ±4%, ±5%, ±10%, or ±15% of the stated number or value, and includes all values ​​within this range of the stated number or value. The actual range of variation can be determined from the context.

[0010]

[0010] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any or all examples or illustrative language (e.g., "for example") provided herein is intended merely to clarify the disclosure and does not limit the scope of the disclosure unless otherwise required. Only limitations described herein as essential to the invention should be construed as such limitations; variations of the invention lacking limitations not described herein as essential are contemplated as aspects of the invention.

[0011]

[0011] Additional features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as aspects of the present invention. Similarly, features of the present invention described herein may be recombined into additional embodiments that are also intended as aspects of the present invention, regardless of whether that combination of features is expressly designated as an aspect or embodiment of the present invention. This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein (including those described in separate sections) are contemplated, even if the combinations of features are not together in the same sentence, paragraph, or section of this document. [Brief explanation of the drawings]

[0012] [Figure 1]

[0012] Figure 1 shows a representative schematic of the NK differentiation process described herein: Step 0 - PSC culture; Step 1 - hematopoietic differentiation; Step 2 - NK differentiation; Step 3A - NK activation; and Step 3B - NK expansion. Steps 0-3B are referenced in the following figure legend. [Figure 2]

[0013] Figures 2A-2C are bar graphs showing the cell yield (Figure 2A), percentage of CD56+CD45+ cells (Figure 2B), and cell viability (Figure 2C) obtained from the method for generating human PSC-derived engineered NK cells (FcR-UDC I-NK cells) described herein. The left bar of each graph represents the condition without UM171, and the right bar of each graph represents the condition with 35 nM UM171. [Figure 3A]

[0014] Figures 3A-D are bar graphs depicting the yield and proliferation of NK cells at step 2 (NK differentiation) in multiple gene-edited clones (Figures 3A-3B) and PSC cell lines (Figures 3C-D). In Figures 3A-3B, NK cells were derived from the NIH3 PSC cell line (wild-type) or genetically engineered NIH3 PSC cell lines: UDC I-PSC, FcR-UDC I-PSC, and UDC I / II-PSC. In Figures 3C-3D, NK cells were derived from genetically engineered NIH3 and W1C1 PSC cell lines (UDC I-PSC and UDC I / II-PSC) or the parental NIH3 cell line (wild-type). [Figure 3B] Figures 3A-D are bar graphs depicting the yield and proliferation of NK cells at step 2 (NK differentiation) in multiple gene-edited clones (Figures 3A-3B) and PSC cell lines (Figures 3C-D). In Figures 3A-3B, NK cells were derived from the NIH3 PSC cell line (wild-type) or genetically engineered NIH3 PSC cell lines: UDC I-PSC, FcR-UDC I-PSC, and UDC I / II-PSC. In Figures 3C-3D, NK cells were derived from genetically engineered NIH3 and W1C1 PSC cell lines (UDC I-PSC and UDC I / II-PSC) or the parental NIH3 cell line (wild-type). [Figure 3C]Figures 3A-D are bar graphs depicting the yield and proliferation of NK cells at step 2 (NK differentiation) in multiple gene-edited clones (Figures 3A-3B) and PSC cell lines (Figures 3C-D). In Figures 3A-3B, NK cells were derived from the NIH3 PSC cell line (wild-type) or genetically engineered NIH3 PSC cell lines: UDC I-PSC, FcR-UDC I-PSC, and UDC I / II-PSC. In Figures 3C-3D, NK cells were derived from genetically engineered NIH3 and W1C1 PSC cell lines (UDC I-PSC and UDC I / II-PSC) or the parental NIH3 cell line (wild-type). [Figure 3D] Figures 3A-D are bar graphs depicting the yield and proliferation of NK cells at step 2 (NK differentiation) in multiple gene-edited clones (Figures 3A-3B) and PSC cell lines (Figures 3C-D). In Figures 3A-3B, NK cells were derived from the NIH3 PSC cell line (wild-type) or genetically engineered NIH3 PSC cell lines: UDC I-PSC, FcR-UDC I-PSC, and UDC I / II-PSC. In Figures 3C-3D, NK cells were derived from genetically engineered NIH3 and W1C1 PSC cell lines (UDC I-PSC and UDC I / II-PSC) or the parental NIH3 cell line (wild-type). [Figure 4]

[0015] FIG. 4 is a graph showing the effect of various combinations of NK receptor agonists on the activation of CD56+ cells, as measured by expression of CD215 / IL-15Rα (left panel) or CD25 / IL-2Rα (right panel). [Figure 5]

[0016] Figures 5A-5B are line graphs showing (Figure 5A) cumulative fold expansion and (Figure 5B) cell viability after steps 3A and 3B of the NK differentiation process (shown in Figure 1), where different cell densities seeded at the start of step 3A were tested. NK cells were generated from NIH3 UDC I-PSCs using the methods described herein. [Figure 6]

[0017] Figure 6 is a graph comparing the cell size of NK cells derived from PSCs ("iPSCs") using the methods described herein and peripheral blood-derived NK cells ("PBNK"), as determined by flow cytometry. NK cells derived from PSCs using the methods described herein were, on average, approximately 18% larger in size (volume) than NK cells isolated from peripheral blood. [Figure 7A]

[0018] Figures 7A-7E are bar graphs of the predicted phenotype of NK cells derived from different gene-edited clones: NIH3 wild-type and gene-edited NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 7B] Figures 7A-7E are bar graphs of the predicted phenotype of NK cells derived from different gene-edited clones: NIH3 wild-type and gene-edited NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 7C] Figures 7A-7E are bar graphs of the predicted phenotype of NK cells derived from different gene-edited clones: NIH3 wild-type and gene-edited NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 7D] Figures 7A-7E are bar graphs of the predicted phenotype of NK cells derived from different gene-edited clones: NIH3 wild-type and gene-edited NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 7E] Figures 7A-7E are bar graphs of the predicted phenotype of NK cells derived from different gene-edited clones: NIH3 wild-type and gene-edited NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 8-1]

[0019] Figures 8A-8D are bar graphs of the predicted phenotypes of NK cells derived from different cell lines: NIH3 and W1C1 wild-type, and gene-edited clonally engineered UDC I-PSCs and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 8-2] 8A-8D are bar graphs of predicted phenotypes of NK cells derived from different cell lines: NIH3 and W1C1 wild-type, and gene-edited clonally engineered UDC I-PSCs and UDC I / II-PSCs. Asterisks indicate data not collected. [Figure 9]

[0020] Figures 9A-9C are bar graphs showing the expression of common NK cell and T cell markers in FcR-UDC I-NK cells derived from engineered FcR-UDC I-PSCs (step 2 (left bar for each marker) and step 3 (middle bar for each marker)) compared to expanded PB-NK (right bar for each marker). Steps 2 and 3 are shown in Figure 1. [Figure 10]

[0021] Figures 10A-10C are bar graphs showing the expression of common activation, exhaustion, and "memory-like" markers in FcR-UDC I-NK cells derived from engineered FcR-UDC I-PSCs (step 2 (left bar for each marker) and step 3 (middle bar for each marker)) compared to expanded PB-NK (right bar for each marker). Steps 2 and 3 are shown in Figure 1. [Figure 11]

[0022] Figures 11A-11B are bar graphs showing cytokine and chemokine receptor expression in FcR-UDC I-NK cells derived from engineered FcR-UDC I-PSCs (step 2 (left bar for each receptor) and step 3 (middle bar for each receptor)) compared to expanded PB-NK (right bar for each receptor). Steps 2 and 3 are shown in Figure 1. [Figure 12]

[0023] Figures 12A-12C are bar graphs showing the expression of adhesion / homing, costimulatory, and apoptotic markers in FcR-UDC I-NK cells derived from engineered FcR-UDC I-PSCs (step 2 (left bar for each receptor) and step 3 (middle bar for each receptor)) compared to expanded PB-NK (right bar for each receptor). Steps 2 and 3 are shown in Figure 1. [Figure 13]

[0024] Figure 13 is a line graph showing the natural cytotoxicity (NCC) of NK cells derived from engineered NIH3 and W1C1 PSCs, UDC I-PSCs (2F7 (NIH) and 1A6 (W1C1)), FcR-UDC I-PSCs (3C1), and UDC I / II-PSCs (10A7 (NIH) and 2A6 (W1C1)) against K562 tumor target cells. The y-axis shows the specific lysis rate, and the x-axis shows the effector-to-target cell ratio (E / T). The UDC-NK cells disclosed herein mediated cell lysis in multiple PSC cell lines and gene-edited clones. [Figure 15]

[0025] Figure 15 is a line graph showing NCC of FcR-UDC I-NK cells compared to expanded PB-NK cells. The y-axis shows the specific lysis rate, and the x-axis shows the effector-to-target cell ratio (E / T). The UDC-NK cell population disclosed herein mediated cell lysis to a similar extent as PB-NK cells. [Figure 16]

[0026] FIG. 16 is a line graph showing that FcR-UDC I-NK do not exhibit fratricide or cytotoxicity against donor PBMCs but exhibit NCC against K562 tumor target cells. [Figure 17]

[0027] FIG. 17 is a line graph showing the dose-dependent killing effect of engineered NK cells (FcR-UDC I-NK cells) on sequentially added K562 tumor cells. [Figure 18]

[0028] Figures 18A-18D are line graphs showing antibody-dependent cellular cytotoxicity (ADCC) activity of engineered FcR-UDC I-NK cells generated by the methods described herein. The y-axis shows the specific lysis rate, and the x-axis shows the effector-to-target cell ratio (E / T). In Figures 18A-18C, the FcR-UDC I-NK cell populations disclosed herein exhibited ADCC activity against three different CD20+ tumor cell lines: Daudi (Figure 18A), Ramos (Figure 18B), and Raji (Figure 18C) using commercially available CD20-binding rituximab compared to the IgG1 control. In Figure 18D, expanded PB-NK cells showed no difference in ADCC activity between rituximab and the IgG1 control. Circles represent no antibody, squares represent human IgG1, and triangles represent rituximab. [Figure 19]

[0029] FIG. 19 is a bar graph showing the NCC and ADCC activity of FcR-UDC I-NK cells in a wide range of B lymphoma cell lines. [Figure 20A]

[0030] Figure 20A is a bar graph identifying inducible cytokines after stimulation with rituximab (R-mab)-coated Raji tumor cells in FcR-UDC I-NK cells (left bars for each condition indicated) compared with expanded PB-NK cells (right bars for each condition indicated). The x-axis of each graph represents "No stimulation," "Raji + hIgG1," and "Raji + R-mab." Significant increases were observed in TNF-alpha, interferon-gamma, GM-CSF, and IP-10. Moderate increases were observed in granzyme-B, MIP-1 alpha, MIP-1 beta, RANTES, IL-8, IL-10, and MCP-1. Undetected cytokines include IL-1 alpha, IL-1 beta, IL-6, and IL-12, as shown in Figures 20B-20E. [Figure 20B]Figure 20A is a bar graph identifying inducible cytokines after stimulation with rituximab (R-mab)-coated Raji tumor cells in FcR-UDC I-NK cells (left bars for each condition indicated) compared with expanded PB-NK cells (right bars for each condition indicated). The x-axis of each graph represents "No stimulation," "Raji + hIgG1," and "Raji + R-mab." Significant increases were observed in TNF-alpha, interferon-gamma, GM-CSF, and IP-10. Moderate increases were observed in granzyme-B, MIP-1 alpha, MIP-1 beta, RANTES, IL-8, IL-10, and MCP-1. Undetected cytokines include IL-1 alpha, IL-1 beta, IL-6, and IL-12, as shown in Figures 20B-20E. [Figure 20C] Figure 20A is a bar graph identifying inducible cytokines after stimulation with rituximab (R-mab)-coated Raji tumor cells in FcR-UDC I-NK cells (left bars for each condition indicated) compared with expanded PB-NK cells (right bars for each condition indicated). The x-axis of each graph represents "No stimulation," "Raji + hIgG1," and "Raji + R-mab." Significant increases were observed in TNF-alpha, interferon-gamma, GM-CSF, and IP-10. Moderate increases were observed in granzyme-B, MIP-1 alpha, MIP-1 beta, RANTES, IL-8, IL-10, and MCP-1. Undetected cytokines include IL-1 alpha, IL-1 beta, IL-6, and IL-12, as shown in Figures 20B-20E. [Figure 20D]Figure 20A is a bar graph identifying inducible cytokines after stimulation with rituximab (R-mab)-coated Raji tumor cells in FcR-UDC I-NK cells (left bars for each condition indicated) compared with expanded PB-NK cells (right bars for each condition indicated). The x-axis of each graph represents "No stimulation," "Raji + hIgG1," and "Raji + R-mab." Significant increases were observed in TNF-alpha, interferon-gamma, GM-CSF, and IP-10. Moderate increases were observed in granzyme-B, MIP-1 alpha, MIP-1 beta, RANTES, IL-8, IL-10, and MCP-1. Undetected cytokines include IL-1 alpha, IL-1 beta, IL-6, and IL-12, as shown in Figures 20B-20E. [Figure 20E] Figure 20A is a bar graph identifying inducible cytokines after stimulation with rituximab (R-mab)-coated Raji tumor cells in FcR-UDC I-NK cells (left bars for each condition indicated) compared with expanded PB-NK cells (right bars for each condition indicated). The x-axis of each graph represents "No stimulation," "Raji + hIgG1," and "Raji + R-mab." Significant increases were observed in TNF-alpha, interferon-gamma, GM-CSF, and IP-10. Moderate increases were observed in granzyme-B, MIP-1 alpha, MIP-1 beta, RANTES, IL-8, IL-10, and MCP-1. Undetected cytokines include IL-1 alpha, IL-1 beta, IL-6, and IL-12, as shown in Figures 20B-20E. [Figure 21]

[0031] 21A-21D are bar graphs identifying cytokines and / or chemokines that were not elevated after co-culture with allogeneic PBMCs versus Raji + rituximab in FcR-UDC I-NK cells (left bar for each condition) compared to expanded PB-NK (right bar for each condition). The x-axis of each graph represents "No stimulation," "Raji + rituximab," and "AlloPBMCs." [Figure 22]

[0032] Figures 22A-22G are bar graphs showing the in vivo persistence of FcR-UDC I-NK cells in the absence of antigen in non-tumor-bearing mice. NK cells (4 x 10) were administered intravenously to mice, followed by IL-2 (30,000 IU, three times a week) and IL-15 (100 ng daily for 7 days). The percentage and absolute number of FcR-UDC I-NK cells in the blood, spleen, and bone marrow were measured. [Figure 23-1]

[0033] Figures 23A-23E are bar graphs showing the in vivo tissue distribution of FcR-UDC I-NK cells in non-tumor-bearing mice. NK cells (5 x 10, 7.5 x 10, and 10 x 10) were administered intravenously, followed by IL-2 (30,000 IU, three times a week) and IL-15 (100 ng daily). The percentage of human CD56+ cells among mouse CD45+ cells (FcR-UDC I-NK cells) was measured in the lung (Figure 23B), liver (Figure 23D), spleen (Figure 23E), blood (Figure 23A), and bone marrow (Figure 23C). [Figure 23-2] Figures 23A-23E are bar graphs showing the in vivo tissue distribution of FcR-UDC I-NK cells in non-tumor-bearing mice. NK cells (5 x 10, 7.5 x 10, and 10 x 10) were administered intravenously, followed by IL-2 (30,000 IU, three times a week) and IL-15 (100 ng daily). The percentage of human CD56+ cells among mouse CD45+ cells (FcR-UDC I-NK cells) was measured in the lung (Figure 23B), liver (Figure 23D), spleen (Figure 23E), blood (Figure 23A), and bone marrow (Figure 23C). [Figure 24]

[0034] Figure 24 is a graph of the efficacy study of FcR-UDC I-NK cells plus rituximab in the Raji intraperitoneal tumor model. NSG mice were irradiated and intraperitoneally administered Raji tumor cells (3 x 10). On day 4, FcR-UDC I-NK cells or PB-NK cells (5 x 10 each) were administered with or without rituximab (300 μg). On day 7, an additional dose of rituximab (300 μg) was administered. NSG mice were imaged / monitored at the indicated time points up to 120 days. [Figure 25-1]

[0035] Figures 25A-25C show (Figure 25A) survival curves and (Figures 25B-25C) tumor burdens after the efficacy study of Figure 24. In Figure 25A, both FcR-UDC I-NK cells + rituximab and PB-NK + rituximab showed a significant increase in median survival time (days) compared to untreated, rituximab alone, FcR-UDC I-NK cells alone, and PB-NK alone. Rituximab alone and PB-NK alone increased median survival time (days) compared to untreated. In Figures 25B-25C, FcR-UDC I-NK cells + rituximab and PB-NK + rituximab showed durable responses that delayed tumor onset by 60 days in 50% of mice. [Figure 25-2] Figures 25A-25C show (Figure 25A) survival curves and (Figures 25B-25C) tumor burdens after the efficacy study of Figure 24. In Figure 25A, both FcR-UDC I-NK cells + rituximab and PB-NK + rituximab showed a significant increase in median survival time (days) compared with untreated, rituximab alone, FcR-UDC I-NK cells alone, and PB-NK alone. Rituximab alone and PB-NK alone increased median survival time (days) compared with untreated. In Figures 25B-25C, FcR-UDC I-NK cells + rituximab and PB-NK + rituximab showed durable responses that delayed tumor onset by 60 days in 50% of mice. [Figure 26]

[0036] Figure 26 is a graph of the efficacy study of FcR-UDC I-NK cells plus rituximab in the Raji intraperitoneal tumor model. NSG mice were irradiated and intraperitoneally administered Raji tumor cells (2 x 10). On day 2, FcR-UDC I-NK cells (10 x 10) were administered with or without rituximab (100 μg). On days 6, 10, and / or 16, additional doses of FcR-UDC I-NK cells or PB-NK cells (10 x 10 each) were administered. NSG mice were imaged / monitored at the indicated time points up to day 86. [Figure 27-1]

[0037] Figures 27A-27B show (Figure 27A) tumor burden and (Figure 27B) survival curves following the efficacy study of Figure 26. In Figure 27A, FcR-UDC I-NK cells plus rituximab (days 2, 10, and 16) demonstrated a sustained effect in delaying tumor onset compared to FcR-UDC I-NK cells plus rituximab (days 2, 6, and 10). In Figure 27B, FcR-UDC I-NK cells plus rituximab (regardless of dosing regimen) demonstrated a significant increase in median survival time (days) compared to untreated, rituximab alone, and FcR-UDC I-NK cells alone. [Figure 27-2] Figures 27A-27B show (Figure 27A) tumor burden and (Figure 27B) survival curves following the efficacy study of Figure 26. In Figure 27A, FcR-UDC I-NK cells plus rituximab (days 2, 10, and 16) demonstrated a sustained effect in delaying tumor onset compared with FcR-UDC I-NK cells plus rituximab (days 2, 6, and 10). In Figure 27B, FcR-UDC I-NK cells plus rituximab (regardless of dosing regimen) demonstrated a significant increase in median survival time (days) compared with untreated, rituximab alone, and FcR-UDC I-NK cells alone. [Figure 28-1]

[0038] Figures 28A-28B relate to efficacy studies using NOGf mice in which mouse FcgR was knocked out. Figure 28A is a graph of a study in which rituximab or FcR-UDC I-NK cells plus rituximab were administered in a Raji tumor model. NOGf mice were intravenously injected with Raji tumor cells (1 x 10). Rituximab (100 μg, intraperitoneal) was administered on days 1, 8, and 15 after tumor cell implantation, and FcR-UDC-I-NK (10 M) was administered intravenously on days 1, 4, 8, and 15. Figure 28B shows the tumor burden after the efficacy study in Figure 28A. Cryopreserved FcR-UDC I-NK cells plus rituximab inhibited tumor growth to a greater extent than rituximab alone. [Figure 28-2] Figures 28A-28B relate to efficacy studies using NOGf mice in which mouse FcgR was knocked out. Figure 28A is a graph of a study in which rituximab or FcR-UDC I-NK cells plus rituximab were administered in a Raji tumor model. NOGf mice were intravenously administered Raji tumor cells (1 x 10). Rituximab (100 μg, intraperitoneal) was administered on days 1, 8, and 15 after tumor cell implantation, and FcR-UDC-I-NK (10 M) was administered intravenously on days 1, 4, 8, and 15. Figure 28B shows the tumor burden after the efficacy study in Figure 28A. Cryopreserved FcR-UDC I-NK cells plus rituximab inhibited tumor growth to a greater extent than rituximab alone. [Figure 29-1]

[0039] Figures 29A-29B relate to efficacy studies using NOGf mice engineered to express human interleukin-15 (IL-15) and knockout mouse FcgR. Figure 29A is a graph of a study in the Raji tumor model in which rituximab (50 or 100 μg, intraperitoneal administration) or FcR-UDC I-NK cells (3 × 10 (3 M) or 15 × 10 (15 M), intravenous administration) plus rituximab (50 or 100 μg, intraperitoneal administration) was administered. Figure 29B shows the tumor burden after the efficacy study in Figure 29A. 15 M cryopreserved FcR-UDC I-NK cells plus rituximab inhibited tumor growth to a greater extent than rituximab alone. [Figure 29-2] Figures 29A-29B relate to efficacy studies using NOGf mice engineered to express human interleukin-15 (IL-15) and with mouse FcgR knockout. Figure 29A is a graph of a study in the Raji tumor model in which rituximab (50 or 100 μg, intraperitoneal administration) or FcR-UDC I-NK cells (3 × 10 (3 M) or 15 × 10 (15 M), intravenous administration) plus rituximab (50 or 100 μg, intraperitoneal administration) was administered. Figure 29B shows tumor burden after the efficacy study in Figure 29A. 15 M cryopreserved FcR-UDC I-NK cells plus rituximab inhibited tumor growth to a greater extent than rituximab alone. [Figure 30]

[0040] Figure 30 is a line graph showing the antitumor effect of FcR-UDC I-NK cells plus anti-claudin 6 (CLDN6) antibodies (ASP1893 mAb, a humanized anti-CLDN6 antibody, and ASP1650, a human / mouse chimeric anti-CLDN6 antibody) against CLDN6-positive tumor cells (ovarian cancer cell line PA-1). The y-axis shows the specific lysis rate (%), and the x-axis shows the amount (ng / mL) of antibody ASP1893 mAb or ASP1650. The anti-CLDN6 antibodies ASP1893 mAb and ASP1650 induced ADCC against CLDN6+ tumor cells comparable to that of FcR-UDC I-NK cells. [Figure 31A]

[0041] Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31B]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31C]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31D]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31E]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31F]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31G]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31H]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31I]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31J]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31K]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31L]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 31M]Figures 31A-31M are line graphs showing the antitumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibody against tumor cells expressing different levels of CLDN6. Assays were performed using FcR-UDC I-NK cells with the antibody ASP1893 mAb, a control IgG1 antibody, or no antibody. Figures 31A-31D show assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E-31G show assays using tumor cells moderately expressing CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H-31I show assays using tumor cells lowly expressing CLDN6 (COV362 and NEC8-Luc cells). Figures 31J-31M show assays using tumor cells that do not express CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). The y-axis shows the specific lysis rate (%), and the x-axis shows the E / T ratio. FcR-UDC I-NK cells mediated ADCC against tumor cells by the ASP1893 monoclonal antibody (mAb) in a CLDN6 expression-dependent manner. [Figure 32]

[0042] Figures 32A-32B show cytokine secretion by FcR-UDC I-NK cells. The figures are bar graphs correlating interferon-gamma (IFN-g; pg / mL) or tumor necrosis factor-alpha (TNF-α) produced by FcR-UDC I-NK cells in the presence of tumor cells (left bar for each cell line tested), tumor cells with human IgG1 (center bar for each cell line tested), and tumor cells with anti-CLDN6 antibody (ASP1893 mAb) (right bar for each cell line tested). SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells show high levels of CLDN6. ABC-1 and OV90 cells show intermediate levels of CLDN6. COV362 and NEC8-Luc cells show low levels of CLDN6. COV318, ES-2, and SKOV3 cells are CLDN6-negative. Upon stimulation with tumor cells and ASP1893 mAb, FcR-UDC I-NK cells produced IFN-γ and TNF-α in a CLDN6-dependent manner. [Figure 33]

[0043] Figure 33 is a schematic diagram of an in vivo assay evaluating the antitumor effect of a combination of UDC-NK cells and an anti-CLDN6 antibody in a PA-1 xenograft solid tumor mouse model. NOGf IL-15 mice express human IL-15 and lack mouse FcgR. [Figure 34-1]

[0044] Figures 34A-34B show the tumor volume (Figure 34A) and survival curve (Figure 34B) after the efficacy study in Figure 33. Combination treatment of FcR-UDC-NK cells and anti-CLDN6 antibody resulted in tumor growth inhibition (Figure 34A) and prolonged mouse survival (Figure 34B) in the PA-1 SC tumor model. [Figure 34-2] Figures 34A-34B show tumor volume (Figure 34A) and survival curves (Figure 34B) after the efficacy study in Figure 33. Combination treatment with FcR-UDC-NK cells and anti-CLDN6 antibody resulted in tumor growth inhibition (Figure 34A) and prolonged survival of mice (Figure 34B) in the PA-1 SC tumor model. [Figure 35-1]

[0045] Figures 35A-35C show a study investigating the antitumor effect of a combination of UDC-NK cells and an anti-CLDN6 antibody in a PA-1 xenograft mouse model using NOGf mice engineered to express human interleukin-15 (IL-15) and knocked out mouse FcgR. Figure 35A shows a schematic diagram of an in vivo assay in which 7.5 μM FcR-UDC-NK cells were administered intravenously on days 16, 20, 23, and 27, and anti-CLDN6 antibody (300 μg, intraperitoneal) was administered on days 16 and 23. Figures 35B-35C show tumor volume (Figure 35B) and survival curves (Figure 35C) after the efficacy study in Figure 35A. Combination treatment with FcR-UDC-NK cells and an anti-CLDN6 antibody resulted in tumor growth inhibition (Figure 35B) and prolonged mouse survival (Figure 35C) in the PA-1 SC tumor model. [Figure 35-2] Figures 35A-35C show a study investigating the antitumor effect of a combination of UDC-NK cells and an anti-CLDN6 antibody in a PA-1 xenograft mouse model using NOGf mice engineered to express human interleukin-15 (IL-15) and knocked out mouse FcgR. Figure 35A shows a schematic diagram of an in vivo assay in which 7.5M FcR-UDC-NK cells were administered intravenously on days 16, 20, 23, and 27, and anti-CLDN6 antibody (300 μg, intraperitoneal) was administered on days 16 and 23. Figures 35B-35C show tumor volume (Figure 35B) and survival curves (Figure 35C) after the efficacy study in Figure 35A. Combination treatment of FcR-UDC-NK cells and anti-CLDN6 antibody resulted in tumor growth inhibition (Fig. 35B) and prolonged mouse survival (Fig. 35C) in the PA-1 SC tumor model. [Figure 36]

[0046] Figures 36A and 36B show the ADCC activity of FcR-UDC-NK cells. Figure 36A is a line graph showing the antitumor effect of FcR-UDC I-NK cells and an anti-claudin 18.2 antibody (zolbetuximab) on BxPC3-CLDN18.2-Luc tumor cells. The y-axis shows the specific lysis rate (%), and the x-axis shows the E:T ratio. Figure 36B is a line graph showing the antitumor effect of FcR-UDC I / II-NK cells and an anti-claudin 18.2 antibody (zolbetuximab) on BxPC3-CLDN18.2-luciferase tumor target cells. The y-axis shows the fluorescence intensity of the remaining target cells normalized to the start of the experiment, and the x-axis shows the measurement time. [Figure 37]

[0047] Figures 37A-37B are bar graphs showing the secretion of interferon gamma (Figure 37A) or tumor necrosis factor alpha (Figure 37B) by FcR-UDC I-NK in samples containing BxPC3-CLDN18.2-2-Luc tumor cells alone, BxPC3-CLDN18.2-2-Luc tumor cells with human IgG, or BxPC3-CLDN18.2-2-Luc tumor cells with zolbetuximab. DETAILED DESCRIPTION OF THE INVENTION

[0013]

[0048] The present disclosure provides populations of natural killer (NK) cells with properties favorable for immune cell therapy. The materials and methods described herein enable the generation of substantially homogeneous populations of NK cells with, for example, reduced generation of unwanted cells, improved safety profiles, robust in vivo persistence, improved ability to engraft in subjects, and / or improved ability to reduce tumor burden.

[0014]

[0049] The present disclosure provides a population of NK cells derived from pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The present disclosure further provides a population of engineered NK cells in which the NK cells express at least one exogenous polypeptide selected from the group consisting of human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The NK cells have been genetically engineered to disrupt one or more copies (e.g., all copies) of the cell's endogenous beta-2 microglobulin (B2M) and / or one or more copies (e.g., all copies) of a human leukocyte antigen (HLA) class II-associated gene selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), and class II transactivator (CIITA). These disruptions allow, for example, the NK cells to reduce or avoid immune detection and to generate NK cell populations that do not require HLA matching for therapeutic applications. Optionally, the NK cells also express a chimeric antigen receptor (CAR) (e.g., a universal CAR), an Fc receptor (FcR or CD16), a non-native NKG2D receptor, or other types of receptors.

[0015]

[0050] NK cells are cytotoxic lymphocytes belonging to the innate immune system and have immune surveillance functions. NK cells have attracted attention as therapeutic agents due to their natural cytotoxicity against exogenous, transformed, or virus-infected cells, antibody-dependent cellular cytotoxicity, and cytokine release profile that can amplify immune responses. NK cells are typically defined by the expression of CD56 or CD16 and the absence of CD3. In various embodiments of the present disclosure, NK cells express one or more (e.g., two or more, three or more, four or more, etc.) cell surface markers selected from CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and / or NKp44. Methods for detecting cell surface markers are known in the art and include, for example, flow cytometry-based methods.

[0016]

[0051] NK cells (or PSCs or their intermediates from which the NK cells are derived) are genetically engineered to disrupt one or more copies (e.g., all copies) of the cell's endogenous B2M gene. B2M is a key component of the HLA class I (HLA-I) complex. HLA is a cell surface complex that mediates leukocyte-leukocyte interactions or interactions between leukocytes and other cells. HLA-I molecules are cell surface complexes that present antigens to CD8+ cytotoxic T cells, thereby mediating cell-mediated immunity. HLA-I molecules contain an HLA-I heavy chain and B2M. Genetic disruption of one or more (e.g., all) copies of the B2M gene reduces or eliminates production of the B2M protein (SEQ ID NO: 5), thereby reducing or eliminating expression of the HLA-I complex on NK cells. A representative nucleic acid sequence encoding B2M is set forth in SEQ ID NO: 1.

[0017]

[0052] Also contemplated are NK cells (or PSCs or intermediates from which NK cells are derived) genetically engineered to destroy one or more copies (e.g., all copies) of a subset of HLA-I. Six HLA class I alpha (α) chains have been identified: three classical (HLA-A, HLA-B, and HLA-C) and three non-classical (HLA-E, HLA-F, and HLA-G), which are involved in peptide binding specificity in the HLA-I binding cleft. In this regard, the present disclosure contemplates NK cells genetically engineered to destroy one or more copies (e.g., all copies) of HLA-I genes selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and combinations thereof. For example, NK cells (or PSCs or intermediates thereof from which NK cells are derived) may be genetically engineered to disrupt one or more copies (e.g., all copies) of HLA-A, HLA-B, and / or HLA-C. HLA molecules are described, for example, in Choo, Yonsei Med. J., 48(1):11-23(2007); www.ebi.ac.uk / ipd / imgt / hla / ; and further described in GenBank accession numbers NG_029217 (HLA-A), XP_041536701 (HLA-A), NG_023187 (HLA-B), NP_005505 (HLA-B), NG_029422 (HLA-C), NP_001229971 (HLA-C), NM_005516 (HLA-E), NP_005507 (HLA-E), NG_012009 (HLA-F), NP_001091949 (HLA-F), NG_029039 (HLA-G), and NP_001371219 (HLA-G).

[0018]

[0053] The NK cells of the present disclosure (or PSCs or intermediates thereof from which the NK cells are derived) are genetically engineered to disrupt one or more (e.g., all) copies of an HLA class II (HLA-II)-associated gene selected from RFXANK, RFX5, RFXAP, and CIITA. HLA-II molecules are transmembrane proteins found on antigen-presenting cells (APCs) and, in some cases, on solid organs. HLA-II molecules contain two homologous subunits, an alpha (α) subunit and a beta (β) subunit. HLA-II-associated genes encode HLA-II regulatory proteins that regulate the expression of HLA class II molecules. Genetic disruption of one or more (e.g., all) copies of one or more HLA-II-associated genes RFXANK, RFX5, RFXAP, and / or CIITA reduces or eliminates the production of the encoded proteins, thereby reducing or eliminating the presentation of HLA-II complexes on NK cells. The NK cells (or PSCs or intermediates thereof from which the NK cells are derived) may comprise one or more copies (e.g., all copies) of a gene encoding one of RFXANK, RFX5, RFXAP, and / or CIITA (e.g., the cells have been engineered to disrupt one or more copies (e.g., all copies) of the RFXANK gene), one or more copies (e.g., all copies) of a gene encoding any combination of two of RFXANK, RFX5, RFXAP, and / or CIITA; one or more copies (e.g., all copies) of a gene encoding any combination of three of RFXANK, RFX5, RFXAP, and / or CIITA; or one or more copies (e.g., all copies) of a gene encoding all of RFXANK, RFX5, RFXAP, and CIITA. RFXANK is encoded, e.g., by the sequences of SEQ ID NOs: 3 and 5; RFX5 is encoded, e.g., by the sequences of SEQ ID NOs: 7 and 9; RFXAP is encoded, e.g., by SEQ ID NO: 11; and CIITA is encoded, e.g., by SEQ ID NO: 13. In various embodiments of the present disclosure, NK cells are engineered to destroy all copies of B2M and RFXANK.

[0019]

[0054] Any suitable technique can be used to introduce disruption into target genes (for example, HLA-II-related genes, B2M genes, or other genes of interest).Many techniques for disrupting endogenous coding sequences are known in the art, including gene editing systems such as CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases, as well as target vectors that cut and / or insert nucleic acid sequences into target sites in cellular genomes.One example of a technique is the use of adeno-associated virus (AAV) vector-based editing, which involves the targeted insertion of an optionally edited sequence (potentially a transgene) into cellular genomes, disrupting endogenous coding sequences and thereby reducing or avoiding the production of endogenous proteins.In various embodiments, serotype 3B AAV vectors are used. The manipulation of stem cells using AAV vectors is described in more detail in, for example, Riolobos et al., Molecular Therapy, 21(6), 1232-1241 (2013), which is incorporated herein by reference in its entirety, and is particularly referred to for its disclosure of the use of AAV vectors and the inhibition of HLA complex production.Methods for confirming the disruption of target genes include those known in the art, including but not limited to PCR detection (e.g., quantitative real-time polymerase chain reaction (qRT-PCR)), RNA sequencing, next-generation sequencing, flow cytometry, and immunostaining.

[0020]

[0055] In various embodiments, the engineered NK cells express at least one polypeptide selected from the group consisting of HLA-E, HLA-F, and HLA-G. In the context of the present disclosure, NK cells are engineered to express HLA-E, HLA-F, and / or HLA-G, i.e., nucleic acids encoding HLA-E, HLA-F, and / or HLA-G are introduced into NK cells (or PSCs or intermediates thereof from which the NK cells are derived), and the encoded proteins are produced and presented on the cell surface. Optionally, the engineered NK cells express HLA-E. Optionally, the engineered NK cells express a single-chain fusion HLA-E comprising at least a portion of B2M linked, directly or via a linker sequence, to at least a portion of an HLA-I alpha chain, e.g., HLA-E. Optionally, the engineered NK cells express a single-chain fusion HLA-F or HLA-G comprising at least a portion of B2M linked, directly or via a linker sequence, to at least a portion of an HLA-I alpha chain, e.g., HLA-F or HLA-G. The engineered NK cells may overexpress a protein naturally produced by the cell, may express a protein encoded by an exogenous nucleic acid after knockout or disruption of the protein's corresponding endogenous coding sequence, or may express a protein not naturally encoded by the wild-type NK cell genome. Representative DNA and protein sequences for HLA-E are set forth in SEQ ID NOs: 15 and 16. Representative DNA and protein sequences for HLA-F are set forth in SEQ ID NOs: 17 and 18. Representative DNA and protein sequences for HLA-G are set forth in SEQ ID NOs: 19 and 20. It should be understood that variants of the sequences set forth herein may be used, and thus the present disclosure also contemplates the use of nucleic acids comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequences disclosed herein.The present disclosure further contemplates the use of nucleic acids encoding peptides comprising amino acid sequences having 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater identity to any of the amino acid sequences described herein. Preferably, the variants maintain HLA-I function, such as forming a functional peptide-binding cleft for peptide presentation and / or engaging inhibitory receptors on NK cells. Cells expressing HLA-E, HLA-F, and / or HLA-G minimize the risk of rejection when used as adoptive cell therapy. In various embodiments of the present disclosure, NK cells are engineered to disrupt all copies of B2M and RFXANK and to express HLA-E, e.g., single-chain fusion HLA-E.

[0021]

[0056] The NK cells of the population may also express an exogenous receptor (i.e., a receptor encoded by an exogenous nucleic acid introduced into the NK cells or the PSCs from which the NK cells were derived, or an intermediate thereof). In various embodiments, the NK cells of the population express a chimeric antigen receptor. "Chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor engineered to recognize and bind to an antigen expressed by a target cell, e.g., a tumor cell. Generally, a CAR is a chimera of the signaling domain of the T cell receptor (TCR) complex and an antigen recognition domain (e.g., a single-chain fragment of an antibody (scFv) or other antibody fragment). See, e.g., Enblad et al., Human Gene Therapy. 2015;26(8):498-505. CARs come in various formats, each containing different components. "First-generation" CARs link the antigen-binding domain to the CD3 zeta intracellular signaling domain of the T cell receptor via a hinge and transmembrane domain. The cytoplasmic or intracellular signaling domain generates a stimulatory signal for proliferation and effector function when the CAR engages with a target antigen. Many intracellular signaling domains contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from CD8, CD3 zeta, CD3 gamma, CD3 epsilon, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, FcγRIλ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG). Intracellular signaling domains include those derived from CD3 zeta (CD3), which are known in the art (TCR zeta, e.g., GenBank accession number BAG36664.1). Exemplary hinge domains include, but are not limited to, a CD28 hinge, a CD8 alpha hinge, a human IgG4 hinge domain, and a combination of a human IgG4 hinge domain and a CH3 human IgG4 domain.Hinge domains are further described, for example, in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Publication WO 2014031687, and U.S. Patent No. 8,822,647. "Second-generation" CARs incorporate additional domains, such as CD28, 4-1BB (41BB), or ICOS, to provide costimulatory signals. Additional costimulatory domains include, but are not limited to, CD27, CD134, OX40, CD149, DAP10, CD30, IL2-R, IL7r6, IL21-R, NKp30, NKp44, CD40, CD137, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or DNAM-1 costimulatory domains (or any combination thereof). "Third generation" CARs contain two costimulatory domains fused to a TCR CD3 zeta chain. Third generation costimulatory domains include, for example, a combination of CD3 zeta, CD27, CD28, 4-1BB, ICOS, or OX40. CARs constructed in this manner can, for example, trigger cell activation upon binding to a target antigen in a manner similar to endogenous T cell receptors but in a major histocompatibility complex (MHC)-independent manner.

[0022]

[0057] Optionally, NK cells can express a fragment crystallized (Fc) region receptor (FcR), such as CD16 (FcγRIII). In this regard, NK cells can be engineered to express FcR, i.e., a nucleic acid encoding an FcR is introduced into NK cells (or PSCs or intermediates thereof from which the NK cells are derived), and the encoded protein is produced and presented on the cell surface. Generally, an FcR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain generally comprises a domain that binds to the immunoglobulin (Ig) Fc region. The intracellular domain comprises a sufficient portion of the Fc receptor to enable ITAMs to initiate cell signaling upon binding of the Ig Fc region to the extracellular domain. Cells expressing FcR mediate both the efficacy of antibody-based therapeutics and, to a lesser extent, B cell-mediated adaptive immune responses. NK cells are mediators of antibody-dependent cell-mediated cytotoxicity (ADCC), which is driven by Fc binding to FcR. In ADCC, the FcR of NK cells binds to antibodies, forming a lytic synapse at the interface with the target cell, triggering the release of cytokines and lytic granules. These granules contain enzymes such as perforin and granzyme B, which induce the destruction of antibody-coated target cells. Engineered NK cells that express Fc region receptors are referred to herein as "FcR-UDC-NK cells" or "FcR-NK cells."

[0023]

[0058] NK cells may express Fc-gamma (γ) receptors, Fc-alpha (α) receptors, or Fc-epsilon (ε) receptors. Receptors of the Fcγ family bind IgG and include FcγRI (CD64), FcγRII (including CD32, CD32a, CD32b, and CD32c), and FcγRIII (including CD16, CD16a, and CD16b). CD16a has two allelic variants, 158V and 158F, which differ in affinity for IgG; the NK cells of the present disclosure may express CD16a, CD16b, or both. Cancer patients homozygous for the 158V high-affinity allele of CD16a have been reported to respond well to monoclonal antibody (mAb) therapy (see, e.g., Cartron et al., Blood. 99, 754-58 (2002)). FcRn also binds IgG. The Fcε receptor family binds IgE and includes FcεRI and FcεRII (CD23). The Fcα family of receptors binds IgA and includes FcαRI (CD89) and Fcα / μR. NK cells may express any one or more of these FcRs. Furthermore, FcRs may be chimeric FcRs, in which part of the FcR structure is derived from one FcR and another part is derived from a different FcR (or is a composite with similar function to the FcR subunit).

[0024]

[0059] NK cells can express non-natural NKG2D receptors, such as receptors comprising non-natural NKG2D receptor ectodomains that bind to engineered NKG2D ligand α1-α2 domains with higher affinity than native α1-α2 domains. Non-natural NKG2D receptors are described in further detail, for example, in U.S. Pat. No. 10,259,858 and U.S. Patent Application Publication No. 2020 / 0138866, which are incorporated by reference in their entireties, particularly for their disclosures regarding engineered NKG2D ectodomains.

[0025]

[0060] The NK cells of the population may express an exogenous polypeptide (i.e., a polypeptide encoded by an exogenous nucleic acid introduced into the NK cells or the PSCs from which the NK cells are derived, or an intermediate thereof). In various embodiments, the NK cells of the population express cytokines, chemokines, etc., including, but not limited to, IL-15 or IL-2.

[0026]

[0061] Optionally, the NK cells further comprise a nucleic acid molecule encoding a suicide gene product. Combinations of nucleic acid molecules encoding different suicide gene products are also contemplated. Suicide genes are useful for selectively killing cells by inducing apoptosis or converting non-toxic compounds into toxic compounds to kill unwanted cells. Examples of suicide gene products include, but are not limited to, caspases (e.g., caspase 9), thymidine kinases, cytosine deaminases, cytochrome P450s, and DNases. See, for example, U.S. Patent Application Publication No. 2022 / 0025001 (incorporated herein by reference). Optionally, the nucleic acid encoding the suicide gene product is operably linked to an inducible promoter. See, for example, Straathof et al., Blood, 105(11), 4247-4254 (2005) (incorporated herein by reference). Optionally, the engineered NK cells comprise a herpes simplex virus thymidine kinase (TK) suicide gene. See, e.g., Bonini et al., Science, 276, 1719-1724 (1997) (incorporated herein by reference). Optionally, the engineered NK cells contain one or more copies of a TK suicide gene. In various embodiments, the NK cells contain a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. For example, the NK cells may contain a nucleic acid encoding an HSV TK and a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. In another embodiment, the NK cells can be depleted by targeted antibody-mediated depletion using any number of NK cell-specific targets, including, but not limited to, CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and / or NKp44.

[0027]

[0062] The method of genetically engineering host cells to stably produce one or more gene products of interest is known in the art.Exemplary method includes using viral vector.Viral vector includes any suitable viral vector, including, for example, retrovirus, adenovirus, parvovirus (for example, adeno-associated virus), coronavirus, orthomyxovirus (for example, influenza virus), rhabdovirus (for example, rabies virus and vesicular cheilitis virus), paramyxovirus (for example, measles virus and Sendai virus), picornavirus, alphavirus, herpesvirus (for example, herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus) and poxvirus (for example, vaccinia virus, fowlpox virus and canarypox virus).Exogenous nucleic acid can also be introduced into host cells by gene editing technology, for example, using the system described herein. Preferably, the nucleic acid encoding CAR or FcR and the nucleic acid encoding HLA-E, HLA-F, or HLA-G are integrated into the cell genome, such as the B2M or HLA-II locus. Thus, in various embodiments, the B2M locus is disrupted by inserting a nucleic acid encoding a different gene product into the B2M locus to replace the expression of endogenous wild-type B2M protein. Alternatively or additionally, a specific HLA-II locus is disrupted by inserting a nucleic acid encoding a different gene product into the HLA-II locus to replace the expression of endogenous wild-type HLA-II protein.

[0028]

[0063] In various embodiments of the present disclosure, NK cells are derived from pluripotent stem cells. The term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are typically functionally defined as stem cells that: (a) have the ability to induce teratomas when transplanted into immunodeficient (SCID) mice; (b) can differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal); and (c) express one or more markers of embryonic stem cells (e.g., express Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). Pluripotent stem cells can be generated, for example, using methods known in the art. Examples of pluripotent stem cells include embryonic stem cells. "Embryonic stem cells" (ESCs) include, for example, cells derived from the inner cell mass of a human blastocyst or morula, including those serially passaged as cell lines. Regardless of their origin or method of generation, embryonic stem cells can be identified based on, for example, (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunodeficient animals.

[0029]

[0064] Another exemplary pluripotent stem cell is an induced pluripotent stem cell (iPSC). iPSCs are generated by reprogramming somatic cells by expressing a combination of factors ("reprogramming factors"), including, but not limited to, Oct4, Sox2, c-Myc, Nanog, Lin28, and / or Klf4. Induced pluripotent stem cells can be generated using virtually any somatic cell at any developmental stage as a starting point. For example, cells can be derived from embryonic, fetal, neonatal, infant, or adult donors. Exemplary somatic cells that can be used include CD34+ umbilical cord blood cells, fibroblasts such as dermal fibroblasts obtained from skin samples or biopsies, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. In certain embodiments, the somatic cells are not fibroblasts.

[0030]

[0065] Pluripotent stem cells can be derived from any species. Embryonic stem cells have been successfully derived from, for example, mice, several non-human primate species, and humans. Therefore, those skilled in the art can generate embryonic stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mice and rats), ungulates (such as cattle, sheep), dogs (domestic and wild dogs), cats (domestic and wild cats, e.g., lions, tigers, and cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (such as dolphins and whales). Similarly, iPSCs can be derived from any species. iPSCs have been successfully generated using, for example, mouse and human cells. Therefore, iPSCs can be easily generated using donor cells from any species, such as those described herein.

[0031]

[0066] The population of NK cells disclosed herein possess various properties that make them advantageous for therapeutic applications, such as, for example, superior engraftment, homing, survival, persistence, immune evasion, and / or cytotoxicity. For example, in various embodiments, the engineered population of NK cells can be transplanted into a subject after administration, e.g., using protocols known to those skilled in the art. In other words, the population of NK cells can home to a target tissue (e.g., bone marrow) and / or persist in a subject for a period of time (e.g., 24 hours to several days, or weeks or months) after administration. In various embodiments, at least a portion of the population of engineered NK cells (e.g., at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or the entire population) has the ability to engraft in a target tissue. For example, in various embodiments, at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the population of engineered NK cells are capable of engrafting in the target tissue (e.g., blood or bone marrow) of the subject. Optionally, upon administration to a subject, the population of engineered NK cells can remain engrafted in the target tissue for at least 1 day (e.g., at least 2 days, at least 5 days, at least 10 days). In this regard, at least a portion of the population of NK cells can remain engrafted in the target tissue for at least 20 days (e.g., at least 50 days, at least 100 days, at least 200 days, at least 500 days, or at least 1000 days) after administration.

[0032]

[0067] Alternatively or additionally, the NK cells in the population can optionally induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or natural cytotoxicity (NCC) of cancer cells. NK cells mediate NCC through adhesion and conjugation to target cells and the release of perforin and granzymes, which induce apoptosis in the target cells. ADCC is a mechanism by which immune cells bearing Fc receptors kill antibody-coated cells when the Fc receptor binds to the Fc portion of the antibody. NK cells mediate ADCC, for example, via CD16, and their engraftment results in the release of cytolytic granules and inflammatory mediators. Methods for characterizing ADCC and NCC activity are known in the art and are described herein and in, for example, U.S. Patent Application Publication No. 2020 / 0131475 (which describes an assay involving the use of Raji cells (derived from Burkitt's lymphoma) pre-incubated with an anti-CD20 antibody as targets for the ADCC assay (Tsirigotis et al., J of Steroid Biochem and Mol Bio 108:267-271 (2008)); U.S. Patent Application Publication No. 2022 / 0040230; and Bhatnagar et al. al., Eur. J. Immunol., 44(11):3368 (2014). In various embodiments, administering a population of NK cells to a subject suffering from cancer reduces the subject's tumor burden or burden by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. Tumor burden is characterized using any suitable method, including, but not limited to, X-ray, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels, cytology, histology, biopsy samples, and / or enumeration of circulating target cells.

[0033]

[0068] Optionally, at least a portion of the NK cells in the population (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) have an increased volume compared to peripheral blood NK cells. Methods for measuring cell volume are known in the art, as set forth in Model, Cytometry, 93(3):281 (2018), which is incorporated by reference in its entirety, particularly with respect to its description of methods for measuring cell volume. In various embodiments of the present disclosure, the NK cells of the population have at least about a 5% increase in cell volume compared to peripheral blood NK cells. For example, the NK cells of the population have at least about a 10% increase, or at least about a 15% increase (e.g., about 5% to about 20%, about 7% to about 18%, about 10% to about 15%, about 10% to about 20%, or about 15% to about 20%) in cell volume compared to peripheral blood NK cells.

[0034]

[0069] The present disclosure also provides kits comprising one or more containers containing a population of NK cells described herein. The kits can further include instructions and written information regarding the efficacy and use of the NK cells. Sterile, sealed containers, such as vials, bottles, vessels, and / or packages, containing a population of NK cells, optionally with appropriate instructions for use, are also contemplated. In a further embodiment, the present disclosure provides an article of manufacture or unit dosage form comprising: (a) a population of NK cells described herein; (b) a container containing the population; and (c) a label affixed to, or package insert included in, the container regarding the use of the NK cells in the treatment of a disease or disorder (e.g., cancer).

[0035]

[0070] Also provided herein are pharmaceutical compositions comprising a population of NK cells and a pharmaceutically acceptable carrier, excipient, or diluent. In exemplary embodiments, the composition is sterile. Pharmaceutical compositions according to the present disclosure can be formulated for delivery by any route of administration. "Route of administration" may refer to any route known in the art, including, but not limited to, aerosol, intranasal, oral, transmucosal, transdermal, or parenteral administration. "Parenteral administration" refers to the route of administration commonly associated with injection and includes intraorbital, infusion, intraarterial, intracystic, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, intratumoral, transmucosal, or intratracheal administration. The composition may be in the form of a solution or suspension for infusion or injection. Each component of the carrier is "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation and safe for in vivo use.

[0036]

[0071] The present disclosure further provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a population of engineered NK cells, or a pharmaceutical composition comprising the population of engineered NK cells, thereby treating the disease or disorder in the subject. Similarly, the present disclosure provides uses of the population of engineered NK cells in the treatment of a disease or disorder, as well as the use of the population of NK cells in the preparation of a medicament for treating a disease or disorder in a subject in need thereof.

[0037]

[0072] In various embodiments, the disease or disorder is cancer. Examples of cancer include, but are not limited to, prostate cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, endometrial cancer, soft tissue carcinoma, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer (e.g., renal cell carcinoma), oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, parathyroid cancer, skin cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), cervical cancer, brain cancer (e.g., glioblastoma), liver cancer (e.g., hepatocellular carcinoma), bone cancer (e.g., osteosarcoma), or ovarian cancer. Hematopoietic cancers are particularly contemplated. Examples of cancer also include leukemias and lymphomas, such as acute myeloid leukemia, hairy cell leukemia, chronic myeloid leukemia, and ovarian cancer. Examples of cancers include, but are not limited to, lymphocytic leukemia, and non-Hodgkin's lymphoma (e.g., diffuse large B-cell lymphoma, Burkitt's lymphoma, Mantel cell lymphoma, and follicular lymphoma). Other cancers include, but are not limited to, alveolar rhabdomyosarcoma, breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, esophageal cancer, gastrointestinal cancer, Hodgkin's lymphoma, malignant mesothelioma, multiple myeloma, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, and ureteral cancer.

[0038]

[0073] In various embodiments, the disease or disorder is an autoimmune disease or disorder. Autoimmunity is an immune response to self-antigens, causing the body to attack normal cells and tissues. There are over 80 types of autoimmune diseases. The body's response to an autoimmune condition is similar to that of an infection, involving immune cell activation, inflammation, and tissue damage. In various embodiments, the autoimmune condition is one of over 80 diseases or syndromes characterized by loss of B cell tolerance and the production of autoantibodies. In various embodiments, the autoimmune condition is graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), multiple sclerosis, Sjögren's syndrome, systemic sclerosis / scleroderma, scleroderma, ulcerative colitis, inflammatory bowel disease, bullous pemphigoid, insulin-dependent diabetes mellitus, diabetes mellitus or type 1 diabetes, Crohn's disease, psoriatic arthritis, or rheumatoid arthritis.

[0039]

[0074] The method can include administering the population of NK cells before, concurrently with, or after other therapies, such as chemotherapy, surgical resection of a tumor, immunotherapy (such as cytokine therapy or administration of an antibody construct), or radiation therapy. For example, the present disclosure contemplates methods of administering the engineered population of NK cells described herein to a subject in need thereof as part of a treatment regimen that also includes administration of one or more cytokines to the subject. In some embodiments, the cytokine is an interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, or IL-33). For example, in various embodiments of the method, the method further comprises administering IL-15 and / or IL-2 to the subject.

[0040]

[0075] In various embodiments, the methods involve the use of engineered NK cells described herein (e.g., FcR-UDC I-NK (e.g., differentiated cells with a disruption of the B2M gene resulting in the loss of HLA class I proteins and expressing exogenous single-chain fusion HLA-E as well as CD16) or FcR-UDC I / II-NK (e.g., FcR-UDC I / II-NK further engineered with a knockout mutation of an HLA class II-associated gene, such as RFXANK, resulting in the disruption of HLA class II proteins). I-NK) as part of a treatment regimen that also includes administering one or more antibody constructs (e.g., antibodies) to the subject. The present disclosure further contemplates treatment regimens in which an antigen-binding antibody fragment or antibody-like protein product (i.e., a protein based on the structural features of an antibody that enable antigen binding) is administered to the subject. An "antibody construct" refers to an antibody, an antigen-binding fragment of an antibody, or an antibody-like protein product. An "antibody" generally refers to an intact immunoglobulin. Disclosures herein that refer to antibodies also apply to antibody fragments and antibody-like protein products. Suitable antigen-binding antibody fragments include, for example, F(ab)2 or Fab fragments. Antibody-like protein products include, but are not limited to, single-chain Fvs (scFvs), diabodies, nanobody constructs, antibody mimetics, and the like. In various embodiments, the method comprises administering an intact antibody, antibody fragment, or antibody-like protein product comprising an Fc region. In various embodiments, the antibody, antibody fragment, or antibody-like protein product binds to a cell surface antigen (e.g., a tumor antigen), such as, but not limited to, 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171 , CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, CLDN3, CLDN18.2, CLDN18.1, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto-1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin-A4, ephrin-B2, ephrin-B4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2) ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GM3, GPC1, GPC2, GPC3, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, interferon receptor, integrin (α4, α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α 11bincluding β3 integrin), KIT, LAGE-la, LAIR1, LAMP-1, LCK, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, MYCN, myostatin, NA17, NCA-90, NCAM, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PD-L1, PD-L2, PDGFR, PDGFR-beta, PIK3CA, PLAC1, polysialic acid, PRSS21, PSCA, PSMA, PTK7, RAGE-1, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, TGF-1, TGF-β2, TGS5, Tie2, TIM-1, Tn The Ag binds to TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1, or a combination thereof.Examples of antibodies for use in connection with the methods disclosed herein include rituximab, cetuximab, trastuzumab, panitumumab, ofatumumab, brentuximab, pertuzumab, ado-trastuzumab emtansine, obinutuzumab, nimotuzumab, bevacizumab, alemtuzumab, gemtuzumab, ranibizumab, olaratumumab, ontuximab, isatuximab, sacituzumab, daratumumab, lintuzumab, balantamab, indatuximab, dinutuximab, alemtuzumab, ibritumomab, tositumomab, Examples of antibodies include, but are not limited to, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, zolbetuximab (e.g., WO2016166124, which is incorporated herein by reference in its entirety, and is particularly incorporated with respect to the description of zolbetuximab), ASP1650 (e.g., as described in WO2012156018, which is incorporated herein by reference in its entirety, and is particularly incorporated with respect to the description of SEQ ID NOS: 35 and 36, the CDR sequences within SEQ ID NOS: 35 and 36, and SEQ ID NOS: 27 and 25), ASP1893 mAb (a humanized version of ASP1650 (i.e., which shares the CDR sequences of ASP1650)), and veltuzumab. Additional examples of antibodies include, but are not limited to, adalimumab, eculizumab, and natalizumab. In some embodiments, the antibody is rituximab, ASP1893 mAb, ASP1650, or zolbetuximab.

[0041]

[0076] For example, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a population of engineered NK cells described herein, and further administering to the subject an antibody that binds to a cell surface antigen on a target cell, the removal of which provides a therapeutic benefit to the subject. In various embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a population of engineered NK cells and an effective amount of an antibody specific for a cancer cell antigen (e.g., any of the anti-cancer antibodies described herein). In various embodiments of the present disclosure, an effective amount refers to treating the patient's cancer by administering multiple doses and / or multiple rounds of NK cells and antibody as a course of treatment. Merely by way of example of embodiments of the present disclosure, a subject may have breast cancer and NK cells may be administered with an anti-HER2 antibody (e.g., trastuzumab), or a subject may have colon carcinoma and NK cells may be administered with an anti-EGFR antibody (e.g., cetuximab or panitumumab); or a subject may have AML and NK cells may be administered with an anti-CD123 antibody or an anti-FLT3 antibody. The subject may also have a CD20-positive cancer. In various embodiments, the subject has a CLDN18.2-positive cancer, such as gastric or gastroesophageal junction (GEJ) adenocarcinoma, and NK cells are administered with an anti-CLDN18.2 antibody.Alternatively, the subject may have a CLDN6-positive cancer (e.g., ovarian cancer, particularly ovarian adenocarcinoma or ovarian teratocarcinoma, lung cancer, such as small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma or sarcomatous carcinoma, bile duct cancer, urinary tract cancer, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, , particularly renal cell carcinoma, e.g., clear cell renal cell carcinoma or papillary renal cell carcinoma, colon cancer, small intestine cancer, e.g., ileal cancer, particularly small intestinal adenocarcinoma and ileal adenocarcinoma, testicular germ cell carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and testicular germ cell carcinoma, uterine cancer, germ cell tumor, e.g., teratoma carcinoma or germ cell carcinoma, particularly testicular germ cell tumor, and metastatic forms thereof), and the NK cells may be administered together with an anti-CLDN6 antibody. Optionally, the antibody is rituximab, zolbetuximab, ASP1893 mAb, or ASP1650.

[0042]

[0077] In various aspects, the present invention provides a method of treating an autoimmune condition in a subject in need thereof, comprising administering to the subject an effective amount of a population of engineered NK cells and an effective amount of an antibody specific for an immune cell antigen, optionally wherein the antibody is rituximab.

[0043]

[0078] In some embodiments of the present disclosure, the engineered NK cells are optionally co-administered with an antibody as part of a treatment regimen that may optionally include a chemotherapeutic agent that is not an antibody. Optionally, the chemotherapeutic agent is selected from fluorouracil, oxaliplatin, and / or capecitabine.

[0044]

[0079] As used herein, the term "treatment" and related terms do not necessarily imply 100% or complete cure or remission. Rather, there are various degrees of treatment that those skilled in the art will recognize as having potential benefit or therapeutic effect. In this regard, a method for treating a disease or disorder can provide any amount or level of treatment. Furthermore, treatment by this method may include treating one or more conditions or symptoms or signs of the disease being treated. For example, the treatment method of the present disclosure may inhibit one or more symptoms of the disease. Treatment by the method of the present disclosure may also include slowing the progression of the disease.

[0045]

[0080] Cancer treatment can be determined in various ways. Any improvement in the subject's health status is contemplated (for example, at least or about 10% reduction, at least or about 20% reduction, at least or about 30% reduction, at least or about 40% reduction, at least or about 50% reduction, at least or about 60% reduction, at least or about 70% reduction, at least or about 80% reduction, at least or about 90% reduction, or at least or about 95% reduction in any parameter described herein). For example, therapeutic effect refers to one or more of the following improvements in disease: (1) reduction in tumor cell count; (2) increase in tumor cell death; (3) inhibition of tumor cell survival; (5) inhibition (i.e., delaying to some extent, preferably stopping) of tumor growth or the appearance of new lesions; (6) reduction in tumor size or amount (i.e., burden); (7) disappearance of clinically detectable disease; (8) reduction in the level of cancer marker; (9) increase in patient survival rate or survival time, and / or (10) some alleviation of one or more symptoms (e.g., pain) associated with disease or condition.

[0046]

[0081] For example, in various embodiments, administration of the engineered NK cells of the present disclosure increases the subject's survival time by at least 20 days (e.g., at least 30 days, at least 45 days, at least 60 days, at least 90 days, or more) compared to the subject's survival time if the population of engineered NK cells were not administered. Alternatively, or additionally, administering the NK cells to the subject reduces the subject's tumor burden. In various embodiments, the methods of the present disclosure further include monitoring the subject's treatment. Disease status can be monitored, for example, by clinical examination, X-ray, computed tomography (CT, e.g., spiral CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels in cancer (e.g., carcinoembryonic antigen (CEA)), cytology, histology, biopsy sampling, and / or enumeration of circulating target cells. Other methods and criteria known in the art include, for example, RECIST (Response Evaluation Criteria in Solid Tumors) and irRC (Immune Response Criteria). The term "effective amount" refers to a quantity of NK cells (or combination therapy, such as antibody therapy) sufficient to achieve a desired biological response in a clinically relevant period of time; for example, "therapeutically effective" refers to a quantity of NK cells sufficient to ameliorate one or more causes or symptoms of a condition or symptom. The amount of engineered NK cells administered to a subject can be, for example, 10 4 ~10 11 , 10 5 ~10 10 , or 10 6 ~10 9 The dose may include cells / kg body weight.

[0047]

[0082] Subjects include, but are not limited to, mammals of the order Rodentia, such as mice and hamsters; mammals of the order Lagomorpha, such as rabbits; mammals of the order Carnivora, including Felines (cats) and Canines (dogs); mammals of the order Artiodactyla, including Bovines (cows) and Swines (pigs); or mammals of the order Persodactyla, including Equines (horses). In some embodiments, the mammal is of the order Primate, Ceboid, or Simoid (monkeys) or Anthropoid (humans and apes). In some embodiments, the mammal is a human. The term "subject in need thereof" includes subjects already suffering from a disease or disorder as well as subjects in which a disease or disorder is to be prevented.

[0048]

[0083] The present disclosure also provides a method for producing natural killer (NK) cells from pluripotent stem cells (PS) under feeder-free, optionally serum-free conditions. The method includes the steps of: (a) culturing pluripotent stem cells, optionally genetically engineering the PSCs to disrupt one or more copies (e.g., all copies) of endogenous B2M and / or one or more copies (e.g., all copies) of an HLA class II-associated gene selected from the group consisting of RFXANK, RFX5, RFXAP, and CIITA, and optionally expressing a non-classical HLA class I protein (e.g., HLA-E) associated with B2M; (b) differentiating the pluripotent stem cells into progenitor cells capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; and (c) differentiating the progenitor cells into NK cells under feeder-free, optionally serum-free conditions in a culture medium containing SCF, IL-7, IL-15, and Flt3L (Flt3 ligand). In various embodiments, step (b) comprises differentiating the pluripotent stem cells using embryoid bodies to obtain progenitor cells that can differentiate into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives. In various embodiments, the PSCs are human PSCs. In various further embodiments, the method comprises (d) culturing NK cells in a culture medium containing IL-15 and IL-18 (and optionally IL-21). In various embodiments, step (d) comprises culturing NK cells in the presence of an antibody or ligand that binds to a receptor on NK cells and promotes NK cell activation and / or proliferation. In various embodiments, the antibody or ligand binds to DNAM-1, OX40, NKG2D, 2B4, NKp30, or NKp46. Optionally, the method includes culturing the NK cells in the presence of an antibody that binds to DNAM-1, an antibody that binds to OX40, an antibody that binds to NKG2D, an antibody that binds to 2B4, an antibody that binds to NKp30, and / or an antibody that binds to NKp46; any combination of the above antibodies can be provided during the culturing step.Alternatively, the method includes culturing NK cells in the presence of a ligand that binds to DNAX accessory molecule 1 (DNAM-1), a ligand that binds to OX40 (e.g., OX40L), a ligand that binds to natural killer group 2D (NKG2D) (e.g., MHC class I chain-related protein A (MICA) or UL16-binding protein 1 (ULBP1)), a ligand that binds to 2B4 (CD244), a ligand that binds to natural killer protein 30 (NKp30) (e.g., B7H6), and / or a ligand that binds to natural killer protein 46 (NKp46, CD355) to activate and / or expand the NK cells. Any combination of the above ligands can be provided during the culturing step. Optionally, the ligand comprises at least a portion of a natural ligand that mediates receptor binding and activation fused to an Fc domain.

[0049]

[0084] In various embodiments, the methods described herein are particularly suited for large-scale production of NK cells derived from human PSCs. Previous methods for NK production under feeder-free conditions have enabled 5- to 500-fold expansion of PBNK cells. See, e.g., Lu et al., J. Hematol. Oncol. 14:7 (2021). Surprisingly, the disclosed methods enable at least 1000-fold expansion of cells. Indeed, in some embodiments, the disclosed methods enable at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, or at least 10,000-fold expansion (or any range incorporating these endpoints). The expansion levels achieved under feeder-free conditions using the materials and methods described herein are superior to those achieved by previous methods.

[0050]

[0085] The method for producing engineered NK cells derived from human PSCs is performed under "feeder-free conditions." "Feeders" are cells that are often co-cultured with cells of interest (e.g., PSCs and / or NK cells); they support and promote the growth of target cells (e.g., PSCs) by releasing growth factors and nutrients into the intracellular environment. Cell types used as feeder cells include, for example, fibroblasts (mouse or human) as well as endothelial cells and other stromal cells (e.g., OP9 cells). Feeder-free conditions refer to culture conditions (or media) that are essentially free of feeder cells (e.g., stromal cells) and / or that have not been preconditioned by culturing feeder cells. Surprisingly, the disclosed method provides robust populations of NK cells without the need for feeder cells or media conditioned with feeder cells. In fact, the disclosed method produces comparable NK cells compared to NK production methods under feeder conditions. The use of feeder-free conditions is cost-effective and minimizes xenogeneic contaminants.

[0051]

[0086] "Culturing" refers to maintaining cells in a culture medium under conditions suitable for cell survival and / or proliferation. The steps described herein can be performed using any suitable container (e.g., microwell plate, flask, bioreactor, etc.) and environmental conditions that allow cell growth and differentiation. Any cell culture medium suitable for stem cell proliferation and differentiation can be used in this method. Exemplary media include, but are not limited to, mTeSR™1, TeSR™2, mTeSR™Plus, TeSR™E8, APEL™, STEMdiff™ APEL™2, or StemSpan™ from STEMCELL Technologies (Vancouver, Canada), primate ES / iPS cell medium from ReproCELL (Boston, Mass.), and StemPro®-34 and StemPro® hESC SFM from Invitrogen (Carlsbad, Calif.), Gibco® PFHM-II medium from ThermoFisher Scientific (Waltham, Mass.), and X-VIVO™ from Lonza (Basel, Switzerland). The medium optionally contains additives such as, for example, amino acids (e.g., glutamine, arginine, or asparagine), hydrolysates, vitamins, nucleosides, hormones and / or other growth factors (e.g., insulin, transferrin, insulin-like growth factor, or epidermal growth factor), ions (e.g., sodium, chloride, calcium, or magnesium), buffers, small molecules (e.g., GSK-3 inhibitors, MEK inhibitors, or Rho kinase (ROCK) inhibitors), and / or sugar(s) (e.g., glucose or galactose). Media lacking one or more of these additives are also contemplated. In various embodiments, the medium is a chemically defined medium, i.e., a cell culture medium in which all chemical components are known and which is typically serum-, serum albumin-, and hydrolysate-free.

[0052]

[0087] The method includes disrupting one or more copies (e.g., all copies) of endogenous B2M and / or one or more copies (e.g., all copies) of RFXANK, RFX5, RFXAP, or CIITA (or a combination thereof), and optionally culturing human pluripotent stem cells that have been genetically engineered to express an engineered non-classical HLA class I protein, e.g., HLA-E, associated with B2M, as described above for generating a population of NK cells. The disclosures regarding PSCs and NK cells described above also apply to the PSCs used in the method. For example, the human PSCs optionally include a nucleic acid molecule encoding one or more suicide gene products, such as HSV thymidine kinase, which may be present in one or more copies, and are optionally embryonic stem cells or induced PS cells, as described above.

[0053]

[0088] Any suitable initial seeding density can be used to establish the culture for differentiation, for example, about 0.5 x 10 5 ~About 10×10 5 An initial seeding density of 100 human pluripotent stem cells / mL can be used (e.g., approximately 1 x 10 5 ~Approx. 5×10 5 or about 1.5 x 10 5 ~Approx. 3.5×10 5 or about 2 x 10 5 ~Approx. 2.5×10 5cells / mL). Human pluripotent stem cells can be cultured for an appropriate period of time, e.g., 1 to 7 days (e.g., 1 to 4 days or 1 to 3 days), before the first differentiation step to obtain a sufficient number of human pluripotent stem cells suitable for differentiation. The method includes differentiating the human pluripotent stem cells to generate progenitor cells capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives. In various embodiments, pluripotent stem cells are differentiated into progenitor cells using embryoid bodies (EBs). See, e.g., Itskovitz-Eldor et al., Mol Med., 6(2):88-95 (2000), incorporated herein by reference in its entirety. EBs are aggregates or clusters of pluripotent cells. EBs are used as an intermediate step in the generation of progenitor cells capable of further differentiation into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives (or as an intermediate step in the production of hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives themselves). In various embodiments, the method does not include differentiating human pluripotent stem cells into hemangioblasts or hemangioblast colony forming cells as progenitor cells.

[0054]

[0089] The differentiation step to generate progenitor cells involves culturing human PSCs (e.g., using EBs) under conditions suitable for generating progenitor cells. In an exemplary embodiment, human pluripotent stem cells are cultured in a medium containing cytokines that promote the formation of EBs. In various embodiments, the medium contains at least one of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), or stem cell factor (SCF). Combinations of BMP4, SCF, and VEGF are contemplated, including the use of all three cytokines to induce differentiation. Cytokines can be used in any appropriate amount. For example, about 1 ng / mL to about 40 ng / mL of BMP4 may be provided (e.g., about 5 ng / mL to about 35 ng / mL, about 10 ng / mL to about 30 ng / mL, about 15 ng / mL to about 25 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, or about 24 ng / mL of BMP4 may be provided). Optionally, about 1 ng / mL to about 40 ng / mL of VEGF may be provided (e.g., about 5 ng / mL to about 35 ng / mL, about 10 ng / mL to about 30 ng / mL, about 15 ng / mL to about 25 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, or about 24 ng / mL of VEGF may be provided). Optionally, about 10 ng / mL to about 60 ng / mL of SCF may also be provided (e.g., about 25 ng / mL to about 55 ng / mL, about 30 ng / mL to about 50 ng / mL, about 35 ng / mL to about 45 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, or about 44 ng / mL of SCF may also be provided). In various embodiments, the method includes exposing the human pluripotent stem cells to fibroblast growth factor (FGF, e.g., bFGF); alternatively, the method does not include the use of FGF (e.g., bFGF).The differentiation step can be carried out for any suitable period, for example, from about 1 to about 21 days, such as from about 1 to about 14 days, from about 7 to about 18 days, from about 10 to about 14 days, or about 14 days. In various embodiments, the first differentiation step is carried out for up to about 14 days. During the differentiation step culture period, the medium may be changed periodically. Generally, at least a portion of the medium (e.g., half of the medium) is changed every two days until the end of the differentiation step. The conditions described herein produce high-quality EBs, which improves differentiation and NK cell yield.

[0055]

[0090] The method further comprises differentiating the progenitor cells (e.g., using EBs) into NK cells under feeder-free, optionally serum-free conditions in a culture medium comprising SCF, IL-7, IL-15, and Flt3L (i.e., step (c) or the second differentiation step). In various embodiments, the progenitor cells are collected and replated in a suitable container for growth, proliferation, and differentiation. The collected cells may be treated with SCF (e.g., about 5 ng / mL to about 40 ng / mL, about 10 ng / mL to about 35 ng / mL, about 15 ng / mL to about 30 ng / mL, about 15 ng / mL to about 25 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, or about 24 ng / mL of SCF), IL-7 (e.g., about 5 ng / mL to about 40 ng / mL, about 10 ng / mL to about 35 ng / mL, about 15 ng / mL to about 30 ng / mL, about 15 ng / mL to about 25 ng / mL, about 16 ng / mL, about 17 ng / mL, about 24 ng / mL of SCF), or IL-8 (e.g., about 5 ng / mL to about 40 ng / mL, about 10 ng / mL to about 35 ng / mL, about 15 ng / mL to about 30 ng / mL, about 15 ng / mL to about 25 ng / mL, about 16 ng / mL, about 17 ng / mL, about Culture the cells in medium containing IL-7 (18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, or about 24 ng / mL), IL-15 (e.g., about 1 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 7 ng / mL to about 15 ng / mL, about 9 ng / mL to about 12 ng / mL, or about 10 ng / mL of IL-15), and Flt3L (e.g., about 1 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 7 ng / mL to about 15 ng / mL, about 9 ng / mL to about 12 ng / mL, or about 10 ng / mL of Flt3L). The second differentiation step can be carried out for any suitable period, for example, from about 1 day to about 35 days, such as from about 1 day to about 21 days, from about 7 days to about 32 days, from about 10 days to about 28 days, from about 14 days to about 25 days, or about 21 days. In various embodiments, this step is carried out for up to about 21 days. The medium may be periodically changed during the second differentiation step culture period. In some embodiments, the culture is not agitated for an initial period (e.g., 1 day, 2 days, 3 days, or 4 days counted from the start of the differentiation step), although this is not required.At least a portion of the medium (eg, half of the medium) may be changed periodically, such as every two days, twice a week, or once a week, until the end of the differentiation step.

[0056]

[0091] In various embodiments, IL-3 is included in the culture medium during the second differentiation step. In this regard, about 1 ng / mL to about 10 ng / mL of IL-3 is optionally provided (e.g., about 3 ng / mL to about 8 ng / mL, about 4 ng / mL to about 6 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, or about 9 ng / mL of IL-3 is provided). IL-3 is optionally provided only for a portion of the second differentiation step culture period, e.g., the first 1, 2, 3, 4, 5, 6, or 7 days of culture, and is not included in the culture medium for the remainder of the second (NK) differentiation step.

[0057]

[0092] In a preferred embodiment of the present disclosure, the culture medium for the second differentiation step (step (c), differentiation of progenitor cells into NK cells) contains UM171. Any appropriate amount of UM171 can be used to achieve the desired effect; typically, about 10 nM to about 60 nM (e.g., about 15 nM to about 55 nM, about 20 nM to about 50 nM, about 25 nM to about 45 nM, about 30 nM to about 40 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, or about 39 nM) is used. UM171 is a pyrimido-indole derivative that significantly increases NK cell yield (i.e., NK cell expansion) and reduces variability in the resulting NK cell population. Therefore, a substantially homogeneous NK cell population can be achieved without the need to purify or filter the final NK cell product. The term "homogeneous" refers to a cell population in which each cell is identical or substantially identical to other cells within the population (i.e., expresses one or more of the same cell surface markers indicative of NK cells, such as CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and / or NKp44). In various embodiments of the present methods, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the resulting human pluripotent stem cell-derived engineered NK cell population are identical or substantially identical to other cells within the population (e.g., display one or more of the above-mentioned cell surface markers). In this regard, the present disclosure contemplates methods that do not include a purification, isolation, or filtration step to remove undesired cell types after differentiating the progenitor cells into NK cells (or, optionally, after culturing to activate the NK cells). In various embodiments, UM171 is present only in the culture medium of step (c) and not in the culture medium of other steps of the method (although this is not required).

[0058]

[0093] The disclosed methods optionally include a further culture step (step (d)) to promote NK cell activation and / or proliferation. While this step is not required, it may be desirable to provide a population of NK cells with particularly desirable characteristics (e.g., improved cytotoxicity, improved in vivo persistence, improved engraftment, etc.). The activation step involves culturing NK cells (from the second differentiation step, step (c)) in a culture medium containing IL-15 and IL-18. IL-15 and IL-18 may be provided in any amount, including, for example, about 1 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 7 ng / mL to about 15 ng / mL, about 9 ng / mL to about 12 ng / mL, or about 10 ng / mL of each of IL-15 and IL-18. Optionally, the NK cells are cultured in the presence of IL-15, IL-18, and IL-21 during at least a portion of the activation step (step (d)). Suitable amounts of IL-21 include, but are not limited to, about 1 ng / mL to about 30 ng / mL, about 5 ng / mL to about 20 ng / mL, about 7 ng / mL to about 15 ng / mL, about 9 ng / mL to about 12 ng / mL, or about 10 ng / mL of IL-21. In various embodiments, the culture medium utilized in the activation step does not contain IL-12.

[0059]

[0094] In various embodiments, NK cells are cultured in the presence of an antibody that binds to DNAM-1 (DNAX accessory molecule, CD222), an antibody that binds to OX40, an antibody that binds to NKG2D, an antibody that binds to 2B4, an antibody that binds to NKp30, an antibody that binds to NKp46, or any combination of the above antibodies. For example, step (d) can include culturing NK cells in the presence of an antibody that binds to NKp30. NKp30, also known as CD337, is a 30-kD type I transmembrane protein that is a stimulatory receptor on NK cells. Alternatively or additionally, step (d) can include culturing NK cells in the presence of an antibody that binds to 2B4. 2B4, also known as CD244, is a member of the signaling lymphocyte activation molecule (SLAM) family that mediates non-MHC-restricted killing. Alternatively or additionally, step (d) can include culturing NK cells in the presence of an antibody that binds to DNAM-1. DNAM-1, also known as CD226, is a 65 kD glycoprotein of the Ig superfamily expressed on the surface of NK cells, T cells, and monocytes / macrophages. For example, step (d) can include culturing NK cells in the presence of an antibody that binds to NKp30 and an antibody that binds to 2B4. In yet another example, step (d) can include culturing NK cells in the presence of an antibody that binds to NKp30 and an antibody that binds to DNAM-1. Anti-NKp30 antibodies, 2B4 antibodies, and anti-DNAM-1 antibodies are commercially available, for example, from SinoBiological (Wayne, PA) and BioLegend (San Diego, CA). In various embodiments, step (d) can include culturing the NK cells in the presence of a ligand that binds to DNAM-1, a ligand that binds to OX40 (e.g., OX40L), a ligand that binds to NKG2D (e.g., MICA or ULBP1), a ligand that binds to 2B4 (CD244), a ligand that binds to NKp30 (e.g., B7H6), and / or a ligand that binds to NKp46 to activate the NK cells. Any combination of the above ligands can be provided in the culturing step.The antibody, antibodies, ligand, or ligands may be provided in the culture medium or coated onto the container.

[0060]

[0095] The activation step (step (d)) can be carried out for any suitable period of time, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In various embodiments, step (d) is carried out for about 5 to about 10 days. Furthermore, the activation step may be carried out in multiple vessels to accommodate expansion of the NK cell population. For example, the activation step may involve culturing NK cells in one vessel (e.g., a flask such as a T75 flask) for an initial period (e.g., 1, 2, 3, or 4 days), and then transferring the cells to a larger vessel (e.g., a bioreactor) for a second period (e.g., 1, 2, 3, or 4 days). Also, in various embodiments, IL-21, if present, is provided for only a portion of the activation step. In this regard, the method can include including IL-21 in the culture medium for an initial period (e.g., one or more of days 0-4 of the activation step), but not including IL-21 in the culture medium for the remainder of the culture period.

[0061]

[0096] NK cells are cultured in a vessel (e.g., a flask or bioreactor) suitable for NK cell growth, proliferation, and activation. The vessel is optionally coated with a substance that promotes cell adhesion or one or more matrix components, such as laminin or laminin fragments, entactin, collagen, gelatin, vitronectin, fibronectin, Synthemax® (Corning Incorporated), Matrigel®, polylysine, thrombospondin, or ProNectin-F™. An ​​example of a matrix component is RetroNectin®, a 63-kD fragment of recombinant human fibronectin fragment, also referred to in the literature as rFN-CH-296. Optionally, a Notch ligand, e.g., DLL4, is provided. In various embodiments, the vessel is coated with a Notch ligand, e.g., DLL4 and RetroNectin®. DLL4 and RetroNectin® are provided in any suitable amount, including, for example, relative amounts that result in a DLL4:RetroNectin® ratio of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5. The coated container can be used in any of the method steps disclosed herein, including step (c) and / or step (d).

[0062]

[0097] Of note, performing the activation step (step (d)) multiple times has been found to not adversely affect the characteristics of the NK cell population (e.g., not significantly reduce the functionality of the cells). If desired, step (d) can be repeated up to five times (i.e., two, three, four, or five times) to further expand and / or activate the NK cells.

[0063]

[0098] In various embodiments of the method, IL-2 and / or IL-6 are not included in the culture medium for one or more (eg, any) steps of the method.

[0064]

[0099] The present disclosure further provides a method for increasing the purity of a population of pluripotent stem cell-derived NK cells. The method includes differentiating embryoid bodies (i.e., differentiated cells within the embryoid bodies) into NK cells under feeder-free, optionally serum-free conditions in the presence of UM171 in a culture medium containing SCF, IL-7, IL-15, and Flt3L. In various embodiments, the culture medium further contains IL-3. Optionally, the method is performed in a vessel coated with RetroNectin® and DLL4. Optionally, the method is performed for about 1 to about 21 days. The result of the method is a substantially pure CD56+, CD45+ NK cell population (i.e., a population of NK cells with low levels of undesired cell types (contaminating cells)). The method enables the generation of PSC-derived NK cells to obtain a substantially pure population of NK cells without the need for extensive further purification of the resulting cell population. In various embodiments, the method produces a cell population in which at least about 75% of the cells are CD56+, CD45+ NK cells (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells are CD56+, CD45+ NK cells).

[0065]

[0100] The disclosed methods yield robust populations of NK cells suitable for, e.g., therapeutic applications. For example, the NK cells optionally express one or more cell surface markers selected from the group consisting of CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and NKp44, e.g., any combination of these cell surface markers. In preferred embodiments, the NK cells also express at least CD56. The NK cells optionally express engineered non-classical HLA class I proteins associated with B2M, e.g., HLA-E, HLA-F, and / or HLA-G, as described above. The NK cells can also be engineered to express a CAR (e.g., a universal CAR), an FcR, a non-native NKG2D receptor, or other types of receptors (e.g., as described above). The resulting NK cell population also preferably exhibits the above characteristics, e.g., engrafts in a subject after administration, optionally remains engrafted for at least 20 days, is capable of inducing ADCC and / or NCC of cancer cells, and is capable of reducing tumor burden. In various embodiments, the method comprises: 9 , at least 10 10 , or at least 10 11 This results in the generation of CD56-positive NK cells.

[0066]

[0101] The following examples are intended to illustrate the present invention but not to limit its scope. [Example]

[0067]

[0102] The Examples describe exemplary methods for generating engineered natural killer (NK) cells from human pluripotent stem cells under feeder-free, serum-free conditions.

[0068] Example 1: Engineered human pluripotent stem cells

[0103] Human pluripotent stem cells (PSCs) (NIH2, NIH3, and W1C1) were genetically engineered to (i) disrupt the B2M gene to eliminate HLA class I protein function and (ii) express exogenous single-chain fusion HLA-E (UDC I-PSCs). The UDC I-PSCs were further engineered to lack HLA class II protein by knockout mutation of the HLA class II-associated gene RFXANK (UDC I / II-PSCs). Alternatively, the UDC I-PSCs or UDC I / II-PSCs were further engineered to express a gene encoding Fc receptor / CD16 (FcR-UDC I-PSCs or FcR-UDC I / II-PSCs). UDC I-PSCs, UDC I / II-PSCs, and / or FcR-UDC I-PSCs are collectively referred to herein as "UDC PSCs." The differentiated cells are referred to herein as, for example, "FcR-UDC I-NK" or "FcR-UDC I / II-NK."

[0069] Example 2: NK differentiation from parentally derived and engineered human pluripotent stem cells

[0104] Parental and engineered UDC PSCs (collectively "PSCs") were thawed, expanded, and cultured on vitronectin in mTeSR™ (STEMCELL Technologies, Cambridge, MA) feeder-free, animal component-free medium for up to 7 days. PSCs were dispersed into single cells, transferred to microwell plates, and cultured in STEMdiff™ APEL™ 2 and Gibco® PFHM-II medium (STEMCELL Technologies, Cambridge, MA; ThermoFisher Scientific, Waltham, MA) for 14 days to differentiate into progenitor cells (via embryoid bodies) capable of differentiating into hematopoietic, endothelial, and / or mesodermal derivatives. The medium was supplemented with BMP4 (20 ng / mL), VEGF (20 ng / mL), and SCF (40 ng / mL). Half of the medium was replaced every 2 days, beginning on day 3. Embryoid bodies were replated onto DLL4 / retronectin-coated T75 flasks and differentiated into NK cells. Embryoid bodies were cultured in a differentiation medium containing SCF (20 ng / mL), IL-7 (20 ng / mL), IL-15 (10 ng / mL), Flt3L (10 ng / mL), IL-3 (5 mg / mL), and 35 nM UM171 in modified DMEM / F12 medium under feeder-free conditions for 21 days. Half of the medium was replaced twice weekly. The resulting NK cells (UDC-NK) were washed and collected. UDC-NK cells were activated and expanded by culturing them in flasks coated with anti-NKp30 antibody, anti-DNAM-1 antibody, and retronectin in CTS™ NK-Xpander™ or RPMI-1640 medium containing IL-15 (10 ng / mL), IL-18 (10 ng / mL), and IL-21 (10 ng / mL) for 3 days. The UDC-NK cells were then transferred to G-REX® or spinner flask bioreactors and cultured in the presence of IL-15 and IL-18 for an additional 3 days. A representative schematic of the NK differentiation process is shown in Figure 1.

[0070]

[0105] This method yielded a substantially homogenous population of UDC NK cells, regardless of the parental cell line or engineered UDC PSCs. At the start of step 0 (as shown in Figure 1), approximately 4 × 10 6 Starting with approximately 5 x 10 iPSCs 10 NK cells were generated and expanded up to 10,000-fold. The addition of UM171 improved the yield of UDC-NK cells during NK differentiation, as shown in Figures 2A-2C. Furthermore, in multiple generation runs and gene-edited clones, at least 90% of the cells were CD56-positive and CD45-positive, without the need for purification steps to remove undesired cell types (see Figure 7A). At least 90% of the CD45-positive cells were obtained from both UDC I-PSCs and FcR-UDC I-PSCs. + CD56 + NK cells were generated. Furthermore, as shown in Figures 3A and 3B, NK cell activation and expansion resulted in consistent, high cell yields in PSC cell lines (NIH3 and W1C1) and gene-edited clones (UDC I-PSC, UDC I / II-PSC, and FcR-UDC I-PSC).

[0071]

[0106] For the activation and proliferation of UDC-NK cells (step 3 or step (d)), a combination of NK agonists was tested (see Figure 4). Single NK agonists were also tested and showed moderate NK activation. UDC-NK cells (step 2) showed robust proliferation and high cell viability. As shown in Figures 5A-5B, at four different cell seeding densities, UDC-NK cells (step 2) cultured on retronectin, anti-NKp30 antibody, and anti-DNAM-1 antibody in medium containing IL-15, IL-18, and IL-21 showed 16- to 32-fold or more proliferation and over 90% cell viability compared to the step 2 control, which only showed 3- to 4-fold proliferation.

[0072]

[0107] Analysis of cell population subsets and cell impurities at the end of the NK differentiation process showed very low levels of CD3+ T cells and few, if any, remaining PSCs. Table 1 shows details of the cell population subsets / impurities.

[0073] [Table 1]

[0074]

[0108] The UDC-NK cells of the present invention were, on average, about 18% larger in volume compared to peripheral blood NK cells (PB-NK) as shown by flow cytometry (see Figure 6).

[0075] Example 3: In vitro characterization of UDC PSC-derived NK cells

[0109] UDC-NK cells derived from UDC I-PSCs, UDC I / II-PSCs, and UDC I-FcR-PSCs exhibited the expected cell phenotypes in the different gene-edited clones. As shown in Figure 7A, parental and UDC PSC-derived UDC I-PSC-derived NK cells all expressed CD45 + CD56 + The positive rate was high. NK cells derived from UDC I-PSCs (UDC I-NK cells) did not express HLA class I, but did express HLA class II and HLA-E, and wild-type levels of CD16 (FcR). See Figures 7B-7E. UDC-NK cells derived from FcR-UDC I-PSCs (FcR-UDC I-NK cells) showed a similar expression profile, except for higher levels of CD16 compared to wild-type cells. Finally, UDC-NK cells derived from UDC I / II-PSCs (UDC I / II-NK cells) did not express HLA class I or II proteins, but expressed HLA-E and wild-type levels of CD16. This data indicates that NK cells derived from gene-edited clones maintain the gene editing even after differentiation from PSCs. The expected cell phenotype was also confirmed in different PSC cell lines (NIH3 and W1C1) (see Figures 8A-8B). Based at least on the above results, the NK differentiation process disclosed herein is robust across different gene-edited clones as well as PSC cell lines.

[0076]

[0110] UDC-NK cells expressed common NK markers and other biomarkers similar to those of PB-NK. As shown in Figure 9C, common NK markers, such as NKp30, NKp46, NKG2D, DNAM-1, 2B4, NKG2A, and CD94, were expressed in FcR-UDC I-NK cells (both steps 2 and 3 in Figure 1, i.e., steps (b) and (c), respectively) similarly to expanded PB-NK cells (PB-NK cells expanded in step 3 of the NK differentiation protocol), with some exceptions. Similarly, the T cell marker CD8 was commonly expressed in FcR-UDC I-NK cells and expanded PB-NK cells, whereas CD4, TCRαβ, or TCRγδ were not expressed, and CD3 was expressed with little or no expression (Figure 9B). The lack of TCRαβ plays an important role in safety.

[0077]

[0111] UDC-NK cells expressed multiple activation markers, and exhausted or "memory-like" markers were rarely, if ever, expressed. Figures 10A–10C show that FcR-UDC I-NK cells (step 2 and step 3) and expanded PB-NK cells were assayed for activated, exhausted, and "memory-like" markers. With the exception of CD69, activation markers were largely similar between FcR-UDC I-NK cells and expanded PB-NK cells. FcR-UDC I-NK cells showed significantly higher levels of CD69 than expanded PB-NK cells. Unlike expanded PB-NK cells, FcR-UDC I-NK cells showed few, if any, exhausted or "memory-like" markers. Expanded PB-NK cells showed higher levels of the exhaustion markers TIGIT, LAG3, KLRG1, and ILT2, and higher levels of the memory-like marker NKG2C. Differential expression of cytokine and chemokine receptors (see Figures 11A-11B) and adhesion / homing, costimulatory, and apoptosis markers (see Figures 12A-12C) is shown for FcR-UDC I-NK cells and expanded PB-NK cells.

[0078]

[0112] UDC-NK cells also demonstrated natural cytotoxicity (NCC) activity in multiple runs, gene-edited clones, and PSC cell lines. Using a 2-hour europium release assay, the NCC activity of UDC-NK cells was measured by their ability to kill K562 tumor target cells. Figure 13 shows that 1 × 10 cells per well were used for the analysis. 4 10 K562-GFP tumor cells were transfected and a corresponding number of UDC-NK cells were added depending on the E:T ratio. The results show consistent and similar in vitro NCC activity (i.e., specific lysis) of UDC-NK cells against K562 tumor cells across multiple runs, regardless of whether they were PSC cell lines or gene-edited clones. Furthermore, FcR-UDC I-NK cells exhibited NCC activity similar to that of expanded PB-NK cells; at the same time, no cytotoxicity or flatricidality was observed against normal allogeneic peripheral blood mononuclear cells (PBMCs) or the FcR-UDC I-NK cells themselves (see Figure 16). Surprisingly, these UDC-NK cells exhibited continuous NCC activity against K562 tumor cells, and UDC-NK cells were able to kill freshly added K562 tumor cells in a dose-dependent manner (see Figure 17). IncuCyte continuous killing assays demonstrated that 1.5 x 10 UDC-NK cells were transfected with 1.5 x 10 UDC-NK cells. 4 (3:1 E:T) or 5 × 10 4 This was performed using UDC-NK cells (FcR-UDC I-NK) (c.3C1) in a 10:1 E:T ratio. 5 × 10 3 K562-GFP target cells were added in a total of six wells, and the wells were imaged every 3 hours for 6 days.

[0079]

[0113] Like NCC, UDC-NK cells express a broad range of CD20 +They demonstrated potent antibody-dependent cellular cytotoxicity (ADCC) against tumor cell lines. Using a 4-hour flow-based ADCC assay, tumor cells were labeled with cell trace violet and the antibody was administered at a concentration of 1 μg / ml. When combined with rituximab, FcR-UDC I-NK cells demonstrated increased ADCC activity against three tumor cell lines (Daudi, Ramos, and Raji) compared with a control IgG1 antibody (see Figures 18A-18C). In contrast, expanded PB-NK cells plus rituximab did not demonstrate increased ADCC activity against Raji tumor cells compared with a control IgG1 antibody (see Figure 18D). Furthermore, FcR-UDC I-NK cells demonstrated NCC and ADCC activity against a broad range of B lymphoma cell lines (see Figure 19). These results suggest that UDC-NK cells express a broad range of CD20 + and have been shown to have ADCC activity against B lymphoma cell lines.

[0080]

[0114] UDC-NK cells produced a cytokine profile indicative of activated NK cells capable of inducing ADCC after stimulation with rituximab-coated Raji tumor cells. Using a standard cytokine secretion assay, 1 × 10 5 FcR-UDC I-NK cells / well were co-cultured with rituximab-coated Raji tumor cells at an E:T ratio of 1:1 for 25 hours. Cytokine secretion was measured using a 15-plex Cytokine Luminex immunoassay (see Figure 20A). Furthermore, FcR-UDC I-NK cells did not increase cytokine / chemokine secretion after co-culture with allogeneic PBMCs, demonstrating the specificity of FcR-UDC I-NK cells (see Figures 20B-E and 21A-D).

[0081] Example 4: In vivo persistence and distribution of UDC-PSC-derived NK cells

[0115] UDC-NK cells demonstrated in vivo persistence in the blood, spleen, and bone marrow, even in the absence of antigen. NSG mice were injected with 4 × 10 6FcR-UDC I-NK cells (c.3C1) were injected intravenously (iv) followed by IL-2 (30,000 IU, 3 times a week) and IL-15 (100 ng, daily for 7 days). The percentage of FcR-UDC I-NK cells among lymphocytes / WBCs in each tissue was determined by flow cytometry and staining for human CD45 and human CD56. Total cell counts were measured by flow cytometry (per mL of blood, per spleen, and per BM cells from two femurs and two tibias), and the percentage of FcR-UDC I-NK cells in each tissue was calculated. As shown in Figures 22A-22G, FcR-UDC I-NK cells persisted in vivo for more than 10 days after i.v. administration in the absence of antigen. Furthermore, FcR-UDC I-NK cells were distributed in multiple tissues: lung > liver > spleen > blood > bone marrow (see Figures 23A-23E).

[0082] Example 5: In vivo antitumor efficacy study in the Raji intraperitoneal tumor model using UDC-NK cells

[0116] The in vivo efficacy of UDC-NK cells against tumors was examined in the Raji intraperitoneal (ip) tumor model. In a representative study, NSG mice were divided into six groups (n=7 or 8) as shown in Table 2 below.

[0083] [Table 2]

[0084]

[0117] In Figure 24, NSG mice were irradiated and injected with Raji (Burkitt's lymphoma) cells (3 x 10 5 Raji cells are resistant to NK cells in the absence of anti-CD20 antibodies. On day 4, NSG mice were intraperitoneally injected with 5 × 10 6NSG mice were administered 1 dose of FcR-UDC I-NK cells and / or rituximab (300 μg). On day 7, a second dose of rituximab (300 μg) was administered. IL-15 (100 ng) was administered daily for 7 days and IL-2 (30,000 IU) was administered three times weekly for 3 weeks. NSG mice were imaged and evaluated to assess survival, BLI (tumor burden), body weight, and other health status at various days up to day 121. As shown in Figures 25A-25B, the combination of FcR-UDC I-NK and rituximab significantly extended the survival of lymphoma-bearing NSG mice compared to that observed with rituximab or FcR-UDC I-NK cells alone (see Figure 25A). The efficacy of FcR-UDC I-NK cells plus rituximab and PB-NK cells plus rituximab was similar (see Figure 25B), although the response rate was approximately 50%.

[0085]

[0118] In another study, mice were divided into 5 groups (n=8) as shown in Table 3 below.

[0086] [Table 3]

[0087]

[0119] In Figure 26, NSG mice were irradiated and injected with Raji (Burkitt's lymphoma) cells (2 x 10 5 On day 2, 10 × 10 6 Mice were administered 10 × 10 FcR-UDC I-NK cells and / or 100 μg rituximab. On days 6, 10, and / or 16, 10 × 10 6Mice were intraperitoneally injected with FcR-UDC I-NK cells. Mice received daily doses of IL-15 (100 ng) and IL-2 (30,000 IU) three times a week, starting on day 2 and continuing until day 21 after the last NK cell injection. NSG mice were imaged at various times up to day 86, and survival, BLI (tumor burden), body weight, and other health status were assessed. FcR-UDC I-NK and rituximab significantly extended the survival of lymphoma-bearing mice compared with that observed using rituximab or FcR-UDC-NK cells alone (see Figure 27B). All mice treated with FcR-UDC-NK + rituximab responded to treatment, and extending the interval between injections prolonged the duration of the response (see Figure 27A).

[0088] Example 6: In vivo antitumor efficacy study using UDC-NK cells in the Raji intravenous tumor model

[0120] The in vivo efficacy of UDC-NK cells against tumors was tested in the Raji intravenous tumor model. The Raji intravenous model is a more aggressive, disseminated model and represents a more clinically relevant physiological model of lymphoma. In a representative study using NOGf mice with mouse FcgR knockout (FcResolv NOG), NOGf mice were divided into three groups as shown in Table 4 below.

[0089] [Table 4]

[0090]

[0121] In Figure 28A, NOGf mice were treated with Raji cells (1 x 10 5 cells) together with rituximab (100 μg, intraperitoneal administration) or FcR-UDC I-NK cells (10 × 10 6 On days 8 and 15, rituximab was administered alone (100 μg, intraperitoneally) or in combination with FcR-UDC I-NK cells (10 × 10 6 In Group 3, mice were administered FcR-UDC I-NK cells alone (10 × 10 6Mice were treated with IL-15 (100 ng, intravenous). Mice were treated with IL-15 (100 ng, intraperitoneal). As shown in Figure 28B, the combination of FcR-UDC I-NK + rituximab achieved a significant reduction in tumor burden compared to rituximab alone and untreated.

[0091]

[0122] In another study, mice were divided into four groups (n=8) as shown in Table 5.

[0092] [Table 5]

[0093]

[0123] In Figure 29A, NOGf mice engineered to express human IL-15 were inoculated with Raji cells (1 x 10 5 cells) were intravenously injected, and on day 1, rituximab alone (100 μg, intraperitoneal administration) or FcR-UDC I-NK cells (3 × 10 6 or 15 x 10 6 On day 4, FcR-UDC I-NK cells were administered alone (3 × 10 6 or 15 x 10 6 On days 8 and 11, rituximab was administered alone (50 μg, intravenous) or in a 3 × 10 6 on day 11 in combination with 15 × 10 FcR-UDC I-NK cells (intravenous administration) or rituximab on day 11. 6 As shown in Figure 29B, 15 x 10 6 The combination of FcR-UDC I-NK cells plus rituximab achieved the greatest reduction in tumor burden.

[0094]

[0124] Example 7: Antitumor efficacy test using UDC-NK cells and anti-claudin 6 (CLDN6) antibody

[0125] Antibody-dependent cellular cytotoxicity (ADCC) was evaluated against the CLDN6+ ovarian cancer cell line PA-1 using the UDC-NK cells described herein in combination with an anti-CLDN6 antibody. Briefly, in a 4-hour flow-based ADCC assay, CellTrace Violet-labeled PA-1 cells were co-cultured with FcR-UDC I-NK cells in the presence of titrated concentrations of the anti-CLDN6 antibodies ASP1650 (also known as IMAB027) and ASP1893 mAb. As shown in Figure 30, FcR-UDC I-NK cells exhibited enhanced cytotoxicity against PA-1 cells in a dose-dependent manner with anti-CLDN6 antibodies. The EC50 using the antibody ASP1893 mAb (humanized IgG1) was approximately 4.32 ng / mL, and the EC50 using the antibody ASP1650 (chimeric IgG1) was approximately 8.49 ng / mL.

[0095]

[0126] A panel of tumor cell lines was also tested for surface CLDN6 expression and divided into four groups: CLDN6hi, CLDN6mid, CLDN6low, and CLDN6-. FcR-UDC I-NK cells were tested in a 4-hour flow-based ADCC assay against a panel of tumor cell lines with different surface CLDN6 levels. As shown in Figures 31A-31M, FcR-UDC I-NK cells exhibited ADCC activity against CLDN6+ tumor cells in a CLDN6 expression-dependent manner. Interestingly, FcR-UDC I-NK cells exhibited natural cytotoxicity (NCC) against a wide range of tumor cell lines, regardless of CLDN6 surface expression, even without anti-CLDN6 antibodies.

[0096]

[0127] The production of TNF-α and IFN-γ by UDC-NK cells after stimulation with tumor cells coated with anti-CLDN6 antibody was also examined. 5 FcR-UDC I-NK cells and 10 5Tumor cells were co-cultured in 96-well plates in the presence or absence of 1 μg / mL of anti-CLDN6 antibody (ASP1893 mAb) or control hIgG1 antibody. After 24 hours of co-culture, culture supernatants were collected and TNF-α and IFN-γ were measured by ELISA (R&D Systems). Figures 32A-32B show that FcR-UDC I-NK cells secreted elevated levels of TNF-α and IFN-γ after stimulation with a panel of tumor cell lines compared to basal cytokine levels without stimulation. FcR-UDC I-NK cells showed enhanced cytokine production upon stimulation with CLDN6+ tumor cells and ASP1893 mAb.

[0097]

[0128] The in vivo efficacy of UDC-NK cells against tumors was tested in a CLDN6+ PA-1 cell xenograft mouse tumor model. In a representative study, mice were divided into four groups (n=6 or 7) as shown in Table 6 below. Briefly, 5×10 6 PA-1 cells were implanted subcutaneously into the flanks of NOGf mice engineered to express human IL-15. As shown in Figure 33, 13 days after tumor implantation, mice received 7.5 x 10 PA-1 cells with or without intraperitoneal administration of anti-CLDN6 (300 μg ASP1650). 6 Cryopreserved FcR-UDC I-NK cells were treated intravenously. The cryopreserved FcR-UDC I-NK cells were thawed and immediately injected into mice. Figures 34A-34B show that the combination treatment of FcR-UDC I-NK cells and anti-CLDN6 antibody demonstrated statistically significant antitumor efficacy and improved overall survival. Monotherapy with FcR-UDC I-NK cells or anti-CLDN6 antibody did not demonstrate significant antitumor efficacy.

[0098] [Table 6]

[0099]

[0129] The above examples demonstrate that UDC-NK cells exhibit natural cytotoxicity in vitro against a wide range of tumor cell lines without anti-CLDN6 antibodies, regardless of CLDN6 surface expression. The examples further demonstrate that the UDC-NK cells of the present disclosure exhibit potent anti-tumor activity in vitro and in vivo when combined with anti-CLDN6 antibodies.

[0100]

[0130] In another study, a PA-1 cell xenograft mouse tumor model was used using NOGf mice expressing human IL-15. As shown in Figure 35A, 7.5 x 10 6 FcR-UDC I-NK cells were administered intravenously and / or anti-CLDN6 antibody (300 μg) was administered intraperitoneally. Combination treatment of FcR-UDC I-NK cells and anti-CLDN6 antibody resulted in tumor growth inhibition (Figure 35B) and prolonged survival (Figure 35C). In mice administered FcR-UDC I-NK cells + anti-CLDN6 antibody, a survival rate of over 50% was observed after approximately 50 days.

[0101]

[0131] Example 8: Antitumor test using UDC-NK cells and anti-claudin 18.2 (CLDN18.2) antibody

[0132] This example demonstrates that UDC-NK cells exhibit antitumor activity when used in combination with the anti-CLDN18.2 antibody, zolbetuximab. Briefly, similar to the assay described in Example 7, an ADCC assay was performed using FcR-UDC I-NK cells and BxPC3-CLDN18.2-Luc tumor target cells as a 4-hour coculture. Zolbetuximab was present at a concentration of 1 μg / mL. The results are shown in Figure 36A, demonstrating zolbetuximab-dependent killing of CLDN18.2-expressing tumor cells. The antitumor effect of FcR-UDC I / II-NK cells in combination with an anti-claudin 18.2 antibody (zolbetuximab) against BxPC3-CLDN18.2-luciferase tumor cells is shown in Figure 36B, demonstrating that the highest cell killing level was achieved using FcR-UDC I / II-NK cells in combination with zolbetuximab.

[0102]

[0133] Cytokine secretion assays were also performed using a method similar to that described in Example 7. BxPC3-CLDN18.2-Luc cells were cultured with FcR-UDC I-NK cells of the present disclosure, FcR-UDC I-NK cells and human IgG, or FcR-UDC I-NK cells and zolbetuximab. After 24 hours of coculture in the presence of IL-15 (10 ng / mL), supernatants were collected and tested for interferon-gamma or tumor necrosis factor-alpha levels. Zolbetuximab was present at a concentration of 1 μg / mL. The results are shown in Figures 37A-37B. Cytokine secretion by FcR-UDC I-NK cells was significantly increased in samples containing zolbetuximab and BxPC3-CLDN18.2-Luc tumor target cells.

[0103]

[0134] The Examples described herein demonstrate that the engineered NK cells of the present disclosure exhibit anti-tumor activity against a wide range of tumor types, including lymphoma, ovarian cancer, and pancreatic cancer. The Examples report that NK cells alone exhibit anti-tumor activity, and that the anti-tumor activity is enhanced by the use of antibody constructs against tumor antigens (e.g., CLDN6, CLDN18.2, and CD20). While tumor cells were used in the Examples to demonstrate the cell-killing activity of the engineered NK cells of the present disclosure, it will be understood that the data can be extrapolated to additional cell types.

[0104]

[0135] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and expressly indicated to be incorporated herein in its entirety.

Claims

1. A population of engineered natural killer (NK) cells, wherein the NK cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G); the NK cells comprise a genetically engineered disruption of one or more copies of endogenous beta-2 microglobulin (B2M); A population of engineered NK cells, wherein the NK cells comprise a genetically engineered disruption of one or more copies of a human leukocyte antigen (HLA) class II-associated gene selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA).

2. 2. The population of engineered NK cells of claim 1, wherein the NK cells further comprise a nucleic acid molecule encoding a suicide gene product.

3. 3. The population of engineered NK cells of claim 2, wherein the NK cells comprise a herpes simplex virus thymidine kinase (TK) suicide gene.

4. 4. The population of engineered NK cells of any one of claims 1 to 3, wherein the NK cells are derived from pluripotent stem cells.

5. 5. The population of engineered NK cells of claim 4, wherein the pluripotent stem cells are embryonic stem cells.

6. 5. The population of engineered NK cells of claim 4, wherein the pluripotent stem cells are induced pluripotent stem cells.

7. 7. The population of engineered NK cells of any one of claims 1 to 6, wherein the NK cells express one or more cell surface markers selected from the group consisting of CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and NKp44.

8. 8. The population of engineered NK cells of any one of claims 1 to 7, which is capable of engrafting in a subject after administration.

9. 9. The population of engineered NK cells of claim 8, wherein at least 50% of said population of engineered NK cells are capable of engrafting in a target tissue of said subject.

10. 10. The population of engineered NK cells of claim 8 or claim 9, which is capable of remaining engrafted in the target tissue of the subject for at least 20 days after administration.

11. The population of engineered NK cells according to any one of claims 8 to 10, wherein the target tissue is blood or bone marrow.

12. 12. The population of engineered NK cells of any one of claims 1 to 11, wherein the NK cells are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells.

13. 13. The population of engineered NK cells of any one of claims 1 to 12, wherein said NK cells are capable of inducing natural cytotoxicity (NCC) against cancer cells.

14. 14. The population of engineered NK cells of claim 12 or claim 13, wherein the NK cells reduce tumor burden by at least 10%.

15. 15. The population of engineered NK cells of any one of claims 1 to 14, wherein the NK cells have at least about a 5% increase in cell volume compared to peripheral blood NK cells.

16. 16. The population of engineered NK cells of any one of claims 1 to 15, wherein the cells express a chimeric antigen receptor or an Fc receptor.

17. 17. The population of engineered NK cells of any one of claims 1 to 16, wherein the NK cells comprise a genetically engineered disruption of all copies of endogenous B2M.

18. 18. The population of engineered NK cells of any one of claims 1 to 17, wherein the NK cells comprise a genetically engineered disruption of all copies of an HLA class II associated gene selected from the group consisting of RFXANK, RFX5, RFXAP and CIITA.

19. 1. A method for producing engineered natural killer (NK) cells from human pluripotent stem cells under feeder-free conditions, comprising: (a) culturing human pluripotent stem cells genetically engineered to disrupt one or more copies of endogenous beta-2 microglobulin (B2M) and one or more copies of a human leukocyte antigen (HLA) class II-associated gene selected from regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA); (b) differentiating the human pluripotent stem cells into progenitor cells capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; (c) differentiating the precursor cells into NK cells under feeder-free conditions in a culture medium containing SCF, IL-7, IL-15, and Flt3L; and (d) culturing the NK cells in a culture medium containing IL-15 and IL-18 in the presence of an antibody or ligand that binds to a receptor on the NK cells and promotes activation of the NK cells; thereby generating engineered NK cells derived from human pluripotent stem cells.

20. 1. A method for producing engineered natural killer (NK) cells from human pluripotent stem cells under feeder-free conditions, comprising: (a) culturing human pluripotent stem cells genetically engineered to disrupt one or more copies of endogenous beta-2 microglobulin (B2M) and one or more copies of a human leukocyte antigen (HLA) class II-associated gene selected from regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA); (b) differentiating the human pluripotent stem cells into progenitor cells capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives using embryoid bodies; and (c) differentiating the precursor cells into NK cells under feeder-free conditions in a culture medium containing SCF, IL-7, IL-15, and Flt3L; thereby generating engineered NK cells derived from human pluripotent stem cells.

21. 21. The method of claim 20, further comprising the step of (d) culturing the NK cells in a culture medium containing IL-15 and IL-18, in the presence of an antibody or ligand that binds to a receptor on the NK cells and promotes activation of the NK cells.

22. 22. The method of claim 19 or claim 21, wherein step (d) comprises culturing the NK cells in the presence of an antibody that binds to DNAM-1, an antibody that binds to OX40, an antibody that binds to NKG2D, an antibody that binds to 2B4, an antibody that binds to NKp30, an antibody that binds to NKp46, or a combination thereof.

23. 23. The method of claim 22, wherein step (d) comprises culturing the NK cells in the presence of an antibody that binds to NKp30 and an antibody that binds to DNAM-1.

24. 22. The method of claim 19 or claim 21, wherein step (d) comprises culturing the NK cells in the presence of a ligand that binds to DNAM-1, a ligand that binds to OX40, a ligand that binds to NKG2D, a ligand that binds to 2B4, a ligand that binds to NKp30, or a ligand that binds to NKp46, or a combination thereof.

25. 25. The method of claim 24, wherein step (d) comprises culturing the NK cells in the presence of a ligand that binds to NKp30 and a ligand that binds to DNAM-1.

26. 26. The method of any one of claims 19 or 21-25, wherein the culture medium in step (d) further comprises IL-21.

27. The method of any one of claims 19 to 26, wherein the human pluripotent stem cells of step (a) further comprise a nucleic acid molecule encoding a suicide gene product.

28. 28. The method of claim 27, wherein the NK cells comprise a herpes simplex virus thymidine kinase (TK) suicide gene.

29. The method according to any one of claims 19 to 28, wherein the human pluripotent stem cells are human embryonic stem cells.

30. The method according to any one of claims 19 to 28, wherein the human pluripotent stem cells are human induced pluripotent stem cells.

31. 31. The method of any one of claims 19 to 30, wherein the culture medium of step (c) further comprises UM171.

32. 32. The method of any one of claims 19 to 31, wherein the NK cells express one or more cell surface markers selected from the group consisting of CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and NKp44.

33. 33. The method of any one of claims 19 to 32, wherein the NK cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G).

34. The method of any one of claims 19 to 33, wherein the NK cells are capable of engrafting in the subject after administration.

35. 35. The method of claim 34, wherein at least 50% of the NK cells are capable of engrafting in the subject.

36. 36. The method of claim 34 or claim 35, wherein the NK cells are capable of remaining engrafted in the subject for at least 20 days after administration.

37. The method of any one of claims 34 to 36, wherein the NK cells are capable of engrafting in blood and bone marrow.

38. The method of any one of claims 19 to 37, wherein the NK cells are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells.

39. The method of any one of claims 19 to 38, wherein the NK cells are capable of inducing natural cytotoxicity (NCC) against cancer cells.

40. 40. The method of claim 38 or claim 39, wherein the NK cells reduce tumor burden by at least 10%.

41. 41. The method of any one of claims 19 and 21 to 40, wherein step (d) is repeated up to five times.

42. A method for increasing the purity of a population of NK cells derived from pluripotent stem cells, comprising the step of differentiating embryoid bodies into NK cells under feeder-free conditions in a culture medium containing SCF, IL-7, IL-15, and Flt3L in the presence of UM171.

43. 43. The method of claim 42, wherein the culture medium further comprises IL-3.

44. 44. The method of claim 42 or 43, wherein the method is carried out in a vessel coated with a recombinant human fibronectin fragment and DLL4.

45. 45. The method of any one of claims 42 to 44, wherein the method is carried out for about 1 day to about 21 days.

46. A pharmaceutical composition comprising the population of engineered NK cells of any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

47. 47. A method of treating a disease or disorder in a subject in need thereof, comprising administering to said subject an effective amount of the population of engineered NK cells of any one of claims 1 to 18, or the pharmaceutical composition of claim 46, thereby treating said disease or disorder in said subject.

48. 48. The method of claim 47, wherein the disease or disorder is cancer.

49. 49. The method of claim 47 or claim 48, further comprising administering one or more cytokines to the subject.

50. 50. The method of claim 49, wherein the one or more cytokines are IL-15 and / or IL-2.

51. 51. The method of any one of claims 47-50, wherein said administering increases survival of the subject by at least 20 days compared to survival of the subject if said population of engineered NK cells was not administered.

52. 51. The method of any one of claims 47-50, wherein said administering reduces tumor burden in said subject.

53. 53. The method of any one of claims 47 to 52, further comprising administering to the subject one or more antibodies.

54. 48. The method of claim 47, wherein the disease or disorder is an autoimmune disorder.

55. 55. The method of claim 54, further comprising administering to the subject one or more antibodies.

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