Method for expanding CD56+ / CD3-cells

Abstract

The present disclosure relates to a method of expanding or producing CD56 + / CD3-cells, said method comprising contacting said CD56 + / CD3-cells with an NKp46 agonist and / or a CD30 agonist.

Description

Cross-references to related applications

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

[0002] Reference to the sequence listing submitted electronically The contents of the sequence list submitted electronically (name: 3817_188PC02_Seqlisting_ST26; size: 4,409 bytes; and creation date: April 19, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to CD56 + / CD3 - Cell expansion. Background Technology

[0004] Natural killer (NK) cells hold therapeutic potential for a variety of human malignancies, including adoptive immunotherapy for cancer treatment and other indications. NK cells do not require a primary immunization phase and are not HLA-restricted, thus representing the potential for a “readily available” and effective anti-tumor response. Induced pluripotent stem cell (iPSC)-derived NK (iNK) cells have been included in several clinical trials for the treatment of hematologic malignancies and solid tumors. Obtaining sufficient cell numbers remains a major obstacle to NK cell immunotherapy due to poor in vitro expansion, limited in vivo lifespan, and the fact that they constitute only a small fraction of human peripheral blood mononuclear cells (PBMCs). Therefore, there is still a need to improve the methods for expanding NK and iNK cells. Summary of the Invention

[0005] Some aspects of this disclosure relate to a method for expanding CD56+ / CD3- cells, the method comprising: culturing the 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 expanding CD56+ / CD3- cells, the method comprising: culturing the CD56+ / CD3- cells in the presence of a CD30 agonist.

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

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

[0008] In some respects, 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 mimics thereof.

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

[0010] Some aspects of this disclosure relate to a method for generating modified CD56+ / CD3- cells, the method comprising: (A) providing an in vitro host cell for generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to generate 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 a CD30 agonist.

[0011] In some aspects, HPCs are derived from one or more pluripotent stem cells. In some aspects, the method further includes transducing one or more pluripotent stem cells with at least one exogenous nucleic acid molecule encoding one or more NKp46 peptides. In some aspects, the pluripotent stem cells are iPSCs.

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

[0013] In some respects, 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 mimics thereof.

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

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

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

[0017] Some aspects of this disclosure relate to methods for treating a disease or ailment in a subject in need, said methods including administering to the subject the CD56+ / CD3- cells disclosed herein or the pharmaceutical compositions disclosed herein. Attached Figure Description

[0018] Figure 1 The image is a graphical representation of the flow cytometry results, illustrating the expression of NKp46 in untransduced induced pluripotent stem cells (iPSCs) and iPSCs transduced with a construct expressing NKp46 via the CAG promoter.

[0019] Figure 2A and Figure 2B The image is a graphical representation of the flow cytometry results, illustrating the expression of NKp46 in iNK cells differentiated from iPSCs that were not transduced with iPSCs and those transduced with CAG-NKp46. Figure 2A Examples of NKp46 expression in transduced cells, and Figure 2B via CD56 + / CD3 -The staining results confirmed the iNK phenotype.

[0020] Figure 3 Graphical representation of the fold expansion of iNK cells differentiated from CAG-NKp46 transduced iPSCs after NKp46 Ab-mediated (anti-NKp46 / anti-CD2) activation.

[0021] Figures 4A to 4D To indicate the expression of NKp46 and CD56 in iNK cells differentiated from iPSCs transduced by CAG-NKp46. Figure 4A CD3 expression Figure 4B ), NKG2D expression ( Figure 4C ) and CD16 expression ( Figure 4D Graphical representation of the flow cytometry results.

[0022] Figure 5A and Figure 5B Graphical representation of the fold expansion of activated iNK cells mediated by CD30 Ab. Figure 5A The data shows the fold expansion of iNK cells after the third expansion, and... Figure 5B The fold expansion of iNK cells after the fourth expansion is shown.

[0023] Figure 6 A graphical representation of the in vivo persistence of iNK cells is shown by the number of human CD45+ cells in peripheral blood each week for four weeks following iNK cell administration.

[0024] Figure 7A and Figure 7B A graphic illustration of the results of bioluminescence imaging (BLI) for assessing tumor burden after administration of iNK-CD19 CAR cells. Figure 7A For example, a graphical representation of the quantification of tumor burden by total BLI throughput is shown. CAR #1 is a CD19 CAR-iNK line cultured in the presence of anti-CD3 antibody, IL-7, and IL15 as described herein; and CAR #2 is a CD19 CAR-iNK line cultured in the presence of anti-CD16 antibody, IL-2, and IL15 as described herein. Figure 7B Here is a series of photographs illustrating tumor burden as assessed by BLI.

[0025] Figure 7C The in vivo activity of CD19-mbIL15 CAR-iNK in the Nalm6 / NSG mouse model was demonstrated. Figure 7C A graphical representation of the change (percentage change) in mouse body weight.

[0026] Figure 8The graphic representation of the results of the in vitro repeated antigen stimulation assay is illustrated by the total RFP-positive surface area of ​​each image per well after adding untransduced iNK cells and iNK-mesothelin (Meso) CAR cells to each well.

[0027] Figure 9A and Figure 9B A graphic representation of BLI results for assessing tumor burden after in vivo administration of iNK-mesothelin CAR cells. Figure 9A For example, a graphical representation of the quantification of tumor burden using total BLI throughput is shown. Figure 9B A series of photographs illustrating the tumor burden assessed by BLI.

[0028] Figure 10A and Figure 10B The following is a graphical representation of the proliferation of iNK-mesothelin CAR cells in vivo. Figure 10A The percentage of live cells that are human CD45+ cells in mouse peripheral blood is shown. Figure 10B The absolute number of human CD45+ cells per μL of mouse peripheral blood is shown. Detailed Implementation

[0029] Some aspects of this disclosure relate to methods for expanding CD56+ / CD3- cells, the methods comprising: culturing the 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. In some aspects, the CD56+ / CD3- cells are derived from pluripotent stem cells. In some aspects, the method further comprises transducing the pluripotent stem cells with an exogenous nucleic acid sequence encoding one or more NKp46 polypeptides.

[0030] Some aspects of this disclosure relate to methods for generating modified CD56+ / CD3- cells, the methods comprising: (a) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (b) differentiating the HPCs to generate 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 expanding CD56+ / CD3- cells, the methods 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 expanding CD56+ / CD3- cells, the methods comprising culturing CD56+ / CD3- cells in the presence of a CD16 agonist (e.g., an anti-CD16 antibody). In some aspects, the methods further comprise culturing cells in IL-7.

[0033] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific compositions or process steps described, and therefore is naturally subject to variation. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and characteristics that can be readily separated from or combined with characteristics of any of the other aspects without departing from the scope or spirit of this disclosure. Any of the enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0034] The headings provided herein are not intended to limit any aspect of this disclosure, which can be defined by referring to the entire specification. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be restrictive.

[0035] I. Terminology To facilitate understanding of this invention, certain terms are first defined. As used herein, unless expressly specified otherwise, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0036] Unless otherwise indicated, as described herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range listed, and, where appropriate, to include fractions (such as one-tenth and one-hundredth of an integer).

[0037] Throughout this disclosure, the term "a / an" entity refers to one or more of the entities; for example, "chimeric polypeptide" should be understood to mean one or more chimeric polypeptides. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0038] Furthermore, the term “and / or” as used herein should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: 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). Additionally, “or” is used to indicate an open list of components. For example, “where X contains A or B” means that X contains A, X contains B, X contains A and B, or X contains A or B and any other component.

[0039] The terms “about” or “consistently of…” refer to a value or composition within an acceptable error range as determined by a person skilled in the art, which will depend in part on the manner in which the value or composition is measured or determined (i.e., the limitations of the measurement system). For example, “about” or “consistently of…” may mean within or greater than one standard deviation according to practice in the art. Alternatively, “about” or “consistently of…” may mean a range up to 10%. Furthermore, particularly for biological systems or processes, the term may mean a value up to an order of magnitude or up to five times. When a specific value or composition is provided in the application and claims, unless otherwise indicated, the meaning of “about” or “consistently of…” should be assumed to be within an acceptable error range of said specific value or composition.

[0040] The terms “activated immune cells,” “activated T cells,” and “activated NK cells” specifically refer to immune cells that are undergoing cell division, such as T cells and / or NK cells.

[0041] An "antigen" is any molecule, such as a peptide, that elicits an immune response or can be bound by a TCR. This immune response may involve antibody production, activation of specific immune-competent cells, or a combination thereof. Those skilled in the art will readily understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Antigens can be expressed endogenously, i.e., through genomic DNA, or can be recombinantly expressed. Antigens and / or antigenic determinants may be specific to a particular tissue (such as cancer cells), or may be widely expressed. Additionally, fragments of larger molecules can act as antigens. In some aspects, the 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 respects, antigens are displayed on the surface of an 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 comprising a framework region derived from an immunoglobulin or a portion thereof that specifically binds to and recognizes an antigen and contains at least one CDR. The use of the term "antigen-binding domain" is intended to include complete antibodies, polyclonal, monoclonal, and recombinant antibodies, portions thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-individual genotype antibodies, antibody constructs such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb, and Fd, disulfide-linked Fv (dsFc), and antibody-associated polypeptides.

[0044] As used in this article, the term "dominant cell generation" refers to the use of cells without the need for cell selection / sorting by magnetic, fluorescent, chemical-based methods, etc.

[0045] As used herein, the term “approximately” when applied to one or more values ​​of interest means a value similar to the stated reference value. In some respects, unless otherwise stated or apparent from the context (other than such a number exceeds 100% of the possible values), the term “approximately” means a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in any direction (greater or less).

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

[0047] As used herein, the term "culturing a cell population in the presence of a substance" means, for example, culturing a cell population in a culture medium containing the substance. Such culturing can be, for example, in a culture medium containing the substance alone or in coexistence with other differentiation-inducing factors. When the substance is added to the culture medium, it can be added directly to the medium, or it can be added to the medium after being dissolved in a suitable solvent at the time of use. The substance can also be immobilized on a matrix or carrier surface for culturing. As used herein, the terms "culturing a cell population in the presence of a substance" and "contacting a cell population with a substance" are used interchangeably.

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

[0049] The terms “exogenous” and “heterogeneous” are used interchangeably and refer to any material derived from another individual or species or artificially created (i.e., not natural to the introduced gene sequence, cell, system, or subject), such as immune cells, nucleic acid sequences, genes, peptides, or other biological substances.

[0050] The term "wildtype" refers to the original, natural, non-mutated, or non-truncated gene or protein sequence of an organism. "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells within the body. Unregulated cell division and growth can lead to the formation of malignant tumors or cells that invade adjacent tissues and can also metastasize to distant sites of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors. Examples of cancers treatable by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other white blood cell malignancies. In some embodiments, the method of the present invention can be used to reduce the size of tumors derived from, for example, cancers, including 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, gastric 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. Diseases, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system 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, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) sarcoma, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including those induced by asbestos), or any combination thereof. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancers are those that cannot be corrected by surgical intervention and that initially do not respond to chemotherapy or radiation therapy or become unresponsive over time.

[0051] It should be understood that where aspects are described in this document using the language "contains", similar aspects described as "composed of" and / or "substantially composed of" are also provided.

[0052] As used herein, a “cytokine” is a non-antibody protein (or a fusion protein comprising (i) a non-antibody protein and (ii) a receptor, a receptor subunit, or a portion thereof) released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by cells, added to cells in a culture, administered to a subject, or any combination thereof. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines (including interleukin (IL) 7 and IL-15) promote immune cell survival and proliferation, and pro-inflammatory cytokines 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 pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, 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 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 class of cytokines that mediate cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eosinophil activation chemokine, eosinophil activation chemokine-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1a (MIP-1a, MIP-1a), MIP-Ib (MIP-1b), γ-induced protein 10 (IP-10), and thymus activation-regulated chemokines (TARC or CCL17).

[0054] Other examples of cytokines include, but are not limited to, chemokine (CC motif) ligands (CCL1), 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 factor (LIF), oncosin M (OSM), CD154, lymphotoxin (LT) β, 4-IBB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD30L, CD70, CD153, CD178, glucocorticoid-induced TNFR-associated ligand (GITRL), and tumor necrosis factor superfamily member 14. (TNFSF14), OX40L, TNF and ApoL-associated leukocyte expression ligand 1 (TALL-1), or TNF-associated apoptosis-inducing ligand (TRAIL). In some embodiments, the cytokines include, but are not limited to, fusion proteins of IL15 and IL-15Rα.

[0055] The term "analyte" refers to any agent that has the same function as another agent, compound, chemical, molecule, etc. In some aspects of this disclosure, cytokine analogs include any agent that has the same function as a specific cytokine.

[0056] The term "engineered autologous cell therapy," abbreviated as "eACT™," also known as adoptive cell transfer, is the process of collecting a patient's own immune cells (e.g., T cells and / or NK cells) and subsequently genetically altering them 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 (e.g., T cells and / or NK cells) may be engineered to express, for example, chimeric antigen receptors (CARs) or T cell receptors (TCRs). CAR-positive (+) immune cells (e.g., T cells or immune cells) are engineered to express extracellular single-chain variable fragments (scFvs) specific to a specific tumor antigen linked to an intracellular signaling motif comprising a co-stimulatory domain and an activation domain. The co-stimulatory domain may be derived from, for example, CD28, and the activation domain may be derived from, for example, CD3-ζ ( Figure 1In some embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. The CAR scFv may be designed to target, for example, CD19, a transmembrane protein expressed by cells in a B-cell lineage, including all normal B cells and B-cell malignancies, including but not limited to NHL, CLL, and non-T-cell ALL. In some aspects, the CAR scFV may bind mesothelin. Mesothelin is a protein expressed in many solid tumors, including but not limited to mesothelioma, ovarian cancer, pancreatic adenocarcinoma, and lung and uterine malignancies. Examples of CAR+ T-cell therapies and constructs are described in U.S. Patent Publications Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in their entirety.

[0057] "Immune response," as understood in the art, and generally refers to a biological response in a vertebrate body against a foreign agent or abnormality (e.g., cancer cells), which protects the organism from damage caused by these agents and the diseases they cause. An immune response is mediated by the action of one or more cells of 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 any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues in a vertebrate body. Immune responses include, for example, the activation or suppression of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or suppression of any other cells of the immune system, such as NK cells. In some contexts, an immune response refers to NK cell-mediated killing of foreign cells, such as allogeneic T-cell therapy.

[0058] As used herein, the term "introduction" or "introduction" refers to the expression of heterologous polynucleotides and / or polypeptides in cells. In some aspects, introduction is achieved by transfecting cells with the polynucleotide of interest. In some aspects, introduction is achieved by genetically modifying said cells to express a heterologous sequence, for example, using gene-editing tools including, but not limited to, CRISPR / Cas, CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas12a, CRISPR / Cpf1, zinc finger, TALEN, Closver-Cas, or variants thereof. In some aspects, introduction is achieved by contacting cells with mRNA encoding the polypeptide of interest, thereby allowing the mRNA to enter the cell or the cell nucleus. In some aspects, introduction includes transfecting or transducing cells with the polynucleotide encoding the polypeptide.

[0059] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, NKT cells, or B cells. NK cells are a class of cytotoxic lymphocytes that represent a major component of the innate immune system. NK cells repel tumor- and virus-infected cells by inducing apoptosis or programmed cell death in target cells. NK cells are generally defined as CD3- / CD56+ / CD7+ cells. In some embodiments, NK cells may also express CD16 (CD16+) and / or NKG2D (NKG2D+). In some embodiments, the immune cells generated using the methods described herein are NK cells characterized as CD56+ / CD3- cells.

[0060] As defined herein, CD56+ / CD3- cells refer to cells containing functional CD56 but not functional CD3. In some embodiments, CD56+ / CD3- cells may be NK cells. In another example, CD56+ / CD3- cells may be progenitor cells of NK cells. CD56+ / CD3- cells may be prepared according to any method known in the art (see, for example, US2021 / 0292713, which is incorporated herein by reference).

[0061] As used herein, the terms "pluripotent stem cells," "PS cells," or "PSCs" refer to cells capable of self-renewing themselves by dividing and developing into the three main cell populations that make up the human body: ectoderm, endoderm, and mesoderm cells. Pluripotent stem cells are able to produce cells from all three basic body layers, and therefore they can potentially generate any cells or tissues needed for the body to repair itself. This property is known 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 immature (e.g., pluripotent) state. Various methods for dedifferentiating cells are known, including but not limited to overexpressing Oct3 / 4, Sox2, Klf4, and c-Myc (“Yamanaka factor”) in cells (see, for example, Takahashi and Yamanaka, Cell 126:663-76 (2006)). In some respects, iPS cells are pluripotent cells, for example, capable of differentiating into a limited number of cell types. In some respects, iPS cells are totipotent cells, for example, capable of differentiating into any cell type. In some respects, iPS cells can redifferentiate into specific cell types, such as immune cells.

[0063] The term "CD3" refers to differentiation cluster 3, a multimeric protein complex called 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 not by native NK cells.

[0064] The term "CD56" refers to the neural cell adhesion molecule (NCAM) and is a member of the immunoglobulin superfamily involved in homophile and heterophile interactions. CD56 is a phenotypic marker of NK cells. Furthermore, defects and phenotypic alterations in the CD56+ immune cell fraction are associated 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 and tumor marker belonging to the tumor necrosis factor receptor family. CD30 is typically expressed on activated T cells, NK cells, and B cells.

[0066] The term "NKp46" (also known as CD335) refers to a receptor belonging to the natural cytotoxic receptor (NCR) family expressed by human NK cells. The NKp46 peptide used in this invention is not limited to wild-type proteins, as long as it is activated by the NKp46 agonist described below. Whether such an NKp46 peptide can be activated by an NKp46 agonist can be determined by confirming phosphorylation of downstream molecules of NKp46 (e.g., PI3K and ERK1 / 2). In some embodiments, the NKp46 peptide comprises the amino acid sequence of 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 listed in SEQ ID NO: 1 (SEQ ID NO: 1; Table 1). In some aspects, 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% sequence identity with the nucleic acid sequence listed in SEQ ID NO: 1. In some aspects, the nucleic acid sequence encodes an NKp46 polypeptide comprising 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% sequence identity with the amino acid sequence listed in SEQ ID NO: 2. In some aspects, the nucleic acid sequence encodes an NKp46 polypeptide comprising the amino acid sequence listed in SEQ ID NO: 2.

[0068] Table 1. NKp46 sequence

[0069] In some embodiments, a vector is provided that contains 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, adenovirus-associated virus (AAV) vector, lentiviral vector, and retroviral vector. As 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 aspects, the vector contains a polynucleotide encoding at least one (e.g., one or more) NKp46. In this case, the vector may contain one or more adapter sequences, such as a cleavable peptide (e.g., E2A). For example, the E2A peptide may be located between two NKp46 segments.

[0070] In some aspects, the vector contains polynucleotides encoding (i) one or more chimeric antigen receptors (CARs) and / or (ii) one or more cytokines or mimics thereof. In some aspects, the vector contains polynucleotides encoding multiple (e.g., two or more) CARs and / or their cytokines or mimics. In this case, the vector may contain one or more adapter sequences, such as cleavable peptides (e.g., E2A). For example, the E2A peptide may be located between the CAR and / or the cytokine.

[0071] 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 used for detection.

[0072] The term "CD16" (FcγRIIIa) refers to a type I transmembrane receptor containing two extracellular Ig-like domains expressed on NK cells. CD16 functions as a low-affinity IgG receptor that mediates antibody-dependent cytotoxicity (ADCC) on NK cells.

[0073] The term "CD34" refers to the E-selectin and P-selectin ligands on human HSPCs, which bind with kinetics comparable to other known selectin ligands. CD34 is commonly used to identify and isolate human hematopoietic stem cells / progenitor cells (HSPCs).

[0074] The term "CD2" refers to a glycoprotein, which is a co-stimulatory receptor mainly expressed on T cells and NK cells.

[0075] As used herein, the term "primary NK cells" refers to NK cells directly taken from a subject or to a culture of NK cells that can be directly isolated from a subject.

[0076] As used herein, the term "iNK cell" or "induced NK cell" refers to an NK cell differentiated from an iPS cell. The iNK cell expresses CD56. In another embodiment, such an iNK cell may express both CD56 and CD16. In yet another example, such an iNK cell may express CD56 and be CD3- (CD56+ / CD3-).

[0077] As used in this article, "hematopoietic stem cell" or "HSC" or "hematopoietic progenitor cell" or "HPC" refers to an immature cell that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets. Hematopoietic stem cells are found in peripheral blood and bone marrow and are also known as "blood stem cells".

[0078] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's dextran, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids, and nutritional supplements, such as similar materials and combinations thereof, as known to those skilled in the art. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0079] As used herein, the terms “recombinant” or “modified” are intended to refer to cells containing nucleic acids not naturally present in cells, such as immune cells, and may also refer to cells in which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to a specific subject cell, but also to the progeny of that cell. While some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny are still included within the scope of the terms “recombinant” or “modified” as used herein.

[0080] As used herein, the terms “subject” and “patient” are used interchangeably and refer to humans or non-humans, such as primates, mammals, and vertebrates. In a particular context, a subject is a human.

[0081] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant fusion protein having an antigen-specific extracellular domain coupled to an intracellular domain, which instructs the cell to perform specific functions after the antigen binds to the extracellular domain.

[0082] As used herein, a “suicide gene” or “suicide switch” refers to a gene that, when expressed by a cell, causes the cell to undergo apoptosis. Non-limiting examples of suicide genes include inducible caspase-9 suicide gene, viral thymidine kinase, cytosine deaminase, intracellular antibodies against antioxidant enzymes (AOE), bacterial nitroreductases, other caspases, and DNases.

[0083] In some respects, suicide genes include the inducible caspase-9 (iCasp9) suicide gene. The inducible caspase-9 (iCasp9) "safety switch" provides a solution that allows the removal of activated CAR T cells. Induction of iCasp9 depends on the administration of the small molecule dimer AP1903, and dimerization leads to rapid induction of apoptosis in transduced cells, preferentially killing activated cells expressing high levels of the transgene. SeeFor example, Gargett, T et al. Front. Pharmacol., October 28, 2014, 5:235.

[0084] In some respects, suicide genes involve viral thymidine kinase (TK). Thymidine kinase is an ATP-thymidine 5'-phosphotransferase that converts deoxythymidine to deoxythymidine 5'-monophosphate, which is further phosphorylated to deoxythymidine diphosphate, and then phosphorylated to deoxythymidine triphosphate by viral thymidine kinase and nucleoside diphosphate kinase, respectively. Deoxythymidine triphosphate is incorporated into the synthesized DNA molecule via DNA polymerase. Some dNTP analogs, such as ganciclovir (GCV), a synthetic analog of 2'-deoxyguanosine, can terminate DNA synthesis upon incorporation into the synthesized DNA. The termination of synthesis triggers an apoptosis signaling cascade. Although GCV is not recognized by human thymidine kinase, it is recognized as a matrix for some viral thymidine kinases, such as herpes simplex virus-1 thymidine kinase (HSV-TK). Therefore, human cells expressing HSV-TK convert GCV to GCV phosphate, which is further phosphorylated and incorporated into the synthesized DNA, leading to the termination of synthesis and apoptosis. Although variants of HSV-TK are not restricted, HSV-TK is, for example, TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug; 21(8): 929-41).

[0085] In some respects, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine into uracil and releases ammonia. Under physiological conditions, the modified site is recognized by a nuclease, followed by the breaking of phosphodiester bonds in the DNA, which is then repaired by incorporating new cytosine. However, cytosine deaminase can also convert 5-fluorocytosine to 5-fluorouracil (5-FU). Thus, after providing the non-toxic prodrug 5-FC, cytosine deaminase converts it into the highly toxic 5-FU (i.e., a suicide inhibitor of thymidine synthase), thereby inhibiting cell growth and apoptosis.

[0086] The term "inducible" refers to a reactive or active state in response to the presence of an activator or initiator. Genes or enzymes can be inducible, meaning they are activated or expressed only in the presence of a specific molecule.

[0087] The term "activator" or "initiator" refers to a molecule, chemical, matrix, or agent that initiates or activates the activity or expression of a gene, protein, or any other biological component.

[0088] The term "CAG promoter" refers to a hybrid nucleic acid construct consisting of a cytomegalovirus (CMV) enhancer fused to the chicken β-actin (CAG) promoter.

[0089] The term "promoter" is a DNA sequence that binds to a protein to initiate the transcription of RNA from downstream DNA.

[0090] "NKp46 agonist" is an agent that activates, upregulates, or stimulates NKp46, whether chemical or biological. NKp46 agonists can be used alone or in combination with two or more NKp46 agonists. In some aspects, NKp46 agonists comprise anti-NKp46 antibodies that can activate, upregulate, or stimulate NKp46 (in this specification, such anti-NKp46 antibodies that can activate, upregulate, or stimulate NKp46 are sometimes referred to as "anti-NKp46 antibodies" or "anti-NKp46 agonist antibodies"). Such anti-NKp46 antibodies may include, but are not limited to, clones 9E2, 4k12, n1D9, B-N40, B-L46, 900, 2B11A3, 8F24, and 195314 (R&D systems, Biolegend, Miltenyi biotec, etc.), NK cell activation / expansion kits for human (Miltenyi biotec), and Cloudz human NK cell expansion kits (R&D systems). These antibodies can be functional fragments, such as Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), biantibodies, triantibodies, F(ab)2, F(ab'2), F(ab'2), single-chain Fv (scFv), biantibodies, triantibodies, tetraantibodies, and microantibodies. Antibodies can be derived from animals such as mice, rats, cattle, rabbits, goats, sheep, and guinea pigs. Antibody isotypes are not restricted, and isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM.

[0091] "CD30 agonists" are agents that activate, upregulate, or stimulate CD30, whether chemical or biological. CD30 agonists can be used alone or in combination with two or more CD30 agonists. In some aspects, CD30 agonists comprise anti-CD30 antibodies (i.e., "anti-CD30 antibodies" or "anti-CD30 agonist antibodies") that can activate, upregulate, or stimulate CD30. Such anti-CD30 antibodies can be, but are not limited to, clones #81316 (R&D systems). These antibodies can be their functional fragments, such as Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), biantibodies, triantibodies, F(ab)2, F(ab'2), F(ab'2), single-chain Fv (scFv), biantibodies, triantibodies, tetraantibodies, and microantibodies. Antibodies can be derived from animals such as mice, rats, cattle, rabbits, goats, sheep, guinea pigs, etc. Antibody isotypes are not restricted, and isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM.

[0092] The antibody can be a monoclonal or polyclonal antibody, preferably a monoclonal antibody, and the antibody can also be a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0093] Such antibodies can be produced by known methods, such as by constructing an expression vector containing nucleic acid encoding the antibody, culturing a transformant containing the nucleic acid, or culturing a hybridoma that produces the antibody.

[0094] The term "caspases inhibitor" refers to any chemical or biological agent that inhibits caspase. Caspase can be controlled in several ways, including through the processing and activation regulation of molecules such as FADD, APAF-1, Bcl-2 family members, FLIP, and IAP. Active caspase can be controlled by a variety of inhibitors, some of which include products of viral and cellular genes, as well as artificial caspase inhibitors developed for research and pharmaceutical purposes (Ekert et al. CellDeath. Differ, November 1999, 6(11):1081-6).

[0095] As used in this article, “medium” or “culture medium” refers to an aqueous solution of nutrients that can be used to grow cells for an extended period of time, i.e., the liquid used in cell culture.

[0096] NK-related diseases can include any lesion or condition associated with abnormal NK function. Such lesions or conditions may include, but are not limited to, cancer, viral infections, including pneumonia and other viruses such as herpesvirus and 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, Sjogren's syndrome, and multiple sclerosis.

[0097] As used herein, the term "treating" or "treatment" for a disease or ailment refers to the implementation of a protocol that may include administering one or more therapies to a patient in an effort to alleviate the signs or symptoms of the disease. In some aspects, treatment reduces the rate of disease progression, improves or alleviates the disease state, and / or promotes remission or improved prognosis. Remission may occur before and after the signs or symptoms of the disease or ailment appear. Therefore, in some aspects, "treating" or "treatment" includes "preventing" or "preventing" a disease or ailment. However, "treating" or "treatment" does not require complete remission of all signs and / or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0098] In various aspects, the modified cell populations described herein can be used to treat diseases in subjects in need or to alleviate symptoms associated with diseases such as cancer. In some aspects, the modified cells are NK or iNK cells that are transduced by CAR or TCR and / or modified to exhibit reduced expression of endogenous MHC class I and MHC class II HLA genes and increased expression of certain heterologous genes in the cells, such that when these cells are administered to a subject, the modified cells are less likely to be killed by the subject's immune system.

[0099] As used in this article, the terms “ug”, “uL” and “uM” are interchangeable with “μg”, ​​“μL” and “μM”, respectively.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms used in this disclosure.

[0101] Units, prefixes, and symbols are represented in their form accepted by the International System of Units (SI). Numerical ranges include numbers within defined ranges. Unless otherwise indicated, as stated herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the listed range, including, where appropriate, fractions (such as tenths and hundredths of integers).

[0102] The abbreviations used herein are defined throughout this disclosure. Various aspects of this disclosure are further described in detail in the following sections.

[0103] The various aspects described herein are further described in detail in the following sections.

[0104] II. Compositions disclosed herein Some aspects of this disclosure relate to a method for expanding CD56+ / CD3- cells, the method comprising: culturing the 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 expanding CD56+ / CD3- cells, the method comprising: culturing the CD56+ / CD3- cells in the presence of a CD30 agonist. In some aspects, the CD56+ / CD3- cells are derived from pluripotent stem cells (iPSCs).

[0105] Some aspects of this disclosure relate to a method for generating modified CD56+ / CD3- cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to generate 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 a CD30 agonist. In some aspects, the HPCs are derived from one or more pluripotent stem cells.

[0106] II.A. Methods of this Disclosure II.A.1. NKp46 agonist Some aspects of this disclosure relate to methods for generating and / or expanding CD56+ / CD3- cells. In some aspects, the method includes culturing CD56+ / CD3- cells in the presence of an NKp46 agonist. Any NKp46 agonist may 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 that specifically binds to NKp46 or its antigen-binding portion (“anti-NKp46 antibody”). In some aspects, the NKp46 agonist comprises a small molecule.

[0107] In some aspects, the NKp46 agonist comprises an anti-NKp46 antibody. Any anti-NKp46 antibody may be used in the methods disclosed herein. In some aspects, CD56+ / CD3- cells are contacted with about 100 ng / ml to about 10 μg / ml of anti-NKp46 antibody. In some aspects, CD56+ / CD3- cells are contacted with about 500 ng / ml to about 5 μg / ml of anti-NKp46 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 100 ng / ml, approximately 150 ng / ml, approximately 200 ng / ml, approximately 250 ng / ml, approximately 300 ng / ml, approximately 350 ng / ml, approximately 400 ng / ml, approximately 450 ng / ml, approximately 500 ng / ml, approximately 600 ng / ml, approximately 700 ng / ml, approximately 800 ng / ml, approximately 900 ng / ml, approximately 1 μg / ml, approximately 2 μg / ml, approximately 3 μg / ml, approximately 4 μg / ml, approximately 5 μg / ml, approximately 6 μg / ml, approximately 7 μg / ml, approximately 8 μg / ml, approximately 9 μg / ml, or approximately 10 μg / ml of anti-NKp46 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 1 μg / ml of anti-NKp46 antibody.

[0108] In some aspects, CD56+ / CD3- cells are further contacted with at least one cytokine. In some aspects, the at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-7. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-12. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-15. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-18. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-21.

[0109] In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2 and IL-7. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2 and IL-12. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2 and IL-15. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2 and IL-18. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2 and IL-21. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2, IL-10, and IL-15. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist and IL-2, IL-7, IL-12, IL-15, and IL-21. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist (preferably, an anti-NKp46 agonist antibody) and IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0110] Some aspects of this disclosure relate to a method for generating iNK cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to generate 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 contacted with an NKp46 agonist. In some aspects, the CD56+ / CD3- cells are contacted with an NKp46 agonist for about 1 day. In some aspects, the CD56+ / CD3- cells are contacted with an NKp46 agonist for about 2 days. In some aspects, the CD56+ / CD3- cells are contacted with an NKp46 agonist for about 3 days. In some aspects, the CD56+ / CD3- cells are contacted with an NKp46 agonist for about 4 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 5 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 6 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 7 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 8 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 9 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 10 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 11 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 12 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 13 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 14 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 15 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 16 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 17 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 18 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 19 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 agonists for approximately 20 days. In some cases, CD56+ / CD3- cells were exposed to an NKp46 agonist for approximately 21 days.

[0111] In some aspects, CD56+ / CD3- cells are contacted with one or more of (i) an NKp46 agonist, IL-2, IL-7, IL-15 and IL-21, IL-12 and IL-18 for a first culture; then, CD56+ / CD3- cells are contacted with (ii) an NKp46 agonist, IL-2, IL-7 and IL-15 for a second culture. In some aspects, the first culture lasts approximately 1 day. In some aspects, the first culture lasts approximately 2 days. In some aspects, the first culture lasts approximately 3 days. In some aspects, the first culture lasts approximately 4 days. In some aspects, the first culture lasts approximately 5 days. In some aspects, the first culture lasts approximately 6 days. In some aspects, the first culture lasts approximately 7 days. In some aspects, the first culture lasts approximately 8 days. In some aspects, the first culture lasts approximately 9 days. In some aspects, the first culture lasts approximately 10 days. In some aspects, the first culture lasts approximately 11 days. In some aspects, the first culture lasts approximately 12 days. In some aspects, the first culture lasts approximately 13 days. In some aspects, the first culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 1 day. In some aspects, the second culture lasts approximately 2 days. In some aspects, the second culture lasts approximately 3 days. In some aspects, the second culture lasts approximately 4 days. In some aspects, the second culture lasts approximately 5 days. In some aspects, the second culture lasts approximately 6 days. In some aspects, the second culture lasts approximately 7 days. In some aspects, the second culture lasts approximately 8 days. In some aspects, the second culture lasts approximately 9 days. In some aspects, the second culture lasts approximately 10 days. In some aspects, the second culture lasts approximately 11 days. In some aspects, the second culture lasts approximately 12 days. In some aspects, the second culture lasts approximately 13 days. In some aspects, the second culture lasts approximately 14 days.

[0112] In some cases, the second culture is repeated to increase the number of cells produced. In some cases, the second culture is performed twice. In some cases, the second culture is performed three times. In some cases, the second culture is performed four times. In some cases, the second culture is performed five times. In some cases, the second culture is performed six times. In some cases, the second culture is performed seven times. In some cases, the second culture is performed eight times. In some cases, the second culture is performed nine times. In some cases, the second culture is performed ten times. In some cases, the second culture is performed more than ten times.

[0113] II.A.2. CD30 agonist In some aspects, the method includes culturing CD56+ / CD3- cells in the presence of a CD30 agonist. Any CD30 agonist may be used in the methods disclosed herein. In some aspects, the CD30 agonist is a polypeptide. In some aspects, the CD30 agonist is an antibody that specifically binds to CD30 or its antigen-binding portion (“anti-CD30 antibody”). In some aspects, the CD30 agonist comprises a small molecule.

[0114] In some aspects, the CD30 agonist comprises an anti-CD30 antibody. Any anti-CD30 antibody may be used in the methods disclosed herein. In some aspects, CD56+ / CD3- cells are contacted with about 100 ng / ml to about 10 μg / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with about 250 ng / ml to about 2.5 μg / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with anti-CD30 antibodies 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 aspects, CD56+ / CD3- cells are contacted with approximately 200 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 225 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 250 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 275 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 300 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 325 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 350 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 375 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 400 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 500 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 600 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 700 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 800 ng / ml of anti-CD30 antibody. In some aspects, CD56+ / CD3- cells are contacted with approximately 900 ng / ml of anti-CD30 antibody.In some cases, CD56+ / CD3- cells are contacted with approximately 1 μg / ml of anti-CD30 antibody.

[0115] In some aspects, CD56+ / CD3- cells are further contacted with at least one cytokine. In some aspects, the at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-7. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-12. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-15. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-18. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-21.

[0116] In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2 and IL-7. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2 and IL-12. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2 and IL-15. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2 and IL-18. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2 and IL-21. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2, IL-10, and IL-15. In some aspects, CD56+ / CD3- cells are contacted with a CD30 agonist and IL-2, IL-7, IL-12, IL-15, and IL-21. In some aspects, CD56+ / CD3- cells are exposed to CD30 agonists (e.g., anti-CD30 agonist antibodies) and IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0117] Some aspects of this disclosure relate to a method for generating iNK cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to generate 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 contacted with a CD30 agonist. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 1 day. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 2 days. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 3 days. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 4 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 5 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 6 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 7 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 8 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 9 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 10 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 11 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 12 days. In some cases, CD56+ / CD3- cells were exposed to a CD30 agonist for 13 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 14 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 15 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 16 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 17 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 18 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 19 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 20 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 21 days.

[0118] Some aspects of this disclosure relate to a method for generating iNK cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs), and (B) differentiating the HPCs to generate 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 contacted with a CD30 agonist. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 1 day. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 2 days. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 3 days. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 4 days. In some aspects, the CD56+ / CD3- cells are contacted with a CD30 agonist for 5 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 6 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 7 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 8 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 9 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 10 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 11 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 12 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 13 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 14 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 15 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 16 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 17 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 18 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 19 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 20 days. In some aspects, CD56+ / CD3- cells were exposed to a CD30 agonist for 21 days.

[0119] In some aspects, CD56+ / CD3- cells are contacted with one or more of (i) a CD30 agonist, IL-2, IL-7, IL-15 and IL-21, IL-12 and IL-18 for a first culture; then, CD56+ / CD3- cells are contacted with (ii) a CD30 agonist, IL-2, IL-7 and IL-15 for a second culture. In some aspects, the first culture lasts approximately 1 day. In some aspects, the first culture lasts approximately 2 days. In some aspects, the first culture lasts approximately 3 days. In some aspects, the first culture lasts approximately 4 days. In some aspects, the first culture lasts approximately 5 days. In some aspects, the first culture lasts approximately 6 days. In some aspects, the first culture lasts approximately 7 days. In some aspects, the first culture lasts approximately 8 days. In some aspects, the first culture lasts approximately 9 days. In some aspects, the first culture lasts approximately 10 days. In some aspects, the first culture lasts approximately 11 days. In some aspects, the first culture lasts approximately 12 days. In some aspects, the first culture lasts approximately 13 days. In some aspects, the first culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 1 day. In some aspects, the second culture lasts approximately 2 days. In some aspects, the second culture lasts approximately 3 days. In some aspects, the second culture lasts approximately 4 days. In some aspects, the second culture lasts approximately 5 days. In some aspects, the second culture lasts approximately 6 days. In some aspects, the second culture lasts approximately 7 days. In some aspects, the second culture lasts approximately 8 days. In some aspects, the second culture lasts approximately 9 days. In some aspects, the second culture lasts approximately 10 days. In some aspects, the second culture lasts approximately 11 days. In some aspects, the second culture lasts approximately 12 days. In some aspects, the second culture lasts approximately 13 days. In some aspects, the second culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 15 days.

[0120] In some cases, the second culture is repeated to increase the number of cells produced. In some cases, the second culture is performed twice. In some cases, the second culture is performed three times. In some cases, the second culture is performed four times. In some cases, the second culture is performed five times. In some cases, the second culture is performed six times. In some cases, the second culture is performed seven times. In some cases, the second culture is performed eight times. In some cases, the second culture is performed nine times. In some cases, the second culture is performed ten times. In some cases, the second culture is performed more than ten times.

[0121] In some aspects, the method includes culturing CD56+ / CD3- cells in the presence of an NKp46 agonist and a CD30 agonist. In some aspects, 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 methods disclosed herein. In some aspects, CD56+ / CD3- cells are amplified with an anti-NKp46 antibody. In some aspects, the anti-NKp46 antibody is plate-bound. In some aspects, CD56+ / CD3- cells are contacted with about 100 ng / ml to about 10 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some aspects, CD56+ / CD3- cells are contacted with about 500 ng / ml to about 5 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some respects, CD56+ / CD3- cells are exposed to approximately 100 ng / ml, approximately 150 ng / ml, approximately 200 ng / ml, approximately 250 ng / ml, approximately 300 ng / ml, approximately 350 ng / ml, approximately 400 ng / ml, approximately 450 ng / ml, approximately 500 ng / ml, approximately 600 ng / ml, approximately 700 ng / ml, approximately 800 ng / ml, approximately 900 ng / ml, approximately 1 μg / ml, approximately 2 μg / ml, approximately 3 μg / ml, approximately 4 μg / ml, approximately 5 μg / ml, approximately 6 μg / ml, approximately 7 μg / ml, approximately 8 μg / ml, approximately 9 μg / ml, or approximately 10 μg / ml of anti-NKp46 antibody and / or CD30 agonist. In some embodiments, CD56+ / CD3- cells are contacted with approximately 1 μg / ml of anti-NKp46 antibody and / or a CD30 agonist. In other embodiments, CD56+ / CD3- cells are contacted with plate-bound NKp46 antibody and approximately 300 ng / ml of anti-CD30 antibody.

[0122] In some aspects, CD56+ / CD3- cells are further contacted with at least one cytokine. In some aspects, the at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-2. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-7. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-12. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-15. In some aspects, CD56+ / CD3- cells are contacted with an NKp46 agonist, a CD30 agonist, and IL-18. In some respects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-21.

[0123] In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2 and IL-7. In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2 and IL-12. In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2 and IL-15. In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2 and IL-18. In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2 and IL-21. In some aspects, CD56+ / CD3- cells are exposed to NKp46 agonists, CD30 agonists, and IL-2, IL-10, and IL-15. In some aspects, 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 aspects, 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.

[0124] Some aspects of this disclosure relate to a method for generating iNK cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs) containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides; and (B) differentiating the HPCs to generate 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 contacted with the NKp46 agonist and the CD30 agonist. In some aspects, the CD56+ / CD3- cells are contacted with the NKp46 agonist and the CD30 agonist for approximately 1 day. In some aspects, the CD56+ / CD3- cells are contacted with the NKp46 agonist and the CD30 agonist for approximately 2 days. In some aspects, the CD56+ / CD3- cells are contacted with the NKp46 agonist and the CD30 agonist for approximately 3 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 4 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 5 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 6 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 7 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 8 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 9 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 10 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 11 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 12 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 13 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 14 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 15 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 16 days. In some aspects, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 17 days. In some cases, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 18 days.In some embodiments, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 19 days. In some embodiments, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 20 days. In some embodiments, CD56+ / CD3- cells were exposed to NKp46 and CD30 agonists for approximately 21 days.

[0125] In some aspects, CD56+ / CD3- cells are contacted with one or more of (i) NKp46 agonists, CD30 agonists, IL-2, IL-7, IL-15 and IL-21, IL-12 and IL-18 for a first culture; then, CD56+ / CD3- cells are contacted with (ii) NKp46 agonists, CD30 agonists, IL-2, IL-7 and IL-15 for a second culture. In some aspects, the first culture lasts approximately 1 day. In some aspects, the first culture lasts approximately 2 days. In some aspects, the first culture lasts approximately 3 days. In some aspects, the first culture lasts approximately 4 days. In some aspects, the first culture lasts approximately 5 days. In some aspects, the first culture lasts approximately 6 days. In some aspects, the first culture lasts approximately 7 days. In some aspects, the first culture lasts approximately 8 days. In some aspects, the first culture lasts approximately 9 days. In some aspects, the first culture lasts approximately 10 days. In some aspects, the first culture lasts approximately 11 days. In some aspects, the first culture lasts approximately 12 days. In some aspects, the first culture lasts approximately 13 days. In some aspects, the first culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 1 day. In some aspects, the second culture lasts approximately 2 days. In some aspects, the second culture lasts approximately 3 days. In some aspects, the second culture lasts approximately 4 days. In some aspects, the second culture lasts approximately 5 days. In some aspects, the second culture lasts approximately 6 days. In some aspects, the second culture lasts approximately 7 days. In some aspects, the second culture lasts approximately 8 days. In some aspects, the second culture lasts approximately 9 days. In some aspects, the second culture lasts approximately 10 days. In some aspects, the second culture lasts approximately 11 days. In some aspects, the second culture lasts approximately 12 days. In some aspects, the second culture lasts approximately 13 days. In some aspects, the second culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 15 days.

[0126] In some cases, the second culture is repeated to increase the number of cells produced. In some cases, the second culture is performed twice. In some cases, the second culture is performed three times. In some cases, the second culture is performed four times. In some cases, the second culture is performed five times. In some cases, the second culture is performed six times. In some cases, the second culture is performed seven times. In some cases, the second culture is performed eight times. In some cases, the second culture is performed nine times. In some cases, the second culture is performed ten times. In some cases, the second culture is performed more than ten times.

[0127] II.A.3. Culture Reagents In some aspects, CD56+ / CD3- cells are contacted with IL-2 at concentrations of about 0.1 to about 100 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-2 at concentrations of about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 1 to about 40 ng / ml, about 1 to about 30 ng / ml, about 1 to about 20 ng / ml, or about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-2 at concentrations of about 1 to about 20 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-2 at concentrations of about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with approximately 10 to approximately 20 ng / ml IL-2. In other aspects, CD56+ / CD3- cells are contacted with approximately 5 to approximately 15 ng / ml IL-2.

[0128] In some cases, CD56+ / CD3- cells were exposed to IL-2 at concentrations of 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 cases, CD56+ / CD3- cells were exposed to approximately 10 ng / ml IL-2.

[0129] In some aspects, CD56+ / CD3- cells are contacted with IL-7 at concentrations of about 0.1 to about 100 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-7 at concentrations of about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 1 to about 40 ng / ml, about 1 to about 30 ng / ml, about 1 to about 20 ng / ml, or about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-7 at concentrations of about 1 to about 20 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-7 at concentrations of about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with approximately 10 to approximately 20 ng / ml IL-7. In other aspects, CD56+ / CD3- cells are contacted with approximately 5 to approximately 15 ng / ml IL-7.

[0130] In some cases, CD56+ / CD3- cells were exposed to IL-7 at concentrations of 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 cases, CD56+ / CD3- cells were exposed to approximately 10 ng / ml IL-7.

[0131] In some aspects, CD56+ / CD3- cells are contacted with IL-15 at concentrations of about 0.1 to about 100 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-15 at concentrations of about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 1 to about 40 ng / ml, about 1 to about 30 ng / ml, about 1 to about 20 ng / ml, or about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-15 at concentrations of about 1 to about 20 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with IL-15 at concentrations of about 1 to about 10 ng / ml. In some aspects, CD56+ / CD3- cells are contacted with approximately 10 to approximately 20 ng / ml IL-15. In other aspects, CD56+ / CD3- cells are contacted with approximately 5 to approximately 15 ng / ml IL-15.

[0132] In some cases, CD56+ / CD3- cells were exposed to IL-15 at concentrations of approximately 1 ng / ml, approximately 2 ng / ml, approximately 3 ng / ml, approximately 4 ng / ml, approximately 5 ng / ml, approximately 6 ng / ml, approximately 7 ng / ml, approximately 8 ng / ml, approximately 9 ng / ml, approximately 10 ng / ml, approximately 11 ng / ml, approximately 12 ng / ml, approximately 13 ng / ml, approximately 14 ng / ml, approximately 15 ng / ml, approximately 16 ng / ml, approximately 17 ng / ml, approximately 18 ng / ml, approximately 19 ng / ml, approximately 20 ng / ml, approximately 30 ng / ml, approximately 40 ng / ml, approximately 50 ng / ml, approximately 60 ng / ml, approximately 70 ng / ml, approximately 80 ng / ml, approximately 90 ng / ml, or approximately 100 ng / ml. In some cases, CD56+ / CD3- cells were exposed to approximately 10 ng / ml IL-15.

[0133] In some aspects, CD56+ / CD3- cells are contacted with about 0.1 to about 100 ng / ml of IL-21. In some aspects, CD56+ / CD3- cells are contacted with about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 1 to about 40 ng / ml, about 1 to about 30 ng / ml, about 1 to about 20 ng / ml, or about 10 to about 20 ng / ml of IL-21. In some aspects, CD56+ / CD3- cells are contacted with about 10 to about 30 ng / ml of IL-21. In some aspects, CD56+ / CD3- cells are contacted with about 10 to about 20 ng / ml of IL-21. In some aspects, CD56+ / CD3- cells are contacted with approximately 20 to approximately 30 ng / ml IL-21. In other aspects, CD56+ / CD3- cells are contacted with approximately 15 to approximately 25 ng / ml IL-21.

[0134] In some cases, CD56+ / CD3- cells were 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 cases, CD56+ / CD3- cells were exposed to approximately 20 ng / ml IL-21.

[0135] In some aspects, CD56+ / CD3- cells are contacted with about 0.1 to about 100 ng / ml of IL-12. In some aspects, CD56+ / CD3- cells are contacted with about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 10 to about 90 ng / ml, about 20 to about 80 ng / ml, about 30 to about 70 ng / ml, or about 40 to about 60 ng / ml of IL-12. In some aspects, CD56+ / CD3- cells are contacted with about 40 to about 60 ng / ml of IL-12. In some aspects, CD56+ / CD3- cells are contacted with about 40 to about 50 ng / ml of IL-12. In some aspects, CD56+ / CD3- cells are contacted with approximately 50 to approximately 60 ng / ml IL-12. In other aspects, CD56+ / CD3- cells are contacted with approximately 45 to approximately 55 ng / ml IL-12.

[0136] In some cases, CD56+ / CD3- cells were 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 cases, CD56+ / CD3- cells were exposed to approximately 50 ng / ml of IL-12.

[0137] In some aspects, CD56+ / CD3- cells are contacted with about 0.1 to about 100 ng / ml of IL-18. In some aspects, CD56+ / CD3- cells are contacted with about 1 to about 100 ng / ml, about 1 to about 90 ng / ml, about 1 to about 80 ng / ml, about 1 to about 70 ng / ml, about 1 to about 60 ng / ml, about 1 to about 50 ng / ml, about 10 to about 90 ng / ml, about 20 to about 80 ng / ml, about 30 to about 70 ng / ml, or about 40 to about 60 ng / ml of IL-18. In some aspects, CD56+ / CD3- cells are contacted with about 40 to about 60 ng / ml of IL-18. In some aspects, CD56+ / CD3- cells are contacted with about 40 to about 50 ng / ml of IL-18. In some aspects, CD56+ / CD3- cells are contacted with approximately 50 to approximately 60 ng / ml IL-18. In other aspects, CD56+ / CD3- cells are contacted with approximately 45 to approximately 55 ng / ml IL-18.

[0138] In some cases, CD56+ / CD3- cells were 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 cases, CD56+ / CD3- cells were exposed to approximately 50 ng / ml of IL-18.

[0139] In some aspects, CD56+ / CD3- cells are further contacted with a caspase inhibitor. Any caspase inhibitor may be used in the methods disclosed herein. In some aspects, the caspase inhibitor comprises Z-VAD-FMK. In some aspects, CD56+ / CD3- cells are contacted with about 1 to about 100 μM Z-VAD-FMK. In some aspects, CD56+ / CD3- cells were contacted with approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, or approximately 100 μM Z-VAD-FMK. In some aspects, CD56+ / CD3- cells were contacted with approximately 10 μM Z-VAD-FMK.

[0140] In some respects, CD56+ / CD3- cells expand in closed systems.

[0141] In some aspects, the method includes expanding 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.

[0142] In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 3 days initially, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 2 days initially, followed by a second culture for approximately 10 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 9 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 8 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 7 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 11 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 12 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 13 days. In some embodiments, CD56+ / CD3- cells are cultured for approximately 4 days initially, followed by a second culture for approximately 14 days.

[0143] II.A.4. A novel method for manufacturing iNK cells Some aspects of this disclosure relate to methods for generating 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 may 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 that specifically binds to CD16 or its antigen-binding moiety (“anti-CD16 antibody”). In some aspects, the CD16 agonist comprises a small molecule.

[0144] In some aspects, the CD16 agonist comprises an anti-CD16 antibody. Any anti-CD16 antibody may be used in the methods disclosed herein. In some aspects, CD56+ / CD3- cells are contacted with a culture vessel coated with an anti-CD16 antibody. In some aspects, the culture vessel is coated with approximately 0.1 μg / cm³ of anti-CD16 antibody. 2 Approximately 1 μg / cm 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.4 μg / cm³.2 Approximately 0.8 μg / cm 2 Anti-CD16 antibody. In some cases, the culture vessel is coated with approximately 0.5 μg / cm³. 2 Approximately 0.7 μg / cm 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.6 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.1 μg / cm³. 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 / cm 2 or 2 μg / cm 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.3 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.4 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.5 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.6 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.7 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.8 μg / cm³. 2 Anti-CD16 antibody. In some cases, the culture vessel was coated with approximately 0.9 μg / cm³. 2 Anti-CD16 antibody.

[0145] In some aspects, CD56+ / CD3- cells are further contacted with at least one cytokine. In some aspects, the at least one cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-2. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-15. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-18. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-21.

[0146] In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7 and / or IL-2. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7 and IL-15. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7 and IL-18. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist and IL-7, IL-15, and IL-18. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist (e.g., an anti-CD16 agonist antibody) and IL-7, IL-15, IL-18, and IL-21.

[0147] Some aspects of this disclosure relate to a method for generating iNK cells, the method comprising: (A) providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs), and (B) differentiating the HPCs to generate one or more CD4- cells. In some aspects, the method further comprises (C) transduction of NK cells. In some aspects, step (C) comprises differentiating CD4- cells to generate one or more NK cells, activating and expanding the NK cells, and transducing the NK cells with gamma retrovirus. In some aspects, step (C) comprises activating and expanding the NK cells and transducing the NK cells with gamma retrovirus.

[0148] In some aspects, the method includes culturing CD56+ / CD3- cells in the presence of a CD16 agonist. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 1 day. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 2 days. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 3 days. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 4 days. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 5 days. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 6 days. In some aspects, CD56+ / CD3- cells are contacted with a CD16 agonist for approximately 7 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 8 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 9 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 10 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 11 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 12 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 13 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 14 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 15 days. In some aspects, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 16 days. In some embodiments, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 17 days. In some embodiments, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 18 days. In some embodiments, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 19 days. In some embodiments, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 20 days. In some embodiments, CD56+ / CD3- cells were exposed to a CD16 agonist for approximately 21 days.

[0149] In some aspects, CD56+ / CD3- cells are contacted with (i) a CD16 agonist, IL-7, IL-15, IL-18, and IL-21 for a first culture; then, CD56+ / CD3- cells are contacted with (ii) IL-7 and IL-15 for a second culture. In some aspects, the first culture lasts approximately 1 day. In some aspects, the first culture lasts approximately 2 days. In some aspects, the first culture lasts approximately 3 days. In some aspects, the first culture lasts approximately 4 days. In some aspects, the first culture lasts approximately 5 days. In some aspects, the first culture lasts approximately 6 days. In some aspects, the first culture lasts approximately 7 days. In some aspects, the first culture lasts approximately 8 days. In some aspects, the first culture lasts approximately 9 days. In some aspects, the first culture lasts approximately 10 days. In some aspects, the first culture lasts approximately 11 days. In some aspects, the first culture lasts approximately 12 days. In some aspects, the first culture lasts approximately 13 days. In some aspects, the first culture lasts approximately 14 days. In some aspects, the second culture lasts approximately 1 day. In some aspects, the second culture lasts approximately 2 days. In some aspects, the second culture lasts approximately 3 days. In some aspects, the second culture lasts approximately 4 days. In some aspects, the second culture lasts approximately 5 days. In some aspects, the second culture lasts approximately 6 days. In some aspects, the second culture lasts approximately 7 days. In some aspects, the second culture lasts approximately 8 days. In some aspects, the second culture lasts approximately 9 days. In some aspects, the second culture lasts approximately 10 days. In some aspects, the second culture lasts approximately 11 days. In some aspects, the second culture lasts approximately 12 days. In some aspects, the second culture lasts approximately 13 days. In some aspects, the second culture lasts approximately 14 days.

[0150] II.B. Cells disclosed herein Any cell type may be used in the compositions and methods disclosed herein. In some aspects, the cells are immune cells. In some aspects, the cells are pluripotent stem cells (PSCs). In some aspects, the cells are induced pluripotent stem cells (iPSCs). In some aspects, the cells are embryonic stem cells (ESCs). In some aspects, the cells are immune cells selected from T cells, NK cells, NKT cells, or tumor-infiltrating lymphocytes.

[0151] In some respects, the cells are cells differentiated from iPSCs. In some respects, the cells are immune cells differentiated from iPSCs. In some respects, the cells are CD56+ / CD3- cells differentiated from iPSCs. In some respects, the cells are NK cells differentiated from iPSCs. In some respects, the cells are NKT cells differentiated from iPSCs.

[0152] In some aspects, the cells are CD56+ / CD3- cells differentiated from iPSCs, wherein the iPSCs are transduced with at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides. In some aspects, the iPSCs are transduced prior to differentiation into CD56+ / CD3- cells. Therefore, some aspects of this disclosure relate to methods for amplifying CD56+ / CD3- cells, wherein the cells comprise at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides.

[0153] In some aspects, CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding one or more chimeric antigen receptors (CARs). In some aspects, CD56+ / CD3- cells further comprise at least one exogenous nucleic acid molecule encoding one or more cytokines or mimics thereof. In some aspects, 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 mimic thereof. In some aspects, CD56+ / CD3- cells differentiate from iPSCs, wherein 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 mimic thereof, (iii) a suicide switch, or (iv) any combination of (i)-(iii).

[0154] In some aspects, at least one exogenous nucleic acid molecule encoding a cytokine or a mimic thereof encodes an IL-15 polypeptide. In some aspects, 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%, or at least about 99% sequence identity with the amino acid sequence of human IL-15 (UniProt P40933). In some aspects, the IL-15 polypeptide comprises human IL-15 (UniProt P40933). In some aspects, at least one exogenous nucleic acid molecule encoding a cytokine or a mimic thereof encodes an IL-15 polypeptide mimic. In some aspects, at least one exogenous nucleic acid molecule encoding a cytokine or a mimic thereof encodes an IL-15 polypeptide containing IL-15, said IL-15 being linked to IL-15Rα to form a fusion protein (IL-15Ra / IL-15 complex) (e.g., the IL-15Ra / IL-15 complex disclosed in US2021 / 0292713, which is incorporated herein by reference).

[0155] II.C. Treatment Methods Some aspects of this disclosure relate to methods for treating a disease or ailment in a subject of need, the methods comprising administering to the subject the compositions disclosed herein. In some aspects, the methods comprise administering the modified or engineered cells disclosed herein. In some aspects, the methods comprise administering the cell populations disclosed herein. In some aspects, the methods comprise administering a composition comprising CD56+ / CD3- cells or iNK cells, or CD56+ / CD3- cells or iNK cells, and a pharmaceutically acceptable carrier.

[0156] In some respects, diseases or ailments include NK-related diseases.

[0157] In some respects, disease or ailment includes cancer; for example, the subject has cancer. In some respects, cancer includes 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, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia. Blood disorders, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, 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 aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory or relapsed after one or more prior anticancer therapies. In some aspects, the one or more prior anticancer therapies include standard of care.

[0158] In some aspects, the compositions disclosed herein are administered in combination with other anticancer therapies. In some aspects, the other anticancer therapies include chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some aspects, the other anticancer therapies include chemotherapy. In some aspects, the other anticancer therapies include immune checkpoint inhibitors. In some aspects, the other anticancer therapies include PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, GITR antagonists, or any combination thereof. In some aspects, the anticancer therapy includes an antibody or its antigen-binding portion that specifically binds to and inhibits PD-1. In some aspects, the anticancer therapy includes an antibody or its antigen-binding portion that specifically binds to and inhibits PD-L1.

[0159] In some aspects, the method further includes pre-treating the subject prior to the administration of the immune cell population. In some aspects, chemotherapy is administered to the subject prior to the administration of the immune cell population. In some aspects, immune-depleting chemotherapy is administered to the subject prior to the administration of the immune cell population. In some aspects, immune-depleting chemotherapy includes cyclophosphamide, fludarabine, or both.

[0160] In some aspects, the method includes administering (i) an expanded cell population and (ii) cytokines to a subject. In some aspects, cytokines include IL-2, its analogues, its variants, or fragments thereof.

[0161] In some respects, the cells of this disclosure are at least about 1 x 10 6 1 cell, at least about 2 x 10 6 1 cell, at least about 3 x 10 6 1 cell, at least approximately 4 x 10 6 1 cell, at least about 5 x 10 6 1 cell, 1 x 10 7 1 cell, at least about 2 x 10 7 1 cell, at least about 3 x 10 7 1 cell, at least approximately 4 x 10 7 1 cell, at least about 5 x 10 7 1 cell, 1 x 10 8 1 cell, at least about 2 x 10 8 1 cell, at least about 3 x 10 8 1 cell, at least approximately 4 x 10 8 1 cell, at least approximately 5 x 10 8 1 cell, 1 x 10 9 1 cell, at least about 2 x 10 9 1 cell, at least about 3 x 10 91 cell, at least approximately 4 x 10 9 One cell or at least about 5 x 10 9 A dose of one cell was administered to the subject.

[0162] The cells disclosed herein are of low toxicity and therefore can be safely administered in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously or intralymphaticly, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0163] In some embodiments of the invention, the method of the invention for clinical use is combined with other agents for the effective treatment of diseases or disorders including NK-related diseases as described above.

[0164] II.D. Cell therapy products Some aspects of this disclosure relate to a cell population comprising a plurality of cells modified as disclosed herein. Some aspects of this disclosure relate to a cell therapy comprising a cell population modified as disclosed herein. In some aspects, the cell population is cryopreserved. Any cryopreservation method for cells (e.g., immune cells) may be used with 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 cell transport.

[0165] The cell therapies and / or cell populations disclosed herein may be further formulated with one or more excipients. Any excipient that can preserve cells may be used in the methods and compositions disclosed herein. In some aspects, the cell therapies and / or cell populations are formulated with one or more excipients that allow for cryopreservation of cells (e.g., DMSO).

[0166] In some aspects, the cell population comprises a plurality of cells modified as disclosed herein and one or more additional cells. In some aspects, 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 cell population comprise a plurality of cells modified as disclosed herein. In some aspects, at least about 10% of the cells in the cell population comprise a plurality of cells modified as disclosed herein. In some aspects, at least about 20% of the cells in the cell population comprise a plurality of cells modified as disclosed herein. In some aspects, at least about 25% of the cells in the cell population comprise a plurality of cells modified as disclosed herein. In some aspects, at least about 30% of the cells in the cell population comprise a plurality of cells modified as disclosed herein. In some aspects, at least about 40% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 50% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 60% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 65% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 70% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 75% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 80% of the cells in the cell population contain a plurality of cells modified as disclosed herein. In some aspects, at least about 90% of the cells in the cell population contain a plurality of cells modified as disclosed herein.

[0167] Unless otherwise stated, the practices disclosed herein will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. Such techniques are well explained in the literature. See, for example, Sambrook et al., eds. (1989) *Molecular Cloning: A Laboratory Manual* (2nd edition; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (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., U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) *Nucleic Acid Hybridization*; Hames and Higgins, ed. (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., NYMiller and Calos (eds., 1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al. (eds.), Methods In Enzymology, Vol. 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, Vol. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooke, Antisense Drug Technology: Principles, Strategies and Applications, 2nd ed., CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0168] All references cited above, as well as all references cited in this paper, are incorporated into this paper in their entirety by way of citation.

[0169] The following examples are provided in an illustrative rather than restrictive manner.

[0170] Example Example 1 Generation of induced pluripotent stem cells transduced by NKp46 To generate NKp46-transduced induced pluripotent stem cells (iPSCs), the iPSC line Ff101s04 was used at a rate of 1 x 10⁻⁶. 4Cells / well were seeded in 24-well iMatrix511 plates containing StemFit AK03N medium (containing 50 μM Y-27632 (a selective p160ROCK inhibitor)). The next day, the medium was replaced with StemFit AK03N, and iPSCs were infected with a lentiviral vector expressing the NKp46 gene (SEQ ID NO: 1) under CAG promoter control to generate FfI01s04 / CAG-NKp46 cells. The proliferated FfI01s04 / CAG-NKp46 cells were stained with an anti-NKp46 antibody (APC, BioLegend) conjugated with anti-phycocyanin, and analyzed by flow cytometry to quantify the number of NKp46-expressing cells. The number of NKp46-expressing cells in untransduced iPSCs and CAG-NKp46-transduced iPSCs is shown in the figure. Figure 1 middle.

[0171] Example 2 iNK cell differentiation from NKp46-derived iPSCs To differentiate invariant natural killer (iNK) cells from NKp46-derived iPSCs, FfI01s04 / NKp46 cells were dispersed in StemFit AK03N medium on day 0 and incubated at 5 x 10⁻⁶ cells per ... 5Cells / well were seeded in 6-well plates treated with ultra-low adhesion. StemFit AK03N medium contained 10 μM CHIR99021 (GSK-3 inhibitor) and 10 μM Y-27632. On Day 1 (Day 2), cells were dispersed in human pluripotent stem cell (HPC) induction medium (Invitrogen) containing bone morphogenetic 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 medium, and the cells were cultured for 2 days. On day 4, cells were redistributed to another 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 another 3 days. On day 7, cells were exposed to another 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 for another 7 days. During this 7-day culture period, the medium was changed every 2–3 days.

[0172] On day 14, cells were harvested and CD34-positive cells were isolated using CD34-microbeads (Miltenyi Biotech) at a concentration of 1.16 x 10⁻⁶.5 Cells / dish were seeded in 10 cm culture dishes. Each 10 cm culture 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. These cells were cultured in 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 stromal cell-derived factor-1α (SDF-1α, Peprotech). On day 21, cells were harvested and cultured at 1 x 10⁻⁶ cells / mL. 6 Cells / dish were seeded in fresh 15 cm culture dishes coated with hDLL4 / RetroNectin, and all cells were harvested on day 35.

[0173] On day 35, harvest cells at 5 x 10⁻⁶ ppm. 5 Cells / dish were seeded in MEMα medium supplemented with 15% FBS, 4 mM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mg / ml ascorbate 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 in 48-well plates. On day 39, half the medium was replaced in each well, and all cells were harvested as differentiated iNK cells on day 42. iNK cells were stained with antibodies listed in Table 2, and the number of cells expressing NKp46 and the number of CD56+ / CD3- phenotype cells were analyzed by flow cytometry, as shown in Table 2. Figure 2A and Figure 2B As shown in the figure. The results indicate that the percentage of cells expressing NKp46 was good, and CD56+ / CD3- accounted for approximately 83.2% of the cell population.

[0174] Table 2. Staining agents used for peptide detection

[0175] Example 3 iNK cell expansion iNK cells were packed at 5 x 10 4 Cells / well were seeded in 96-well plates and administered via anti-NKp46 / CD2 antibody-conjugated beads (Miltenyi) supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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 (a caspase inhibitor, R&D systems). Cells were expanded in Iscove Modified Dalbergian Medium (IMDM) using [systems]. After 4 days of culture, cells were sputtered at 1 x 10⁻⁶ cells / mL. 6 Cells / well were transferred to G-Rex24 medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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. During the subsequent 10-day culture period, the medium was changed every 2–3 days. The repeatability and scalability of iNK cells were plotted as follows: Figure 3 As shown in Table 3, iNK cells differentiated from NKp46 untransduced iPSCs were expanded using the same method. Under these conditions, the expansion rate of iNK cells was relatively low, as indicated in Table 3.

[0176] As indicated in Table 4, the expanded iNK cells were stained with antibodies, and the expression of NKG2D, CD56, CD16, and CD3 was analyzed by flow cytometry. Figures 4A to 4D As shown. Figure 4A As shown, NKp46+ and CD56+ cells accounted for approximately 98.3% of the cell population. Figure 4B As shown, NKp46+ and CD3- cells accounted for approximately 97.2% of the cell population. Figure 4C As shown, NKp46+ and NKG2D+ cells accounted for approximately 52% of the cell population. Figure 4D As shown, NKp46+ and CD16+ cells accounted for approximately 50.9% of the cell population.

[0177] Table 3. Fold change (iNK cells differentiated from NKp46 without iPSC transduction)

[0178] Table 4. Staining agents used for peptide detection

[0179] Example 4 Effects of anti-CD30 antibody on iNK cell expansion On day 1, after two expansions using the method described in Example 3, the cryopreserved and harvested day 13 iNK cells were cultured for 1 day in Iscove modified Dalberg's medium (IMDM) supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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 then cultured at a density of 2 x 10⁻⁶ cells / mL. 6 Cells / well were seeded in T25 flasks and, on day 0, with or without 300 ng / ml anti-CD30 antibody (81316, R&D systems), supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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&Dsystems) was amplified in the IMDM by plate-bound anti-NKp46 antibody (monoclonal mouse IgG2B clone #195314, R&Dsystems).

[0180] After culturing for 3 days, the cells were cultured at 5 x 10⁻⁶ cells / year. 6 Cells / well were transferred to G-Rex6 medium supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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. The medium was changed every 2–3 days during an additional 11 days of culture, and scalability was checked on day 14 (3rd amplification). Repeatability was checked using the same protocol from day 0 to day 14 of Example 4 (4th amplification).

[0181] The expandability of iNK cells, with or without anti-CD30 antibodies, is shown in... Figure 5A and Figure 5B As shown in Table 5, the expansion rate of iNK cells without anti-CD30 antibodies was lower compared to those with anti-CD30 antibodies.

[0182] Table 5. Fold changes in the 3rd and 4th amplifications

[0183] Example 5 Effects of anti-CD30 antibody on the in vivo persistence of iNK cells On day 0 of the fourth expansion, the iNK cells harvested on day 14 of the third expansion were used at a dose of 1 x 10⁻⁶. 7Cells / well were seeded in T75 flasks and, with or without 300 ng / ml anti-CD30 antibody (81316, R&D systems), supplemented with 15% FBS, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml ascorbate 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 (a caspase inhibitor, R&D systems). Activation of anti-NKp46 antibody (R&D systems) via plate binding in Iscove modified Dalberg medium (IMDM).

[0184] After 3 days of culture, cells were cultured at 5 x 10⁻⁶ cells / ml with or without 300 ng / ml anti-CD30 antibody (81316, R&D systems). 6 10 cells / well were transferred to G-Rex6 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).

[0185] On day 5, after activation, the cells were placed at 2 x 10 7 Cells / well were transferred to G-Rex6 and harvested on day 7. These cells were cryopreserved in CS10 (BioLife Solutions) and were to be administered intravenously to 8-week-old NOG mice (0.5 or 1 x 10⁻⁶ cells / well). 7Before administration, cells were thawed (N=3) per mouse. Peripheral blood was collected weekly after administration and fixed with FACS lysis buffer (BD), then 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 by flow cytometry while iNK cells persisted in vivo. Figure 6 As shown, iNK cells expanded with anti-CD30 antibody exhibited higher in vivo persistence.

[0186] Example 6 iPS cells differentiate into HP cells as the main body. QHJI cells are an iPS cell line derived from peripheral blood mononuclear cells from healthy individuals. On day 0, QHJI cells dispersed in StemFit complete medium were incubated at 6 x 10⁻⁶ cells / day under hypoxic (5% O₂) conditions. 5 Cells / well were seeded in 6-well plates treated with ultra-low adhesion. 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).

[0187] 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 redistributed to 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 another 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 another 7 days. During this 7-day culture period, the culture medium was changed every 2-3 days.

[0188] Example 7 The main body of HP cells differentiates into a population containing CD4- cells. On day 14, the cell population obtained from Example 6 (“HP cell body”) without any cell separation was measured at 3.12 x 10⁻⁶. 6 Cells / dish were seeded in 15 cm culture dishes and cultured at 37°C and 5% O2. It is worth noting that various seeding densities can be used, and the aforementioned seeding density is an example. Each 15 cm culture 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 passaged into fresh 15 cm culture dishes coated with hDLL4 / RetroNectin. On day 28, the cells were further passaged into fresh 15 cm culture dishes coated with hDLL4 / RetroNectin. On day 35, all cells, including CD4-cells, were harvested.

[0189] Example 8 NK cell transduction A. The main body differentiates into NK cell main body. On day 35, a cell population containing CD4- cells (i.e., cells obtained from Example 7 without undergoing any cell separation) was divided into 1 x 10⁻⁶ cells. 6 Cells / well were seeded in 48-well plates and cultured at 37°C for three days at 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.

[0190] On day 38, cells were dispersed in another 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. On day 42, all cells, including NK cells (“NK cell body”), were harvested.

[0191] B. Activation and expansion of NK cells On day 41, at 4°C, retrotronectin (0.15 ug / cm) was used. 2 ) and anti-CD3 antibody (UCHT1, R&D Systems) (0.6 μg / cm2) or anti-CD16 antibody (3G8, Biogems) (0.6 μg / cm2) 2 The cells were spread into tissue culture containers and left for 16 hours. On day 42, the NK cells harvested from Example 8A were resuspended in IMDM medium (Thermo Fisher Scientific (Gibco)) 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), 10 ng / mL IL-7, 50 ng / mL IL-15, 50 ng / mL IL-18, 20 ng / mL IL-21, 10 μM Z-VAD-FMK caspase inhibitor, and 300 ng / mL anti-CD30 antibody (81316, R&D Systems) to a final volume of 1 x 10⁻⁶. 5 10 cells / mL and transferred to anti-CD3 coated containers for three days of incubation.

[0192] On day 45, cells were collected and transferred to γ-retroviral-coated containers 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 ascorbate 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 then transferred to G-Rex containers (Wilson Wolf) for further culture for 11 days. IL-7 was used with anti-CD3 antibody, and IL-2 was used with anti-CD16 antibody. The medium was replaced with 50-80% fresh medium every 2-3 days using the same composition, and if the cell concentration exceeded 4 x 10⁻⁶ cells / mL, the medium was replenished. 7 cells / cm 2 The cells are then diverted to additional G-Rex containers. On day 56, the cells are harvested for final use.

[0193] If a sufficient number of cells is required, the 15-day process described herein (days 41-56) is repeated multiple times to increase the total number of NK cells produced. For iNK-CD19 CAR cells, cells undergo 5 rounds of activation (5 x 15-day process), with CAR transduction occurring in the second activation. For iNK-Meso CAR cells, cells undergo 4 rounds of activation (4 x 15-day process), with CAR transduction occurring in the third activation.

[0194] C. NK cell gamma retrovirus transduction.

[0195] For NK cell gamma retroviral transduction, on day 44, tissue culture vessels were incubated at 4°C with retrotronectin (10.5 μg / cm³). 2Spread the cells for 16 hours. On day 45, incubate the γ-retrovirus at 32°C on the retrotronectin-spread container for 2 hours, and then harvest. On day 45, centrifuge the NK cells described in Example 8B at 300 xg at 32°C onto the retrotronectin / γ-retrovirus-spread container for 5 minutes and incubate overnight. If a higher percentage of transduced cells is required, repeat the steps described on days 44 and 45 to perform γ-retrovirus transduction twice. On day 46 (or day 47 if two γ-retroviral transductions were performed), 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 ascorbate-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). Subsequently, the medium was replaced 75% every 2–3 days with the same composition, and if the cell concentration exceeded 4 x 10⁻⁶ cells / mL, the cells were transferred to IMDM medium. 7 cells / cm 2 The cells are then diverted to additional G-Rex containers. On day 56, the cells are harvested for final use.

[0196] If necessary, the 15-day process described in Example 8B (days 41 to 56) may be repeated multiple times to increase the total number of iNK cells produced. For iNK-CD19 CAR cells, the cells undergo 5 rounds of activation (5 x 15-day process - the same process as day 41 to 56), and CAR and IL-15Ra / IL-15 gene transduction is performed in the second activation. For iNK-Meso CAR cells, the cells undergo 4 rounds of activation (4 x 15-day process - the same process as day 41 to 56), and CAR and IL-15Ra / IL-15 gene transduction is performed in the third activation.

[0197] Table 6 shows the different culture conditions and the CAR+ and total cell counts based on each culture condition. Those cells were used in the in vivo studies in Example 10.

[0198] Table 6. Cultivation conditions.

[0199] Example 9 CAR-NK cell preparation NK cells are further modified to express one or more CARs. NK cells are modified by: (1) synthesizing an anti-CD19 CAR gene or an anti-mesothelin CAR gene and an IL-15Rα / IL-15 gene; (2) preparing a retroviral vector containing an anti-CD19 CAR gene or an anti-mesothelin CAR gene and an IL-15Rα / 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-15Rα / IL-15 gene.

[0200] CAR / IL15-NK cell preparation The anti-CD19 CAR gene was prepared by synthesizing oligopeptides designed to be arranged from the N-terminus, as shown in Table 6.

[0201] Table 7. Oligopeptides.

[0202] The anti-CD19 CAR was constructed according to US2021 / 0292713, which is incorporated herein by reference in its entirety. The anti-mesothelin CAR was constructed according to WO2023 / 009700, which is incorporated herein by reference in its entirety.

[0203] Example 10 In vivo antitumor activity of iNK-CD19 CAR Nalm6 cells (ATCC; cancer cells) expressing luciferase (5 x 10⁻⁶) were transported via the tail vein. 5 (10 x 10^6 cells) were transplanted into NOD / Shi-scid, IL-2Rγ empty mice (“NSG mice”). 8-9 week old female NSG mice were obtained from The Jackson Laboratory. On day 4, four days after Nalm6 cell transplantation, the Nalm6-transplanted NSG mice were administered iNK-CD19 CAR cells (10 x 10^6 cells) dispersed in 0.2 ml PBS via the tail vein. 6 0.2 ml of PBS (1 CAR+ cells) or cell-free PCR. Following administration of iNK-CD19 CAR cells or PBS, luciferase was administered to mice via the tail vein. Luciferase activity was measured using an IVIS imaging system (PerkinElmer) for 42 days.

[0204] Figure 7A and Figure 7BThe following are shown: (1) PBS treatment (mediator control), (2) iNK-CD19 CAR cells activated with anti-CD3 antibody using the cell cultures described in Examples 6 to 8 (“iNK-CD19 CAR #1”), and (3) iNK-CD19 CAR cells activated with anti-CD-16 antibody using the cell culture methods described in Examples 6 to 8, but excluding Example 8A (“iNK-CD19 CAR #2”). Although detectable tumor growth was observed in the PBS mediator treatment group up to day 14 after tumor cell infusion and gradually increased from that time point, significant tumor cell expansion was not observed until day 28 in both iNK-CD19 CAR groups. Figure 7C The graph illustrates the percentage change in body weight of mice during the 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. In conclusion, these data demonstrate the ability of iNK-CD19 CAR cells to reduce the proliferation of Nalm6 tumor cells in vivo.

[0205] Example 11 In vitro repeat killing ability of iNK-mesothelin CAR cells Repeated antigen stimulation (RAS) assays were performed using GSU tumor cells expressing high levels of mesothelin and red fluorescent protein (RFP) and iNK-mesothelin CAR cells (iNK-Meso CAR cells). GSU-RFP cells were spaced at 1 x 10⁻⁶ cells per well. 5 One cell was seeded into a 96-well plate and allowed to grow overnight. The next day, (1) cell-free, (2) 1 x 10 cells were seeded into the wells. 5 One untransduced (UTD) iNK or (3) 1 x 10 5 One iNK-Meso CAR cell was added to each well. The RFP-positive surface area of ​​each well was measured every 4 hours using Incucyte.

[0206] To repeatedly attack iNK cells, an additional 1 x 10⁻⁶ cells will be added every 2-3 days. 5 One GSU-GFP cell was added to each well. This process is referred to as "attack" in this paper. A total of four attacks were performed.

[0207] Figure 8The growth of GSU tumor cells during the study is illustrated, measured by the total RFP-positive surface area per well per image. GSU cells were continuously grown under tumor-only conditions until approximately 120 hours, at which point the wells became over-confluent. In contrast, UTD and iNK-Meso CAR cells significantly reduced the tumor cell growth rate. UTD iNK cells completely suppressed tumor growth in one attack, while iNK-Meso CAR cells controlled tumor cell growth in two attacks. These results demonstrate that while UTD iNK cells possess some innate tumor control capabilities, CAR transduction further enhances the ability of iNK cells to kill tumor cells.

[0208] Example 12 In vivo antitumor activity of iNK-Meso CAR GSU cells expressing luciferase (1 x 10⁻⁶ cells) were injected intraperitoneally. 6 (1 cell) was transplanted into NOD / Shi-scid, IL-2R γ empty mice (“NSG mice”). 8-9 week old female NSG mice were obtained from The Jackson Laboratory.

[0209] Four days after Nalm6 cell transplantation, iNK-Meso CAR cells (20 x 10⁻⁶) dispersed in 0.2 ml PBS were administered via tail vein to NSG mice transplanted with GSU. 6 0.2 ml of PBS (with or without iNK-Meso cells) or cell-free PBS was administered to mice via intraperitoneal injection after administration of iNK-Meso cells or PBS. Luciferase activity was measured using an IVIS imaging system (PerkinElmer) for 31 days. Table 8 below shows the two study groups: PBS alone and iNK-Meso CAR cells.

[0210] Figure 9A and Figure 9B Tumor growth is shown by luciferase activity in mice treated with (1) PBS (mediator control) or (2) iNK-Meso CAR cells. Treatment with iNK-Meso CAR cells significantly delayed tumor growth compared to the PBS control, indicating that iNK-Meso CAR cells have antitumor activity.

[0211] In addition, peripheral blood samples were collected to assess in vivo iNK-Meso CAR cell proliferation of human CD45, which is present only on the introduced iNK-Meso CAR cells, by flow cytometry. Figure 10A and Figure 10BThe cell growth kinetics of iNK-Meso CAR cells over time are shown, indicated by the percentage of total viable human CD45+ cells and the absolute number of human CD45+ cells per μL of mouse peripheral blood. Some iNK-Meso CAR cells were detectable on day 4 (the earliest time point assessed). However, iNK-Meso CAR cell levels continued to increase, ultimately peaking on day 11. The detectable increase in iNK cells from day 4 to day 11 indicates that iNK-Meso CAR cells can proliferate in an in vivo mouse model system.

[0212] Table 8.

[0213] It should be understood that the detailed description section, rather than the summary and abstract section, is intended to interpret the claims. The summary and abstract section may set forth one or more, but not all, exemplary embodiments of this disclosure as contemplated by the inventors, and is therefore not intended to limit this disclosure and the appended claims in any way.

[0214] The foregoing description of specific embodiments so fully reveals the general nature of this disclosure that others can readily modify and / or adapt various applications of such specific embodiments by applying knowledge within the art without departing from the general concept of this disclosure. Therefore, such modifications and adaptations are intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that the wording or terminology used herein is for descriptive and not limiting purposes, and that the terminology or terminology of this specification should be interpreted by those skilled in the art based on the teachings and guidance.

[0215] The breadth and scope of this disclosure should not be limited to any of the exemplary embodiments described above, but should be defined solely by the appended claims and their equivalents.

[0216] All references cited throughout this application (including bibliographic references, U.S. or foreign patents or patent applications, and websites) are hereby expressly incorporated by way of reference as if they were written in their entirety herein for any purpose. In the event of any inconsistency, the material disclosed herein shall prevail.

[0217] While various specific aspects have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the invention. Many changes will be apparent to those skilled in the art upon reading this description.

Claims

1. A method for amplifying CD56+ / CD3- cells, the method comprising: The CD56+ / CD3- cells are cultured 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 of claim 1, wherein the CD56+ / CD3- cells are derived from pluripotent stem cells.

3. The method of 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 of claim 2 or 3, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

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

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

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

8. The method of 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 mimics thereof.

9. The method of claim 8, wherein the cytokine or its mimic is IL-15.

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

11. A method for producing modified CD56+ / CD3- cells, the method comprising: (A) Providing ex vivo host cell generation comprising hematopoietic progenitor cells (HPCs), said HPCs containing at least one exogenous nucleic acid molecule encoding one or more NKp46 polypeptides, and (B) Differentiate the HPC to produce one or more CD56+ / CD3- cells.

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

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

14. The method of 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 of claim 13 or 14, wherein the pluripotent stem cell is an iPSC.

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

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

19. The method of 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 mimics thereof.

20. The method of claim 19, wherein the cytokine or its mimic is IL-15.

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

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

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

24. A CD56+ / CD3- cell, said CD56+ / CD3- cell obtained by the method of any one of claims 1 to 23.

25. A pharmaceutical composition comprising the CD56+ / CD3- cells and excipients as described in claim 24.

26. A method of treating a disease or ailment of a subject in need, the method comprising administering to the subject the CD56+ / CD3- cells of claim 24 or the pharmaceutical composition of claim 25.

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

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

29. The method of claim 28, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

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

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

33. A CD56+ / CD3- cell, said CD56+ / CD3- cell obtained by the method of any one of claims 27 to 32.

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

35. A method of treating a disease or ailment of a subject in need, the method comprising administering to the subject the CD56+ / CD3- cells of claim 33 or the pharmaceutical composition of claim 34.

Images