Engineered natural killer cells and related methods

Abstract

The present disclosure provides an engineered population of 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 cell comprises a genetically modified disruption of one or more copies of an endogenous beta-2 microglobulin and a genetically modified disruption of one or more copies of a human leukocyte antigen class II-related gene, the human leukocyte antigen class II-related gene is selected from a protein containing regulatory factor X-related ankyrin, a regulatory factor 5, a regulatory factor X-related protein and a class II trans-activator. Also provided are a method of producing human pluripotent stem cell-derived engineered natural killer cells without feeder cells, and a method of treating a disease or condition in a subject by administering the NK cells to the subject.

Description

Technical Field

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

[0002] Cross - Reference to Related Applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,785, filed on December 29, 2022, which is hereby incorporated by reference in its entirety.

[0004] Incorporation by Reference of Electronically Submitted Materials

[0005] A computer - readable nucleotide / amino acid sequence listing, submitted concurrently herewith and identified as follows, is incorporated by reference in its entirety: a 58,824 - byte file named "57819P_Seqlisting.XML"; created on December 18, 2022. Background of the Invention

[0006] Engineering patient - derived immune cells to express chimeric antigen receptors (CARs) has revolutionized the landscape of adoptive cell therapy, providing scientists and clinicians with the ability to harness the powerful cytolytic capacity of immune cells and direct them, in an MHC - independent manner, to targets expressing a specific antigen. Natural killer (NK) cells, which are generated from the lymphoid lineage, are part of the innate immune system and are attractive for adoptive cell therapy because they have been found to detect and kill certain types of tumor cells. Typical sources of NK cells include peripheral blood NK cells (PB - NK), cord blood NK cells, and NK cell lines such as NK - 92. Despite their promise, these sources suffer from genetic instability, inconsistent cytotoxicity, limited expansion, product heterogeneity, and / or host immunogenicity. To address the limitations of current therapy options, there is a need in the art for improved NK - cell - based therapies. Summary of the Invention

[0007] The present disclosure provides populations of natural killer (NK) cells derived from pluripotent stem cells. The present disclosure further provides engineered NK cell populations, wherein the NK cells express at least one polypeptide selected from human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The NK cells further comprise genetic disruption of one or more copies (e.g., all copies) of endogenous β-2 microglobulin (B2M) and / or genetic disruption of one or more copies (e.g., all copies) of human leukocyte antigen (HLA) class II-associated genes selected from regulator of factor X-associated ankyrin-containing protein (RFXANK), regulator factor 5 (RFX5), regulator of factor X-associated protein (RFXAP), and class II transactivator (CIITA). For example, the NK cells comprise genetic disruption of endogenous B2M and RFXANK and express HLA-E. In another example, the NK cells comprise genetic disruption of all copies of endogenous B2M and all copies of RFXANK and express HLA-E. In various aspects, the NK cells further comprise a nucleic acid molecule encoding a suicide gene product (e.g., 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 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. Patent No. 10,259,858 and U.S. Patent Publication No. 2020 / 0138866), which in some aspects is capable of binding an antibody-based bispecific molecule (e.g., MicAbody); or other types of receptors.

[0008] The present disclosure further provides a method for generating pluripotent stem cell-derived NK cells under feeder cell-free and optionally serum-free conditions. The method comprises (a) culturing pluripotent stem cells (PSCs) which are optionally genetically engineered to disrupt one or more copies (e.g., all copies) of B2M and / or one or more copies (e.g., all copies) of HLA class II-associated genes selected from RFXANK, RFX5, RFXAP, and CIITA, and optionally expressing HLA class I proteins, such as HLA-E; (b) differentiating the pluripotent stem cells into precursor cells (e.g., using embryoid bodies), the precursor cells being capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; and (c) differentiating the precursor cells into NK cells in a feeder cell-free and optionally serum-free medium comprising one or more of SCF, IL-7, IL-15, and Flt3L. Optionally, the medium of step (c) further comprises a pyrimido[4,5-b]indole derivative, such as (1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine dihydrobromide dihydrate (“UM171”). In various aspects, the PSCs are human PSCs. In other various aspects, the method further comprises (d) culturing the NK cells in a 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 the NK cells to promote NK cell activation and expansion. In one aspect, the antibody or ligand binds to a receptor selected from 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 may be included in step (d). For example, step (d) may comprise culturing the NK cells in the presence of an antibody that binds NKp30 and / or an antibody that binds DNAM-1.

[0009] The present disclosure also provides a method for 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 of the subject. In some aspects of the present disclosure, the disease is cancer. Optionally, one or more cytokines, such as 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.

[0010] Although the term "comprising" is used in the specification to provide various embodiments in a variety of situations, the related embodiments may also be described using the language of "consisting of" or "consisting essentially of". This disclosure contemplates embodiments described in terms of "comprising" a feature to include embodiments "consisting of" or "consisting essentially of" that feature. The term "a" or "an" means one or more. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein. The term "or" should be understood to include items in the alternative or in combination, unless the context clearly dictates otherwise.

[0011] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each end value, unless otherwise indicated, and each separate value and end value is incorporated into the specification as if it were individually recited herein. However, this description also contemplates ranges where the lower and / or upper values are not included. When the term "about" is used with respect to a number or a general value, it means that number or value plus or minus 1%, 2%, 3%, 4%, 5%, 10%, or 15% of that number or value, including all values within that range. The actual intended variation can be determined from the context.

[0012] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the disclosure and does not impose a limitation on the scope of the disclosure, unless otherwise claimed. Only limitations described herein as critical to the invention should be considered as such; variations of the invention lacking limitations described herein as critical are intended to be aspects of the invention.

[0013] Additional features and variations of the 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 to be aspects of the invention. Similarly, the features of the invention described herein can be recombined into additional embodiments, which are also intended to be aspects of the invention, whether or not the combination of features is specified as an aspect or embodiment of the invention. The entire document is intended to be read as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features do not appear together in the same sentence or paragraph or section of the document. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Depicts a representative scheme 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 referred to in the following figure descriptions.

[0015] Figures 2A - 2C Illustrates the cell yields ( Figure 2A ) resulting from the method of generating human PSC-derived engineered NK cells (FcR-UDC I-NK cells) described herein, the percentage of + CD56 + CD45 Figure 2B ) cells ( Figure 2C ), and the cell viability ( ) of the cells. The bars on the left side of each graph represent the conditions without UM171, and the bars on the right side of each graph represent the conditions in which 35 nM UM171 is present.

[0016] Figure 3A -D is a bar graph illustrating the Step 2 (NK differentiation) yields and NK cell expansion in multiple gene-edited clones ( Figures 3A - 3B ) and PSC cell lines ( Figure 3C -D). In Figures 3A - 3B Figures 3C - 3D

[0017] Figure 4

[0018] Figures 5A - 5B Figure 1 Figure 5A Figure 5B

[0019] Figure 6 ), and the cell viability ( Figure 5B ) of the cells. The bars on the left side of each graph represent the conditions without UM171, and the bars on the right side of each graph represent the conditions in which 35 nM UM171 is present.

[0019] Figure 6Graph comparing the cell size of NK cells derived from PSCs (“iPSC”) using the methods described herein and NK cells from peripheral blood (“PBNK”), as measured by flow cytometry. The size (by volume) of NK cells derived from PSCs using the methods described herein is on average approximately 18% larger than NK cells isolated from peripheral blood.

[0020] Figures 7A - 7E Bar graph of the expected phenotypes of NK cells derived from the following different gene-edited clones: NIH3 wild-type and engineered NIH3 UDC I-PSCs, FcR-UDC I-PSCs, and UDC I / II-PSCs. Asterisks indicate that data was not collected.

[0021] Figures 8A - 8D Bar graph of the expected phenotypes of NK cells derived from the following different cell lines: NIH3 and W1C1 wild-type, and engineered UDC I-PSCs and UDC I / II-PSCs from gene-edited clones. Asterisks indicate that data was not collected.

[0022] Figures 9A - 9C Bar graph illustrating the expression of common NK and T cell markers in FcR-UDC I-NK cells (step 2 (left bars for each marker) and step 3 (middle bars for each marker)) derived from engineered FcR-UDC I-PSCs compared to expanded PB-NK (right bars for each marker). Steps 2 and 3 are described in Figure 1 herein.

[0023] Figures 10A - 10C Bar graph illustrating the expression of common activation, exhaustion, and “memory-like” markers in FcR-UDC I-NK cells (step 2 (left bars for each marker) and step 3 (middle bars for each marker)) derived from engineered FcR-UDC I-PSCs compared to expanded PB-NK (right bars for each marker). Steps 2 and 3 are described in Figure 1 herein.

[0024] Figures 11A - 11B Bar graph illustrating the expression of cytokine and chemokine receptors in FcR-UDC I-NK cells (step 2 (left bars for each receptor) and step 3 (middle bars for each receptor)) derived from engineered FcR-UDC I-PSCs compared to expanded PB-NK (right bars for each receptor). Steps 2 and 3 are described in Figure 1 herein.

[0025] Figures 12A - 12CBar graph showing the expression of adhesion / homing, co - stimulatory, and apoptosis 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 described in Figure 1 in.

[0026] Figure 13 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 percentage of specific lysis is provided on the y - axis and the effector - to - target cell ratio (E / T) is provided on the x - axis. Among multiple PSC cell lines and gene - edited clones, the UDC - NK cells disclosed herein mediated cell lysis.

[0027] Figure 15 Line graph showing the NCC of FcR - UDC I - NK cells compared to expanded PB - NK. The percentage of specific lysis is provided on the y - axis and the effector - to - target cell ratio (E / T) is provided on the x - axis. The UDC - NK cell population disclosed herein mediated a comparable degree of cell lysis to PB - NK.

[0028] Figure 16 Line graph showing that FcR - UDC I - NK did not show killing and cytotoxicity against donor PBMCs, but showed NCC against K562 tumor target cells.

[0029] Figure 17 Line graph showing the potency of engineered NK cells (FcR - UDC I - NK cells) to kill continuously added K562 tumor cells in a dose - dependent manner.

[0030] Figures 18A - 18D Line graph showing the antibody - dependent cell cytotoxicity (ADCC) activity of engineered FcR - UDC I - NK cells generated using the methods described herein. The percentage of specific lysis is provided on the y - axis and the effector - to - target cell ratio (E / T) is provided on the x - axis. In Figures 18A - 18C it is shown that the FcR - UDC I - NK cell population disclosed herein showed ADCC activity against 3 different CD20 + tumor cell lines: Daudi (Figure 18A )、Ramos( Figure 18B ) and Raji( Figure 18C ). In Figure 18D , the expanded PB-NK did not show any difference in ADCC activity between rituximab and IgG1 control. Circles represent no antibody, squares represent human IgG1, and triangles represent rituximab.

[0031] Figure 19 is a bar graph showing the NCC and ADCC activities of FcR-UDC I-NK cells in a wide range of B lymphoma cell lines.

[0032] Figure 20A is a bar graph identifying the inducible cytokines in FcR-UDC I-NK cells (left bars for each condition) compared to expanded PB-NK (right bars for each condition) after stimulation with rituximab (R-mab)-coated Raji tumor cells. The x-axis of each graph describes "no stimulation", "Raji + hIgG1", and "Raji + R-mab". A significant increase was observed for TNF-α, interferon-γ, GM-CSF, and IP-10. A moderate increase was observed for granzyme-B, MIP-1α, MIP-1β, RANTES, IL-8, IL-10, and MCP-1. The cytokines not detected included IL-1α, IL-1β, IL-6, and IL-12, as Figures 20B - 20E shown.

[0033] Figures 21A - 21D is a bar graph identifying the cytokines and / or chemokines that were not elevated in FcR-UDC I-NK cells (left bars for each condition) compared to expanded PB-NK (right bars for each condition) after co-culture with allogeneic PBMC compared to co-culture with Raji plus rituximab. The x-axis of each graph describes "no stimulation", "Raji + rituximab", and "AlloPBMC".

[0034] Figures 22A - 22G is a bar graph showing the in vivo persistence of FcR-UDC I-NK cells in non-tumor-bearing mice in the absence of any antigen. NK cells (4x 10 6 ) were injected intravenously into mice, and then IL-2 (30,000 IU, 3x / wk.) and IL-15 (100 ng daily for 7 days) were administered. The percentage and absolute number of FcR-UDC I-NK cells in the blood, spleen, and bone marrow were measured.

[0035] Figures 23A - 23Eis a bar graph illustrating the in vivo tissue distribution of FcR-UDC I-NK cells in non-tumor bearing mice. NK cells (5 x 10 6 , 7.5x 10 6 and 10x 10 6 ) were injected intravenously, followed by IL-2 (30,000 IU, 3x / wk.) and IL-15 (100 ng daily). Figure 23B ),liver( Figure 23D ),spleen( Figure 23E ),blood( Figure 23A ) and bone marrow ( Figure 23C ) to measure human CD56 in mouse plus human CD45 + The percentage of cells (FcR-UDC I-NK cells).

[0036] Figure 24 Figure 1 is a graphic representation of the efficacy study of FcR-UDC I-NK cells plus rituximab in the Raji intraperitoneal tumor model. NSG mice were irradiated and treated with Raji tumor cells (3 x 10 5 On day 4, FcR-UDC I-NK cells or PB-NK cells (5 x 10 6 ), with or without rituximab (300 μg). On day 7, an additional dose of rituximab (300 μg) was administered. Imaging / monitoring of NSG mice was performed at the indicated time points until day 120.

[0037] Figures 25A - 25C It explains Figure 24 After the efficacy study ( Figure 25A ) survival curve and ( Figures 25B - 25C ) tumor burden. Figure 25A In the study, FcR-UDC I-NK cells plus rituximab and PB-NK plus rituximab both had a significant increase in median survival (days) compared to untreated, rituximab, FcR-UDC I-NK cells, and PB-NK alone. Rituximab and PB-NK alone increased median survival (days) compared to untreated. Figures 25B - 25C In the present study, FcR-UDC I-NK cells plus rituximab as well as PB-NK plus rituximab provided durable responses that delayed tumor onset until day 60 in 50% of mice.

[0038] Figure 26 Figure 1 is a graphic representation of the efficacy study of FcR-UDC I-NK cells plus rituximab in the Raji intraperitoneal tumor model. NSG mice were irradiated and treated with Raji tumor cells (2 x 10 5) Injection (intraperitoneal). On day 2, administer FcR-UDC I-NK cells (10x10 6 ), with or without rituximab (100 μg). On days 6, 10, and / or 16, administer additional doses of FcR-UDC I-NK cells or PB-NK cells (each 10x10 6 ). Image / monitor NSG mice at the specified time points until day 86.

[0039] Figures 27A - 27B Illustrates the ( Figure 26 ) tumor burden and ( Figure 27A ) survival curves after the potency study in Figure 27B . In Figure 27A , FcR-UDC I-NK cells plus rituximab (D2, 10, and 16) had a longer durable response with delayed tumor onset compared to FcR-UDC I-NK cells plus rituximab (D2, 6, and 10). In Figure 27B , FcR-UDC I-NK cells plus rituximab (both regardless of the dosing regimen) had a significant increase in median survival (days) compared to untreated, rituximab alone, and FcR-UDC I-NK cells alone.

[0040] Figures 28A - 28B Involves a potency study using mouse FcgR knockout NOGf mice. Figure 28A Is a study illustration in which rituximab or FcR-UDC I-NK cells plus rituximab are administered in a Raji tumor model. Inject (intravenously) NOGf mice with Raji tumor cells (1x10 5 ). Administer rituximab (100 μg, intraperitoneal) on days 1, 8, and 15 after tumor cell transplantation, and administer FcR-UDC-I-NK (10M) intravenously on days 1, 4, 8, and 15. Figure 28B Illustrates the tumor burden after the potency study in Figure 28A . Cryopreserved FcR-UDC I-NK cells plus rituximab showed a greater degree of tumor growth inhibition compared to administration of rituximab alone.

[0041] Figures 29A - 29B Involves a potency study using NOGf mice engineered to express human interleukin 15 (IL-15) and having a mouse FcgR knockout. Figure 29A Is one in which rituximab (50 or 100 μg, intraperitoneal) or FcR-UDC I-NK cells (3x10 6 (3M) or 15x10 6(15M), intravenous) Rituximab (50 or 100 μg, intraperitoneal) study illustration. Figure 29B Illustrates the tumor burden after Figure 29A potency study. 15M cryopreserved FcR-UDC I-NK cells plus rituximab showed a great degree of tumor growth inhibition compared to rituximab alone.

[0042] Figure 30 Is a line graph showing the anti-tumor effects 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). Specific lysis (%) is provided on the y-axis, and the amount (ng / mL) of antibody ASP1893 mAb or ASP1650 is provided on the x-axis. Anti-CLDN6 antibodies ASP1893 mAb and ASP1650 induced ADCC against CLDN6+ tumor cells comparable to that of FcR-UDC I-NK cells.

[0043] Figures 31A - 31M Is a line graph showing the anti-tumor effects of FcR-UDC I-NK cells plus anti-CLDN6 antibodies against tumor cells expressing various levels of CLDN6. Assays were performed with FcR-UDC I-NK cells together with antibody ASP1893mAb and control IgG1 antibody or without antibody. Figures 31A - 31D Represents assays using tumor cells highly expressing CLDN6 (SKOV3-CLDN6, ES-2-CLDN6, PA-1, and NEC14 cells). Figures 31E - 31G Represents assays using tumor cells expressing moderate levels of CLDN6 (ABC-1, OV90, and OVCAR3 cells). Figures 31H - 31I Represents assays using tumor cells expressing low levels of CLDN6 (COV362 and NEC8-Luc cells). Figures 31J - 31M Represents assays using tumor cells not expressing CLDN6 (COV318, ES-2, SKOV3, and NCI-H1373 cells). Specific lysis (%) is provided on the y-axis, and the E / T ratio is provided on the x-axis. FcR-UDC I-NK cells and ASP1893 mAb mediated ADCC against tumor cells in a CLDN6 expression-dependent manner.

[0044] Figures 32A - 32BCytokine secretion of FcR-UDC I-NK cells is illustrated. The figure is a bar graph showing interferon-γ (IFN-γ; pg / mL) or tumor necrosis factor-α (TNF-α) produced by FcR-UDC I-NK cells in the presence of tumor cells (left bars for each cell line tested), tumor cells and human IgG1 (middle bars for each cell line tested), and tumor cells and anti-CLDN6 antibody (ASP1893 mAb) (right bars 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 moderate levels of CLDN6. COV362 and NEC8-Luc cells express low levels of CLDN6. COV318, ES-2, and SKOV3 cells are negative for CLDN6. FcR-UDC I-NK cells show IFN-γ and TNF-α production in a CLDN6 expression-dependent manner after stimulation with tumor cells and ASP1893 mAb.

[0045] Figure 33 It is a schematic diagram of an in vivo assay examining the anti-tumor efficacy of UDC-NK cells conjugated with anti-CLDN6 antibody in a PA-1 xenograft solid tumor mouse model. NOGfIL-15 mice express human IL-15 and lack murine FcγR.

[0046] Figures 34A - 34B Illustrates after Figure 33 efficacy study of tumor volume ( Figure 34A ) and survival curve ( Figure 34B ). The combined treatment of FcR-UDC-NK cells and anti-CLDN6 antibody results in tumor growth inhibition ( Figure 34A ) and prolongs the survival of mice in the PA-1 SC tumor model ( Figure 34B ).

[0047] Figures 35A - 35C A study was conducted to examine the anti-tumor efficacy of UDC-NK cells conjugated with anti-CLDN6 antibody in a PA-1 xenograft solid tumor mouse model using NOGf mice that were engineered to express human interleukin 15 (IL-15) and have a murine FcγR knockout. Figure 35A It is a schematic diagram of an in vivo assay in which 7.5 × 10^6 FcR-UDC-NK cells were intravenously administered on days 16, 20, 23, and 27, and anti-CLDN6 antibody (300 μg, intraperitoneally) was administered on days 16 and 23. Figures 35B - 35C Illustrates after Figure 35A efficacy study of tumor volume ( Figure 35B ) and survival curve ( Figure 35C)。The combined treatment of FcR-UDC-NK cells and anti-CLDN6 antibody results in tumor growth inhibition( Figure 35B ) and prolongs the survival of mice in the PA-1SC tumor model( Figure 35C ).

[0048] Figures 36A - 36B Illustrates the ADCC activity of FcR-UDC-NK cells. Figure 36A Is a line graph illustrating the anti-tumor effect of FcR-UDC I-NK cells plus anti-claudin 18.2 antibody (Zolbetuximab) against BxPC3-CLDN18.2-Luc tumor cells. Specific lysis (%) is provided on the y-axis and the E:T ratio is provided on the x-axis. Figure 36B Is a line graph illustrating the anti-tumor effect of FcR-UDC I / II-NK cells plus anti-claudin 18.2 antibody (Zolbetuximab) against BxPC3-CLDN18.2-luciferase tumor target cells. The remaining target cell fluorescence intensity normalized to the start of the experiment is provided on the y-axis and the measurement time is provided on the x-axis.

[0049] Figures 37A - 37B Is a bar graph illustrating the interferon-γ( Figure 37A ) or tumor necrosis factor-α( Figure 37B ) secretion by FcR-UDC I-NK in samples containing BxPC3-CLDN18.2-Luc tumor cells alone, BxPC3-CLDN18.2-Luc tumor cells and human IgG, or BxPC3-CLDN18.2-Luc tumor cells and Zolbetuximab. DETAILED DESCRIPTION

[0050] The present disclosure provides natural killer (NK) cell populations that have favorable properties for immunotherapy. The materials and methods described herein are capable of generating, for example, substantially homogeneous NK cell populations in which the occurrence of unwanted cells is reduced, the safety profile is improved, the in vivo persistence is strong, the ability to engraft in a subject is increased, and / or the ability to reduce tumor burden is increased.

[0051] The present disclosure provides populations of NK cells derived from pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The present disclosure further provides engineered populations of NK cells, wherein the NK cells express at least one exogenous polypeptide selected from human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The NK cells are 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 human leukocyte antigen (HLA) class II-associated genes selected from the protein containing regulator of X-associated ankyrin (RFXANK), regulator factor 5 (RFX5), regulator of X-associated protein (RFXAP), and class II transactivator (CIITA). The disruption allows the NK cells to, for example, reduce or evade immune detection and allows for the generation of HLA-mismatched NK cell populations 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.

[0052] NK cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. NK cells are attractive for therapy because of their natural cytotoxicity against foreign, transformed, or virus-infected cells, their antibody-dependent cellular cytotoxicity, and their 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 aspects of the present disclosure, the 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.

[0053] The NK cell (or the PSC or an intermediate from which the NK cell is derived) is genetically engineered to disrupt one or more copies (e.g., all copies) of the endogenous B2M gene of the cell. B2M is a key component of the HLA class I (HLA-I) complex. HLA is a cell surface complex that mediates leukocyte-leukocyte or leukocyte-other cell interactions. HLA-I molecules are cell surface complexes that present antigens to CD8+ cytotoxic T cells, thereby mediating cellular immunity. HLA-I molecules comprise an HLA-I heavy chain and B2M. Disruption of one or more (e.g., all) copies of the B2M gene reduces or abolishes the production of the B2M protein (SEQ ID NO:5), thereby reducing or abolishing the display of the HLA-I complex on the NK cell. A representative nucleic acid sequence encoding B2M is provided in SEQ ID NO:1.

[0054] NK cells (or the PSC or an intermediate from which the NK cell is derived) are also contemplated that are genetically engineered to disrupt one or more copies (e.g., all copies) of a subset of HLA-I. Six HLA class I alpha (α) chains have been identified, including three classical ones (HLA-A, HLA-B, and HLA-C) and three non-classical ones (HLA-E, HLA-F, and HLA-G), which are responsible for the specificity of peptide binding in the HLA-I binding groove. In this regard, the present disclosure contemplates NK cells that are genetically engineered to disrupt one or more copies (e.g., all copies) of an HLA-I gene selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and combinations thereof. For example, the NK cell (or the PSC or an intermediate from which the NK cell is derived) can 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 further described, for example, in Choo, Yonsei MedJ., 48(1):11–23 (2007);

[0055] www.ebi.ac.uk / ipd / imgt / hla / ; and 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).

[0056] The NK cells of the present disclosure (or the PSCs or intermediates from which the NK cells are derived) are genetically engineered to disrupt one or more (e.g., all) copies of HLA class II (HLA-II)-associated genes selected from RFXANK, RFX5, RFXAP, and CIITA. HLA-II molecules are transmembrane proteins present on antigen-presenting cells (APCs) and sometimes on solid organs. HLA-II molecules comprise 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. Disruption of one or more copies (e.g., all copies) of one or more of the HLA-II-associated genes RFXANK, RFX5, RFXAP, and / or CIITA reduces or abolishes the production of the encoded proteins, thereby reducing or abolishing the display of the HLA-II complex on the NK cells. The NK cells (or the PSCs or intermediates from which the NK cells are derived) may comprise disruption of one or more copies (e.g., all copies) of the gene encoding one of RFXANK, RFX5, RFXAP, and / or CIITA (e.g., the cells are engineered to disrupt one or more copies (e.g., all copies) of the RFXANK gene); disruption of one or more copies (e.g., all copies) of any combination of two of RFXANK, RFX5, RFXAP, and / or CIITA; disruption of one or more copies (e.g., all copies) of any combination of three of RFXANK, RFX5, RFXAP, and / or CIITA; or disruption of one or more copies (e.g., all copies) of the genes encoding all of RFXANK, RFX5, RFXAP, and CIITA. RFXANK is encoded by sequences such as SEQ ID NO:3 and 5; RFX5 is encoded by sequences such as SEQ ID NO:7 and 9; RFXAP is encoded by sequences such as SEQ ID NO:11; and CIITA is encoded by sequences such as SEQ ID NO:13. In various aspects of the present disclosure, the NK cells are engineered to disrupt all copies of B2M and RFXANK.

[0057] Any suitable technique for introducing disruption in a target gene (e.g., an HLA-II related gene, B2M gene, or any other target gene) can be used. Many techniques for disrupting endogenous coding sequences are known in the art, including the use of gene editing systems such as the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases, as well as targeting vectors that cut and / or insert nucleic acid sequences into target sites in the cell genome. Exemplary techniques employ adeno-associated virus (AAV)-vector-based editing, which optionally includes targeting the insertion of an editing sequence (possibly, a transgene) into the cell genome to truncate the endogenous coding sequence, thereby resulting in reduced or avoided production of the endogenous protein. In various aspects, serotype 3B AAV vectors are used. The use of AAV vectors to engineer stem cells is further described, for example, in Riolobos et al., Molecular Therapy, 21(6), 1232-1241 (2013), which is incorporated herein by reference in its entirety and particularly for the disclosure regarding the use of AAV vectors and disruption of the HLA complex. Methods for confirming target gene disruption are known in the art and include, but are not limited to, PCR-based assays (e.g., quantitative real-time polymerase chain reaction (qRT-PCR)), RNA sequencing, next-generation sequencing methods, flow cytometry, and immunostaining.

[0058] In various aspects, the engineered NK cells express at least one polypeptide selected from 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 the NK cells (or the PSCs or intermediates from which the NK cells are derived), and the encoded proteins are produced and displayed on the cell surface. Optionally, the engineered NK cells express HLA-E. Optionally, the engineered NK cells express a single-chain fusion HLA-E that comprises at least a portion of B2M (e.g., HLA-E) directly or via a linker sequence linked to at least a portion of the HLA-I α-chain. Optionally, the engineered NK cells express a single-chain fusion HLA-F or HLA-G that comprises at least a portion of B2M directly or via a linker sequence linked to at least a portion of the HLA-I α-chain (e.g., HLA-F or HLA-G). Engineered NK cells can overexpress proteins naturally produced by the cell, can express proteins encoded by exogenous nucleic acids after knockout or disruption of the corresponding endogenous coding sequences of the proteins, or can express proteins not naturally encoded by the wild-type NK cell genome. Representative DNA and protein sequences of HLA-E are provided in SEQ ID NO:15 and 16. Representative DNA and protein sequences of HLA-F are provided in SEQ ID NO:17 and 18. Representative DNA and protein sequences of HLA-G are provided in SEQ ID NO:19 and 20. It will be understood that variants of the sequences provided herein can be used, and thus, the present disclosure contemplates the use of nucleic acids comprising at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequences disclosed herein. The present disclosure further contemplates the use of nucleic acids encoding peptides that comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the amino acid sequences disclosed herein. Preferably, the variants retain HLA-I function, such as forming a functional peptide-binding groove to display peptides and / or engaging inhibitory receptors on NK cells. When used as adoptive cell therapy, cells expressing HLA-E, HLA-F, and / or HLA-G minimize the risk of rejection. In various aspects of the present disclosure, NK cells are engineered to disrupt all copies of B2M and RFXANK and are engineered to express HLA-E, such as single-chain fusion HLA-E.

[0059] The NK cells of the population can also express exogenous receptors (i.e., receptors encoded by exogenous nucleic acids introduced into the NK cells or the PSCs or intermediates from which the NK cells are derived). In various aspects, the NK cells of the population express chimeric antigen receptors. A "chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor that has been engineered to recognize and bind an antigen expressed by a target cell, such as a tumor cell. Generally, a CAR is a chimera of the signal transduction domain and the antigen recognition domain (e.g., a single-chain fragment (scFv) of an antibody or other antibody fragment) of the T cell receptor (TCR) complex. See, e.g., Enblad et al., Human Gene Therapy. 2015;26(8):498-505. There are various forms of CARs, each containing different components. A "first-generation" CAR links the antigen-binding domain to the CD3ζ intracellular signal transduction domain of the T cell receptor via a hinge and transmembrane domain. When the CAR engages the target antigen, the cytoplasmic or intracellular signal transduction domain generates a stimulatory signal for proliferation and effector functions. Many intracellular signal transduction domains contain signal transduction motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs containing cytoplasmic signal transduction sequences include those derived from CD8, CD3ζ, CD3γ, CD3ε, CD32 (FcγRIIa), DAP10, DAP12, CD79a, CD79b, FcγRlγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG). Intracellular signal transduction domains, including those derived from CD3ζ (CD3), are known in the art (TCRζ, e.g., GenBank accession number BAG36664.1). Exemplary hinge domains include, but are not limited to, the CD28 hinge, the CD8α hinge, the human IgG4 hinge domain, and the human IgG4 hinge domain combined with the CH3 human IgG4 domain. Hinge domains are further described, e.g., in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Publication No. WO 2014031687, and U.S. Patent No. 8,822,647. A "second-generation" CAR contains additional domains, such as CD28, 4-1BB (41BB), or ICOS, to provide co-stimulatory signals. Additional co-stimulatory 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 co-stimulatory domains (or any combination thereof).The "third generation" CAR contains two co-stimulatory domains fused to the TCR CD3ζ chain. The third generation co-stimulatory domains can include, for example, a combination of CD3ζ, CD27, CD28, 4-1BB, ICOS, or OX40. The CAR constructed in this way can trigger, for example, cell activation in a manner similar to the endogenous T cell receptor after binding to the target antigen, but independent of the major histocompatibility complex (MHC).

[0060] Optionally, the NK cell-expressing fragment crystallizable (Fc) region receptor (FcR), such as CD16 (FcγRIII). In this regard, the NK cells can be engineered to express FcR, that is, the nucleic acid encoding FcR is introduced into the NK cells (or the PSC or intermediate from which the NK cells are derived) and the encoded protein is produced and displayed on the cell surface. Generally, FcR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain generally includes a domain that binds to the immunoglobulin (Ig) Fc region. The intracellular domain contains a portion of the Fc receptor sufficient to allow ITAM to initiate cell signaling when the Ig Fc region binds to the extracellular domain. Cells expressing FcR mediate the efficacy of antibody-based therapies and, to a lesser extent, B cell-mediated adaptive immune responses. NK cells are mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) driven by Fc binding to FcR. In ADCC, the NK cell FcR binds the antibody and forms 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 expressing the Fc region receptor are referred to herein as "FcR-UDC-NK cells" or "FcR-NK cells".

[0061] The NK cells can 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 (CD32, including CD32a, CD32b, and CD32c), and FcγRIII (CD16, including CD16a and CD16b). CD16a has two allelic variants, 158V and 158F, which have different IgG affinities; the NK cells of the present disclosure can express CD16a, CD16b, or both. Cancer patients homozygous for the 158V higher-affinity allele of CD16a are reported to respond better to monoclonal antibody (mAb) therapy (see, e.g., Cartron et al. Blood. 99, 754-58 (2002)). FcRn also binds IgG. Receptors of the Fcε family bind IgE and include FcεRI and FcεRII (CD23). Receptors of the Fcα family bind IgA and include FcαRI (CD89) and Fcα / μR. The NK cells can express any one or more of these FcRs. Additionally, the FcR can be a chimeric FcR, where a portion of the FcR structure is derived from one type of FcR and another portion is derived from a different FcR (or is synthetic in a manner that functions similarly to an FcR subunit).

[0062] The NK cells can express non-native NKG2D receptors, such as receptors comprising a non-native NKG2D receptor extracellular domain that binds a modified NKG2D ligand α1-α2 domain with greater affinity than it binds the native α1-α2 domain. Non-native NKG2D receptors are further described, for example, in U.S. Patent No. 10,259,858 and U.S. Patent Publication No. 2020 / 0138866, each of which is incorporated herein by reference in its entirety and particularly with respect to the disclosure of its modified NKG2D extracellular domain.

[0063] The population of NK cells can also express exogenous polypeptides (i.e., polypeptides encoded by exogenous nucleic acids that are introduced into the NK cells or the PSCs or intermediates from which the NK cells are derived). In various aspects, the population of NK cells expresses cytokines, chemokines, etc., including but not limited to IL-15 or IL-2.

[0064] 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 can be used to selectively kill cells by inducing apoptosis or converting non-toxic compounds into toxic compounds, resulting in unwanted cell death. Examples of suicide gene products include, but are not limited to, caspases (e.g., caspase 9), thymidine kinase, cytosine deaminase, cytochrome P450, and DNase. See, e.g., U.S. Patent Publication No. 2022 / 0025001, which is incorporated herein by reference. Optionally, the nucleic acid encoding the suicide gene product is operably linked to an inducible promoter. See, e.g., Straathof et al., Blood, 105(11), 4247-4254 (2005), which is incorporated herein by reference. Optionally, the engineered NK cells comprise the herpes simplex virus thymidine kinase (TK) suicide gene. See, e.g., Bonini et al., Science, 276, 1719-1724 (1997), which is incorporated herein by reference. Optionally, the engineered NK cells comprise one or more copies of the TK suicide gene. In various aspects, the NK cells comprise a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. For example, the NK cells can comprise a nucleic acid encoding HSV TK and a nucleic acid encoding a hygromycin-thymidine kinase fusion protein. In another embodiment, the NK cells can be eliminated 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.

[0065] Methods for genetically engineering host cells to stably produce one or more target gene products are known in the art. Exemplary methods include the use of viral vectors. Viral vectors can include any suitable viral vector, including, for example, retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), picornaviruses, alphaviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Exogenous nucleic acids can also be introduced into host cells by gene editing techniques, such as using the systems described herein. Preferably, the nucleic acids encoding CAR or FcR and the nucleic acids 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 aspects, the B2M locus is disrupted by inserting nucleic acids encoding different gene products into the B2M locus to replace the expression of the endogenous wild-type B2M protein. Alternatively or additionally, certain HLA-II loci are optionally disrupted by inserting nucleic acids encoding different gene products into the HLA-II locus to replace the expression of the endogenous wild-type HLA-II protein.

[0066] In various aspects of the present disclosure, the 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 obtained. Pluripotent stem cells are generally defined functionally as: (a) stem cells that are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; (b) stem cells that are capable of differentiating into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types); and (c) stem cells that express one or more markers of embryonic stem cells (e.g., express Oct 4, 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 using, for example, methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells. "Embryonic stem cells" (ESC) include, for example, cells derived from the inner cell mass of a human blastocyst or morula, including those that have been continuously passaged as a cell line. Regardless of their source or the specific method used to generate them, embryonic stem cells can be identified based on, for example: (i) the ability to differentiate into cells of all three germ layers, (ii) the expression of at least Oct-4 and alkaline phosphatase, and (iii) the ability to produce teratomas when transplanted into immunodeficient animals.

[0067] Another exemplary pluripotent stem cell is an induced pluripotent stem cell (iPSC). For example, iPSCs are generated by reprogramming somatic cells with a combination of factors (“reprogramming factors”), such as but not limited to Oct4, Sox2, c-Myc, Nanog, Lin28, and / or Klf4. Induced pluripotent stem cells can be generated using almost any somatic cell at any stage of development as a starting point. For example, the cells can be from an embryo, fetus, neonate, adolescent, or adult donor. Exemplary somatic cells that can be used include CD34+ cord blood cells, fibroblasts (e.g., dermal fibroblasts obtained from a skin sample or biopsy), synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. In certain embodiments, the somatic cells are not fibroblasts.

[0068] Pluripotent stem cells can be from any species. Embryonic stem cells have been successfully generated from, for example, multiple species of mice, non-human primates, and humans. Thus, one of ordinary skill 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 (cows, sheep, etc.), canids (domestic and wild canids), felids (domestic and wild felids, such as lions, tigers, and cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including the giant panda), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and so on. Similarly, iPSCs can be from any species. iPSCs have been successfully generated using, for example, mouse and human cells. Thus, iPSCs can be readily generated using donor cells from any species, such as any of the species described herein.

[0069] The NK cell populations of the present disclosure have multiple attributes that render the populations advantageous for therapeutic applications, such as superior engraftment, homing, viability, persistence, immune evasion, and / or cytotoxicity. For example, in various aspects, the engineered NK cell populations are capable of being engrafted into a subject using, for example, protocols known to those of skill in the art after administration. In other words, the NK cell populations are capable of homing to a target tissue (e.g., bone marrow) and / or persisting in a subject for a period of time (e.g., 24 hours to several days or weeks or months) after administration. In various aspects, at least a portion (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 population) of the engineered NK cell populations is capable of being engrafted into a target tissue. For example, in various aspects, 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 engineered NK cell populations is capable of being engrafted into a target tissue (e.g., blood or bone marrow) of a subject. Optionally, once administered to a subject, the engineered NK cell populations are capable of remaining engrafted in a 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 NK cell populations is capable of remaining engrafted in a 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.

[0070] Alternatively or additionally, the NK cells of the population are optionally capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or natural cytotoxicity (NCC) of cancer cells. NK cells mediate NCC by adhering to and binding to target cells and releasing perforin and granzyme, which induce apoptosis in the target cells. ADCC is a mechanism by which immune cells carrying Fc receptors kill antibody-coated cells after the Fc receptors bind to the Fc portion of the antibody. NK cells mediate ADCC, for example, via CD16, and ligation of CD16 results in the release of cytolytic granules and inflammatory mediators. Methods for characterizing ADCC activity and NCC activity are known in the art and are described herein and, for example, in U.S. Patent Publication No. 2020 / 0131475, which describes an assay (Tsirigotis et al., J of Steroid Biochem and Mol Bio 108:267-271 (2008)) that includes using Raji cells (derived from Burkitt lymphoma) pre-incubated with an anti-CD20 antibody as the target in an ADCC assay; U.S. Patent Publication No. 2022 / 0040230; and Bhatnagar et al., Eur. J. Immunol., 44(11):3368 (2014). In various aspects, administration of the population of NK cells to a subject with cancer reduces the tumor burden or load of the subject 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 can be 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 sampling, and / or circulating target cell counts.

[0071] Optionally, at least a portion (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%) of the NK cells in the population have a greater volume than peripheral blood NK cells. Methods for determining cell volume are known in the art, as described in Model, Cytometry, 93(3):281(2018), which is incorporated herein by reference in its entirety and particularly for the description of methods for measuring cell volume. In various aspects of the present disclosure, the cell volume of the NK cells in the population is increased by at least about 5% compared to peripheral blood NK cells. For example, the cell volume of the NK cells in the population is increased by at least about 10% or at least about 15% compared to peripheral blood NK cells (e.g., an increase of 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%).

[0072] The present disclosure also provides a kit comprising one or more containers containing the NK cell population described herein. The kit may further comprise instructions and written information regarding the indication and use of the NK cells. A sterile, sealed container (e.g., vial, bottle, vessel, and / or package) containing the NK cell population is also contemplated, optionally together with suitable instructions for use. In a further aspect, the present disclosure provides an article or unit dosage form comprising: (a) the NK cell population described herein; (b) a container containing the population; and (c) a label affixed to the container or a package insert included in the container that pertains to the use of the NK cells for treating a disease or disorder (e.g., cancer).

[0073] The present disclosure also provides a pharmaceutical composition comprising an NK cell population and a pharmaceutically acceptable carrier, excipient, or diluent. In an exemplary aspect, the composition is a sterile composition. The pharmaceutical composition according to the present disclosure can be formulated for delivery by any route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, or parenteral. "Parenteral" refers to a route of administration typically associated with injection, including intracavitary, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intralung, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, intratumoral, transmucosal, or transtracheal. The composition can be in the form of a solution or suspension for infusion or for injection. The components of the carrier are "pharmaceutically acceptable" because it must be compatible with the other ingredients of the formulation and safe for in vivo use.

[0074] The present disclosure further provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a population of engineered NK cells or a pharmaceutical composition comprising a population of engineered NK cells, thereby treating the disease or disorder of the subject. Similarly, the present disclosure provides the use of a population of engineered NK cells in the treatment of a disease or disorder, and the use of a population of NK cells in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

[0075] In various aspects, 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 sarcoma, 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, but are not limited to, leukemia and lymphoma, such as acute myeloid leukemia, hairy cell leukemia, chronic lymphocytic leukemia and non-Hodgkin lymphoma (e.g., diffuse large B cell lymphoma, Burkitt lymphoma, mantle cell lymphoma and follicular lymphoma). Other cancers include, but are not limited to, alveolar rhabdomyosarcoma, breast cancer; cancer of the anus, anal canal or anorectum; eye cancer, intrahepatic bile duct cancer, joint cancer; cancer of the nose, nasal cavity or middle ear; esophageal cancer, gastrointestinal cancer, Hodgkin 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.

[0076] In various aspects, the disease or disorder is an autoimmune disease or disorder. Autoimmunity is an immune response to self-antigens that causes the body to attack normal cells and tissues. There are over 80 types of autoimmune diseases. The body's response to an autoimmune condition mimics the response to an infection and involves immune cell activation, inflammation and tissue damage. In various aspects, the autoimmune condition is one of over 80 diseases or syndromes characterized by a loss of B cell tolerance and the production of autoantibodies. In various aspects, the autoimmune condition is graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), multiple sclerosis, Sjogren's syndrome, systemic sclerosis / scleroderma, cutaneous sclerosis, ulcerative colitis, inflammatory bowel disease, bullous pemphigoid, insulin-dependent diabetes mellitus or type 1 diabetes, Crohn's disease, psoriatic arthritis or rheumatoid arthritis.

[0077] The method can include administering a population of NK cells before, concurrently with, or after different therapies such as chemotherapy, surgical resection of a tumor, immunotherapy (e.g., cytokine therapy or administration of an antibody construct), or radiotherapy. For example, the present disclosure contemplates a method in which the engineered population of NK cells described herein is administered to a subject in need as part of a treatment regimen that also includes administering to the subject one or more cytokines. In some aspects, 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 aspects of the method, the method further includes administering IL-15 and / or IL-2 to the subject.

[0078] In various aspects, the method can include administering, as part of a treatment regimen, a population of engineered NK cells described herein (e.g., FcR-UDC I-NK (e.g., differentiated cells in which the B2M gene is disrupted, resulting in abrogation of HLA class I proteins and expression of an exogenous single-chain fusion HLA-E as well as CD16) or FcR-UDC I / II-NK (e.g., FcR-UDC I-NK further engineered by knockout mutations of HLA class II-related genes such as RFXANK, resulting in disruption of HLA class II proteins)), the treatment regimen further including administering to a subject one or more antibody constructs (e.g., antibodies). The present disclosure further contemplates a treatment regimen in which an antigen-binding antibody fragment or antibody-like protein product (i.e., a protein based on antibody structural features that permit antigen binding) is administered to a 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 a complete immunoglobulin. The disclosures herein regarding 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 Fv (scFv), diabodies, nanobody constructs, antibody mimetics, and the like. In various aspects, the method includes administering a complete antibody or antibody fragment or antibody-like protein product that comprises an Fc region. In various aspects, 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, EphrinA4, EphrinB2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, Folic acid receptor α, Folic acid receptor β, 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-11Ra, Interferon receptor, Integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α 11bβ3 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-β, PIK3CA, PLAC1, polysialic acid, PRSS21, PSCA, PSMA, PTK7, RAGE-1, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, TGF-1, TGF-β2, TGS5, Tie 2, TIM-1, Tn Ag, 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 combination with the methods disclosed herein include, but are not limited to, rituximab, cetuximab, trastuzumab, panitumumab, ofatumumab, brentuximab vedotin, pertuzumab, ado-trastuzumab emtansine, obinutuzumab, nimotuzumab, bevacizumab, alemtuzumab, gemtuzumab ozogamicin, ranibizumab, olaparib, Ontuximab, isatuximab, certolizumab pegol, daratumumab, lintuzumab, Balantamab, inotuzumab ozogamicin, detuximab vedotin, alemtuzumab, ibritumomab tiuxetan, tositumomab, panitumumab, tremelimumab, ticilimumab, catumaxomab, Oregovomab, zotuximab (described, for example, in International Patent Publication No. WO 2016166124, which is incorporated herein by reference in its entirety and specifically with respect to the description of zotuximab), ASP1650 (described, for example, in International Patent Publication No. WO2012156018, which is incorporated herein by reference in its entirety and specifically with respect to the description of SEQ ID NO:35 and 36, the CDR sequences within SEQ ID NO:35 and 36, and SEQ ID NO:27 and 25), ASP1893 mAb (a humanized form of ASP1650 (i.e., sharing the CDR sequences of ASP1650)), and veltuzumab. Additional examples of antibodies include, but are not limited to, adalimumab, eculizumab, and natalizumab. In some aspects, the antibody is rituximab, ASP1893 mAb, ASP1650, or zotuximab.

[0079] For example, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to the subject an effective amount of the engineered NK cell population described herein and further administering to the subject an antibody that binds to a cell surface antigen on a target cell, the elimination of which provides a therapeutic benefit to the subject. In various aspects, the present disclosure provides methods of treating cancer in a subject in need thereof, wherein the methods comprise administering to the subject an effective amount of the engineered NK cell population and an effective amount of a cancer cell antigen-specific antibody (e.g., any of the anti-cancer antibodies described herein). In various aspects of the present disclosure, an effective amount refers to treating a patient's cancer by administering multiple doses and / or multiple rounds of doses of NK cells and antibodies as part of a treatment course. Merely as examples of aspects of the present disclosure, the subject may have breast cancer and the NK cells are administered with an anti-HER2 antibody (e.g., trastuzumab); or the subject may have colon cancer and the NK cells are administered with an anti-EGFR antibody (e.g., cetuximab or panitumumab); or the subject may have AML and the NK cells are administered with an anti-CD123 antibody or an anti-FLT3 antibody. The subject may have a CD20-positive cancer. In various aspects, the subject has a CLDN18.2-positive cancer, such as gastric or gastroesophageal junction (GEJ) adenocarcinoma, and the 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, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma; gastric cancer, breast cancer, liver cancer, pancreatic cancer; skin cancer, particularly basal cell carcinoma or squamous cell carcinoma; malignant melanoma; head and neck cancer, particularly malignant pleomorphic adenoma; sarcoma, particularly synovial sarcoma or carcinosarcoma; cholangiocarcinoma; cancer of the bladder, particularly transitional cell carcinoma and papillary carcinoma; kidney cancer, particularly renal cell carcinoma, including clear cell renal cell carcinoma or papillary renal cell carcinoma; colon cancer; cancer of the small intestine, including cancer of the ileum, particularly small intestinal adenocarcinoma and ileal adenocarcinoma; testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer; testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer; uterine cancer; germ cell tumors, such as teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis; and metastatic forms thereof), and the NK cells are administered with an anti-CLDN6 antibody. Optionally, the antibody is rituximab, zotuximab, ASP1893 mAb or ASP1650.

[0080] In various aspects, the present disclosure provides methods of treating an autoimmune condition in a subject in need thereof, wherein the methods comprise administering to the subject an effective amount of the engineered NK cell population and an effective amount of an immune cell antigen-specific antibody. Optionally, the antibody is rituximab.

[0081] In some aspects of the present disclosure, the engineered NK cells are optionally co-administered with an antibody as part of a treatment regimen, which may optionally include chemotherapy that is not an antibody. Optionally, the chemotherapeutic agent is selected from fluorouracil, oxaliplatin, and / or capecitabine.

[0082] As used herein, the term "treatment" and words related thereto do not necessarily mean 100% or complete treatment or alleviation. Rather, there are varying degrees of treatment that are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, a method of treating a disease or disorder can provide any amount or level of treatment. Additionally, the treatment provided by the method can include treatment of one or more conditions or symptoms or precursors of the disease being treated. For example, the treatment methods of the present disclosure can inhibit one or more symptoms of the disease. Additionally, the treatment provided by the methods of the present disclosure can include slowing the progression of the disease.

[0083] Treatment of cancer can be determined in any of a variety of ways. Any improvement in the health of the subject is contemplated (e.g., at least or about a 10% reduction, at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, or at least or about a 95% reduction of any of the parameters described herein). For example, a therapeutic response refers to one or more of the following disease improvements: (1) a decrease in the number of tumor cells; (2) an increase in tumor cell death; (3) inhibition of tumor cell survival; (5) inhibition (i.e., slowing to some extent, preferably stopping) of tumor growth or the appearance of new lesions; (6) a decrease in tumor size or burden (i.e., load); (7) the absence of clinically detectable disease; (8) a decrease in the level of cancer markers; (9) an increase in patient survival rate or survival time; and / or (10) some alleviation of one or more symptoms (e.g., pain) associated with the disease or disorder.

[0084] For example, in various aspects, relative to the survival time of a subject without administration of a modified NK cell population, administration of the modified NK cells of the present disclosure increases the survival time of the subject by at least 20 days (e.g., at least 30 days, at least 45 days, at least 60 days, at least 90 days, or longer). Alternatively or additionally, administration of NK cells to a subject reduces the tumor burden of the subject. In various aspects, the methods of the present disclosure further include monitoring the treatment of the subject. The disease state can be monitored by, for example, 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 the case of cancer (e.g., carcinoembryonic antigen (CEA)), cytology, histology, biopsy sampling, and / or circulating target cell counts. 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 an amount of NK cells (or combination therapy, e.g., antibody therapy) sufficient to achieve the desired biological response within a clinically relevant time period; for example, "therapeutically effective" refers to a sufficient amount of NK cells that ameliorate one or more causes or symptoms of a condition or disease. The amount of modified NK cells administered to a subject can comprise, for example, 10 4 to 10 11 、10 5 to 10 10 or 10 6 to 10 9 cells / kg body weight of the subject.

[0085] The subject is a mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters; mammals of the order Lagomorpha, such as rabbits; mammals from the order Carnivora, including felines (cats) and canines (dogs); mammals from the order Artiodactyla, including bovines (cows) and suids (pigs); or mammals of the order Perissodactyla, including equines (horses). In some aspects, the mammal belongs to the order Primates, the order Ceboidae or Simoidae (monkeys) or the order Anthropoidea (humans and apes). In some aspects, the mammal is a human. The term "subject in need" includes subjects that have a disease or disorder, as well as subjects that are to be prophylactically treated for a disease or disorder.

[0086] The present disclosure also provides a method for generating pluripotent stem (PS) cell-derived natural killer (NK) cells under feeder-free and optionally serum-free conditions. The method includes (a) culturing pluripotent stem cells, optionally the PSCs are genetically modified to disrupt one or more copies (e.g., all copies) of endogenous B2M and / or one or more copies (e.g., all copies) of HLA class II-associated genes, the HLA class II-associated genes are selected from RFXANK, RFX5, RFXAP, and CIITA, and optionally expressing a modified non-classical HLA class I protein associated with B2M, such as HLA-E; (b) differentiating the pluripotent stem cells into precursor cells that are capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; and (c) differentiating the precursor cells into NK cells in a medium containing SCF, IL-7, IL-15, and Flt3L (Flt3 ligand) under feeder-free and optionally serum-free conditions. In various aspects, step (b) includes using embryoid bodies to differentiate the pluripotent stem cells to obtain precursor cells that are capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives. In various aspects, the PSCs are human PSCs. In other various aspects, the method further includes (d) culturing the NK cells in a medium containing IL-15 and IL-18 (and optionally, IL-21). In various aspects, step (d) includes culturing the NK cells in the presence of an antibody or ligand that binds to a receptor on the NK cells to promote activation and / or expansion of the NK cells. In various aspects, 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 foregoing antibodies can be provided during the culturing step. Alternatively, the method can include culturing the 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 foregoing ligands can be provided during the culturing step. Optionally, the ligand at least comprises a portion of the native ligand that mediates receptor binding and activation fused to an Fc domain.

[0087] In every aspect, the methods described herein are particularly suitable for the large-scale generation of human PSC-derived NK cells. Previous methods for generating NKs under feeder-free conditions allowed for at most a 5- to 500-fold expansion of PBNK cells. See, e.g., Lu et al., J. Hematol. Oncol. 14:7 (2021). Apparently, the methods of the present disclosure allow for at least a 1000-fold expansion of the cells. In fact, in some embodiments the methods of the present disclosure allow for 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 10000-fold expansion (or any range having these values as endpoints). The expansion levels achieved under feeder-free conditions using the materials and methods described herein are superior to previous methods.

[0088] The method for generating human PSC-derived engineered NK cells is carried out under "feeder-free conditions". A "feeder cell" is a cell that is typically co-cultured with target cells, such as PSCs and / or NK cells; feeder cells release growth factors and nutrients into the cell environment, which support and promote the growth of the target cells (e.g., PSCs). Cell types used as feeder cells include, for example, fibroblasts (mouse or human) and endothelial cells and other stromal cells (e.g., OP9 cells). Feeder-free conditions refer to culture conditions (or media) that are substantially free of feeder (e.g., stromal) cells and / or not pre-conditioned by culturing with feeder cells. The methods of the present disclosure surprisingly provide a robust NK cell population without the need for feeder cells or media conditioned by feeder cells. In fact, even compared to methods for generating NKs under feeder cell conditions, the methods of the present disclosure allow for comparable NK production. Using feeder-free conditions is cost-effective and minimizes xenogeneic contaminants.

[0089] "Culture" refers to maintaining cells in a medium under conditions suitable for the survival and / or proliferation of the cells. The steps described herein can be carried out using any suitable container (e.g., microtiter plate, culture flask, bioreactor, etc.) and environmental conditions that allow for cell growth and differentiation. Any suitable cell culture medium for growing and differentiating stem cells can be used for the methods. Examples of media include, but are not limited to, mTeSR from STEMCELL Technologies (Vancouver, Canada) TM 1, TeSR TM 2, mTeSR TM Plus, TeSR TM E8, APEL TM , STEMdiff TM APEL TM 2 or StemSpanTM ; Primate ES / iPS cell medium from ReproCELL (Boston, MA) and -34 and hESC SFM, from ThermoFisher Scientific (Waltham, MA) PFHM-II medium, and X-VIVO from Lonza (Basel, Switzerland) TM . The medium optionally contains supplements such as 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 inhibitor, MEK inhibitor, or Rho kinase (ROCK) inhibitor), and / or sugars (e.g., glucose or galactose). Media lacking one or more of these supplements are also contemplated. In various aspects, the medium is a chemically defined medium, i.e., a cell culture medium in which all chemical components are known and typically do not include serum, serum albumin, and hydrolysates.

[0090] The method includes culturing human pluripotent stem cells, which are optionally genetically engineered to disrupt 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 combinations thereof), and optionally express a modified non-classical HLA class I protein related to B2M, such as HLA-E, as described above regarding the generation of NK cell populations. The disclosures provided above regarding PSCs and NK cells are also applicable to the PSCs used in the method. For example, the human PSCs optionally contain nucleic acid molecules encoding one or more suicide gene products that can be present in one or more copies, such as HSV thymidine kinase, and are optionally embryonic stem cells or induced PS cells, as described above.

[0091] Any suitable initial seeding density can be used to establish the culture for differentiation. For example, an initial seeding density of about 0.5x10 5 to about 10x10 5 human pluripotent stem cells / mL can be used (e.g., about 1x10 5 to about 5x10 5 or about 1.5x10 5 to about 5x10 5 or about 2x10 5to about 2.5 x 10 5 cells / mL). Prior to the first differentiation step, the human pluripotent stem cells can be cultured for any suitable time, such as 1 to 7 days (e.g., 1 to 4 days or 1 to 3 days), to provide a sufficient amount of human pluripotent stem cells suitable for differentiation. The method then comprises differentiating the human pluripotent stem cells to produce precursor cells that are capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives. In various aspects, embryoid bodies (EBs) are used to differentiate the pluripotent stem cells into precursor cells. See, e.g., Itskovitz-Eldor et al., Mol Med., 6(2):88-95 (2000), which is incorporated herein by reference in its entirety. An EB is an aggregate or cluster of pluripotent cells. EBs can be used as an intermediate stage in producing precursor cells having the potential to further differentiate into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives (or as an intermediate stage in producing hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives themselves). In various aspects, the method does not include differentiating the human pluripotent stem cells into angioblasts or vascular colony-forming cells as precursor cells.

[0092] The differentiation steps to generate precursor cells include culturing human PSCs under conditions suitable for generating precursor cells (e.g., using EBs). In an exemplary aspect, human pluripotent stem cells are cultured in a medium containing cytokines that promote EB formation. In various aspects, 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 envisioned, including using all three cytokines to induce differentiation. The cytokines can be used in any suitable amount. For example, BMP4 can be provided at about 1 ng / mL to about 40 ng / mL (e.g., BMP4 can be provided at 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). Optionally, VEGF can be provided at about 1 ng / mL to about 40 ng / mL (e.g., VEGF is provided at 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). Additionally, optionally, SCF can be provided at about 10 ng / mL to about 60 ng / mL (e.g., SCF is provided at 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). In various aspects, the method includes exposing human pluripotent stem cells to fibroblast growth factor (FGF, e.g., bFGF); alternatively, the method does not include using FGF (e.g., bFGF). The differentiation steps can be carried out for any suitable time, e.g., about 1 day to about 21 days, such as about 1 day to about 14 days, about 7 days to about 18 days, about 10 days to about 14 days, or about 14 days. In various aspects, the first differentiation step is carried out for at most about 14 days. During the culture in the differentiation step, the medium can be changed periodically. Generally, at least a portion of the medium (e.g., half of the medium) is replaced every other day until the end of the differentiation step. The conditions described herein produce high-quality EBs, which results in improved differentiation and NK cell yield.

[0093] The method further includes differentiating precursor cells (e.g., using EBs) into NK cells (i.e., step (c) or the second differentiation step) in a culture medium comprising SCF, IL-7, IL-15, and Flt3L under feeder-free and optionally serum-free conditions. In various aspects, the precursor cells are harvested and replated in a container suitable for growth, expansion, and differentiation. The harvested cells are cultured in a medium comprising SCF (e.g., SCF at 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), IL-7 (e.g., IL-7 at 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), IL-15 (e.g., IL-15 at 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), and Flt3L (e.g., Flt3L at 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). The second differentiation step can be carried out for any suitable time, e.g., about 1 day to about 35 days, such as 1 day to about 21 days, about 7 days to about 32 days, about 10 days to about 28 days, about 14 days to about 25 days, or about 21 days. In various aspects, this step is carried out for up to about 21 days. During the second differentiation step culture, the medium can be changed periodically. In some aspects, the culture is not disturbed for a first time 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 (e.g., half of the medium) can be replaced every other day, twice a week, once a week, etc., until the end of the differentiation step.

[0094] In various aspects, IL-3 is included in the culture medium during the second differentiation step. In this regard, IL-3 is optionally provided at about 1 ng / mL to about 10 ng / mL (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). Optionally, IL-3 is provided only for a portion of the second differentiation step culture period, e.g., only for 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.

[0095] In a preferred aspect of the present disclosure, the culture medium for the second differentiation step (step (c), differentiation of precursor cells into NK cells) contains UM171. Any suitable 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). UM171 is a pyrimidoindole derivative that significantly increases the yield of NK cells (i.e., NK cell expansion) and reduces the variability of the resulting NK population. Thus, a substantially homogeneous NK cell population can be achieved without the need for purification or filtration of the final NK cell product. The term "homogeneous" refers to a population of cells in which each cell is the same or substantially the same as the other cells in 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 aspects of the method, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the cells of the resulting population of human pluripotent stem cell-derived engineered NK cells are the same or substantially the same as the other cells in the population (e.g., display one or more of the above cell surface markers). In this regard, the present disclosure contemplates methods that do not include purification, isolation, or filtration steps to remove unwanted cell types after the differentiation of precursor cells into NK cells (or optionally after culturing the NK cells to promote activation). In various aspects, UM171 is present only in the culture medium of step (c) and is not present in the culture medium of any other step of the method (although this is not required).

[0096] The method of the present disclosure optionally includes a further culturing step (step (d)) to promote activation and / or expansion of NK cells. Although this step is not required, it may be desirable for providing a population of NK cells with particularly desired characteristics (e.g., improved cytotoxicity, improved in vivo persistence, improved engraftment, etc.). The activation step includes culturing NK cells (from the second differentiation step, step (c)) in a medium containing IL-15 and IL-18. IL-15 and IL-18 can be provided in any suitable amounts, 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 IL-15 and IL-18 each. Optionally, 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 aspects, the medium used in the activation step does not contain IL-12.

[0097] In various aspects, NK cells are cultured in the presence of an antibody that binds DNAM-1 (DNAX accessory molecule CD222), an antibody that binds OX40, an antibody that binds NKG2D, an antibody that binds 2B4, an antibody that binds NKp30, an antibody that binds NKp46, or a combination of any of the foregoing antibodies. For example, step (d) can include culturing NK cells in the presence of an antibody that binds NKp30. NKp30, also known as CD337, is a 30 kD type I transmembrane protein that functions as a stimulatory receptor on NK cells. Alternatively or additionally, step (d) can include culturing NK cells in the presence of an antibody that binds 2B4. 2B4, also known as CD244, is a member of the signaling lymphocytic 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 DNAM-1. DNAM-1, also known as CD226, is a 65 kD glycoprotein member 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 NKp30 and an antibody that binds 2B4. In a further example, step (d) can include culturing NK cells in the presence of an antibody that binds NKp30 and an antibody that binds DNAM-1. Anti-NKp30 antibodies, 2B4 antibodies, and anti-DNAM-1 antibodies are commercially available from, for example, SinoBiological (Wayne, PA) and BioLegend (San Diego, CA). In various aspects, step (d) can include culturing NK cells in the presence of a ligand that binds DNAM-1, a ligand that binds OX40 (e.g., OX40L), a ligand that binds NKG2D (e.g., MICA or ULBP1), a ligand that binds 2B4 (CD244), a ligand that binds NKp30 (e.g., B7H6), and / or a ligand that binds NKp46 to activate the NK cells. A combination of any of the foregoing ligands can be provided during the culturing step. The one or more antibodies or one or more ligands can be provided in the culture medium or can be coated on the container.

[0098] The activation step (step (d)) can be carried out for any suitable time, such as 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 aspects, step (d) is carried out for about 5 days to about 10 days. Additionally, the activation step can be carried out in multiple containers to accommodate the proliferation of the NK cell population. For example, the activation step can include culturing NK cells in one container (e.g., a culture flask, such as a T75 culture flask) for an initial period of time (e.g., 1 day, 2 days, 3 days or 4 days), and then transferring the cells to a larger container (e.g., a bioreactor) for a second period of time (e.g., 1 day, 2 days, 3 days or 4 days). Further, in various aspects, if present, IL-21 is provided only for a portion of the activation step. In this regard, the method can include including IL-21 in the culture medium for an initial period of time (e.g., one or more days out of days 0-4 of the activation step), but not including IL-21 in the culture medium for the remainder of the culture period.

[0099] NK cells are cultured in a container (e.g., a culture flask or a bioreactor) suitable for the growth, expansion and activation of NK cells. The container is optionally coated with a substance that promotes cell adhesion or one or more matrix components, such as laminin or laminin fragments, nidogen, collagen, gelatin, vitronectin, fibronectin, (Corning Incorporated), polylysine, thrombospondin, ProNectin-F TM and the like. An exemplary matrix component is a 63-kD fragment of a recombinant human fibronectin fragment, also known in the art as rFN-CH-296. Optionally, a Notch ligand, such as DLL4, is provided. In various aspects, the container is coated with a Notch ligand (e.g., DLL4) and coated. DLL4 and can be provided in any suitable amount, including relative amounts that result in a DLL4: 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 for any of the method steps disclosed herein, including step (c) and / or step (d).

[0100] Apparently, it has been determined that the activation step (step (d)) can be carried out multiple times without negatively affecting the characteristics of the NK cell population (e.g., not significantly reducing the functionality of the cells). If desired, step (d) can be repeated up to 5 times (i.e., repeated 2 times, 3 times, 4 times or 5 times) to further expand and / or activate the NK cells.

[0101] In various aspects of the method, IL-2 and / or IL-6 are not included in the culture medium for one or more (e.g., any) steps of the method.

[0102] The present disclosure further provides a method for increasing the purity of a population of NK cells derived from pluripotent stem cells. The method includes differentiating embryoid bodies (i.e., the cells within the embryoid bodies) into NK cells in a feeder-free, optionally serum-free condition in a culture medium containing SCF, IL-7, IL-15, and Flt3L and in the presence of UM171. In various aspects, the culture medium further contains IL-3. Optionally, the method is carried out in a container coated with and DLL4. Optionally, the method is carried out for about 1 day to about 21 days. The result of the method is a substantially pure population of CD56+, CD45+ NK cells (i.e., a population of NK cells with low levels of unwanted cell types (contaminating cells)). The method allows for the generation of PSC-derived NK cells without the need for extensive further purification of the resulting cell population to obtain a substantially pure population of NK cells. In various aspects, the method generates 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).

[0103] The method of the present disclosure generates a robust population of NK cells that is suitable for, for example, therapeutic applications. For example, the NK cells optionally express one or more cell surface markers selected from CD56, CD45, CD25, DNAM-1, NKp30, NKG2D, and NKp44, including any combination of the cell surface markers. In a preferred aspect, the NK cells express at least CD56. The NK cells also optionally express engineered non-classical HLA class I proteins associated with B2M, such as 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 population of NK cells also preferably exhibits the characteristics described above, such as being able to engraft into a subject after administration and optionally remaining engrafted for at least 20 days, being able to induce ADCC and / or NCC of cancer cells, and being able to reduce tumor burden. In various aspects, the method results in the generation of at least 10 9 、at least 10 10 or at least 10 11 CD56-positive NK cells.

[0104] The following examples are provided solely to illustrate the present invention and do not limit its scope in any way.

[0105] Example

[0106] The examples describe exemplary methods for generating human pluripotent stem cell-derived engineered natural killer (NK) cells under feeder-free, serum-free conditions.

[0107] Example 1: Engineered human pluripotent stem cells

[0108] Human pluripotent stem cells (PSCs) (NIH2, NIH3, and W1C1) were genetically engineered to (i) disrupt the B2M gene, resulting in the abolition of HLA class I proteins, and (ii) express the exogenous single-chain fusion HLA-E (“UDC I-PSC”). UDC I-PSC was further genetically engineered by knockout mutation of the HLA class II-associated gene RFXANK, resulting in the disruption of HLA class II proteins (“UDC I / II-PSC”). Alternatively, UDC I-PSC or UDC I / II-PSC was further genetically engineered to express the gene encoding the Fc receptor / CD16 (“FcR-UDC I-PSC” or “FcR-UDC I / II-PSC”). UDC I-PSC, UDC I / II-PSC, and / or FcR-UDC I-PSC are collectively referred to herein as “UDC PSC”. Differentiated cells are referred to herein as, for example, “FcR-UDC I-NK” or “FcR-UDC I / II-NK”.

[0109] Example 2: NK differentiation from parental and engineered human pluripotent stem cells

[0110] The parental and engineered UDC PSCs (collectively referred to as “PSCs”) were thawed, expanded, and cultured on vitronectin in mTeSR TM (STEMCELL Technologies, Cambridge, MA) feeder-free, animal component-free medium for up to 7 days. The PSCs were dissociated into single cells and transferred to a microwell plate and cultured in STEMdiff TM APEL TM 2 and Cultured in PFHM-II medium (STEMCELL Technologies, Cambridge, MA; ThermoFisher Scientific, Waltham, MA) for 14 days to differentiate into progenitor cells (via embryoid bodies), which are capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives. The medium was supplemented with BMP4 (20 ng / mL), VEGF (20 ng / mL), and SCF (40 ng / mL). Half-medium changes were performed every other day starting on day 3. The embryoid bodies were re-plated onto DLL4 / Retronectin-coated T75 flasks to differentiate into NK cells. The 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 without feeder cells for 21 days. Half-medium changes were performed twice a week. The resulting NK cells (“UDC-NK”) were washed and harvested. UDC-NK cell activation and expansion were carried out by culturing the UDC-NK cells in flasks coated with anti-NKp30 antibody, anti-DNAM-1 antibody, and Retronectin in CTS TM NK-Xpander TM or in RPMI-1640 medium containing IL-15 (10 ng / mL), IL-18 (10 ng / mL), and IL-21 (10 ng / mL) for 3 days, after which the UDC-NK cells were transferred to G- or a spin flask bioreactor and cultured for an additional 3 days in the presence of IL-15 and IL-18. A representative schematic diagram of the NK differentiation process is shown in Figure 1 as shown.

[0111] Regardless of the parental cell line or the engineered UDC PSC, the method produces a substantially homogeneous population of UDC NK cells. At the start of step 0 (as Figure 1 described), starting with approximately 4 x 10 6 iPSCs, approximately 5 x 10 10 NK cells were produced, achieving a 10,000-fold expansion. The addition of UM171 during NK differentiation increased the UDC-NK cell yield, as shown in Figures 2A - 2C as shown. Additionally, in multiple production runs and gene-edited clones, at least 90% of the cells were CD56 and CD45 positive, and no purification steps were required to remove unwanted cell types (see Figure 7A ). Both UDC I-PSCs and FcR-UDC I-PSCs produced at least 90% CD45 +CD56 + NK cells. In addition, as Figure 3A and 3B shown, activation and expansion of NK cells led to 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).

[0112] For activation and expansion of UDC-NK cells (step 3 or step (d)), combinations of NK agonists were tested (see Figure 4 ). Single NK agonists were also tested and showed moderate NK activation. UDC-NK cells (step 2) showed robust expansion and high cell viability. As Figures 5A - 5B shown, among four different cell seeding densities, UDC NK cells (step 2) cultured on Retronectin, anti-NKp30 antibody, anti-DNAM-1 antibody with a medium containing IL-15, IL-18, and IL-21 showed more than 16 to 32-fold expansion compared to step 2 controls with only 3 to 4-fold expansion, and greater than 90% cell viability.

[0113] Analysis of subsets of the cell population 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 provides details of the subsets / impurities of the cell population.

[0114] Table 1

[0115]

[0116] ND = Not detected

[0117] The UDC-NK cells of the present invention are on average approximately 18% larger by volume than peripheral blood NK cells (PB-NK), as demonstrated by flow cytometry (see Figure 6 ).

[0118] Example 3: In vitro characterization of UDC PSC-derived NK cells

[0119] Among different gene-edited clones, UDC-NK cells derived from UDC I-PSC, UDC I / II-PSC, and UDC I-FcR-PSC expressed the expected cell phenotypes. As Figure 7A shown, both parental and UDC PSC-derived UDC I-PSC-derived NK cells were positive for CD45 + CD56 +Highly positive. UDC I-PSC-derived NK cells (UDC I-NK cells) do not express HLA class I, but express HLA class II and HLA-E, as well as wild-type levels of CD16 (FcR). See Figures 7B - 7E . FcR-UDC I-PSC-derived UDC-NK cells (FcR-UDC I-NK cells) have a similar expression profile, except for expressing higher levels of CD16 compared to wild type. Finally, UDC I / II-PSC-derived UDC-NK cells (UDC I / II-NK cells) do not express either HLA class I or class II proteins, express HLA-E, and express wild-type levels of CD16. This data demonstrates that NK cells derived from gene-edited clones maintain the gene editing after differentiation from PSCs. In different PSC cell lines (NIH3 and W1C1), the expected cell phenotypes were also confirmed (see Figures 8A - 8B ). At least based on the above, the NK differentiation process disclosed herein is robust in different gene-edited clones as well as PSC cell lines.

[0120] UDC-NK cells express common NK markers and other biomarkers, similar to PB-NK. As Figure 9C shown, common NK markers, such as NKp30, NKp46, NKG2D, DNAM-1, 2B4, NKG2A, and CD94, with some exceptions, are expressed on FcR-UDC I-NK cells ( Figure 1 Steps 2 and 3 described in, namely steps (b) and (c) respectively), as well as expanded PB-NK cells (PB-NK cells are expanded by step 3 of the NK differentiation protocol). Similarly, the T cell marker CD8 is usually expressed in FcR-UDC I-NK cells and expanded PB-NK, but CD4, TCRαβ, or TCRγδ are not expressed, and CD3 is expressed rarely or not at all. Figure 9B . The lack of TCRαβ plays an important role in terms of safety.

[0121] UDC-NK cells express several activation markers and few (if any) exhaustion or "memory-like" markers. In Figures 10A - 10CIn this study, the activation, exhaustion, and "memory-like" markers of FcR-UDC I-NK cells (Steps 2 and 3) and expanded PB-NK were measured. Except for CD69, most of the activation markers were similar between FcR-UDC I-NK cells and expanded PB-NK. Compared with expanded PB-NK, FcR-UDC I-NK cells showed a higher level of CD69. Different from expanded PB-NK, FcR-UDC I-NK cells showed little (if any) exhaustion or "memory-like" markers. Expanded PB-NK had higher levels of the exhaustion markers TIGIT, LAG3, KLRG1, and ILT2, as well as higher levels of the memory-like marker NKG2C. Differential expression of cytokines and chemokine receptors (see Figures 11A - 11B ) and adhesion / homing, co-stimulatory, and apoptosis markers (see Figures 12A - 12C ) was shown for FcR-UDC I-NK cells and expanded PB-NK.

[0122] UDC-NK cells also showed natural cell 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. In Figure 13 , 1 x 10 4 K562-GFP tumor cells were added per well, and the corresponding number of UDC-NK cells was added according to the E:T ratio. The results confirmed the consistent and similar in vitro NCC activity (i.e., specific lysis) of UDC-NK cells against K562 tumor cells in multiple runs, regardless of the PSC cell line or gene-edited clone. In addition, FcR-UDC I-NK cells showed NCC activity similar to that of expanded PB-NK; meanwhile, no cytotoxicity or killing against normal allogeneic peripheral blood mononuclear cells (PBMCs) or FcR-UDC I-NK cells themselves was observed (see Figure 16 ). Surprisingly, these UDC-NK cells showed continuous NCC activity against K562 tumor cells, enabling UDC-NK cells to kill freshly added K562 tumor cells in a dose-dependent manner (see Figure 17 ). The IncuCyte continuous killing assay was performed using 1.5 x 10 4 (3:1 E:T) or 5 x 10 4 UDC-NK cells (FcR-UDC I-NK) (c.3C1). Every 24 hours, 5 x 10 3 K562-GFP target cells were added, for a total of 6 times. The wells were imaged every 3 hours for 6 days.

[0123] Similar to NCC, UDC-NK cells display potent antibody-dependent cellular cytotoxicity (ADCC) against a broad range of CD20 + tumor cell lines. Using a 4-hour flow-based ADCC assay, tumor cells were labeled with CellTrace Violet dye and 1 μg / ml antibody was added. FcR-UDC I-NK cells showed increased ADCC activity in combination with rituximab against three (3) tumor cell lines (Daudi, Ramos, and Raji) compared to control IgG1 antibody (see Figures 18A - 18C ). In contrast, expanded PB-NK cells plus rituximab showed no increase in ADCC activity against Raji tumor cells compared to control IgG1 antibody (see Figure 18D ). Additionally, FcR-UDC I-NK cells also showed NCC and ADCC activity against a broad range of B lymphoma cell lines (see Figure 19 ). These results confirm the ADCC activity of UDC-NK cells against a broad range of CD20 + and B lymphoma cell lines.

[0124] UDC-NK cells produce a cytokine profile indicative of activated NK cells that are capable of inducing ADCC upon stimulation with rituximab-coated Raji tumor cells. Using a standard cytokine secretion assay, 1 x 10 5 FcR-UDC I-NK cells / well were co-cultured with rituximab-coated Raji tumor cells at a 1:1 E:T ratio for 25 hours. Cytokine secretion was measured using a 15-Plex Cytokine Luminex immunoassay (see Figure 20A ). Additionally, FcR-UDC I-NK cells did not enhance cytokine / chemokine secretion after co-culture with allogeneic PBMCs, confirming the specificity of FcR-UDC I-NK cells (see Figure 20B -E and 21A-D).

[0125] Example 4: In Vivo Persistence and Distribution of UDC-PSC-Derived NK Cells

[0126] UDC-NK cells also show in vivo persistence in blood, spleen, and bone marrow in the absence of antigen. In NSG mice, 4 x 10 6One FcR-UDC I-NK cell (c.3C1), followed by IL-2 (30,000 IU, 3x / week) and IL-15 (100 ng daily for 7 days). For each tissue, the percentage of FcR-UDC I-NK cells in lymphocytes / WBC was determined by flow cytometry and human CD45 and human CD56 staining. Total cell counts were determined using flow cytometry counting methods (per mL of blood, each spleen, BM cells in 2 femurs + 2 tibias), and the % FcR-UDC I-NK was applied to this number to calculate the number of FcR-UDC I-NK in each tissue. As Figures 22A - 22G shown, in the absence of antigen, FcR-UDC I-NK cells persisted in vivo for more than 10 days after i.v. injection. Additionally, FcR-UDC I-NK cells distributed to multiple tissues: lung > liver > spleen > blood > bone marrow (see Figures 23A - 23E ).

[0127] Example 5: In vivo anti-tumor efficacy study using UDC-NK cells in the Rajii.p. tumor model

[0128] The in vivo efficacy of UDC-NK cells against tumors was studied in the Raji intraperitoneal (i.p.) tumor model. In a representative study, NSG mice were divided into 6 groups (n = 7 or 8) as shown in Table 2 below.

[0129] Table 2

[0130]

[0131] In Figure 24 , NSG mice were irradiated and i.p. injected with Raji (Burkitt lymphoma) cells (3x10 5 cells). Raji cells are resistant to NK cells in the absence of anti-CD20 antibody. On day 4, 5x10 6 FcR-UDC I-NK cells and / or rituximab (300 μg) were given to NSG mice. On day 7, a second dose of rituximab (300 μg) was given. NSG mice were given a daily dose of IL-15 (100 ng) for 7 days and IL-2 (30,000 IU) three times a week for 3 weeks. NSG mice were imaged and evaluated for survival, BLI (tumor burden), body weight, and other health conditions on different dates until day 121. As Figures 25A - 25B shown, the combination of FcR-UDC I-NK and rituximab significantly prolonged the survival of lymphoma-bearing NSG mice beyond that observed with rituximab alone or FcR-UDC I-NK cells (see Figure 25A)。The potency between FcR-UDC I-NK cells plus rituximab and PB-NK cells plus rituximab was similar (see Figure 25B ). However, the response rate was approximately 50%.

[0132] In another study, mice were divided into 5 groups (n = 8) as shown in Table 3 below.

[0133] Table 3

[0134]

[0135] In Figure 26 , NSG mice were irradiated and injected i.p. with Raji (Burkitt lymphoma) cells (2 x 10 5 cells). On day 2, 10 x 10 6 FcR-UDC I-NK cells and / or 100 μg rituximab were given to the mice. On days 6, 10, and / or 16, 10 x 10 6 FcR-UDC I-NK cells were given i.p. to the mice. Starting from day 2 after the last NK dose, the mice were given daily doses of IL-15 (100 ng) and IL-2 (30,000 IU) three times a week until day 21. The NSG mice were imaged and evaluated for survival, BLI (tumor burden), body weight, and other health conditions on different dates until day 86. FcR-UDC I-NK and rituximab significantly prolonged the survival of lymphoma-bearing mice beyond that observed with rituximab alone or FcR-UDC-NK cells (see Figure 27B ). All mice treated with FcR-UDC-NK plus rituximab responded to the treatment, and increasing the interval between doses prolonged the duration of the response (see Figure 27A ).

[0136] Example 6: In vivo anti-tumor efficacy study of UDC-NK cells in the Rajii.v. tumor model

[0137] The in vivo efficacy of UDC-NK cells against tumors was studied in the Rajii.v. tumor model. The Rajii.v. model is a more aggressive dissemination model and represents a more clinically relevant lymphoma physiological model. In a representative study using NOGf mice with mouse FcgR knockout (FcResolvNOG), the NOGf mice were divided into 3 groups as shown in Table 4 below.

[0138] Table 4

[0139]

[0140]

[0141] In Figure 28A , Raji cells (1 x 10 5 cells) were injected i.v. into NOGf mice, and on day 1, rituximab (100 μg, i.p.) or a combination with FcR-UDC I-NK cells (10 x 10 6 , i.v.) was injected. On days 8 and 15, rituximab alone (100 μg, i.p.) or a combination with FcR-UDC I-NK cells (10 x 10 6 , i.v.) was given to the mice. For the third group, FcR-UDC I-NK cells alone (10 x 10 6 , i.v.) were given to the mice on day 4. The mice were given a daily dose of IL-15 (100 ng, i.p.). As Figure 28B shown, the combination of FcR-UDC I-NK plus rituximab achieved a significantly greater reduction in tumor burden compared to rituximab alone and untreated.

[0142] In another study, mice were divided into 4 groups (n = 8) as shown in Table 5 below.

[0143] Table 5

[0144]

[0145] In Figure 29A , Raji cells (1 x 10 5 cells) were injected i.v. into NOGf mice engineered to express human IL-15, and on day 1, rituximab alone (100 μg, i.p.) or a combination with FcR-UDC I-NK cells (3 x 10 6 or 15 x 10 6 ) was injected. On day 4, FcR-UDC I-NK cells alone (3 x 10 6 or 15 x 10 6 , i.v.) were given. On days 8 and 11, rituximab alone (50 μg, i.p.) was given or a combination with 3 x 10 6 FcR-UDC I-NK cells (i.v.) was given, or on day 11, a combination of rituximab with 15 x 10 6 FcR-UDC I-NK cells (i.v.) was given. As Figure 29B shown, the combination of 15 x 10 6 FcR-UDC I-NK cells + rituximab achieved a greater reduction in tumor burden.

[0146] Example 7: Anti-tumor efficacy study using UDC-NK cells and anti-Claudin 6 (CLDN6) antibodies

[0147] The combination of UDC-NK cells and anti-CLDN6 antibodies described herein was evaluated for antibody-dependent cellular cytotoxicity (ADCC) against the CLDN6+ ovarian cancer cell line PA-1. Briefly, in a 4-hour flow-based ADCC assay, cell tracer purple dye-labeled PA-1 cells were co-cultured with FcR-UDC I-NK cells in the presence of titrated concentrations of anti-CLDN6 antibodies ASP1650 (also known as IMAB027) and ASP1893 mAb. As Figure 30 shown, FcR-UDC I-NK cells demonstrated increased cytotoxicity against PA-1 cells in an anti-CLDN6 antibody dose-dependent manner. 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.

[0148] The surface CLDN6 expression of a panel of tumor cell lines was also tested and classified into CLDN6hi, CLDN6mid, CLDN6low, and CLDN6- groups. FcR-UDC I-NK cells were tested against a panel of tumor cell lines with varying levels of surface CLDN6 in a 4-hour flow-based ADCC assay. As Figures 31A - 31M shown, FcR-UDC I-NK cells demonstrated ADCC activity against CLDN6+ tumor cells in a CLDN6 expression-dependent manner. Interestingly, regardless of CLDN6 surface expression, FcR-UDC I-NK cells showed natural cytotoxicity (NCC) against a broad panel of tumor cell lines in the absence of anti-CLDN6 antibodies.

[0149] TNF-α and IFN-γ production by UDC-NK cells after stimulation with anti-CLDN6 antibody-coated tumor cells was also examined. Briefly, 10 5 FcR-UDC I-NK cells and 10 5 tumor cells were co-cultured in a 96-well plate in the presence or absence of 1 μg / mL of anti-CLDN6 antibody (ASP1893 mAb) or control hIgG1 antibody. Twenty-four hours after co-culture, the culture supernatants were harvested for TNF-α and IFN-γ measurement by ELISA (R&D Systems). Figures 32A - 32BFcR-UDC I-NK cells showed elevated TNF-α and IFN-γ secretion after stimulation with a panel of tumor cell lines, compared to the basal cytokine levels without stimulation. FcR-UDC I-NK cells demonstrated superior cytokine production after stimulation with CLDN6+ tumor cells and ASP1893 mAb.

[0150] The in vivo efficacy of UDC-NK cells against tumors was investigated in a CLDN6+ PA-1 cell xenograft mouse tumor model. In a representative study, mice were divided into 4 groups (n = 6 or 7) as shown in Table 6 below. Briefly, 5 x 10 6 PA-1 cells were transplanted subcutaneously into the flanks of NOGf mice engineered to express human IL-15. Thirteen days after tumor transplantation, the mice were treated i.v. with 7.5 x 10 6 cryopreserved FcR-UDC I-NK cells, with or without anti-CLDN6 (300 μg ASP1650) i.p., as Figure 33 described. The cryopreserved FcR-UDC-I-NK cells were thawed and injected directly into the mice. Figures 34A - 34B Combination therapy with FcR-UDC I-NK cells and anti-CLDN6 antibody demonstrated statistically significant anti-tumor efficacy and improved overall survival. Monotherapy with FcR-UDC I-NK cells or anti-CLDN6 antibody did not show significant anti-tumor efficacy.

[0151] Table 6

[0152]

[0153] The above examples confirmed that regardless of CLDN6 surface expression, UDC-NK cells showed natural cytotoxicity against a wide range of tumor cell lines in vitro without anti-CLDN6 antibody. The examples further confirmed that when used in combination with anti-CLDN6 antibody, the UDC-NK cells of the present disclosure showed effective anti-tumor activity in vitro and in vivo.

[0154] In another study, a PA-1 cell xenograft mouse tumor model using NOGf mice expressing human IL-15 was used. As Figure 35A described, 7.5 x 10 6 FcR-UDC I-NK cells were administered i.v. and / or anti-CLDN6 antibody (300 μg) was administered i.p. Combination therapy with FcR-UDC-NK cells and anti-CLDN6 antibody resulted in tumor growth inhibition ( Figure 35B ) and extended survival ( Figure 35C)。In mice given FcR-UDC 1-NK cells + anti-CLDN6 antibody, greater than 50% survival was observed at approximately 50 days.

[0155] Example 8: Anti-tumor study using UDC-NK cells and anti-Claudin 18.2 (CLDN18.2) antibody

[0156] This example demonstrated that UDC-NK cells showed anti-tumor activity when used in combination with the anti-CLDN18.2 antibody zotuximab. Briefly, an ADCC assay was performed as a 4-hour co-culture of FcR-UDC I-NK cells and BxPC3-CLDN18.2-Luc tumor target cells, similar to the assay described in Example 7. Zotuximab was present at 1 mg / mL. The results are shown in Figure 36A which showed zotuximab-dependent killing of tumor cells expressing CLDN18.2. The anti-tumor effect of the combination of FcR-UDC I / II-NK cells and anti-claudin 18.2 antibody (zotuximab) against BxPC3-CLDN18.2-luciferase tumor cells is shown in Figure 36B which showed that the highest level of cell killing was achieved using the combination of FcR-UDC I / II-NK cells and zotuximab.

[0157] Cytokine secretion assays were also performed using a method similar to Example 7. BxPC3-CLDN18.2-Luc cells were cultured with the FcR-UDC I-NK cells of the present disclosure, FcR-UDC I-NK cells and human IgG, or FcR-UDC I-NK cells and zotuximab. Supernatants were collected after 24-hour co-culture in the presence of IL-15 (10 ng / mL), and the levels of interferon-γ or tumor necrosis factor-α were examined. Zotuximab was present at 1 mg / mL. The results are shown in Figures 37A - 37B which showed a significant increase in cytokine secretion by FcR-UDC I-NK cells in samples containing zotuximab and BxPC3-CLDN18.2-Luc tumor target cells.

[0158] The examples provided herein demonstrated that the engineered NK cells of the present disclosure showed anti-tumor activity against a wide range of tumor types, including lymphoma, ovarian cancer, and pancreatic cancer. The examples reported that NK cells alone showed anti-tumor activity, and the anti-tumor activity was enhanced by using antibody constructs against tumor antigens (such as CLDN6, CLDN18.2, and CD20). Although the working examples utilized tumor cells to confirm the cell killing activity of the engineered NK cells of the present disclosure, it should be understood that the data can be extended to additional cell types.

[0159] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference herein and were set forth in its entirety herein.

Claims

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

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

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

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

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

6. The engineered NK cell population according to claim 4, wherein the pluripotent stem cells are induced pluripotent stem cells.

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

8. The engineered NK cell population according to any one of claims 1-7, wherein the engineered NK cell population is capable of being transplanted into a subject after administration.

9. The engineered NK cell population according to claim 8, wherein at least 50% of the engineered NK cell population is capable of being transplanted into the target tissue of the subject.

10. The engineered NK cell population according to claim 8 or 9, wherein the engineered NK cell population is capable of remaining transplanted in the target tissue of the subject for at least 20 days after administration.

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

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

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

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

15. The engineered NK cell population according to any one of claims 1-14, wherein the cell volume of the NK cells is increased by at least about 5% compared to peripheral blood NK cells.

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

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

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

19. A method for generating human pluripotent stem cell-derived engineered natural killer (NK) cells under feeder-free conditions, the method 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 human leukocyte antigen (HLA) class II-associated genes selected from regulator of factor X-associated ankyrin-containing protein (RFXANK), regulator of factor 5 (RFX5), regulator of factor X-associated protein (RFXAP), and class II transactivator (CIITA); (b) differentiating the human pluripotent stem cells into precursor cells capable of differentiating into hematopoietic cell types, endothelial cell types, and / or mesodermal derivatives; (c) differentiating the precursor cells into NK cells in a feeder-free condition in a medium comprising SCF, IL-7, IL-15, and Flt3L; and (d) culturing the NK cells in a medium comprising IL-15 and IL-18 and in the presence of an antibody or ligand that binds to a receptor on the NK cells to promote NK cell activation, thereby generating human pluripotent stem cell-derived engineered NK cells.

20. A method for generating human pluripotent stem cell-derived engineered natural killer (NK) cells under feeder-free conditions, the method 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 human leukocyte antigen (HLA) class II-associated genes selected from regulator of factor X-associated ankyrin-containing protein (RFXANK), regulator of factor 5 (RFX5), regulator of factor X-associated protein (RFXAP), and class II transactivator (CIITA); (b) differentiating the human pluripotent stem cells into precursor 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 in a medium containing SCF, IL-7, IL-15 and Flt3L under feeder-free conditions; thereby generating human pluripotent stem cell-derived engineered NK cells.

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

22. The method according to claim 19 or 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 or an antibody that binds to NKp46, or a combination thereof.

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

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

28. The method according to 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 19-28, wherein the human pluripotent stem cells are human embryonic stem cells.

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

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

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

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

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

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

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

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

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

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

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

41. The method according to claim 19 and any one of claims 21 - 40, wherein step (d) is repeated up to 5 times.

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

43. The method according to claim 42, wherein the medium further comprises IL - 3.

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

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

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

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

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

49. The method according to claim 47 or 48, wherein the method further comprises administering to the subject one or more cytokines.

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

51. The method according to any one of claims 47 - 50, wherein the administration increases the survival time of the subject by at least 20 days relative to the survival time of the subject in the absence of administration of the population of engineered NK cells.

52. The method according to any one of claims 47 - 50, wherein said administration reduces the tumor burden of the subject.

53. The method according to any one of claims 47 - 52, wherein the method further comprises administering to the subject one or more antibodies.

54. The method according to claim 47, wherein the disease or disorder is an autoimmune disorder.

55. The method according to claim 54, wherein the method further comprises administering to the subject one or more antibodies.

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