Method for Proliferating CD56+ / CD3- Cells

Abstract

This disclosure relates to a method for growing or producing CD56+ / CD3- cells, comprising contacting CD56+ / CD3- cells with an NKp46 agonist and / or an aCD30 agonist.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the priority and benefit of U.S. Provisional Application No. 63 / 572,039, filed on March 29, 2024, and U.S. Provisional Application No. 63 / 498,773, filed on April 27, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronically Filed Sequence Listing The content of the electronically filed sequence listing (name: 3817_188PC02_Seqlisting_ST26; size: 4,409 bytes; and creation date: April 19, 2024) is incorporated herein by reference in its entirety.

[0003] Field This disclosure relates to the proliferation of CD56 + / CD3 - cells.

Background Art

[0004] Background Natural killer (NK) cells have therapeutic potential for a variety of human malignancies, including adoptive immunotherapy for cancer treatment and other applications. NK cells do not require a priming phase and are not HLA - restricted, indicating the potential for a "readily available" potent anti - tumor response. Inducible pluripotent stem cell (iPSC) - derived NK (iNK) cells are included in multiple clinical trials for the treatment of not only solid tumors but also hematological malignancies. NK cells do not proliferate sufficiently in vitro, have a limited lifespan in vivo, and occupy only a small fraction of human peripheral blood mononuclear cells (PBMCs), so obtaining a sufficient number of cells is a major obstacle in NK cell immunotherapy. Thus, there remains a need for improvement in methods for expanding NK cells and iNK cells.

Summary of the Invention

[0005] Brief Summary Some aspects of this disclosure relate to methods for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of an NKp46 agonist, wherein the CD56+ / CD3- cells comprise at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. Some aspects of this disclosure relate to methods for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of a CD30 agonist.

[0006] In some embodiments, CD56+ / CD3- cells are derived from pluripotent stem cells. In some embodiments, the method further comprises transducing pluripotent stem cells with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0007] In some embodiments, the NKp46 agonist is an anti-NKp46 antibody. In some embodiments, the CD30 agonist is an anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are cultured in the presence of the agonist and at least one cytokine. In some embodiments, at least one cytokine includes interleukin-(IL)2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof. In some embodiments, CD56+ / CD3- cells are cultured in the presence of the CD30 agonist and at least one cytokine. In some embodiments, at least one cytokine includes interleukin-(IL)2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

[0008] In some embodiments, the CD56+ / CD3- cell further comprises at least one exogenous nucleic acid molecule encoding (i) one or more chimeric antigen receptors (CARs), and / or (ii) one or more cytokines or their mimetic counterparts.

[0009] In some embodiments, the cytokine or its mimetic is IL-15. In some embodiments, IL-15 is linked to IL-15Rα to form a fusion protein.

[0010] Several aspects of the present disclosure relate to a method for producing modified CD56+ / CD3- cells, comprising: (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising one or more exogenous nucleic acid molecules encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises: (C) culturing CD56+ / CD3- cells in the presence of an NKp46 agonist and / or a CD30 agonist.

[0011] In some embodiments, the HPC is derived from one or more pluripotent stem cells. In some embodiments, the method further comprises transducing one or more pluripotent stem cells with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. In some embodiments, the pluripotent stem cells are iPSCs.

[0012] In some embodiments, the NKp46 agonist is an anti-NKp46 antibody. In some embodiments, the CD30 agonist is an anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are cultured in the presence of an NKp46 agonist and at least one cytokine. In some embodiments, at least one cytokine includes IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof. In some embodiments, CD56+ / CD3- cells are cultured in the presence of a CD30 agonist and at least one cytokine. In some embodiments, at least one cytokine includes IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

[0013] In some embodiments, CD56+ / CD3- cells further comprise (i) one or more chimeric antigen receptors (CARs), and / or (ii) one or more cytokines or at least one exogenous nucleic acid molecule encoding their mimetic.

[0014] In some embodiments, the cytokine or its mimetic is IL-15. In some embodiments, IL-15 is linked to IL-15Rα to form a fusion protein.

[0015] Some aspects of this disclosure relate to CD56+ / CD3- cells obtained through the methods disclosed herein.

[0016] Some aspects of this disclosure relate to pharmaceutical compositions comprising CD56+ / CD3- cells and excipients disclosed herein.

[0017] Some aspects of this disclosure relate to methods for treating diseases or conditions in subjects in need, comprising administering CD56+ / CD3- cells or pharmaceutical compositions disclosed herein to a subject. [Brief explanation of the drawing]

[0018] Brief explanation of the drawing / diagram [Figure 1] Figure 1 is a flow cytometry graph illustrating NKp46 expression in untransduced induced pluripotent stem cells (iPSCs) and iPSCs transduced with a construct expressing NKp46 via a CAG promoter.

[0019] [Figure 2] Figures 2A and 2B are graphical representations of flow cytometry results illustrating NKp46 expression in untransduced iPSCs and iNK cells differentiated from transduced iPSCs containing CAG-NKp46. Figure 2A illustrates NKp46 expression in transduced cells, and Figure 2B confirms the iNK phenotype based on CD56+ / CD3- staining results.

[0020] [Figure 3] Figure 3 is a graph showing the fold expansion rate of iNK cells differentiated from iPSCs transduced with CAG-NKp46 after activation via NKp46Ab (anti-NKp46 / anti-CD2).

[0021] [Figure 4] Figures 4A-4D are graphical representations of flow cytometry results showing NKp46 expression, CD56 expression (Figure 4A), CD3 expression (Figure 4B), NKG2D expression (Figure 4C), and CD16 expression (Figure 4D) in iNK cells differentiated from iPSCs transduced with CAG-NKp46.

[0022] [Figure 5] Figures 5A and 5B are graphs showing the proliferation rate (fold expansion) of iNK cells after CD30Ab-mediated activation. Figure 5A shows the proliferation rate (fold expansion) of iNK cells after the third proliferation cycle, and Figure 5B shows the proliferation rate (fold expansion) of iNK cells after the fourth proliferation cycle.

[0023] [Figure 6] Figure 6 is a graphical representation of the in vivo persistence of iNK cells, shown by the number of human CD45+ cells in peripheral blood weekly over a four-week period after iNK cell administration.

[0024] [Figure 7]Figures 7A and 7B show the results of bioluminescence imaging (BLI) evaluating tumor volume after administration of iNK-CD19 CAR cells. Figure 7A is a graphical representation illustrating the quantification of tumor volume as depicted by the total flux of BLI. CAR#1 is a CD19 CAR-iNK lineage cultured in the presence of anti-CD3 antibody, IL-7, and IL15, as described herein; CAR#2 is a CD19 CAR-iNK lineage IL15 cultured in the presence of anti-CD16 antibody, IL-2, and IL15, as described herein. Figure 7B is a series of photographs illustrating the tumor volume assessed by BLI.

[0025] Figure 7C shows the in vivo activity of CD19-mbIL15 CAR-iNKs in the Nalm6 / NSG mouse model. Figure 7C is a graph showing the percentage change in mouse body weight.

[0026] [Figure 8] Figure 8 is a graphical representation of the results of an in vitro repeated antigen stimulation assay, illustrating the tumor cell abundance, measured as total RFP-positive surface area per well image, after adding untransduced iNK cells and iNK-mesothelin (Meso)CAR cells to each well.

[0027] [Figure 9] Figures 9A and 9B show the results of BLI for evaluating tumor volume after administration of iNK-mesothelin CAR cells in vivo. Figure 9A is a graph illustrating the quantification of tumor volume as depicted by the total flux of BLI. Figure 9B is a series of photographs illustrating the tumor volume assessed by BLI.

[0028] [Figure 10]Figures 10A and 10B are graphical representations illustrating in vivo iNK-mesothelin CAR cell proliferation. Figure 10A shows the percentage of viable human CD45+ cells in mouse peripheral blood. Figure 10B shows the absolute number of human CD45+ cells per μL of mouse peripheral blood.

[0029] Detailed explanation Several aspects of this disclosure relate to a method for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of an NKp46 agonist, wherein the CD56+ / CD3- cells contain at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. In some aspects, the CD56+ / CD3- cells are derived from pluripotent stem cells. In some aspects, the method further comprises transducing pluripotent stem cells with an exogenous nucleic acid sequence encoding one or more NKp46 polypeptides.

[0030] Several aspects of the present disclosure relate to a method for producing modified CD56+ / CD3- cells, comprising: (a) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (b) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (c) culturing the CD56+ / CD3- cells in the presence of an NKp46 agonist.

[0031] Some aspects of this disclosure relate to methods for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of a CD30 agonist (e.g., an anti-CD30 antibody).

[0032] Some aspects of this disclosure relate to methods for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of a CD16 agonist (e.g., an anti-CD16 antibody). In some aspects, the method further comprises culturing the cells in IL-7.

[0033] Prior to a more detailed description of this disclosure, it should be understood that this disclosure is not limited to the specific compositions or process steps described, and that these are, of course, subject to change. As will be apparent to those skilled in the art when reading this disclosure, each of the individual embodiments described and explained herein has separate components and features that can be readily separated from or combined with features of any of the other embodiments without deviating from the scope or spirit of this disclosure. Any of the methods described may be performed in the order of the events described, or in any other order that is logically possible.

[0034] The headings provided herein may be defined by reference to the entire specification, rather than by any limitation of different aspects of the disclosure. It should also be understood that the terms used herein are for illustrative purposes only and not intended to be limiting.

[0035] I. Terminology To facilitate understanding of this disclosure, certain terms are defined first. Unless otherwise expressly provided herein, each of the following terms used in this application shall have the meanings set forth below. Additional definitions are provided throughout this application.

[0036] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer within the range described, and, where appropriate, fractions thereof (e.g., one-tenth or one-hundredth of an integer), unless otherwise specified.

[0037] Throughout this disclosure, the term “a” or “an” entity refers to one or more such entities; for example, “chimeric polypeptide” is understood to represent one or more chimeric polypeptides. Thus, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0038] Furthermore, as used herein, “and / or” should be treated as a specific disclosure for each of the two identified features or components, with or without the other. Thus, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In addition, “or” is used to mean an open list of components in a list. For example, “wherein X comprises A or B” means that X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other component.

[0039] The terms “approximately” or “essentially include” refer to a value or composition that is within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and will depend in part on how the value or composition is measured or determined, i.e., on the limitations of the measuring system. For example, “approximately” or “essentially include” may mean within or exceeding one standard deviation according to practice in the art. Alternatively, “approximately” or “essentially include” may mean a range of up to 10%. Furthermore, particularly in the context of biological systems or processes, the terms may mean up to one order of magnitude or up to five times the value. Where a particular value or composition is provided herein and in the claims, unless otherwise specified, the meaning of “approximately” or “essentially include” should be considered to be within an acceptable margin of error for the particular value or composition.

[0040] The terms "activated immune cells," "activated T cells," and "activated NK cells" refer to a variety of immune cells that are undergoing cell division, such as T cells and / or NK cells.

[0041] An "antigen" refers to any molecule, such as a peptide, that has the ability to trigger an immune response or to be bound by a TCR. An immune response may include antibody production, activation of specific immunologically qualified cells, or a combination thereof. Those skilled in the art will readily understand that virtually any macromolecule, including proteins or peptides, can serve as an antigen. Antigens can be expressed endogenously, i.e., by genomic DNA, or recombinantly. Antigens and / or epitopes can be specific to a particular tissue, such as cancer cells, or they can be expressed broadly. In addition, fragments of larger molecules can act as antigens. In some embodiments, an antigen is a hematopoietic stem cell (HSC) antigen.

[0042] As used herein, “antigen-presenting cell” or “APC” refers to a cell or cell-like antigen-presenting surface that expresses one or more antigens. In some embodiments, the antigens are displayed on the surface of the APC.

[0043] As used herein, the term “antigen-binding domain” refers to any protein having a binding domain homologous to an immunoglobulin-binding domain. “Antigen-binding domain” and “antibody” further include polypeptides that specifically bind to and recognize an antigen, and that include a framework region from an immunoglobulin protein or a portion thereof, comprising at least one CDR. The use of the term “antigen-binding domain” means including whole antibodies, polyclonal, monoclonal, and recombinant antibodies, their portions, and further including single-chain antibodies, humanized antibodies, mouse antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody constructs, e.g., scFv, (scFv)2, Fab, Fab′, and F(ab′)2, F(ab1)2, Fv, dAb, and Fd, disulfide-linked Fvs(dsFcs), and antibody-associated polypeptides.

[0044] As used herein, the term “bulk cell product” refers to the use of cells without selection / sorting by magnetic, fluorescence, chemical, or similar methods.

[0045] As used herein, the term “approximately” means a value that is similar to a given reference value when applied to one or more values ​​of interest. In certain embodiments, unless otherwise specified or evident from the context (except where such a number exceeds 100% of the possible values), the term “approximately” means a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the given reference value.

[0046] The term “autologous” refers to any material, such as immune cells, nucleic acid sequences, polypeptides, or other biological materials, that originates from the same source and is subsequently reintroduced. For example, autologous T-cell therapy involves administering T cells isolated from the same subject to the subject. The term “allogeneic” refers to any material originating from one individual that is then introduced into another individual of the same species. For example, allogeneic T-transplantation involves administering T cells obtained from a donor other than the subject to the subject.

[0047] As used herein, the term "culturing a cell population in the presence of a substance" refers, for example, to culturing a cell population in a medium containing the substance. Such cultivation may be in a medium containing the substance alone or in combination with other differentiation-inducing factors, etc. When the substance is added to the culture medium, it may be added directly to the medium or dissolved in a suitable solvent at the time of use and then added to the medium. The substance may also be immobilized on a culture substrate or carrier. As used herein, the terms "culturing a cell population in the presence of a substance" and "a cell population coming into contact with a substance" are used interchangeably.

[0048] The term “endogenous” refers to an agent, cell, gene, polypeptide, or part thereof that grows or develops within an organism. In this disclosure, endogenous HSCs are HSCs or hematopoietic stem cells that originate from or within a subject, or that are currently present within or within a subject.

[0049] The terms “exogenous” and “heterogeneous” are used interchangeably and refer to any material, such as immune cells, nucleic acid sequences, genes, polypeptides, or other biological substances that originate from another individual or species, or that are artificially produced, i.e., gene sequences, cells, systems, or anything unnatural to the target being introduced.

[0050] The term “wild-type” refers to the original, natural, unmutated, or uncut gene or protein sequence of an organism. “Cancer” refers to a broad group of diseases characterized by the unregulated growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. “Cancer” or “cancer tissue” may include tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other leukocyte malignancies. In some embodiments, the methods of the present invention are used for, for example, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, breast cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), Hodgkin's disease, and non-Hodgkin's disease. It can be used to reduce tumor size in tumors originating from cancers including lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system neoplasms (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers, or any combination thereof. Certain cancers may respond to chemotherapy or radiotherapy, or the cancer may be refractory. Refractory cancer refers to cancer that cannot be corrected by surgical intervention, and the cancer either initially responds to chemotherapy or radiation therapy, or becomes unresponsive over time.

[0051] Where an aspect is described herein using the word “including,” it is understood that similar aspects described using the terms “consisting of” and / or “essentially consisting of” are also provided. Where an aspect is described herein using the expression “including,” it is understood that similar aspects described using the expressions “consisting of” and / or “essentially consisting of” are also provided.

[0052] As used herein, “cytokines” refer to non-antibody proteins (or fusion proteins comprising (i) a non-antibody protein and (ii) a receptor, receptor subunit, or part of a receptor, etc.) released from one cell in response to contact with a specific antigen, and cytokines interact with a second cell to mediate a response in the second cell. Cytokines can be endogenously expressed by cells, added to cultured cells, administered to a subject, or any combination thereof. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, which propagate immune responses. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-21, and interferon (IFN)γ. Examples of inflammatory cytokines include, but are not limited to, IL-a, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0053] Chemokines are a type of cytokine that mediates cell chemotaxis, or directional migration. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein la (MIP-la, MIP-la), MIP-Ib (MIP-lb), γ-inducing protein 10 (IP-10), and thymic and activation-regulating chemokines (TARC or CCL17).

[0054] Other examples of cytokines include chemokine (CC motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemotactic protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, IL-23, granulocyte colony-stimulating factor (G-CSF), leukemia suppressor (LIF), oncostatin M (OSM), CD154, and phosphorus. Examples of cytokines include, but are not limited to, fotoxin (LT)β, 4-IBB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD30L, CD70, CD153, CD178, glucocorticoid-inducible TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF and ApoL-related leukocyte expression ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL). In some embodiments, cytokines include, but are not limited to, IL15 and IL-15Rα fusion proteins.

[0055] The term "mimetic" refers to any agent, composition, chemical substance, molecule, or any agent having the same function as another agent, composition, chemical substance, molecule, or analogous agent. In some aspects of this disclosure, a cytokine mimetic includes any agent having the same function as a particular cytokine.

[0056] eACT TMThe term "modified autologous cell therapy," abbreviated as "modified autologous cell therapy" and also known as adoptive cell transfer, is a process in which a patient's own immune cells, such as T cells and / or NK cells, are collected and then genetically modified to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. Immune cells, such as T cells and / or NK cells, can be modified to express, for example, a chimeric antigen receptor (CAR) or T cell receptor (TCR). CAR-positive (+) immune cells, such as T cells or other immune cells, are modified to express an extracellular single-chain variable fragment (scFv) that is specific to a particular tumor antigen and links to an intracellular signaling moiety containing a costimulatory domain and an activation domain. The costimulatory domain may be derived from, for example, CD28, and the activation domain may be derived from, for example, CD3-ζ (Figure 1). In certain embodiments, CARs are constructed to have two, three, four or more costimulatory domains. CAR scFv can be constructed to target, for example, CD19, a transmembrane protein expressed by cells in the B cell lineage, which includes, but is not limited to, all normal B cells, as well as NHL, CLL, and non-T cell ALL. In some embodiments, CAR scFV can conjugate mesothelin, a protein expressed in many solid tumors, including, but not limited to, mesothelioma, ovarian cancer, pancreatic adenocarcinoma, and lung and uterine cancer. Exemplary CAR+ T cell therapies and constructs are described in U.S. Patent Application Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, these references are incorporated by reference throughout.

[0057] "Immune response," as understood in the art, generally refers to the biological reaction within a vertebrate to foreign substances or abnormal cells, such as cancerous cells, which protect the organism from these substances and the diseases they cause. The immune response is mediated by the movement of one or more cells in the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver, thereby resulting in the selective targeting, binding to, damaging, destroying, and / or eliminating pathogens, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues that have invaded the body of an infected vertebrate. An immune response includes, for example, the activation or inhibition of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or any other cells of the immune system, such as NK cells. In some embodiments, an immune response refers to NK cell-mediated killing of foreign cells, such as allogeneic T cell therapy.

[0058] As used herein, the terms “introduce” or “transform” refer to the expression of a heterogeneous polynucleotide and / or polypeptide in a cell. In some embodiments, introduction is achieved by transfecting a cell with the polynucleotide of interest. In some embodiments, introduction is achieved by genetically modified cells that express a heterogeneous sequence, using gene editing tools including, but not limited to, CRISPR / Cas, CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas12a, CRISPR / Cpf1, zinc fingers, TALEN, Closver-Cas, or variants thereof. In some embodiments, introduction is achieved by contact between a cell and mRNA encoding the polypeptide of interest, such that the mRNA enters the cell or cell nucleus. In some embodiments, introduction includes transfecting or transducing a cell with a polynucleotide encoding a polypeptide.

[0059] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, NKT cells, or B cells. NK cells are a type of cytotoxic lymphocyte that is a major component of the innate immune system. NK cells reject virus-infected tumors and cells by inducing apoptosis or programmed cell death in target cells. NK cells are typically defined as CD3- / CD56+ / CD7+ cells. In certain embodiments, NK cells may also express CD16 (CD16+) and / or NKG2D (NKG2D+). In certain embodiments, the immune cells produced using the methods described herein are NK cells characterized as CD56+ / CD3- cells.

[0060] As defined herein, CD56+ / CD3- cells refer to cells that contain functional CD56 but do not contain functional CD3. In certain embodiments, CD56+ / CD3- cells may be NK cells. In another example, CD56+ / CD3- cells may be NK cell progenitors. CD56+ / CD3- cells can be prepared according to any method known in the art (see, for example, US2021 / 0292713 incorporated herein by reference).

[0061] As used herein, the terms “pluripotent stem cell,” “PS cell,” or “PSC” refer to cells that can self-regenerate by dividing and developing into the three main cell groups that make up the human body, including ectoderm, endoderm, and mesoderm cells. Because pluripotent stem cells can create cells from all three layers of the body, they can potentially produce any cells or tissues that the body needs repairing. This characteristic may be referred to as pluripotency.

[0062] As used herein, the terms “induced pluripotent stem cells” or “iPS cells” or “iPSCs” refer to cells that have been dedifferentiated (or reprogrammed) into a more naive, e.g., pluripotent state. Various methods of dedifferentiating cells are known, including, but not limited to, Oct3 / 4, Sox2, Klf4, and c-Myc (“Yamanaka factors”) which are overexpressed in cells (see, e.g., Takahashi and Yamanaka, Cell 126:663-76 (2006)). In some embodiments, iPS cells are pluripotent cells that have the ability to differentiate into a limited number of cell types. In some embodiments, iPS cells are totipotent cells that have the ability to differentiate into, for example, any cell type. In some embodiments, iPS cells can be redifferentiated into specific cell types, such as immune cells.

[0063] The term "CD3" refers to surface antigen classification 3, a multimeric protein complex known as the T3 complex, which serves as a T cell co-receptor and is a defining characteristic of T cells. The specificity of the CD3 antigen to T cells makes it a useful marker for T cells at all developmental stages. CD3 is also weakly expressed by some macrophages, including cells derived from the B cell lineage, but is not expressed in native NK cells.

[0064] The term "CD56" refers to a neuronal adhesion molecule (NCAM), a member of the immunoglobulin superfamily involved in both homophilic and heterophilic interactions. CD56 is a phenotypic marker for NK cells. Furthermore, deletion and phenotypic alterations of the CD56+ immune cell fraction correlate with increased susceptibility to various infections, autoimmune diseases, and malignant diseases.

[0065] The term "CD30," also known as "tumor necrosis factor receptor superfamily member 8" or "TNFRSF8," refers to a cell membrane protein of the tumor necrosis factor receptor family and a tumor marker. CD30 is normally expressed on activated T cells, NK cells, and B cells.

[0066] The term "NKp46," also known as CD335, refers to a receptor expressed by human NK cells belonging to the natural cytotoxicity receptor (NCR) family. The NKp46 polypeptide used in this invention is not limited to the wild-type protein, as long as it is activated by the NKp46 agonist described below. To determine whether such an NKp46 polypeptide can be activated by an NKp46 agonist, this can be detected by confirming that downstream molecules of NKp46 (e.g., PI3K and ERK1 / 2) are phosphorylated. In some embodiments, the NKp46 polypeptide contains an amino acid sequence identical to SEQ ID NO: 2, or an amino acid sequence that is at least about 75%, 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% identical to SEQ ID NO: 2.

[0067] Exemplary nucleic acid sequences encoding NKp46 may include the nucleic acid sequence described in SEQ ID NO: 1 (SEQ ID NO: 1; Table 1). In some embodiments, the polynucleotide comprises a nucleotide sequence having at least about 75%, 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% identical to the nucleic acid sequence described in SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding the NKp46 polypeptide comprises an amino acid sequence having 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% identical to the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding the NKp46 polypeptide comprises the amino acid sequence described in SEQ ID NO: 2.

[0068] Table 1. NKp46 sequences

[0069] [Table 1]

[0070] In some embodiments, vectors are provided that contain any polynucleotide encoding NKp46. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as an adenovirus vector, an adenovirus-associated virus (AAV) vector, a lentiviral vector, or a retroviral vector. In a non-limiting example, the vector may contain the polynucleotide of SEQ ID NO: 1. The vector may contain a promoter upstream of the portion encoding NKp46. An exemplary promoter used herein may be a CAG promoter. In some embodiments, the vector contains at least one (e.g., one or more) polynucleotides encoding NKp46. In such cases, the vector may contain one or more linker sequences, such as a cleavable peptide (e.g., E2A). For example, the E2A peptide may be located between two NKp46s.

[0071] In some embodiments, the vector comprises a polynucleotide encoding (i) one or more chimeric antigen receptors (CARs) and / or (ii) one or more cytokines or their mimetic. In some embodiments, the vector comprises a polynucleotide encoding multiple (e.g., two or more) CARs and / or cytokines or their mimetic. In such cases, the vector may contain one or more linker sequences, such as cleavable peptides (e.g., E2A). For example, the E2A peptide may be located between the CARs and / or cytokines.

[0072] The term "NKG2D" refers to a type C lectin-like receptor expressed on NK cells, γδ T cells, CD8+ T cells, and some autoreactive or immunosuppressive CD4+ T cells, and represents the primary recognition receptor for detection.

[0073] The term "CD16" (FcγRIIIa) refers to a type I transmembrane receptor containing two extracellular Ig-like domains that is expressed in NK cells. The function of CD16 is that of a low-affinity IgG receptor that mediates antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells.

[0074] The term "CD34" refers to E- and P-selectin ligands on human HSPCs that bind with comparable kinetics to other known selectin ligands. CD34 is routinely used to identify and isolate human hematopoietic stem cells / progenitor cells (HSPCs).

[0075] The term "CD2" refers to a glycoprotein that is a costimulatory receptor primarily expressed on T cells and NK cells.

[0076] As used herein, the term “primary NK cells” may refer to NK cells directly taken from a subject or may be the result of culturing NK cells directly isolated from a subject.

[0077] As used herein, the terms “iNK cells” or “induced NK cells” refer to NK cells differentiated from iPS cells. iNK cells express CD56. In further embodiments, such iNK cells may express both CD56 and CD16. In another example, such iNK cells may express CD56 and be CD3-(CD56+ / CD3-).

[0078] As used herein, “hematopoietic stem cells,” “HSCs,” “hematopoietic progenitor cells,” “HPCs,” or “HPC” refers to immature cells that can develop into all types of blood cells, including leukocytes, erythrocytes, and platelets. Hematopoietic stem cells are found in peripheral blood and bone marrow and are also called “blood stem cells.”

[0079] As used herein, “pharmaceutically acceptable carriers” include any and all aqueous solvents (e.g., water, alcohol solutions / aqueous solutions, physiological saline, sodium chloride, parenteral vehicles such as Ringer’s dextrose, etc.), non-aqueous solvents (e.g., injectable organic esters such as propylene glycol, polyethylene glycol, vegetable oil, and ethyl oleate), dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, pigments, liquids and effect supplements, and combinations thereof, as known to those skilled in the art. The pH and precise concentrations of various components in pharmaceutical compositions are prepared according to well-known parameters.

[0080] As used herein, the terms “recombinant” or “modified” cells are intended to refer to cells that may have recombinant expression vectors introduced into them, such as immune cells containing nucleic acids that are not naturally present in the cell. It will be understood that such terms are intended to refer not only to the cells of a particular subject but also to the offspring of such cells. Certain modifications may occur in subsequent generations due to either mutation or environmental influences, but such offspring are still included within the scope of the terms “recombinant” or “modified” as used herein.

[0081] As used herein, the terms “subject” and “patient” are used interchangeably and refer to either human or non-human beings, such as primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0082] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to a recombinant fusion protein having an antigen-specific extracellular domain that binds to an intracellular domain that instructs the cell to perform a specific function when an antigen binds to the extracellular domain.

[0083] As used herein, “suicide gene” or “suicide switch” refers to a gene that, when expressed by a cell, causes the cell to undergo apoptosis. Non-exclusive examples of suicide genes include the inducible caspase-9 suicide gene, viral thymidine kinase, cytosine deaminase, intracellular antibodies against antioxidant enzymes (AOEs), bacterial nitroreductase, other caspases, and DNases.

[0084] In some embodiments, the suicide gene includes the inducible caspase-9 suicide gene. The inducible caspase-9 (iCasp9) "safety switch" provides a solution that enables the removal of activated CART cells. Induction of iCasp9 is achieved by administration of the small molecule dimerizer drug AP1903, and dimerization leads to rapid induction of apoptosis in transduced cells, preferentially killing activated cells that express high levels of the transgene (see, e.g., Gargett, T, et al. Front. Pharmacol., 2014, October 28, 5:235).

[0085] In some embodiments, suicide genes include viral thymidine kinase (TK). Thymidine kinase is an ATP-thymidine 5'-phosphotransferase that converts deoxythymidine to deoxythymidine 5'-monophosphate, which is then phosphorylated to deoxythymidine diphosphate, and subsequently to deoxythymidine triphosphate by viral thymidine kinase and nucleoside diphosphate kinase, respectively. Deoxythymidine triphosphate is incorporated into DNA molecules synthesized by DNA polymerase. Some dNTP analogs, such as ganciclovir (GCV) and a synthetic analog of 2'-deoxyguanosine, have the ability to terminate DNA synthesis when incorporated into the DNA being synthesized. Termination of synthesis triggers the apoptotic signaling cascade. GCVs are not recognized by human thymidine kinases, but they are recognized as substrates for several viral thymidine kinases, such as herpes simplex virus-1 thymidine kinase (HSV-TK). As a result, human cells expressing HSV-TK convert GCVs to GCV phosphate, which is further phosphorylated and incorporated into synthesized DNA, leading to the termination of synthesis and apoptosis. While HSV-TK variants are not limited, one example is TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug;21(8):929-41).

[0086] In some embodiments, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine to uracil, accompanied by the release of ammonia. Under physiological conditions, the alteration site is recognized by an endonuclease, which then breaks the phosphate diester bond in the DNA and initiates repair by incorporating new cytosine. However, cytosine deaminase can also convert 5-fluorocytosine to 5-fluorouracil (5-FU). Therefore, upon offering 5-FC, a non-toxic prodrug, cytosine deaminase converts it to the highly toxic 5-FU (a suicide inhibitor of thymidylate synthetase), leading to inhibition of cell proliferation and apoptosis.

[0087] The term "inducible" refers to a reaction or state of activity that responds to the presence of an activator or initiator. A gene or enzyme can be inducible, meaning it is activated or expressed only in the presence of a specific molecule.

[0088] The terms “activator” or “initiator” refer to any agent that initiates or activates the activity or expression of a molecule, chemical, substrate, or gene, protein, or any other biological component.

[0089] The term "CAG promoter" refers to a hybrid nucleic acid construct consisting of a cytomegalovirus (CMV) enhancer fused with a chicken β-actin (CAG) promoter.

[0090] The term "promoter" refers to a DNA sequence to which a protein binds to initiate RNA transcription from downstream of the promoter's DNA.

[0091] An "NKp46 agonist" is an agent, whether chemical or biological, that activates, increases, or stimulates NKp46. NKp46 agonists can be used alone or in combination with two or more other NKp46 agonists. In some embodiments, an NKp46 agonist comprises an anti-NKp46 antibody that can activate, increase, or stimulate NKp46 (in this specification, such an anti-NKp46 antibody that can activate, increase, or stimulate NKp46 is sometimes referred to as an "anti-NKp46 antibody" or "anti-NKp46 agonist antibody"). Such anti-NKp46 antibodies may include, but are not limited to, Clone 9E2, 4k12, n1D9, B-N40, B-L46, 900, 2B11A3, 8F24 and 195314 (R&D systems, Biolegend, Miltenyi biotec, etc.), NK Cell Activation / Expansion Kit Human (Miltenyi biotec), and Cloudz Human NK Cell Expansion Kit (R&D systems). Such antibodies may be their functional fragments, such as Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv(scFv), diabody, triabody, F(ab)2, F(ab'2), F(ab'2), single-chain Fv(scFv), diabody, triabody, tetrabody and minibody. Antibodies may be derived from animals such as mice, rats, cattle, rabbits, goats, sheep, and guinea pigs. Antibody isotypes include, but are not limited to, IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM.

[0092] A “CD30 agonist” is an agent, whether a chemical or biological agent, that activates, increases, or stimulates CD30. CD30 agonists can be used alone or in combination with two or more other CD30 agonists. In some embodiments, a CD30 agonist contains an anti-CD30 antibody that activates, increases, or stimulates CD30 (i.e., an “anti-CD30 antibody” or “anti-CD30 agonist antibody”). Such an anti-CD30 antibody may, but is not limited to, Clone#81316(R&D systems). Such antibodies may be functional fragments such as Fd, Fv, Fab, F(ab′), F(ab)2, F(ab′)2, single-chain Fv(scFv), diabody, triabody, F(ab)2, F(ab′2), F(ab′2), single-chain Fv(scFv), diabody, triabody, tetrabody, and minibody. Antibodies may be derived from animals such as mice, rats, cattle, rabbits, goats, sheep, guinea pigs, etc. Antibody isotypes include, but are not limited to, IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM.

[0093] The antibody may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody, and may also be a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0094] Such antibodies can be produced by known methods, for example, by constructing an expression vector containing a nucleic acid encoding the antibody, culturing a transformant into which the nucleic acid has been introduced, or culturing a hybridoma that produces the antibody.

[0095] The term "caspase inhibitor" refers to any chemical or biological agent that inhibits caspases. Caspases can be regulated in several ways, including processing and activation control by molecules such as FADD, APAF-1, Bcl-2 family members, FLIP, and IAPs. Activated caspases can be regulated by a variety of inhibitors, including artificial caspase inhibitors developed for research and pharmaceutical purposes (Ekert, et al. Cell Death. Differ, Nov. 1999, 6(11):1081-6) and products of both viral and cellular genes.

[0096] As used herein, “culture medium” or “culture medium” refers to a nutrient solution that can be used to culture cells for an extended period of time, i.e., a liquid used in cell culture.

[0097] NK-related disorders may include any condition or disease associated with abnormal NK function. Such conditions or diseases may include, but are not limited to, cancer, pneumonia and viral infections including herpesviruses and other viruses such as HIV, asthma, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (ulcerative colitis and Crohn's disease), celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, psoriasis, scleroderma, Sjögren's syndrome, and multiple sclerosis.

[0098] As used herein, the terms “treating” a disease or condition or “treatment” a disease or condition mean performing a protocol that may include administering one or more therapies to a patient in order to alleviate the signs or symptoms of the disease. In some embodiments, treatment reduces the rate of disease progression, improves or mitigates a disease state, and / or promotes remission or improves the prognosis. Alleviation may occur before or after the signs or symptoms of the disease or condition appear. Thus, in some embodiments, “treating” or “treatment” includes “preventing” or “prevention” of a disease or undesirable condition. However, “treating” or “treatment” specifically includes protocols that do not require complete elimination of all signs and / or symptoms, do not require a cure, and have only marginal effects in the patient.

[0099] In various embodiments, subjects requiring it may be treated using populations of modified cells described herein to alleviate a disease or symptoms associated with a disease (e.g., cancer). In some embodiments, the modified cells are NK or iNK cells that are transduced with a CAR or TCR and / or modified to reduce the expression of endogenous MHC class I and MHC class II HLA genes in combination with, or by increasing the expression of, specific heterologous genes in the cells, so that when such cells are administered to a subject, the modified cells are less likely to be killed by the subject's immune system.

[0100] As used herein, the terms "ug," "uL," and "uM" are interchangeable with "μg," "μL," and "μM," respectively.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide general dictionaries of many of the terms used herein.

[0102] Units, prefixes, and symbols are expressed in the form recognized by their International System of Units (SI). Numerical ranges include the number defining the range. As described herein, any concentration range, percentage range, proportion range, or integer range should be understood to include, unless otherwise specified, any integer values ​​within the listed range and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer).

[0103] The abbreviations used herein are defined throughout this disclosure. Various aspects of this disclosure are described in further detail in the following subsections.

[0104] The various embodiments described herein are described in further detail in the following subsections.

[0105] II. Compositions of the Disclosure Some aspects of this disclosure relate to a method for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of an NKp46 agonist, wherein the CD56+ / CD3- cells comprise at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. Some aspects of this disclosure relate to a method for growing CD56+ / CD3- cells, comprising culturing CD56+ / CD3- cells in the presence of a CD30 agonist. In some aspects, the CD56+ / CD3- cells are derived from pluripotent stem cells.

[0106] Several aspects of this disclosure relate to a method for producing modified CD56+ / CD3- cells, comprising (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (C) culturing the CD56+ / CD3- cells in the presence of an NKp46 agonist and / or an aCD30 agonist. In some aspects, the HPCs are derived from one or more pluripotent stem cells.

[0107] II.A. Method of Disclosure II.A.1.NKp46 Agonist Several aspects of this disclosure relate to methods for producing and / or growing CD56+ / CD3- cells. In some aspects, the method comprises culturing CD56+ / CD3- cells in the presence of an NKp46 agonist. Any NKp46 agonist can be used in the methods disclosed herein. In some aspects, the NKp46 agonist is a polypeptide. In some aspects, the NKp46 agonist is an antibody or its antigen-binding portion that specifically binds to NKp46 ("anti-NKp46 antibody"). In some aspects, the NKp46 agonist comprises a small molecule.

[0108] In some embodiments, the NKp46 agonist comprises an anti-NKp46 antibody. Any anti-NKp46 antibody can be used in the methods described herein. In some embodiments, CD56+ / CD3- cells are contacted with anti-NKp46 antibody at a concentration of approximately 100 ng / ml to approximately 10 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-NKp46 antibody at a concentration of approximately 500 ng / ml to approximately 5 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-NKp46 antibody at concentrations of approximately 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, or 10 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-NKp46 antibody at approximately 1 μg / ml.

[0109] In some embodiments, CD56+ / CD3- cells are further contacted with at least one cytokine. In some embodiments, at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-7. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-12. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-15. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist and IL-21.

[0110] In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2 and IL-7. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2 and IL-12. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2 and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2 and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2 and IL-21. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist as well as IL-2, IL-10, and IL-15. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist as well as IL-2, IL-7, IL-12, IL-15, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist (preferably an anti-NKp46 agonist antibody) as well as IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0111] Several aspects of this disclosure relate to a method for producing iNK cells, comprising (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (C) culturing CD56+ / CD3- cells in the presence of an NKp46 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist for about 1 day. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist for about 2 days. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist for about 3 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 4 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 5 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 6 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 7 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 8 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 9 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 10 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 11 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 12 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 13 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 14 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 15 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 16 days.In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 17 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 18 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 20 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist for approximately 21 days.

[0112] In some embodiments, CD56+ / CD3- cells are (i) exposed to an NKp46 agonist, IL-2, IL-7, IL-15, and one or more IL-21, IL-12, and IL-18 during a first culture; then the CD56+ / CD3- cells are (ii) exposed to an NKp46 agonist, IL-2, IL-7, and IL-15 during a second culture. In some embodiments, the first culture is continued for about 1 day. In some embodiments, the first culture is continued for about 2 days. In some embodiments, the first culture is continued for about 3 days. In some embodiments, the first culture is continued for about 4 days. In some embodiments, the first culture is continued for about 5 days. In some embodiments, the first culture is continued for about 6 days. In some embodiments, the first culture is continued for about 7 days. In some embodiments, the first culture is continued for about 8 days. In some embodiments, the first culture is continued for approximately 9 days. In some embodiments, the first culture is continued for approximately 10 days. In some embodiments, the first culture is continued for approximately 11 days. In some embodiments, the first culture is continued for approximately 12 days. In some embodiments, the first culture is continued for approximately 13 days. In some embodiments, the first culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 1 day. In some embodiments, the second culture is continued for approximately 2 days. In some embodiments, the second culture is continued for approximately 3 days. In some embodiments, the second culture is continued for approximately 4 days. In some embodiments, the second culture is continued for approximately 5 days. In some embodiments, the second culture is continued for approximately 6 days. In some embodiments, the second culture is continued for approximately 7 days. In some embodiments, the second culture is continued for approximately 8 days. In some embodiments, the second culture is continued for approximately 9 days. In some embodiments, the second culture is continued for approximately 10 days. In some embodiments, the second culture is continued for approximately 11 days. In some embodiments, the second culture is continued for approximately 12 days. In some embodiments, the second culture is continued for approximately 13 days. In some embodiments, the second culture is continued for approximately 14 days.

[0113] In some embodiments, the second culture is repeated to increase the number of cells produced. In some embodiments, the second culture is performed twice. In some embodiments, the second culture is performed three times. In some embodiments, the second culture is performed four times. In some embodiments, the second culture is performed five times. In some embodiments, the second culture is performed six times. In some embodiments, the second culture is performed seven times. In some embodiments, the second culture is performed eight times. In some embodiments, the second culture is performed nine times. In some embodiments, the second culture is performed ten times. In some embodiments, the second culture is performed more than ten times.

[0114] II.A.2.CD30 Agonist In some embodiments, the method comprises culturing CD56+ / CD3- cells in the presence of a CD30 agonist. Any CD30 agonist can be used in the methods disclosed herein. In some embodiments, the CD30 agonist is a polypeptide. In some embodiments, the CD30 agonist is an antibody or its antigen-binding portion that specifically binds to CD30 ("anti-CD30 antibody"). In some embodiments, the CD30 agonist comprises a small molecule.

[0115] In some embodiments, the CD30 agonist comprises an anti-CD30 antibody. Any anti-CD30 antibody can be used in the methods disclosed herein. In some embodiments, CD56+ / CD3- cells are contacted with anti-CD30 antibody at a concentration of approximately 100 ng / ml to approximately 10 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-CD30 antibody at a concentration of approximately 250 ng / ml to approximately 2.5 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-CD30 antibody at concentrations of approximately 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, or 10 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with anti-CD30 antibody at approximately 200 ng / ml. In some embodiments, CD56+ / CD3- cells are contacted with approximately 225 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 250 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 275 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 300 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 325 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 350 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 375 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 400 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 500 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 600 ng / ml of anti-CD30 antibody.In some embodiments, CD56+ / CD3- cells are contacted with approximately 700 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 800 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 900 ng / ml of anti-CD30 antibody. In some embodiments, CD56+ / CD3- cells are contacted with approximately 1 μg / ml of anti-CD30 antibody.

[0116] In some embodiments, CD56+ / CD3- cells are further contacted with at least one cytokine. In some embodiments, at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2. In some embodiments, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-7. In some embodiments, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-12. In some embodiments, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-15. In some embodiments, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist and IL-21.

[0117] In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2 and IL-7. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2 and IL-12. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2 and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2 and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2 and IL-21. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2, IL-10, and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist as well as IL-2, IL-7, IL-12, IL-15, and IL-21. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD30 agonist (e.g., an anti-CD30 agonist antibody) as well as IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0118] Several aspects of this disclosure relate to a method for producing iNK cells, comprising (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (C) culturing CD56+ / CD3- cells in the presence of a CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 1 day. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 2 days. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 3 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 4 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 5 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 6 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 7 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 8 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 9 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 10 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 11 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 12 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 13 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 14 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 15 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 16 days.In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 17 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 18 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 20 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 21 days.

[0119] Several aspects of this disclosure relate to a method for producing iNK cells, comprising (A) providing hematopoietic progenitor cell (including HPC) ex vivo bulk cell products, and (B) differentiating the HPC to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (C) culturing CD56+ / CD3- cells in the presence of a CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 1 day. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 2 days. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 3 days. In some aspects, the CD56+ / CD3- cells are brought into contact with the CD30 agonist for about 4 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 5 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 6 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 7 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 8 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 9 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 10 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 11 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 12 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 13 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 14 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 15 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 16 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD30 agonist for approximately 17 days.In some embodiments, CD56+ / CD3- cells are in contact with a CD30 agonist for approximately 18 days. In some embodiments, CD56+ / CD3- cells are in contact with a CD30 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells are in contact with a CD30 agonist for approximately 20 days. In some embodiments, CD56+ / CD3- cells are in contact with a CD30 agonist for approximately 21 days.

[0120] In some embodiments, CD56+ / CD3- cells are (i) exposed to a CD30 agonist, IL-2, IL-7, IL-15, and one or more IL-21, IL-12, and IL-18 during a first culture; then the CD56+ / CD3- cells are (ii) exposed to a CD30 agonist, IL-2, IL-7, and IL-15 during a second culture. In some embodiments, the first culture is continued for about 1 day. In some embodiments, the first culture is continued for about 2 days. In some embodiments, the first culture is continued for about 3 days. In some embodiments, the first culture is continued for about 4 days. In some embodiments, the first culture is continued for about 5 days. In some embodiments, the first culture is continued for about 6 days. In some embodiments, the first culture is continued for about 7 days. In some embodiments, the first culture is continued for about 8 days. In some embodiments, the first culture is continued for approximately 9 days. In some embodiments, the first culture is continued for approximately 10 days. In some embodiments, the first culture is continued for approximately 11 days. In some embodiments, the first culture is continued for approximately 12 days. In some embodiments, the first culture is continued for approximately 13 days. In some embodiments, the first culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 1 day. In some embodiments, the second culture is continued for approximately 2 days. In some embodiments, the second culture is continued for approximately 3 days. In some embodiments, the second culture is continued for approximately 4 days. In some embodiments, the second culture is continued for approximately 5 days. In some embodiments, the second culture is continued for approximately 6 days. In some embodiments, the second culture is continued for approximately 7 days. In some embodiments, the second culture is continued for approximately 8 days. In some embodiments, the second culture is continued for approximately 9 days. In some embodiments, the second culture is continued for approximately 10 days. In some embodiments, the second culture is continued for approximately 11 days. In some embodiments, the second culture is continued for approximately 12 days. In some embodiments, the second culture is continued for approximately 13 days. In some embodiments, the second culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 15 days.

[0121] In some embodiments, the second culture is repeated to increase the number of cells produced. In some embodiments, the second culture is performed twice. In some embodiments, the second culture is performed three times. In some embodiments, the second culture is performed four times. In some embodiments, the second culture is performed five times. In some embodiments, the second culture is performed six times. In some embodiments, the second culture is performed seven times. In some embodiments, the second culture is performed eight times. In some embodiments, the second culture is performed nine times. In some embodiments, the second culture is performed ten times. In some embodiments, the second culture is performed more than ten times.

[0122] In some embodiments, the method comprises culturing CD56+ / CD3- cells in the presence of an NKp46 agonist and a CD30 agonist. In some embodiments, the NKp46 agonist comprises an anti-NKp46 antibody, and the CD30 agonist comprises an anti-CD30 antibody. Any anti-NKp46 antibody or anti-CD30 antibody may be used in the method disclosed herein. In some embodiments, CD56+ / CD3- cells are enlarged with the anti-NKp46 antibody. In some embodiments, the anti-NKp46 antibody is conjugated to a plate. In some embodiments, CD56+ / CD3- cells are contacted with approximately 100 ng / ml to approximately 10 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some embodiments, CD56+ / CD3- cells are contacted with approximately 500 ng / ml to approximately 5 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some embodiments, CD56+ / CD3- cells are contacted with an anti-NKp46 antibody and / or CD30 agonist at approximately 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, or 10 μg / ml. In some embodiments, CD56+ / CD3- cells are contacted with approximately 1 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some embodiments, CD56+ / CD3- cells are contacted with plate-bound NKp46 antibody and approximately 300 ng / ml of anti-CD30 antibody.

[0123] In some embodiments, CD56+ / CD3- cells are further contacted with at least one cytokine. In some embodiments, at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-2. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-7. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-12. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-21.

[0124] In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-2 and IL-7. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-2 and IL-12. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-2 and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-2 and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with an NKp46 agonist, a CD30 agonist, and IL-2 and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-2, IL-10, and IL-15. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-2, IL-7, IL-12, IL-15, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with an NKp46 agonist (preferably an anti-NKp46 agonist antibody), a CD30 agonist (preferably an anti-CD30 agonist antibody), and IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0125] Several aspects of this disclosure relate to a method for producing iNK cells, comprising (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) differentiating the HPCs to produce one or more CD56+ / CD3- cells. In some aspects, the method further comprises (C) culturing CD56+ / CD3- cells in the presence of an NKp46 agonist and a CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist and the CD30 agonist. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist and the CD30 agonist for about 1 day. In some aspects, the CD56+ / CD3- cells are brought into contact with the NKp46 agonist and the CD30 agonist for about 2 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 3 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 4 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 5 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 6 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 7 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 8 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 9 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 10 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 11 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 12 days.In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 13 days. In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 14 days. In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 15 days. In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 16 days. In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 17 days. In some embodiments, CD56+ / CD3- cells are exposed to NKp46 agonists and CD30 agonists for approximately 18 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 20 days. In some embodiments, CD56+ / CD3- cells are exposed to an NKp46 agonist and a CD30 agonist for approximately 21 days.

[0126] In some embodiments, CD56+ / CD3- cells are (i) exposed to an NKp46 agonist, a CD30 agonist, IL-2, IL-7, IL-15, and one or more IL-21, IL-12, and IL-18 during a first culture; then the CD56+ / CD3- cells are (ii) exposed to an NKp46 agonist, a CD30 agonist, IL-2, IL-7, and IL-15 during a second culture. In some embodiments, the first culture is continued for about 1 day. In some embodiments, the first culture is continued for about 2 days. In some embodiments, the first culture is continued for about 3 days. In some embodiments, the first culture is continued for about 4 days. In some embodiments, the first culture is continued for about 5 days. In some embodiments, the first culture is continued for about 6 days. In some embodiments, the first culture is continued for about 7 days. In some embodiments, the first culture is continued for approximately 8 days. In some embodiments, the first culture is continued for approximately 9 days. In some embodiments, the first culture is continued for approximately 10 days. In some embodiments, the first culture is continued for approximately 11 days. In some embodiments, the first culture is continued for approximately 12 days. In some embodiments, the first culture is continued for approximately 13 days. In some embodiments, the first culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 1 day. In some embodiments, the second culture is continued for approximately 2 days. In some embodiments, the second culture is continued for approximately 3 days. In some embodiments, the second culture is continued for approximately 4 days. In some embodiments, the second culture is continued for approximately 5 days. In some embodiments, the second culture is continued for approximately 6 days. In some embodiments, the second culture is continued for approximately 7 days. In some embodiments, the second culture is continued for approximately 8 days. In some embodiments, the second culture is continued for approximately 9 days. In some embodiments, the second culture is continued for approximately 10 days. In some embodiments, the second culture is continued for approximately 11 days. In some embodiments, the second culture is continued for approximately 12 days. In some embodiments, the second culture is continued for approximately 13 days. In some embodiments, the second culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 15 days.

[0127] In some embodiments, the second culture is repeated to increase the number of cells produced. In some embodiments, the second culture is performed twice. In some embodiments, the second culture is performed three times. In some embodiments, the second culture is performed four times. In some embodiments, the second culture is performed five times. In some embodiments, the second culture is performed six times. In some embodiments, the second culture is performed seven times. In some embodiments, the second culture is performed eight times. In some embodiments, the second culture is performed nine times. In some embodiments, the second culture is performed ten times. In some embodiments, the second culture is performed more than ten times.

[0128] IIA3 Culture Reagent In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 1 to approximately 40 ng / ml, approximately 1 to approximately 30 ng / ml, approximately 1 to approximately 20 ng / ml, or approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 1 to approximately 20 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to approximately 10 to 20 ng / ml of IL-2. In some embodiments, CD56+ / CD3- cells are exposed to approximately 5 to 15 ng / ml of IL-2.

[0129] In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-2 at approximately 10 ng / ml.

[0130] In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 1 to approximately 40 ng / ml, approximately 1 to approximately 30 ng / ml, approximately 1 to approximately 20 ng / ml, or approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 1 to approximately 20 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are brought into contact with IL-7 at a concentration of approximately 10 to 20 ng / ml. In some embodiments, CD56+ / CD3- cells are brought into contact with IL-7 at a concentration of approximately 5 to 15 ng / ml.

[0131] In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-7 at approximately 10 ng / ml.

[0132] In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at concentrations of approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at concentrations of approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 1 to approximately 40 ng / ml, approximately 1 to approximately 30 ng / ml, approximately 1 to approximately 20 ng / ml, or approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at concentrations of approximately 1 to approximately 20 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at concentrations of approximately 1 to approximately 10 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to approximately 10 to 20 ng / ml of IL-15. In some embodiments, CD56+ / CD3- cells are exposed to approximately 5 to 15 ng / ml of IL-15.

[0133] In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at approximately 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-15 at approximately 10 ng / ml.

[0134] In some embodiments, CD56+ / CD3- cells are exposed to IL-21 at concentrations of approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-21 at concentrations of approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 1 to approximately 40 ng / ml, approximately 1 to approximately 30 ng / ml, approximately 1 to approximately 20 ng / ml, or approximately 10 to approximately 20 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-21 at concentrations of approximately 10 to approximately 30 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-21 at concentrations of approximately 10 to approximately 20 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to approximately 20-30 ng / ml of IL-21. In some embodiments, CD56+ / CD3- cells are exposed to approximately 15-25 ng / ml of IL-21.

[0135] In some embodiments, CD56+ / CD3- cells are exposed to IL-21 at concentrations of approximately 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml. In some embodiments, CD56+ / CD3- cells are brought into contact with approximately 20 ng / ml of IL-21.

[0136] In some embodiments, CD56+ / CD3- cells are exposed to IL-12 at concentrations of approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-12 at concentrations of approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 10 to approximately 90 ng / ml, approximately 20 to approximately 80 ng / ml, approximately 30 to approximately 70 ng / ml, or approximately 40 to approximately 60 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-12 at concentrations of approximately 40 to approximately 60 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-12 at concentrations of approximately 40 to approximately 50 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to approximately 50–60 ng / ml of IL-12. In some embodiments, CD56+ / CD3- cells are exposed to approximately 45–55 ng / ml of IL-12.

[0137] In some embodiments, CD56+ / CD3- cells are exposed to IL-12 at concentrations of approximately 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml, 50 ng / ml, 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, 59 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 400 ng / ml. In some embodiments, CD56+ / CD3- cells are brought into contact with approximately 50 ng / ml of IL-12.

[0138] In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at concentrations of approximately 0.1 to approximately 100 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at concentrations of approximately 1 to approximately 100 ng / ml, approximately 1 to approximately 90 ng / ml, approximately 1 to approximately 80 ng / ml, approximately 1 to approximately 70 ng / ml, approximately 1 to approximately 60 ng / ml, approximately 1 to approximately 50 ng / ml, approximately 10 to approximately 90 ng / ml, approximately 20 to approximately 80 ng / ml, approximately 30 to approximately 70 ng / ml, or approximately 40 to approximately 60 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at concentrations of approximately 40 to approximately 60 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at concentrations of approximately 40 to approximately 50 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at approximately 50–60 ng / ml. In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at approximately 45–55 ng / ml.

[0139] In some embodiments, CD56+ / CD3- cells are exposed to IL-18 at concentrations of approximately 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml, 50 ng / ml, 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, 59 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 400 ng / ml. In some embodiments, CD56+ / CD3- cells are brought into contact with approximately 50 ng / ml of IL-18.

[0140] In some embodiments, CD56+ / CD3- cells are further contacted with a caspase inhibitor. Any caspase inhibitor can be used in the methods disclosed herein. In some embodiments, the caspase inhibitor comprises Z-VAD-FMK. In some embodiments, CD56+ / CD3- cells are contacted with about 1 to about 100 μM of Z-VAD-FMK. In some embodiments, CD56+ / CD3- cells are contacted with approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM of Z-VAD-FMK. In some embodiments, CD56+ / CD3- cells are contacted with approximately 10 μM of Z-VAD-FMK.

[0141] In some embodiments, CD56+ / CD3- cells are grown in a closed system.

[0142] In some embodiments, the method includes growing CD56+ / CD3- cells for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days.

[0143] In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 3 days, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 2 days, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 9 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 8 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 7 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 11 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 12 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 13 days. In some embodiments, CD56+ / CD3- cells undergo a first culture for approximately 4 days, followed by a second culture for approximately 14 days.

[0144] II.A.4. New Methods for Producing iNK Cells Some aspects of the present disclosure relate to methods for producing and / or expanding CD56+ / CD3− cells. In some aspects, the method includes culturing CD56+ / CD3− cells in the presence of a CD16 agonist. Any CD16 agonist can be used in the methods disclosed herein. In some aspects, the CD16 agonist is a polypeptide. In some aspects, the CD16 agonist is an antibody or an antigen-binding portion thereof that specifically binds CD16 (an “anti-CD16 antibody”). In some aspects, the CD16 agonist includes a small molecule.

[0145] In some aspects, the CD16 agonist includes an anti-CD16 antibody. Any anti-CD16 antibody can be used in the methods disclosed herein. In some aspects, the CD56+ / CD3− cells are contacted with a culture vessel coated with an anti-CD16 antibody. In some aspects, the culture vessel is coated with about 0.1 μg / cm 2 to about 1 μg / cm 2 of an anti-CD16 antibody. In some aspects, the culture vessel is coated with about 0.4 μg / cm 2 to about 0.8 μg / cm 2 of an anti-CD16 antibody. In some aspects, the culture vessel is coated with about 0.5 μg / cm 2 to about 0.7 μg / cm 2 of an anti-CD16 antibody. In some aspects, the culture vessel is coated with about 0.6 μg / cm 2 of an anti-CD16 antibody. In some aspects, the culture vessel is coated with about 0.1 μg / cm 2 , 0.2 μg / cm 2 , 0.3 μg / cm 2 , 0.4 μg / cm 2 , 0.5 μg / cm 2 , 0.6 μg / cm 2 , 0.7 μg / cm 2 , 0.8 μg / cm 2 , 0.9 μg / cm 2 , 1 μg / cm 2, 1.5 μg / cm³ 2 , or 2 μg / cm³ 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.3 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.4 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.5 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.6 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.7 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.8 μg / cm³ of antibody. 2 The culture vessels are coated with an anti-CD16 antibody. In some embodiments, the culture vessels are coated with approximately 0.9 μg / cm³ of antibody. 2 It is coated with an anti-CD16 antibody.

[0146] In some embodiments, CD56+ / CD3- cells are further contacted with at least one cytokine. In some embodiments, at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some embodiments, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-2. In some embodiments, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7. In some embodiments, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-15. In some embodiments, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-18. In some embodiments, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-21.

[0147] In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist and IL-7 and / or IL-2. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist and IL-7 and IL-15. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist and IL-7 and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist and IL-7 and IL-21. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist and IL-7, IL-15, and IL-18. In some embodiments, CD56+ / CD3- cells are brought into contact with a CD16 agonist (e.g., an anti-CD16 agonist antibody) and IL-7, IL-15, IL-18, and IL-21.

[0148] Several aspects of this disclosure relate to a method for producing iNK cells, comprising (A) providing an ex vivo bulk cell product comprising hematopoietic progenitor cells (HPCs), and (B) differentiating the HPCs to produce one or more CD4- cells. In some aspects, the method further comprises (C) transduction of NK cells. In some aspects, the steps of the method comprise (C) differentiating CD4- cells to produce one or more NK cells, activating and proliferating the NK cells, and transduction of NK cells with a gamma retrovirus. In some aspects, the steps of the method comprise (C) activating and proliferating the NK cells, and transduction of NK cells with a gamma retrovirus.

[0149] In some embodiments, the method includes culturing CD56+ / CD3- cells in the presence of a CD16 agonist. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 1 day. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 2 days. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 3 days. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 4 days. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 5 days. In some embodiments, the CD56+ / CD3- cells are brought into contact with the CD16 agonist for about 6 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 7 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 8 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 9 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 10 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 11 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 12 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 13 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 14 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 15 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 16 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 17 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 18 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells are exposed to a CD16 agonist for approximately 20 days.In some embodiments, CD56+ / CD3- cells are kept in contact with a CD16 agonist for approximately 21 days.

[0150] In some embodiments, CD56+ / CD3- cells are (i) exposed to the CD16 agonist, IL-7, IL-15, IL-18, and IL-21 during a first culture; then the CD56+ / CD3- cells are (ii) exposed to IL-7 and IL-15 during a second culture. In some embodiments, the first culture is continued for about 1 day. In some embodiments, the first culture is continued for about 2 days. In some embodiments, the first culture is continued for about 3 days. In some embodiments, the first culture is continued for about 4 days. In some embodiments, the first culture is continued for about 5 days. In some embodiments, the first culture is continued for about 6 days. In some embodiments, the first culture is continued for about 7 days. In some embodiments, the first culture is continued for about 8 days. In some embodiments, the first culture is continued for about 9 days. In some embodiments, the first culture is continued for about 10 days. In some embodiments, the first culture is continued for approximately 11 days. In some embodiments, the first culture is continued for approximately 12 days. In some embodiments, the first culture is continued for approximately 13 days. In some embodiments, the first culture is continued for approximately 14 days. In some embodiments, the second culture is continued for approximately 1 day. In some embodiments, the second culture is continued for approximately 2 days. In some embodiments, the second culture is continued for approximately 3 days. In some embodiments, the second culture is continued for approximately 4 days. In some embodiments, the second culture is continued for approximately 5 days. In some embodiments, the second culture is continued for approximately 6 days. In some embodiments, the second culture is continued for approximately 7 days. In some embodiments, the second culture is continued for approximately 8 days. In some embodiments, the second culture is continued for approximately 9 days. In some embodiments, the second culture is continued for approximately 10 days. In some embodiments, the second culture is continued for approximately 11 days. In some embodiments, the second culture is continued for approximately 12 days. In some embodiments, the second culture is continued for approximately 13 days. In some embodiments, the second culture is continued for approximately 14 days.

[0151] II.B. Cells in this Disclosure Any cell type may be used in the compositions and methods disclosed herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are pluripotent stem cells (PSCs). In some embodiments, the cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cells are embryonic stem cells (ESCs). In some embodiments, the cells are immune cells selected from T cells, NK cells, NKT cells, or tumor-infiltrating lymphocytes.

[0152] In some embodiments, the cells are cells differentiated from iPSCs. In some embodiments, the cells are immune cells differentiated from iPSCs. In some embodiments, the cells are CD56+ / CD3- cells differentiated from iPSCs. In some embodiments, the cells are NK cells differentiated from iPSCs. In some embodiments, the cells are NKT cells differentiated from iPSCs.

[0153] In some embodiments, the cells are CD56+ / CD3- cells differentiated from iPSCs, which are transduced with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. In some embodiments, the iPSC cells are transduced before the iPSC cells differentiate into CD56+ / CD3- cells. Thus, some embodiments of the present disclosure relate to a method for growing CD56+ / CD3- cells, wherein the cells contain at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides.

[0154] In some embodiments, CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding one or more chimeric antigen receptors (CARs). In some embodiments, CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding one or more cytokines or their mimetic. In some embodiments, CD56+ / CD3- cells further comprise (i) at least one exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR), and (ii) at least one exogenous nucleic acid molecule encoding a cytokine or its mimetic. In some embodiments, CD56+ / CD3- cells are differentiated from iPSCs, the iPSCs are transduced with (i) at least one exogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR), (ii) at least one exogenous nucleic acid molecule encoding a cytokine or its mimetic, (iii) a suicide switch, or (iv) any combination of (i)-(iii).

[0155] In some embodiments, at least one exogenous nucleic acid molecule encoding a cytokine or its mimetic encodes an IL-15 polypeptide. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, 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%, and at least about 99% identical to the amino acid sequence of human IL-15 (UniProt P40933). In some embodiments, the IL-15 polypeptide comprises human IL-15 (UniProt P40933). In some embodiments, at least one exogenous nucleic acid molecule encoding a cytokine or its mimetic encodes an IL-15 polypeptide mimetic. In some embodiments, at least one exogenous nucleic acid molecule encoding a cytokine or its mimetic encodes an IL-15 polypeptide comprising IL-15, which is linked with IL-15Rα to form a fusion protein (IL-15Ra / IL-15 complex) (e.g., the IL-15Ra / IL-15 complex disclosed in US2021 / 0292713 incorporated herein by reference).

[0156] II.C. Treatment method Several aspects of this disclosure relate to methods for treating diseases or conditions in subjects requiring such treatment, including administering compositions disclosed herein to subjects. In some aspects, the method includes administering modified or constructed cells disclosed herein. In some aspects, the method includes administering populations of cells disclosed herein. In some aspects, the method includes administering compositions comprising CD56+ / CD3- cells or iNK cells, or CD56+ / CD3- cells or iNK cells contained in a pharmaceutically acceptable carrier.

[0157] In some embodiments, the disease or condition includes NK-related diseases.

[0158] In some embodiments, disease or condition includes cancer, for example, the subject suffering from cancer. In some embodiments, cancer includes bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia This includes hematological cancers, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, central nervous system neoplasms (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some embodiments, the cancer progresses locally. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer recurs. In some embodiments, the cancer becomes refractory or recurs after one or more prior anticancer therapies. In some embodiments, this includes one or more prior standard therapies.

[0159] In some embodiments, the compositions disclosed herein are administered in combination with additional anticancer therapies. In some embodiments, the additional anticancer therapy includes chemotherapy, immunotherapy, radiotherapy, surgery, or a combination thereof. In some embodiments, the additional anticancer therapy includes chemotherapy. In some embodiments, the additional anticancer therapy includes immune checkpoint inhibitors. In some embodiments, the additional anticancer therapy includes PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, GITR antagonists, or any combination thereof. In some embodiments, the anticancer therapy includes an antibody or its antigen-binding moiety that specifically binds to and inhibits PD-1. In some embodiments, the anticancer therapy includes an antibody or its antigen-binding moiety that specifically binds to and inhibits PD-L1.

[0160] In some embodiments, the method further includes pre-treatment of the subject before administering a population of immune cells. In some embodiments, the subject is subjected to chemotherapy before administering a population of immune cells. In some embodiments, the subject is subjected to immunodepletion chemotherapy before administering a population of immune cells. In some embodiments, immunodepletion chemotherapy includes cyclophosphamide, fludarabine, or both.

[0161] In some embodiments, the method comprises administering (i) a population of proliferated cells and (ii) a cytokine to a target. In some embodiments, the cytokine includes IL-2, its analogues, its variants, or fragments thereof.

[0162] In some embodiments, the cells of this disclosure are at least about 1 x 10 6 Cells, at least about 2 x 10 6 Cells, at least approximately 3 x 10 6 Cells, at least about 4 x 10 6 Cells, at least about 5 x 10 6 Cells, 1 x 10⁷ cells, at least approximately 2 x 10⁷ 7 Cells, at least approximately 3 x 10 7 Cells, at least about 4 x 10 7 Cells, at least about 5 x 10 7 cells, 1x10 8 Cells, at least about 2 x 10 8 Cells, at least approximately 3 x 10 8 Cells, at least about 4 x 10 8 Cells, at least about 5 x 10 8 cells, 1x10 9 Cells, at least about 2 x 10 9 Cells, at least approximately 3 x 10 9 Cells, at least about 4 x 10 9 Cells, or at least about 5 x 10 9 It is administered to the target in cellular doses.

[0163] The cells of this disclosure are low in toxicity and can therefore be safely administered by any convenient method, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodulately, intramedullarily, intramuscularly, intravenously or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0164] In certain embodiments of the present invention, the method of the present invention in a clinical context is combined with other agents that are effective in treating diseases or conditions, including the above-mentioned NK-related diseases.

[0165] II.D. Cell Therapy Products Some aspects of this disclosure relate to a population of cells, including many of the modified cells disclosed herein. Some aspects of this disclosure relate to a cell therapy, including a population of modified cells disclosed herein. In some aspects, the population of cells is cryopreserved. Any method of cryopreserving cells, such as immune cells, can be used in the methods and compositions disclosed herein. In some aspects, the cells are cryopreserved in the presence of DMSO. In some aspects, the cell therapy is cryopreserved to facilitate the shipment of cells.

[0166] The cell therapies and / or cell populations disclosed herein can be further formulated with one or more excipients. Any excipient capable of preserving cells can be used in the methods and compositions disclosed herein. In some embodiments, the cell therapies and / or cell populations are formulated with one or more excipients that enable cryopreservation of cells, such as DMSO.

[0167] In some embodiments, the population of cells includes many of the modified cells disclosed herein and one or more additional cells. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 10% of the cells in the population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 20% of the cells in the population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 25% of the cells in the population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 30% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 40% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 50% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 60% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 65% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 70% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 75% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 80% of the cells in a population of cells include many of the modified cells disclosed herein. In some embodiments, at least about 90% of the cells in a population of cells include many of the modified cells disclosed herein.

[0168] The practices described herein, unless otherwise noted, utilize conventional techniques within the scope of the art, including cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. Such techniques are well described in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DN Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. US Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986)); Crooke, Antisense drug See Technology: Principles, Strategies and Applications, 2nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0169] All references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

[0170] The following examples are for illustrative purposes only and are not limiting. [Examples]

[0171] Example 1 Generation of induced pluripotent stem cells transduced with NKp46 To generate induced pluripotent stem cells (iPSCs) transduced with NKp46, iPSCs, strain Ff101s04, were 1x10 in 24-well plates coated with iMatrix511 in StemFitAK03N medium containing 50 μM Y-27632, a selective p160 ROCK inhibitor. 4Cells were seeded in wells. The following day, the culture medium was replaced with StemFit AK03N, and a lentiviral vector expressing the NKp46 gene (SEQ ID NO: 1) under the control of the CAG promoter was used to infect iPSCs to produce FfI01s04 / CAG-NKp46 cells. The grown FfI01s04 / CAG-NKp46 cells were stained with an anti-NKp46 antibody conjugated with anti-allophycocyanin (APC, BioLegend), and the number of NKp46-expressing cells was analyzed by flow cytometry to quantify the number of cells. The number of NKp46-expressing cells in untransduced iPSCs and iPSCs transduced with CAG-NKp46 is shown in Figure 1.

[0172] Example 2 Differentiation of iNK cells from iPSCs transduced with NKp46 To differentiate iPSCs transduced with NKp46 into variant natural killer (iNK) cells, FfI01s04 / NKp46 cells were dispersed in StemFit AK03N medium and, on day 0, 5x10⁶ cells were placed in a 6-well plate treated with ultra-low adhesion under hypoxic (5% O2) conditions. 5Cells were seeded in wells. StemFit AK03N medium contained the GSK-3 inhibitors 10μMCHIR99021 and 10μM Y-27632. On the following day (day 1), the cells were dispersed in human pluripotent stem cell (HPC) induction medium (Invitrogen Bone Morphogenesis Protein 4 (BMP4) (50 ng / ml, R&D systems), Vascular Endothelial Growth Factor (VEGF, 50 ng / ml, R&D systems), Basic Fibroblast Growth Factor (bFGF, 50 ng / ml, Wako), and Ascorbic Acid 2-phosphate (50 mg / ml, Sigma Aldrich)). The HPC induction medium contained StemPro34 supplemented with L-glutamine (4 mM, Sigma Aldrich), penicillin (100 U / ml, Sigma Aldrich), streptomycin (100 mg / ml, Sigma Aldrich), human insulin (10 mg / ml, Invitrogen), human transferrin (5.5 mg / ml, Invitrogen), sodium selenite (6.7 ng / ml, Invitrogen), Glutamax (1x, Invitrogen), and α-monothioglycerol (0.4 mM, Invitrogen). On day 2, the activin receptor-like kinase receptor SB431542 inhibitor (6 μM in the medium, FUJIFILM Wako Pure Chemical Corporation) was added directly to the culture medium, and the cells were cultured for 2 days. On day 4, the cells were dispersed in a different HPC induction medium containing VEGF (50 ng / ml), bFGF (50 ng / ml), SCF (50 ng / ml, R&D systems), and ascorbic acid 2-phosphate (50 mg / ml), and cultured for a further 3 days. On day 7, the cells were exposed to a different HPC induction medium containing VEGF (50 ng / ml), bFGF (50 ng / ml), SCF (50 ng / ml), ascorbic acid 2-phosphate (50 mg / ml), TPO (30 ng / ml, Peprotech), and Flt3L (10 ng / ml, Peprotech), and cultured in this medium for a further 7 days. The medium was changed every 2-3 days during the 7-day culture period.

[0173] On day 14, the cells were harvested and CD34-positive cells were isolated using CD34-microbeads (Miltenyi Biotech), resulting in 1.16 x 10⁻⁶ cells. 5 Cells were seeded in 10cm dishes using a cell / dish method. Each 10cm dish was coated with rh-DLL4 / Fc chimera (Sino Biological) and RetroNectin (Takara Bio Inc.). The culture medium was changed every 2-3 days during the culture period. MEMα medium (Gibco, Thermo Fisher Scientific) supplemented with 15% fetal bovine serum (FBS, Hyclone), 1x Glutamax, 4 mM L-glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin, 55 μM 2-mercaptoethanol, 50 mg / ml ascorbic acid 2-phosphate, 10 mg / ml human insulin, 5.5 mg / ml human transferrin, 6.7 ng / ml sodium selenite, 50 ng / ml SCF, 50 ng / ml IL-7 (Peprotech), 50 ng / ml Flt3L, 100 ng / ml TPO, 15 μM SB203580 (Tocris), and 30 nM stroma cell-derived factor-1α (SDF-1α, Peprotech) was used as the culture medium for these cells. On day 21, the cells were harvested and placed in a new 15cm dish coated with hDLL4 / RetroNectin in a 1x10 ratio. 6 Cells were seeded in a cell / dish, and all cells were harvested on day 35.

[0174] On day 35, the harvested cells were placed in 5x10 48-well plates in MEMα medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mg / ml ascorbic acid 2-phosphate, 10 mg / ml human insulin, 5.5 mg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2 (Peprotech), and 10 ng / ml IL-7. 5Cells were seeded per well. On day 39, half of the medium in each well was changed, and on day 42, all cells were harvested as differentiated iNK cells. The iNK cells were stained with the antibodies listed in Table 2, and the number of cells expressing NKp46 and the number of CD56+ / CD3- phenotypic cells were analyzed by flow cytometry as shown in Figures 2A and 2B, respectively. The results show that cells expressing NKp46 account for a significant percentage of the cell population, and that CD56+ / CD3- phenotypes comprise approximately 83.2% of the cell population.

[0175] Table 2. Staining used to detect peptides

[0176] [Table 2]

[0177] Example 3 iNK cell proliferation iNK cells were placed in 5x10⁶ 96-well plates. 4Cells were seeded in wells and supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-21 (Peprotech), 50 ng / ml IL-12 (Peprotech), 50 ng / ml IL-18 (MBL), 300 ng / ml anti-CD30 antibody (R&D systems), and 10 μM Z-VAD-FMK (caspase inhibitor, R&D systems) in Iscove's Modified Dulbecco's. Cells were grown in IMDM medium (IMDM) using beads (Miltenyi) conjugated with anti-NKp46 / CD2 antibody. After 4 days of culture, cells were cultured in IMDM medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, and 10 ng / ml IL-15 (1x10⁻¹ 6 The cells were transferred to G-Rex24 at a cell / well ratio. The culture medium was changed every 2-3 days during a further 10 days of culture. The recurrent proliferation of iNK cells was plotted as shown in Figure 3. iNK cells redifferentiated from iPSCs that had not been transduced with NKp46 were proliferated using the same method. As shown in Table 3, the proliferation rate of iNK cells was low under these conditions.

[0178] The proliferated iNK cells were stained with the antibodies shown in Table 4, and the expression of NKG2D, CD56, CD16, and CD3 was analyzed by flow cytometry as shown in Figures 4A-4D. As shown in Figure 4A, NKp46+ and CD56+ cells comprised approximately 98.3% of the cell population. As shown in Figure 4B, NKp46+ and CD3- cells comprised approximately 97.2% of the cell population. As shown in Figure 4C, NKp46+ and NKG2D+ cells comprised approximately 52% of the cell population. As shown in Figure 4D, NKp46+ and CD16+ cells comprised approximately 50.9% of the cell population.

[0179] Table 3. Fold change (iNK cells differentiated from iPSCs that have not been transduced with NKp46)

[0180] [Table 3]

[0181] Table 4. Staining used to detect peptides

[0182] [Table 4]

[0183] Example 4 The effect of anti-CD30 antibodies on iNK cell proliferation On day 1, 13-day iNK cells, which had been cryopreserved and recovered after two proliferation cycles according to the method of Example 3, were cultured for 1 day in Iscove's Modified Dulbecco's Media Media (IMDM) supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, and 10 ng / ml IL-15 (Peprotech). The iNK cells were cultured in 2 x 10⁶ T25 flasks. 6Cells were seeded in wells, and on day 0, in or without 300 ng / ml anti-CD30 antibody (81316, R&D systems), 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-21 (Peprotech), 50 ng / ml IL-12 (Peprotech), 50 ng / ml IL-18 (MBL), and 10 μM Z-VAD-FMK (caspase inhibitor, R&D The cells were grown in IMDM supplemented with (systems) using plate-bound anti-NKp46 antibody (monoclonal mouse IgG2B Clone#195314, R&D systems).

[0184] After 3 days of culture, cells were transferred to IMDM medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, and 10 ng / ml IL-15 in 5x10 cells. 6 The cells were transferred to G-Rex6 in wells. The culture medium was changed every 2-3 days during a further 11 days of incubation, and proliferation was examined on day 14 (third proliferation). Repeat proliferation was examined using the same protocol as in Example 4 from day 0 to day 14 (fourth proliferation).

[0185] Figures 5A and 5B show the proliferation of iNK cells in the presence and absence of anti-CD30 antibody. As shown in Table 5, the proliferation rate of iNK cells without anti-CD30 antibody was lower compared to those with anti-CD30 antibody.

[0186] Table 5. Change in fold ratio during the 3rd and 4th propagation cycles.

[0187] [Table 5]

[0188] Example 5 The effect of anti-CD30 antibodies on the in vivo persistence of iNK cells. On day 0 of the fourth growth cycle, iNK cells harvested from day 14 of the third growth cycle were placed in a T75 flask at a rate of 1 x 10⁶ 7 Cells were seeded in wells and treated with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2 (Peprotech), 10 ng / ml IL-7 (Peprotech), 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-21 (Peprotech), 50 ng / ml IL-12 (Peprotech), 50 ng / ml IL-18 (MBL), and 10 μM Z-VAD-FMK (caspase inhibitor, R&D) in or without 300 ng / ml anti-CD30 antibody (81316, R&D Systems). The antibody was activated with anti-NKp46 antibody (R&D systems) conjugated to a plate in Iscove's Modified Dulbecco's Media (IMDM) supplemented with (R&D systems).

[0189] After 3 days of culture, cells were cultured in IMDM medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 6.7 ng / ml sodium selenite, 10 ng / ml IL-2, 10 ng / ml IL-7, 10 ng / ml IL-15 (Peprotech), 20 ng / ml IL-21 (Peprotech), 50 ng / ml IL-12 (Peprotech), and 50 ng / ml IL-18 (MBL), either in the presence or absence of 300 ng / ml anti-CD30 antibody (81316, R&D systems), in 5x10 cells. 6 The cells were transferred to G-Rex6 in a well-by-well format.

[0190] On day 5, after activation, the cells were divided into 2x10 7 Cells were transferred to G-Rex6 in cells / well and harvested on day 7. These cells were cryopreserved in CS10 (BioLife Solutions) and thawed immediately before intravenous administration to 8-week-old NOG mice (0.5 or 1x10). 7 Cells / mouse, N=3). Peripheral blood was collected weekly after administration, fixed with FACS lysing solution (BD), and stained with mouse CD45-PE antibody (BioLegend) and human CD45-BV510 antibody (BioLegend). The number of mouse CD45-negative and human CD45-positive cells was counted as in vivo sustained iNK cells by flow cytometry. As shown in Figure 6, iNK cells proliferated with anti-CD30 antibody showed higher in vivo persistence.

[0191] Example 6 Differentiation of iPS cells into HP cell bulk cells The iPS cell line, QHJI cells, were derived from peripheral blood mononuclear cells of healthy individuals. On day 0, QHJI cells dispersed in StemFit complete medium were placed in 6x10⁶ 6-well plates under hypoxic (5% O₂) conditions for ultra-low adhesion treatment. 5Cells were seeded in cells / well. StemFit complete medium contained 10 μM CHIR99021 and 50 μM Y-27632. On day 1, QHJI cells were dispersed in hematopoietic progenitor cell (HPC) differentiation medium containing BMP4 (50 ng / mL), VEGF (50 ng / mL), bFGF (50 ng / mL), and ascorbic acid 2-phosphate (50 μg / mL). HPC induction medium contained StemPro34 supplemented with human insulin (10 μg / mL), human transferrin (5.5 μg / mL), sodium selenite (6.7 ng / mL), L-glutamine (2 mM), and α-monothioglycerol (0.4 mM).

[0192] On day 2, SB431542 (6 μM in the medium) was added to the culture medium (i.e., HPC differentiation medium containing cells), and the cells were cultured for 2 days. On day 4, the cells were redispersed into another medium containing VEGF (50 ng / mL), bFGF (50 ng / mL), SCF (50 ng / mL), and ascorbic acid 2-phosphate (50 μg / mL), and cultured for a further 3 days. On day 7, the cells were exposed to another medium containing VEGF (50 ng / mL), bFGF (50 ng / mL), SCF (50 ng / mL), ascorbic acid 2-phosphate (50 μg / mL), TPO (30 ng / mL), and Flt3L (10 ng / mL), and cultured in this medium for a further 7 days. The medium was changed every 2-3 days during this 7-day culture period.

[0193] Example 7 Differentiation of HP cell bulk into a population containing CD4- cells On day 14, the cell population obtained from Example 6 ("HP cell bulk") was divided into 3.12 x 10⁻¹⁰ cells without any cell separation. 6Cells were seeded in 15cm dishes and cultured at 37°C under 5% O2. Note that various seeding densities may be used, and the seeding densities described above are examples only. Each 15cm dish was coated with rh-DLL4 / Fc chimera (Sino Biological) and RetroNectin (Takara Bio Inc). The culture medium was changed every 2-3 days during this culture period. MEMα (Thermo Fisher Scientific (Gibco)) supplemented with 15% FBS, 4 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 55 μM 2-mercaptoethanol, 50 μg / mL ascorbic acid 2-phosphate, 10 μg / mL human insulin, 5.5 μg / mL human transferrin, 6.7 ng / mL sodium selenite, 50 ng / mL SCF, 50 ng / mL IL-7, 50 ng / mL Flt3L, 100 ng / mL TPO, 15 μM SB203580, and 30 nM SDF-1α was used as the culture medium for these cells. On day 21, the cells were subcultured into fresh 15 cm dishes newly coated with hDLL4 / RetroNectin. On day 28, the cells were further subculturised in new 15cm dishes newly coated with hDLL4 / RetroNectin. On day 35, all other cells, including CD4- cells, were harvested.

[0194] Example 8 Transduction of NK cells A. Differentiation of bulk NK cells into bulk cells On day 35, the cell population containing CD4- cells, i.e., the cells obtained from Example 7 without any cell separation, was divided into 1x10⁻¹⁴ cells. 6Cells were seeded in 48-well plates at a rate of one cell / well and cultured for 3 days at 37°C under 5% CO2. MEMα medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / mL penicillin, 100 ng / mL streptomycin, 50 μg / mL ascorbic acid 2-phosphate, 10 μg / mL human insulin, 5.5 μg / mL human transferrin, 6.7 ng / mL sodium selenite, 500 ng / mL anti-CD3 antibody (UCHT1, R&D Systems), 10 ng / mL IL-2, and 10 ng / mL IL-7 was used as the culture medium.

[0195] On day 38, the cells were dispersed in a separate MEMα medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / mL penicillin, 100 ng / mL streptomycin, 50 μg / mL ascorbic acid 2-phosphate, 10 μg / mL human insulin, 5.5 μg / mL human transferrin, 6.7 ng / mL sodium selenite, 10 ng / mL IL-2, and 10 ng / mL IL-7, and this medium was used as the culture medium. On day 42, all cells, including NK cells ("NK cell bulk"), were harvested.

[0196] Activation and proliferation of B.NK cells On day 41, the tissue culture vessels were treated with retronectin (0.15 ug / cm³) at 4°C for 16 hours. 2 ) and anti-CD3 antibody (UCHT1, R&D Systems) (0.6 μg / cm2) or anti-CD16 antibody (3G8, Biogems) (0.6 μg / cm2) 2On day 42, NK cells recovered from Example 8A were coated with either ) or ). 5 The cells were resuspended at a concentration of cells / mL and transferred to anti-CD3 coated containers for 3 days of culture.

[0197] On day 45, cells were harvested and transferred to a container coated with gamma retrovirus for viral transduction as described in Example 8C below, or dispersed in fresh IMDM medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / mL penicillin, 100 ng / mL streptomycin, 50 μg / mL ascorbic acid 2-phosphate, 10 μg / mL human insulin, 5.5 μg / mL human transferrin, 6.7 ng / mL sodium selenite, 10 ng / mL IL-7 or 10 ng / mL IL-2, and 10 ng / mL IL-15. These cells were then transferred to G-Rex containers (Wilson Wolf) for a further 11 days of culture. IL-7 was used with an anti-CD3 antibody, and IL-2 was used with an anti-CD16 antibody. 50-80% fresh medium changes were performed every 2-3 days with the same medium composition until the cell concentration reached 4x10⁶. 7 cells / cm 2 If the number of cells exceeded a certain limit, they were divided into additional G-Rex containers. On day 56, the cells were harvested for final use.

[0198] If necessary to obtain a sufficient number of cells, the 15-day process (days 41-56) described herein was repeated multiple times to increase the total number of NK cells produced. For iNK-CD19 CAR cells, the cells underwent five activation rounds (5 x 15-day process), with CAR transduction occurring during the second activation. For iNK-MesoCAR cells, the cells underwent four activation rounds (4 x 15-day process), with CAR transduction occurring during the third activation.

[0199] Gamma-retroviral transduction of C.NK cells To induce gamma retroviral transduction of NK cells, on day 44, the tissue culture vessels were treated with retronectin (10.5 μg / cm³) at 4°C for 16 hours. 2 The cells were coated with ). On day 45, the gamma retrovirus was incubated at 32°C for 2 hours on a retronectin-coated container and then harvested. On day 45, the NK cells described in Example 8B were centrifuged at 32°C for 5 minutes in a 300xg retronectin / gamma retrovirus-coated container and cultured overnight. If necessary to achieve a higher percentage of transduced cells, the steps described on days 44-45 were repeated twice to perform gamma retrovirus transduction. On day 46 (or day 47 if gamma retrovirus transduction was performed twice), cells were collected and dispersed in IMDM medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / mL penicillin, 100 ng / mL streptomycin, 50 μg / mL ascorbic acid 2-phosphate, 10 μg / mL human insulin, 5.5 μg / mL human transferrin, 6.7 ng / mL sodium selenite, 10 ng / mL IL-7 or 10 ng / mL IL-2, and 10 ng / mL IL-15, and transferred to G-Rex containers (Wilson Wolf). Next, 75% of the medium was changed with the same medium composition every 2-3 days until the cell concentration reached 4 x 10⁶. 7 cells / cm 2 If the number of cells exceeded a certain limit, they were divided into additional G-Rex containers. On day 56, the cells were harvested for final use.

[0200] If necessary, the 15-day process (days 41-56) described in Example 8B was repeated multiple times to increase the total number of NK cells produced. For iNK-CD19 CAR cells, the cells underwent five activation rounds (same process as 5 x 15-day process - days 41-56), with CAR and IL-15Ra / IL-15 gene transduction performed in the second activation. For iNK-Meso CAR cells, the cells underwent four activation rounds (same process as 4 x 15-day process - days 41-56), with CAR and IL-15Ra / IL-15 gene transduction performed in the third activation.

[0201] Table 6 shows the different culture conditions and the CAR+ and total cell counts based on each condition. These cells were used in the in vivo study in Example 10.

[0202] Table 6. Culture status

[0203] [Table 6]

[0204] Example 9 CAR-NK cell preparation NK cells were further modified to express one or more CARs. NK cells were modified by: (1) synthesizing an anti-CD19 CAR gene or an anti-mesothelin CAR gene and an IL-15Ra / IL-15 gene; (2) preparing a retroviral vector containing an anti-CD19 CAR gene or an anti-mesothelin CAR gene and an IL-15Ra / IL-15 gene; and (3) transducing NK cells with a retroviral vector containing an anti-CD19 CAR gene or an anti-mesothelin CAR gene and an IL-15Ra / IL-15 gene.

[0205] CAR / IL15-NK cell preparation The anti-CD19 CAR gene was prepared by synthesizing an oligopeptide designed to be positioned from the N-terminus, as shown in Table 6.

[0206] Table 7. Oligopeptides

[0207] [Table 7]

[0208] The anti-CD19 CAR was constructed in accordance with US2021 / 0292713, which is incorporated herein by reference in its entirety. The anti-mesothelin CAR was constructed in accordance with WO2023 / 009700, which is incorporated herein by reference in its entirety.

[0209] Example 10 In vivo antitumor activity of iNK-CD19 CAR Luciferase-expressing Nalm6 cells (ATCC; cancer cells) (5x10 5 The cells were transplanted via the tail vein into NOD / Shi-scid, IL-2Rγnull mice ("NSG mice"). Female NSG mice aged 8-9 weeks were obtained from Jackson Laboratory. On day 4, four days after Nalm6 cell transplantation, iNK-CD19 CAR cells (10x10) dispersed in 0.2 ml of PBS were transplanted. 6 Nalm6-planted NSG mice were administered either iNK-CD19 CAR cells or 0.2 ml of PBS without cells via the tail vein. After administration of iNK-CD19 CAR cells or PBS, luciferin was administered to the mice via the tail vein. Luciferase activity was measured for 42 days using the IVIS imaging system (PerkinElmer).

[0210] Figures 7A and 7B demonstrate the antitumor efficacy of iNK-CD19 CAR cells produced by (1) PBS treatment (vehicle control), (2) cell culture and anti-CD3 antibody activation ("iNK-CD19 CAR#1") as described in Examples 6-8, and (3) cell culture methods as described in Examples 6-8, excluding Example 8A, and anti-CD-16 antibody activation ("iNK-CD19 CAR#2"). Detectable tumor growth was observed in the PBS vehicle-treated group 14 days after tumor cell injection and gradually increased from that point, whereas significant tumor cell proliferation was not observed in either iNK-CD19 CAR group until day 28. Figure 7C depicts the percentage change in mouse body weight throughout this study. While the PBS control group showed significant (>20%) weight loss by day 28, the iNK-CD19 CAR#1 group did not reach this endpoint until day 42, and the iNK-CD19 CAR#2 group never reached this level of weight loss. Collectively, these data demonstrate the ability of iNK-CD19 CAR cells to reduce the proliferation of Nalm6 tumor cells in vivo.

[0211] Example 11 In vitro repeated killing capacity of iNK mesothelin CAR cells Repeated antigen stimulation (RAS) assays were performed using red fluorescent protein (RFP)-expressing GSU tumor cells expressing high levels of mesothelin protein, and iNK-mesothelin CAR cells (iNK-Meso CAR cells). GSU-RFP cells were placed in 96-well plates at a rate of 1 x 10⁶ cells per well. 5 Cells were seeded and cultured overnight. The next day, (1) no cells, (2) 1 x 10 5 (UTD)iNK that has not been transduced, or (3) 1x10 5 iNK-Meso CAR cells were added to each well. The RFP-positive surface area of ​​each well was measured every 4 hours using an Incucyte.

[0212] To repeatedly challenge iNK cells, an additional 1x10 5GSU-GFP cells were added to each well every 2-3 days. This step is referred to as "challenge" in this specification. A total of four challenges were performed.

[0213] Figure 8 depicts the growth of GSU tumor cells, measured via the total RFP-positive surface area per well image throughout the study. When wells became overconfluent, GSU ​​cells continued to grow in a tumor-only state for approximately 120 hours. In comparison, UTD and iNK-Meso CAR cells showed a significantly reduced rate of tumor cell proliferation. While iNK-Meso CAR cells regulated tumor cell proliferation with two challenges, UTD iNK cells completely inhibited tumor growth with one challenge. These results demonstrate that UTD iNK cells possess some innate tumor-modulating ability, while CAR transduction further enhances the ability of iNK cells to kill tumor cells.

[0214] Example 12 In vitro antitumor activity of iNK-MesoCAR Luciferase-expressing GSU cells (1x10) 6 Cells were transplanted into NOD / Shi-scid, IL-2Rγnull mice ("NSG mice") via intraperitoneal injection. Female, 8-9 week old NSG mice were obtained from Jackson Laboratory.

[0215] Four days after Nalm6 cell transplantation, iNK-Meso CAR cells (20x10) dispersed in 0.2 ml of PBS were used. 6 0.2 ml of PBS (either iNK-Meso cells or no cells) was administered to GSU-planted NSG mice via the tail vein. Following administration of iNK-Meso cells or PBS, luciferin was administered to the mice via intraperitoneal injection. Luciferase activity was measured for 31 days using the IVIS imaging system (PerkinElmer). Table 8 below shows the two study groups: PBS only and iNK-Meso CAR cells.

[0216] Figures 9A and 9B show tumor growth measured by luciferase activity in mice treated with (1) PBS (vehicle control) or (2) iNK-Meso CAR cells. Treatment with iNK-Meso CAR cells significantly slowed tumor growth compared to the PBS control, demonstrating that iNK-Meso CAR cells possess antitumor activity.

[0217] In addition, peripheral blood samples were collected to evaluate iNK-Meso CAR cell proliferation in vivo by flow cytometry of human CD45, which is present only in injected iNK-Meso CAR cells. Figures 10A and 10B show the time-course growth dynamics of iNK-Meso CAR cells, indicated by the percentage of whole-cell human CD45+ cells and the absolute number of human CD45+ cells per μL of mouse peripheral blood. Some iNK-Meso CAR cells were detectable at the earliest point in time evaluated, day 4. However, the levels of iNK-Meso CAR cells continued to increase, eventually peaking at day 11. The increase in detectable iNK cells from day 4 to day 11 indicates that iNK-Meso CAR cells can proliferate in an in vivo mouse model system.

[0218] [Table 8]

[0219] It should be understood that the detailed description section, rather than the summary and abstract, is intended to be used to interpret the claims. The summary and abstract section may describe one or more exemplary embodiments, though not all, of the disclosure intended by the inventor(s), and is therefore not intended to limit the scope of the disclosure and the appended claims in any way.

[0220] The foregoing descriptions of specific embodiments are intended to fully illustrate the general nature of the disclosure, and by applying knowledge within the scope of skill in the art, others can easily modify and / or adapt such specific embodiments for various uses without excessive experimentation and without departing from the general concepts of the disclosure. Therefore, such adaptations and modifications are intended to be equivalent in meaning and scope to the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that the language or terminology used herein is for illustrative purposes only and not limiting, and therefore, the language or terminology used herein should be interpreted by those skilled in the art in light of the teachings and guidance.

[0221] The scope and breadth of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims and their equivalents.

[0222] The content of all cited references throughout this application (including documents, U.S. or foreign patents or patent applications, and websites) is thus expressly incorporated by reference, as if the entirety of those references were included herein for any purpose. In the event of any inconsistency, the content disclosed herein literally shall prevail.

[0223] While various specific embodiments are described and presented, the above specification is not limiting. It will be understood that various modifications are possible without departing from the spirit and scope of the invention. Many variations will become apparent to those skilled in the art through the examination of this specification.

Claims

1. A method for increasing CD56+ / CD3- cells: A method comprising culturing the CD56+ / CD3- cells in the presence of an NKp46 agonist, wherein the CD56+ / CD3- cells contain at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides.

2. The method according to claim 1, wherein the CD56+ / CD3- cells are derived from pluripotent stem cells.

3. The method according to claim 2, further comprising transducing the pluripotent stem cells with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides.

4. The method according to claim 2 or 3, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

5. The method according to any one of claims 1 to 4, wherein the NKp46 agonist is an anti-NKp46 antibody.

6. The method according to any one of claims 1 to 5, wherein the CD56+ / CD3- cells are cultured in the presence of an NKp46 agonist and at least one cytokine.

7. The method according to claim 6, wherein the at least one cytokine includes interleukin-(IL)2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

8. The method according to any one of claims 1 to 7, wherein the CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding (i) one or more chimeric antigen receptors (CARs), and / or (ii) one or more cytokines or their mimetic counterparts.

9. The method according to claim 8, wherein the cytokine or the mimetic thereof is IL-15.

10. The method according to claim 9, wherein IL-15 is linked to IL-15Rα to form a fusion protein.

11. A method for producing modified CD56+ / CD3- cells, which is: (A) To provide an ex vivo bulk cell product containing hematopoietic progenitor cells (HPCs) comprising at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) Differentiating the HPC to produce one or more CD56+ / CD3- cells. Methods that include...

12. (C) Culturing the CD56+ / CD3- cells in the presence of an NKp46 agonist. The method according to claim 11, further comprising:

13. The method according to claim 11 or 12, wherein the HPC is derived from one or more pluripotent stem cells.

14. The method according to claim 13, further comprising transducing the one or more pluripotent stem cells with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides.

15. The method according to claim 13 or 14, wherein the pluripotent stem cells are iPSCs.

16. The method according to any one of claims 11 to 15, wherein the NKp46 agonist is an anti-NKp46 antibody.

17. The method according to any one of claims 11 to 16, wherein the CD56+ / CD3- cells are cultured in the presence of the NKp46 agonist and at least one cytokine.

18. The method according to claim 17, wherein the at least one cytokine includes IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

19. The method according to any one of claims 11 to 18, wherein the CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding (i) one or more chimeric antigen receptors (CARs), and / or (ii) one or more cytokines or their mimetic counterparts.

20. The method according to claim 19, wherein the cytokine or the mimetic thereof is IL-15.

21. The method according to claim 20, wherein IL-15 is linked to IL-15Rα to form a fusion protein.

22. The method according to any one of claims 1 to 21, further comprising culturing the CD56+ / CD3- cells in the presence of a CD30 agonist.

23. The method according to claim 22, wherein the CD30 agonist is an anti-CD30 antibody.

24. CD56+ / CD3- cells obtained by the method described in any one of claims 1 to 23.

25. A pharmaceutical composition comprising the CD56+ / CD3- cells and excipients according to claim 24.

26. A method for treating a disease or condition in a subject requiring such treatment, comprising administering CD56+ / CD3- cells as described in claim 24 or the pharmaceutical composition as described in claim 25 to the subject.

27. A method for growing CD56+ / CD3- cells, comprising culturing the CD56+ / CD3- cells in the presence of a CD30 agonist.

28. The method according to claim 27, wherein the CD56+ / CD3- cells are derived from pluripotent stem cells.

29. The method according to claim 28, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

30. The method according to any one of claims 27 to 29, wherein the CD30 agonist is an anti-CD30 antibody.

31. The method according to any one of claims 27 to 30, wherein the CD56+ / CD3- cells are cultured in the presence of the CD30 agonist and at least one cytokine.

32. The method according to claim 31, wherein the at least one cytokine includes IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, or any combination thereof.

33. CD56+ / CD3- cells obtained by the method described in any one of claims 27 to 32.

34. A pharmaceutical composition comprising the CD56+ / CD3- cells and excipients described in claim 33.

35. A method for treating a disease or condition in a subject requiring such treatment, comprising administering to a subject the CD56+ / CD3- cells described in claim 33 or the pharmaceutical composition described in claim 34.

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