WO2023078288A1

WO2023078288A1 - Systems and methods for enhanced immunotherapies

Info

Publication number: WO2023078288A1
Application number: PCT/CN2022/129205
Authority: WO
Inventors: Xiangjun HE; Yangbin Gao; Luhan Yang
Original assignee: Hangzhou Qihan Biotechnology Co., Ltd.
Priority date: 2021-11-03
Filing date: 2022-11-02
Publication date: 2023-05-11
Also published as: TW202325843A

Abstract

Provided are immune cells that can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell). The immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell, can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo and are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors).

Description

SYSTEMS AND METHODS FOR ENHANCED IMMUNOTHERAPIES

CROSS-REFERENCE

This application claims the benefit International Application No. PCT/CN2021/128464, filed November 03, 2021, and International Application No. PCT/CN2022/124454, filed October 10, 2022, which are entirely incorporated herein by reference.

BACKGROUND

Cancer (e.g., neoplasm, tumor) is a leading cause of death worldwide, accounting for about 10 million deaths annually. Cancer continues to bring increasing health, economic, and emotional burden on individuals, families, communities, and countries. Increase understanding of cancer biology (e.g., specifically cancer immune biology) and genetic engineering has encouraged development of adoptive cell therapies (e.g., cellular immunotherapy) , with a goal to treat or control a number of different cancers.

SUMMARY

The present disclosure provides methods and systems for treating cancer. Some aspects of the present disclosure provide engineered immune cells (e.g., engineered natural killer (NK) cells) and methods of use thereof for treatment of cancer, such as, e.g., as hematologic malignancies or solid tumors.

In an aspect, the present disclosure provides an engineered NK cell, comprising: (1) enhanced expression or activity level of an activating NK receptor as compared to a control NK cell; and (2) one or more members selected from the group consisting of:

(i) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a NKG2D ligand;

(ii) reduced expression and/or activity level of an endogenous CD38 as compared to the control NK cell;

(iii) a reduced expression of one or more genes selected from the group consisting ofBCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70;

(iv) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(v) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and

(vi) a safety switch capable of effecting death of the engineered NK cell.

In another aspect, the present disclosure provides an engineered NK cell, comprising enhanced expression or activity level of an activating NK receptor as compared to a control NK cell, wherein the activating NK receptor is configured to bind an antigen of NKG2D, NKP30, NKP44, NKP46, NKP80, and/or DNAM1, and wherein the activating NK receptor does not comprise a heterologous intracellular signaling domain.

In another aspect, the present disclosure provides an engineered NK cell, comprising a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is selected from the group consisting of CD70, CLEC12A, TIM-3, CD9, CD26, TRAIL-R4 (DCR2) , and NKG2D ligand, wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the chimeric polypeptide receptor.

In another aspect, the present disclosure provides an engineered NK cell, comprising: (1) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is CD38 and/or BCMA; and (2) one or more members selected from the group consisting of:

(i) enhanced expression or activity level of an activating NK receptor as compared to a control NK cell;

(ii) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(iii) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and

(iv) a safety switch capable of effecting death of the engineered NK cell,

wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the one or more members.

In another aspect, the present disclosure provides an engineered NK cell, comprising: (1) reduced expression or activity level of a suppressor of cytokine (SOCS) protein; and (2) one or more members selected from the group consisting of:

(i) enhanced expression or activity level of endogenous activating NK receptor as compared to a control NK cell;

(ii) a heterologous interleukin (IL) cytokine and/or receptor for enhanced interleukin signaling as compared to the control NK cell;

(iii) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a NKG2D ligand;

(iv) reduced expression and/or activity level of an endogenous CD38 as compared to the control NK cell;

(v) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(vi) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59;

(vii) a safety switch capable of effecting death of the engineered NK cell; and

(viii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not a NKG2D ligand.

In some embodiments of any one of the compositions disclosed herein, the engineered NK cell exhibits enhanced cytotoxicity against a target cell as compared to a control cell. In some embodiments of any one of the compositions disclosed herein, the engineered NK cell induces reduced immune response in a host cell as compared to a control cell.

In some embodiments of any one of the compositions disclosed herein, the host cell is an immune cell.

In another aspect, the present disclosure provides a method comprising: administering to a subject in need thereof a subject composition of any one of the compositions disclosed herein. In some embodiments, the separate therapeutic agent comprises a chemotherapeutic agent.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG. ” herein) , of which:

FIG. 1 illustrates nucleic acid vectors encoding one or more genes, such as an activating NK receptor.

FIG. 2A illustrates nucleic acid vectors encoding an activating NK receptor and/or a DAP protein. FIGs. 2B and 2C illustrate expression level of the activating NK receptor in cells treated with one of the nucleic acid vectors from FIG. 2A.

FIG. 3A illustrates nucleic acid vectors encoding a chimeric polypeptide receptor (e.g., a chimeric antigen receptor) exhibiting specific binding to an antigen, such as CLL-1. FIG. 3B illustrates examples of transgenes encoding a CD16 variant that can be inserted into the genome of a host cell. FIG. 3C illustrates different endogenous genes that can be suppressed in an engineered immune cell for immunotherapies.

FIG. 4A illustrates a structural of anti-CLL-1 chimeric polypeptide receptor (e.g., anti-CLL-1 chimeric antigen receptor (CAR) ) . FIG. 4B shows cytotoxicity level of anti-CLL-1 CAR NK cells and non-modified control NK cells, against CLL-1 expressing HL60 tumor cells. FIG. 4C shows expression level of CD107A in anti-CLL-1 CAR NK cells and non-modified control NK cells, upon being in contact with the CLL-1 expressing HL60 tumor cells.

FIG. 5A shows CD56 expression of engineered NK cells from iPSCs engineered to enhance expression and/or activity level of an activating NK receptor. FIG. 5B shows FACS fluoresces intensity of engineered NK cells overexpressing an activating NK receptor.

FIG. 6A shows cytotoxicity analysis of engineered NK cells with enhanced NK receptor and NK cells derived from

iPSCs, against solid tumor cells, Calu-3. FIG. 6B shows serial cytotoxicity analysis with engineered NK cells and NK cells derived from

iPSCs against THP1 and SKOV3. FIG. 6C shows cytotoxicity analysis with engineered NK cells and non-modified control NK cells against THP1 and MOLM13. FIG. 6D shows expression profile of NKG2D and NKP30 in engineered NK cells compared to NK cells derived from

iPSCs during in vitro killing assay.

FIG. 7 shows tumor burden of mice injected with engineered NK cells expressing NK receptors compared to mice injected NK cells without engineering.

FIG. 8A shows change in mice body weight post injection of engineered NK cells expressing NK receptors using SKOV3 tumor model. FIG. 8B shows survival proportions of mice harboring SKOV3 tumor with or without injection of engineered NK cells. FIG. 8C shows pharmacokinetics of hCD56+ cells in peripheral blood of mice injected with engineered NK cells. FIG. 8D shows tumor burden of mice with SKOV3 injected with different dose of engineered NK cells.

FIG. 9A shows cytotoxicity analysis of NK cells with TGFb-R2 knock out and control NK cells incubated with GFP labeled THP1 cells, with different TGF-b dosage at 24 hours and 72 hours. FIG. 9B shows serial cytotoxicity analysis of NK cells with TGFb-R2 knock out and control NK cells incubated with GFP labeled THP1 cells with different TGF-b dosage.

FIG. 10A shows knockout efficiency of TGFb-R2 in NK cells. FIG. 10B shows FACS analysis of NKG2D and NKP30 expression in NK cells with TGFb-R2 knocked out, with or without TGF-b treatment.

FIG. 11A shows cytotoxicity analysis of cord blood natural killer (CBNK) incubated with different ratio of wild type K562 or HLA-E K562. FIG. 11B shows result of CD107a assay of CBNK incubated with different ratio of K562, HLA-E K562, or CBNK alone. FIG. 11C shows FACS analysis of NKG2A expression in CBNK with knockout of NKG2A and with HS2 knockout as control. FIG. 11D shows cytotoxicity analysis of CBNK with NKG2A knockout and CBNK with H2S knockout incubated different ratio of wild type K562 or HLA-E K562.

FIG. 12A shows quantification of CD45+/CD46+ NK cells differentiated from iPSCs engineered with SOCS2 knockout. FIG. 12B shows FACS fluorescence intensity of engineered NK cells with SOCS2 knockout. FIG. 12C shows number of hCD56+/hC45+ NK cells cultured in presence of peripheral blood cells with IL-2 or IL-15 supplementation, to compare persistency level of NK cells with SOCS2 knockout, that of unmodified NK cells, and that of NK cells with CISH knockout.

FIG. 13 illustrates nucleic acid vectors encoding an DNAM1 and TIGIT and shows FACS analysis of TIGIT expression in 293T cells transfected with the nucleic acid vectors.

FIG. 14 illustrates nucleic acid vector encoding NKP80 and shows FACS analysis of NKP80 expression in iPSCs transfected with the nucleic acid vector.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used in the specification and claims, the singular forms “a, ” “an, ” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “achimeric transmembrane receptor” includes a plurality of chimeric transmembrane receptors.

The term “about” or “approximately” generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

The use of the alternative (e.g., “or” ) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives.

The term “cell” generally refers to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses) , an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like) , seaweeds (e.g. kelp) , a fungal cell (e.g., a yeast cell, a cell from a mushroom) , an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc. ) , a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal) , a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc. ) , and etcetera. Sometimes a cell is not originating from a natural organism (e.g. a cell can be a synthetically made, sometimes termed an artificial cell) .

The term “reprogramming, ” “dedifferentiation, ” “increasing cell potency, ” or “increasing developmental potency, ” as used interchangeable herein, generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.

The term “differentiation” generally refers to a process by which an unspecialized ( “uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized ( “committed” ) position within the lineage of a cell. The term “committed” generally refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.

The term “pluripotent” generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) . For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .

The term “induced pluripotent stem cells” (iPSCs) generally refers to stem cells that are derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature. In some cases, iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells. In some cases, iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .

The term “embryonic stem cell” (ESCs) generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In some cases, ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) . In some cases, ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .

The term “isolated stem cells” generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism. For example, HSCs can be isolated from a mammal’s body, such as a human body. In another example, an embryonic stem cells can be isolated from an embryo.

The term “isolated” generally refers to a cell or a population of cells, which has been separated from its original environment. For example, a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist. An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample. The term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.

The term “hematopoietic stem and progenitor cells, ” “hematopoietic stem cells, ” , “hematopoietic progenitor cells, ” or “hematopoietic precursor cells, ” as used interchangeably herein, generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) , and lymphoid lineages (T cells, B cells, NK cells) . In some cases, HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.

The term “immune cell” generally refers to a differentiated hematopoietic cell. Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.

The term “NK cell” or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) . In some cases, NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some cases, isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.

The term “nucleotide, ” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) . The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4-(4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) . Specific examples of fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .

The term “polynucleotide, ” “oligonucleotide, ” or “nucleic acid, ” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or flurescein linked to the sugar) , thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

The term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′and 3′ends. In some uses, the term encompasses the transcribed sequences, including 5′and 3′untranslated regions (5′-UTR and 3′-UTR) , exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .

The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product. ” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated, ” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

The term “peptide, ” “polypeptide, ” or “protein, ” as used interchangeably herein, generally refers to a polymer of at least two amino acid residues joined by peptide bond (s) . This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) . The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids, ” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

The term “derivative, ” “variant, ” or “fragment, ” as used herein with reference to a polypeptide, generally refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.

The term “engineered, ” “chimeric, ” or “recombinant, ” as used herein with respect to a polypeptide molecule (e.g., a protein) , generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule. The term “engineered” or “recombinant, ” as used herein with respect to a polynucleotide molecule (e.g., a DNA or RNA molecule) , generally refers to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

The term “gene editing moiety” generally refers to a moiety which can edit a nucleic acid sequence, whether exogenous or endogenous to a cell comprising the nucleic acid sequence. In some embodiments, a gene editing moiety regulates expression of a gene by editing a nucleic acid sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing genomic DNA sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.

Alternatively or in addition to, a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operatively coupled to the gene (or a target sequence within the gene) , and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. In some cases, a gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. A gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template. A gene editing moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template. In some cases, a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript.

The term “antibody” generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) , as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc. ) . A derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab′, F (ab′) 2, variable fragment (Fv) , single chain variable fragment (scFv) , minibodies, diabodies, and single-domain antibodies ( “sdAb” or “nanobodies” or “camelids” ) . The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies) .

The term “chimeric polypeptide receptor” generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each antigen binding moiety capable of binding to a specific antigen. A chimeric polypeptide receptor can be monospecific (i.e., capable of binding to one type of specific antigen) . Alternatively, a chimeric polypeptide receptor can be multi-specific (i.e., capable of binding to two or more different types of specific antigens) . A chimeric polypeptide receptor can be monovalent (i.e., comprising a single antigen binding moiety) . Alternatively, a chimeric polypeptide receptor can be multivalent (i.e., comprising a plurality of antigen binding moieties) . In some cases, a chimeric polypeptide receptor can comprise a T-cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR) .

The term “antigen binding domain” generally refers to a construct exhibiting preferential binding to a specific target antigen. An antigen binding domain can be a polypeptide construct, such as an antibody, modification thereof, fragment thereof, or a combination thereof. The antigen binding domain can be any antibody as disclosed herein, or a functional variant thereof. Non-limiting examples of an antigen binding domain can include a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy-chain-only antibody (VNAR) , Ig NAR, a chimeric antibody, a recombinant antibody, or antibody fragment thereof. Non-limiting examples of antibody fragment include Fab, Fab′, F (ab) ′2, F (ab) ′3, Fv, single chain antigen binding fragment (scFv) , (scFv) 2, disulfide stabilized Fv (dsFv) , minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody) , recombinant heavy-chain-only antibody (VHH) , and other antibody fragments that maintain the binding specificity of the whole antibody.

The term “safety switch” generally refers to an engineered polypeptide construct designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. When expressed in a cell, the safety switch can induce death of the host cell, thereby inactivating activity of the cell in a host (e.g., in a subject’s body) . Thus, the safety switch can be a suicide moiety. In some cases, the cell can be programmed to express the suicide moiety at certain stage of its life-cycle (e.g., time-programmed) . In some cases, expression of the suicide moiety in a cell can be conditional or inducible. In some examples, conditional regulation (e.g., expression) of a suicide moiety can include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation. Thus, the safety switch can be an inducible suicide moiety. A safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion. In some cases, a safety switch can be activated by an exogenous molecule (e.g., a drug or a prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., engineered NK cell as disclosed herein) .

The term “hypo-immunity regulator” generally refers to a polypeptide construct in a cell, wherein either enhanced expression (e.g., via knock-in of a heterologous gene) or reduced expression (e.g., via knock-out or knock-down of an endogenous gene) of the hypo-immunity regulator in the cell can help the cell to reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration to the host’s body. In some cases, cells (e.g., engineered NK cells as disclosed herein) can be modified to exhibit either enhanced expression or reduced expression of the hypo-immunity regulator, such that the cells can evade the host immune attack upon second or further infusion of the cells into the host (i.e., recipient) . As such, the cells (i) would not be rejected by the host’s immune system (e.g., antibody-mediated complement cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC) ) and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator. A cell exhibiting the enhanced expression or reduced expression of the hypo-immunity regulator can be referred to as exhibiting “hypo-immunity” or being “immune-privileged. ” For example, enhanced hypo-immunity (e.g., enhanced resistance against ADCC) of a population of engineered immune cells (e.g., a population of engineered NK cells) as disclosed herein can be ascertained in vitro (e.g., in the presence of human serum or human complement and an antibody (e.g., SSEA-4 antibody) ) or in vivo (e.g., upon administration to a subject’s bloodstream) .

In some cases, engineering of an immune cell (e.g., NK cells) as disclosed herein can enhance the immune cell’s resistance against immune rejection (e.g., ADCC) by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%.

In some cases, the enhanced resistance against immune rejection (e.g., ADCC) can be ascertained in vitro in a medium comprising at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, or at least or up to about 80%, human complement.

The term “immune checkpoint inhibitor” generally refers to a group of molecules presented on a cell surface of an immune cell (e.g., T cells, myeloid cells, NK cells, B cells, etc. ) that can modulate immune response of the cell by down-regulating or inhibiting the immune response of the immune cell against a target cell, such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) . The target cell can express a receptor or a ligand of the immune checkpoint inhibitor presented on the surface of the immune cell, to engage with the immune checkpoint inhibitor and down-regulate or inhibit the immune response of the immune cells against the target cell. As such, down-regulating or inhibiting expression of the immune checkpoint inhibitor in the immune cell can, in some cases, enhance or prolong the immune response of the immune cell against a target cell.

An exogenous interleukin, as disclosed herein, can comprise an interleukin that is not secreted by the cell or the population of cells (e.g., the engineered immune cell (s) , such as the engineered NK cells as disclosed herein) , and can be artificially added to the environment. In an in vitro environment, an exogenous interleukin may comprise a recombinant interleukin protein that is added to a medium. In an in vivo environment (e.g., in plasma) , an exogenous interleukin may comprise a recombinant interleukin protein that is administered to a subject in need thereof.

The term “persistence” as used herein may generally refer to a presence of at least a portion of a population of cells (e.g., a population of engineered immune cells, such as a population of engineered NK cells as disclosed herein) remaining in an environment after introducing the population of cells to the environment (e.g., in an in vitro medium, in the serum after intravenous (IV) administration, etc. ) . A persistence may be ascertained by a duration of time that at least a portion of the population of cells remain in the environment at a detectable level. In some examples, persistence of a population of cells may correlate to the half-life of the population of cells in the environment (e.g., medium, blood stream, etc. ) . In some cases, a population of cells of interest (e.g., ., a population of engineered immune cells, such as a population of engineered NK cells) may not grow (e.g., proliferate) over time, and a persistence level of the population of cells as disclosed herein can be ascertained by measuring a decrease (or a rate thereof) in a size of the population of cells over time. For example, a population of cells having a greater persistence level (e.g., at least 5%greater) in an environment after a period of time (e.g., after at least about 5 days) than a control population of cells in a comparable environment after a comparable period of time may indicate that a greater proportion (e.g., at least 5%greater) of the size of the population of cells have survived as compared to the control population of cells.

The term “immune response” generally refers to T cell mediated and/or B cell mediated immune responses from a host’s immune system to an object (e.g., a foreign object) . An example of an immune response include T cell responses, e.g., cytokine production and cellular cytotoxicity. IN some cases, an immune response can be indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, such as macrophages.

The term “enhanced expression, ” “increased expression, ” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.

The term “enhanced activity, ” “increased activity, ” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

The term “reduced expression, ” “decreased expression, ” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.

The term “reduced activity, ” “decreased activity, ” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.

The term “subject, ” “individual, ” or “patient, ” as used interchangeably herein, generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

The term “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

A. Overview

T cells are part of the adaptive immune system and can be primed to recognize a specific threat by recognizing immune proteins (i.e., antigens) on a foreign cell surface. In contrast, NK cells are part of the innate immune response and can respond to a broad range of objects that consider to be “non-self. ” Generally, unlike T cells, NK cells can attack their target cells without sensitization (i.e., antigen-specific priming) to eliminate foreign substances.

Unmodified NK cells derived from a subject (e.g., derived from HSCs of the subject) can be cultured and expanded ex vivo, then administered to the subject as a treatment to attack their target cells, e.g., cancer cells. However, NK cell-based therapies can be limited due to short half-life and/or poor proliferation of NK cells ex vivo or in vivo. In addition, unmodified NK cells can be ineffective in targeting harder-to-treat cancers, such as myeloma or solid tumors. Furthermore, ex vivo production of NK cells based on blood-derived stem cells (e.g., HSCs) can yield a limited supply of NK cells for effective adoptive immunotherapy.

Thus, there remains a significant unmet need for immune cells (e.g., NK cells, T cells, etc. ) sourced and engineered to exhibit, for example, enhanced proliferation, half-life, and cytotoxic activity against target cells.

B. Engineered immune cells

The present disclosure describes systems and methods for immunotherapies. Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell) . Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell. Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered Immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered Immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can be derived from an isolated stem cell (e.g., isolated ESCs) . In some cases, engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSCs) .

In some cases, the stem cell disclosed herein (e.g., isolated stem cell, induced stem cell) can be an autologous cell or derived from the autologous cell. The autologous cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the autologous cell can be obtained from the subject before the subject is found to have the condition. In some cases, the autologous cell can be an allogeneic cell, e.g., a universal stem cell with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The autologous cell can be obtained from a healthy donor.

In some cases, the engineered immune cell (e.g., engineered NK cell) can be an autologous cell. The engineered immune cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the engineered immune cell can be obtained from the subject before the subject is found to have the condition. In some cases, the engineered immune cell can be an allogeneic cell, e.g., for a universal allogenic immunotherapy with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The engineered immune cell can be obtained from a healthy donor.

In some aspects, T cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered T cell) . T cells can be engineered to exhibit enhanced proliferation as compared to a control cell. T cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered T cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered T cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered T cells can be autologous to the subject. Alternatively, the engineered T cells can be allogeneic to the subject.

In some aspects, NK cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered NK cell) . NK cells can be engineered to exhibit enhanced proliferation as compared to a control cell. NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered NK cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise or exhibit one or more members of the following:

(#1) an enhanced expression and/or activity level of an activating NK receptor as compared to a control NK cell; and/or

(#2) a chimeric polypeptide receptor (e.g., a chimeric antigen receptor (CAR) , an engineered T cell receptor (TCR) , etc. ) comprising an antigen binding moiety capable of binding to an antigen, (e.g., optionally wherein the antigen is not a NKG2D ligand) ; and/or

(#3) a reduced expression and/or activity level of a suppressor of cytokine (SOCS) protein; and/or

(#4) an enhanced expression and/or activity level of a dead-associated protein (DAP) ; and/or

(#5) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a ligand of the activating NK receptor (e.g., NKG2D ligand) ; and/or

(#6) a reduced expression and/or activity level of endogenous CD38 as compared to the control NK cell; and/or

(#7) reduced expression and/or activity level of an immune checkpoint inhibitor (e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc. ) ; and/or

(#8) reduced expression and/or activity level of a hypo-immunity regulator (e.g., B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, etc. ) ; and/or

(#9) a safety switch capable of effecting death of the engineered NK cell (e.g., upon a stimulus, such as exposure to an activating moiety, such as light or antibody) ; and/or

(#10) a heterologous cytokine (e.g., interleukin (IL) ) and/or a heterologous receptor thereof for enhanced cytokine signaling as compared to the control NK cell.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#1) . The engineered immune cell can exhibit (#1) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#1) and all members of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#2) . The engineered immune cell can exhibit (#2) and one or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and two or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and three or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and four or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and five or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and six or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and seven or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and eight or more members selected from the group consisting of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#2) and all members of (#1) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#3) . The engineered immune cell can exhibit (#3) and one or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and two or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and three or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and four or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and five or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and six or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and seven or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and eight or more members selected from the group consisting of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#3) and all members of (#2) , (#1) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#4) . The engineered immune cell can exhibit (#4) and one or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and two or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and three or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and four or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and five or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and six or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and seven or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and eight or more members selected from the group consisting of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#4) and all members of (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#5) . The engineered immune cell can exhibit (#5) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#5) and all members of (#2) , (#3) , (#4) , (#1) , (#6) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#6) . The engineered immune cell can exhibit (#6) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#6) and all members of (#2) , (#3) , (#4) , (#5) , (#1) , (#7) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#7) . The engineered immune cell can exhibit (#7) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) . The engineered immune cell can exhibit (#7) and all members of (#2) , (#3) , (#4) , (#5) , (#6) , (#1) , (#8) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#8) . The engineered immune cell can exhibit (#8) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) . The engineered immune cell can exhibit (#8) and all members of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#1) , (#9) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#9) . The engineered immune cell can exhibit (#9) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) . The engineered immune cell can exhibit (#9) and all members of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#1) , and (#10) .

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise or exhibit at least (#10) . The engineered immune cell can exhibit (#1) and one or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and two or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and three or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and four or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and five or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and six or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and seven or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and eight or more members selected from the group consisting of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) . The engineered immune cell can exhibit (#10) and all members of (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#1) .

An engineered immune cell (e.g., an engineered NK cell) comprising one or more members selected from the group constating of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can exhibit enhanced the activity (e.g., persistence or viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, maintenance of stemness, etc. ) , as compared to a control immune cell lacking at least one of the one or more members. For example, an engineered NK cell can comprise at least the enhanced expression and/or activity level of the activating NK receptor, and the engineered NK cell can exhibit enhanced cytotoxicity against a cancer or tumor cell, as compared to a control cell lacking enhanced expression/activity level of the activating NK receptor. In another example, an engineered NK cell can comprise a chimeric antigen receptor (e.g., a CAR) comprising an antigen binding moiety capable of binding CD70, CLEC12A, CD9, CD26, TRAIL-R4 (DCR2) , NKG2D ligand, and/or TIM-3, and the engineered NK cell can exhibit enhanced cytotoxicity against a cancer or tumor cell, as compared to a control cell lacking the CAR.

An engineered immune cell (e.g., an engineered NK cell) comprising two or more members selected from the group constating of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can exhibit enhanced activity (e.g., persistence of viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, maintenance of stemness, etc. ) , as compared to a control immune cell lacking at least one of the two or more members. For example, an engineered NK cell can comprise (A) the enhanced expression and/or activity level of the activating NK receptor and (B) at least one or more members selected from the group consisting of (#5) , (#6) , (#7) , and (#8) , as disclosed herein, and the engineered NK cell can exhibit enhanced cytotoxicity against a cancer or tumor cell, as compared to a control cell lacking (A) and/or (B) . In another example, an engineered NK cell can comprise (A) a chimeric antigen receptor (e.g., a CAR) comprising an antigen binding moiety capable of binding CD70, CLEC12A, and/or TIM-and (B) at least one or more members selected from the group consisting of (#1) , (#7) , (#8) , and (#9) , as disclosed herein, and the engineered NK cell can exhibit enhanced cytotoxicity against a cancer or tumor cell, as compared to a control cell lacking (A) and/or (B) .

Any combination of two or more members selected from the group constating of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can induce a synergistic effect on (e.g., can synergistically enhance) an activity of the engineered immune cell (e.g., persistence or viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, maintenance of stemness, etc. ) , as compared to (i) an individual effect on (e.g., an individual enhancement of) the activity of a control immune cell comprising only one of the two or more members, and/or (ii) a sum of the individual effect on (e.g., the sum of the individual enhancements of) the activity thereof. For example, an engineered NK cell can exhibit at least (#1) the enhanced expression and/or activity level of an activating NK receptor and (#2) the chimeric polypeptide receptor (e.g., a CAR) comprising one or more antigen binding moieties capable of binding to an antigen that is, optionally, not a NKG2D ligand, such as BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10. In such example, (#1) and (#2) can yield a synergistic effect in the engineered NK cell, such that an activity of the engineered NK cell (e.g., cytotoxicity against a cancer cell) is greater than that of (i) an individual enhancement of the activity in a control NK cell engineered to comprise only one of (#1) and (#2) and/or (ii) a sum of the individual enhancements thereof.

Non-limiting examples of the enhanced activity of the engineered immune cell (e.g., the engineered NK cell) , as disclosed herein, can include viability of the engineered immune cell, proliferation (or growth rate) of the engineered immune cell, survival of the engineered immune cell, cytotoxicity against a target cell such as a cancer cell, and maintenance of stemness of the engineered immune cell (e.g., stem-like T cells) . Such enhancement in activity can occur (or can be ascertained) in vitro, ex vivo, or in vivo. The enhanced activity can be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, 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 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more, as compared to that in a control immune cell. The enhanced activity can be at most about 100%, at most about 99%, 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 2%, at most about 1%, or less, as compared to that in a control immune cell. The enhanced activity can be at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more, as compared to a control immune cell. The enhanced activity can be at most about 500-fold, at most about 200-fold, at most about 100-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 25-fold, at most about 20-fold, at most about 15-fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.5-fold, at most about 0.2-fold, at most about 0.1-fold, or less, as compared to a control immune cell.

The synergistic effect as disclosed herein (e.g., via any combination of (#1) - (#10) ) can be greater than (i) and/or (ii) by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, 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 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The synergistic effect can be greater than (i) and/or (ii) by at most about 100%, at most about 99%, 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 2%, at most about 1%, or less. The synergistic effect can be greater than (i) and/or (ii) by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more. The synergistic effect can be greater than (i) and/or (ii) by at most about 500-fold, at most about 200-fold, at most about 100-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 25-fold, at most about 20-fold, at most about 15-fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.5-fold, at most about 0.2-fold, at most about 0.1-fold, or less.

Any combination of two or more members selected from the group constating of (#1) , (#2) , (#3) , (#1) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can induce a synergistic effect on (e.g., can synergistically enhance) each other (e.g., A having a synergistic effect on B, B having as synergistic effect on A, or both) . An engineered immune cell (e.g., an engineered NK cell) can comprise at least a first member and a second member (e.g., that is different than the first member) selected from the group constating of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein. The synergistic effect between the first member and the second member can be such that the engineered immune cell exhibit first member in a greater degree than (i) an individual degree of the first member in a control cell comprising only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof. Alternatively or in addition to, the synergistic effect between the first member and the second member can be such that the engineered immune cell exhibit first member in a greater degree than (i) an individual degree of the first member in a control cell comprising only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof. The degree of the first member and/or the second member can be a degree of enhancement in a target moiety’s expression, activity, and/or signaling thereof (e.g., (#1) , (#4) , (#5) , and/or (#10) , as disclosed herein) . The degree of the first member and/or the second member, as disclosed herein, can be a degree of reduction in a target moiety’s expression, activity, and/or signaling thereof (e.g., (#3) , (#6) , (#7) , and/or (#8) , as disclosed herein) . The degree of the first member and/or the second member, as disclosed herein, can be a degree of expression and/or activity of a target moiety (e.g., (#2) , (#5) , (#9) , and/or (#10) , as disclosed herein) .

For example, an engineered NK cell as disclosed herein can comprise the enhanced expression and/or activity level of the activating NK receptor (first member) and the enhanced expression and/or activity level of DAP (second member) . The synergistic effect between the first member and the second member can be such that the engineered NK cell exhibits a greater degree of enhancement in the expression and/or activity level of the activating NK receptor, as compared to (i) an individual degree of enhancement in the expression and/or activity level of the activating NK receptor in a control NK cell engineered to exhibit only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof. Alternatively or in addition to, the synergistic effect between the first member and the second member can be such that the engineered NK cell exhibits a greater degree of enhancement in the expression and/or activity level of the DAP, as compared to (i) an individual degree of enhancement of the expression and/or activity level in the DAP in a control NK cell engineered to exhibit only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof.

In another example, an engineered NK cell as disclosed herein can comprise the enhanced expression and/or activity level of the activating NK receptor (first member) and the reduced expression and/or activity level of the SOCS protein (second member) . The synergistic effect between the first member and the second member can be such that the engineered NK cell exhibits a greater degree of enhancement of the expression and/or activity level of the activating NK receptor, as compared to (i) an individual degree of enhancement in the expression and/or activity level of the activating NK receptor in a control NK cell engineered to exhibit only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof. Alternatively or in addition to, the synergistic effect between the first member and the second member can be such that the engineered NK cell exhibits a greater degree of reduction in the expression and/or activity level of the SOCS protein, as compared to (i) an individual degree of reduction in the expression and/or activity level of the SOCS protein in a control NK cell engineered to exhibit only one of the first member and the second member and/or (ii) a sum of the individual degrees thereof.

The degree of the synergistic effect on each other between two members (or among two or more members) as disclosed herein can be greater than (i) and/or (ii) by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, 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 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The degree of the synergistic effect on each other between two members (or among two or more members) as disclosed herein can be greater than (i) and/or (ii) by at most about 100%, at most about 99%, 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 2%, at most about 1%, or less. The degree of the synergistic effect on each other between two members (or among two or more members) as disclosed herein can be greater than (i) and/or (ii) by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more. The degree of the synergistic effect on each other between two members (or among two or more members) as disclosed herein can be greater than (i) and/or (ii) by at most about 500-fold, at most about 200-fold, at most about 100-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 25-fold, at most about 20-fold, at most about 15-fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.5-fold, at most about 0.2-fold, at most about 0.1-fold, or less.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise/exhibit the enhanced expression and/or activity level of the activating NK receptor as compared to a control NK cell, as disclosed herein. The activating NK receptor can comprise NKG2D, NKP30 (e.g., NKP30A and/or NKP30B isoforms) , NKP44, NKP46, a modification thereof, a functional variant thereof, and/or a combination thereof. The activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKG2D or a modification thereof. The activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP30 or a modification thereof. The activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP44 or a modification thereof. The activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP46 or a modification thereof.

In some cases, the activating NK receptor as disclosed herein may not comprise NKG2C. Alternatively, the activating NK receptor may comprise NKG2C.

Without wishing to be bound by theory, in some cases, the activating NK receptor as disclosed herein may comprise CD100 (SEMA4D) , CD16 (FcgRIIIA) , CD160 (BY55) , CD244 (2B4, SLAMF4) , CD27, CD94–NKG2C, CD94–NKG2E, CD94-NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226) , KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49, NCR, NKG2D (KLRK1, CD314) , NKp30 (NCR3) , NKp44 (NCR2) , NKp46 (NCR1) , NKp80 (KLRF1, CLEC5C) , NTB-A (SLAMF6) , PSGL1, SLAMF7 (CRACC, CS1, CD319) , CD161 (NKR-P1A, NK1.1) , CD94–NKG2A, CD96, CEACAM1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KLRG1, LAIR1, LIR1 (ILT2, LILRB1) , Ly49a, Ly49b, NKR-P1A (KLRB1) , SIGLEC-10, SIGLEC-11, SIGLEC-14, SIGLEC-16, SIGLEC-3 (CD33) , SIGLEC-5 (CD170) , SIGLEC-6 (CD327) , SIGLEC-7 (CD328) , SIGLEC-8, SIGLEC-9 (CD329) , SIGLEC-E, SIGLEC-F, SIGLEC-G, SIGLEC-H, TIGIT, a modification thereof, a functional variant thereof, and/or a combination thereof.

In some cases, the activating NK receptor can be an endogenous activating NK receptor. For example, an endogenous gene encoding the activating NK receptor can be activated (e.g., via a CRISPR-Cas system as disclosed herein) to enhance the expression and/or activity of the NK receptor. In some cases, the activating NK receptor can be a heterologous receptor. The heterologous activating NK receptor can be delivered to the engineered immune cell, and/or a heterologous gene (or a heterologous polynucleotide sequence) encoding an activating NK receptor can be delivered to the engineered immune cell. Such heterologous gene may or may not be integrated into the genome of the engineered immune cell. In some cases, the activating NK receptor may be a fusion protein, e.g., at least a portion (or all) of the activating NK receptor can be fused with at least a portion (or all) of a heterologous protein. For example, the activating NK receptor can comprise a chimeric antigen receptor (CAR) comprising (i) at least a portion of an antigen binding domain of the activating NK receptor (e.g., at least a portion of an extracellular domain of the activating NK receptor) and (ii) an intracellular signaling domain of a CAR (e.g., a first-, second-, or third-regeneration CAR) . In some cases, the activating NK receptor may not be a fusion protein. In some cases, the activating NK receptor may not comprise (i) a heterologous transmembrane and/or (ii) a heterologous signaling domain (e.g., a heterologous intracellular signaling domain) .

In some embodiments, the activating NK receptor can be configured to bind (or capable of binding to) an antigen or ligand of NKG2D, NKP30, NKP44, and/or NKP46, NKP80, and/or DNAM1. Non-limiting examples of a ligand of NKG2D can include a MIC family protein (e.g., MICA, MICB, etc. ) and RAET1/ULBP family protein (e.g., RAET1E/ULBP4, RAET1G/ULBP5, RAET1H/ULBP2, RAET1/ULBP1, RAET1L/ULBP6, RAET1N/ULBP3, Rae-1, H60, MULT-1, etc. ) . Non-limiting examples of a ligand of NKP30 can include BAT3, BAG6, B7-H6, and galectin (e.g., galenctin-3) . Non-limiting examples of a ligand of NKP44 can include mixed-lineage leukemia protein (MLL) (e.g., MLL5, such as 21spe-MLL5) , heparan sulfate (HS) proteoglycans (HSPG) , Nidogen glycoprotein (NID) (e.g., NID1) , Proliferating Cell Nuclear Antigen (PCNA) , Platelet-Derived Growth Factor (PDGF) (e.g., PDGF-DD) , and exosomes. Non-limiting examples of a ligand of NKP46 can include a cytokine such as interleukin (IL) (e.g., IL-2, IL-15, etc. ) , glycoproteins such as HSPG and/or complement factor P (CFP) . Non-limiting examples of a ligand of NKP80 can include AICL and CLEC2B. Non-limiting examples of a ligand of DNAM1 can include CD112 and CD155.

The enhanced expression and/or activity level of the activating NK receptor in the engineered immune cell (e.g., the engineered NK cell) can be greater than that in a control immune cell by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, 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 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The enhanced expression and/or activity level of the activating NK receptor in the engineered immune cell (e.g., the engineered NK cell) can be greater than that in the control immune cell by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more. The control immune cell can be an immune cell (e.g., an NK cell) that is not induced to exhibit an endogenous gene encoding the activating NK receptor. The control immune cell can be an immune cell (e.g., an NK cell) that does not comprise a heterologous activating NK receptor or a gene encoding thereof.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise/exhibit the chimeric polypeptide receptor (e.g., CAR, engineered TCR, etc. ) comprising the antigen binding moiety capable of binding to the antigen. In some embodiments, the antigen may not be a ligand of the activating NK receptor as disclosed herein. For example, the antigen of the antigen binding moiety of the chimeric polypeptide receptor may not be a ligand of NKG2D. In another example, the antigen of the antigen binding moiety of the chimeric polypeptide receptor may not be a ligand of NKP30. In another example, the antigen of the antigen binding moiety of the chimeric polypeptide receptor may not be a ligand of NKP44. In another example, the antigen of the antigen binding moiety of the chimeric polypeptide receptor may not be a ligand of NKP46.

In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10.

In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can be selected from the group consisting of CD70, C-type lectin domain family protein (e.g., CLEC12A) , TIM-3, CD38, CD9, CD26, TRAIL-R4 (DCR2) , NKG2D ligand, and BCMA.

In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein comprise CD70. In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein comprise a C-type lectin domain family protein, e.g., C-type lectin domain family 12 member A (CLEC12A or CLL-1) . In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein comprise TIM-3. In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein comprise CD38. In some embodiments, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein comprise BCMA.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can exhibit the reduced expression and/or activity level of the SOCS protein. Non-limiting examples of the SOCS protein can include CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7. For example, the SOCS protein can comprise SOCS2. The SOCS protein can be an endogenous protein. For example, an endogenous gene encoding the SOCS protein can be hindered or modified (e.g., via a CRISPR-Cas system as disclosed herein, or via a small molecule or protein inhibitor of the SOCS2 protein itself) to reduce the expression and/or activity of SOCS protein.

The reduced expression and/or activity level of the SOCS protein (e.g., SOCS2) in the engineered immune cell (e.g., the engineered NK cell) can be less than that in a control immune cell (e.g., that is not contacted by a CRISPR/Cas protein to hinder or modify a gene encoding the SOCS protein) by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, 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 00%, or about 100%. The reduced expression and/or activity level of the SOCS protein (e.g., SOCS2) in the engineered immune cell (e.g., the engineered NK cell) can be less than that in the control immune cell by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise (or exhibit) the enhanced expression and/or activity level of DAP. Non-limiting examples of the DAP can include DAP-10 and DAP-12. In some cases, the DAP can be an endogenous DAP, and the engineered immune cell can be induced (e.g., via action of a nuclease system) to exhibit the enhanced expression or activity level of the endogenous DAP. For example, the engineered immune cell can be contacted with (e.g., transfected or transduced with) as a CRISPR-Cas system comprising a Cas protein and a guide nucleic acid molecule that are configured to form a complex capable of binding a gene that (i) encodes the endogenous DAP or (ii) controls expression and/or activity of the endogenous DAP. In some cases, the DAP can be a heterologous DAP. The heterologous DAP or a gene encoding thereof can be delivered (e.g., via transfection, viral delivery vehicles, non-viral delivery vehicles, etc. ) to the engineered immune cell.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise (or exhibit) the heterologous cytokine (e.g., IL) and/or the heterologous receptor thereof for enhanced signaling of the cytokine in the engineered immune cell, as compared to that in a control NK cell.

The heterologous cytokine (e.g., the heterologous IL) and/or the heterologous receptor thereof, as disclosed herein, can be of the same species as that of the engineered immune cell (e.g., the engineered NK cell) . For example, both the heterologous cytokine (and/or the heterologous receptor thereof) and the engineered immune cell can be of human origin. Alternatively, the heterologous cytokine (and/or the heterologous receptor thereof) can be of a different species than that of the engineered immune cell.

A heterologous cytokine (e.g., the heterologous IL) and/or the heterologous receptor thereof, as disclosed herein, can be introduced to the engineered immune cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine and/or the heterologous receptor thereof to the engineered immune cell. The heterologous polynucleotide can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) . Alternatively, the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell. In an example, a mRNA encoding a heterologous cytokine can be introduced (or inserted into) the engineered immune cell.

In some cases, the cytokine as disclosed herein can be IL. An IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of ILs. An IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of ILs. Alternatively, the IL can be a single type of IL. Non-limiting examples of the IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. In some examples, the IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof. For example, the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise at least a portion of heterologous variant of any one of the IL as disclosed herein, such as human IL-15 (or a gene encoding thereof) . In another example, the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise at least a portion of heterologous variant of a receptor of any one of the IL as disclosed herein, such as human IL-15 receptor (IL-15R) (or a gene encoding thereof) .

The heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine. Alternatively, the heterologous cytokine may not and need not be secreted by the engineered immune cell. In such a case, for example, the heterologous cytokine can be bound to a cell surface of the engineered immune cell.

In some cases, the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine. An expression cassette encoding the heterologous cytokine can be introduced to the engineered immune cell. The expression cassette can further encode an additional heterologous polypeptide, e.g., a heterologous receptor. Within the expression cassette, a first polynucleotide sequence encoding the heterologous cytokine and a second polynucleotide sequence encoding the additional heterologous polypeptide (e.g., the heterologous receptor) can be coupled to each other via a polynucleotide linker encoding a cleavage linker. In some examples, the heterologous receptor can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) . Alternatively, the expression cassette may not and need not encode any additional heterologous polypeptide other than the heterologous cytokine.

A cleavable linker as disclosed herein can comprise a self-cleaving peptide, such as a self-cleaving 2A peptide. Self-cleaving peptides can be found in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , Thosea asigna virus (TaV) and porcine tescho virus-1 (PTV-I) , and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses. Non-limiting examples of the self-cleaving 2A peptide can include “F2A” , “E2A” , “P2A” , “T2A” , and functional variants thereof.

In some cases, the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be bound to a cell surface the engineered immune cell (e.g., the engineered NK cell) . In some examples, the engineered immune cell can be genetically modified such that a heterologous polynucleotide sequence encoding the heterologous cytokine is coupled to a gene encoding an endogenous transmembrane protein of the engineered immune cell. In an example, the endogenous transmembrane protein can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) . In some examples, an expression cassette encoding a heterologous fusion polypeptide comprising (i) the heterologous cytokine that is coupled to (ii) a heterologous receptor can be introduced to the engineered immune cell. Here, the heterologous cytokine may not and need not be cleavable from the heterologous receptor. Non-limiting examples of the heterologous receptor can include a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) , or a different receptor such as a common gamma chain (γ _C) receptor or a modification thereof.

An expression cassette as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein. Alternatively, the expression cassette may not and need not be integrated into the genome of the engineered cell.

The engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine (e.g., the heterologous IL, such as the heterologous IL-15) and/or the heterologous receptor (e.g., the heterologous IL receptor, such as the heterologous IL-15R) as disclosed herein. The enhanced signaling of the endogenous signaling pathway as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., JAK3, STAT3, STAT5, etc. for IL-15/IL-15R) or (ii) expression of a downstream gene (e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng, etc. for IL-15/IL-15R) via Western blotting or polymerase chain reaction (PCR) techniques.

In some cases, enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R as disclosed herein) in the engineered immune cell of the present disclosure can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

In some cases, enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R as disclosed herein) in the engineered immune cell of the present disclosure can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

Enhanced CD16 signaling (e.g., constitutively activated signaling of CD16) of the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be achieved by having non-cleavable CD16 variant in the subject cell. CD16 (e.g., CD16a) is a transmembrane protein expressed by immune cells (e.g., NK cells) , which binds monomeric IgG attached to target cells to activate the immune cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC) . In a non-engineered immune cell, the binding between CD16 and the monomeric IgG can induce cleavage of the CD16 protein at a cleavage site near the transmembrane domain, to regulates the cell surface density of CD16 upon immune cell activation. Thus, the endogenous CD16 of the engineered immune cell can be modified to enhance its signaling. Alternatively, an enhanced signaling variant of CD16 can be artificially introduced to the engineered immune cell.

In some cases, the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified in its ectodomain (e.g., F176V) via action of a gene editing moiety as disclosed herein, such that the modified CD16 exhibits higher binding affinity to its target (e.g., monomeric IgG) as compared to a natural CD16. In some cases, a heterologous gene encoding such modified CD16 can be introduced to the cell.

In some cases, the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified via action of a gene editing moiety as disclosed herein, such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling. In some examples, the cleavage site (e.g., position 195-198) in the membrane-proximal region (position 189-212) of CD16 can be modified or eliminated (e.g., CD16 S197P variant as a non-cleavable CD16 variant) . In some cases, a heterologous gene encoding such modified CD16 can be introduced to the cell.

In some cases, a heterologous gene encoding a heterologous CD16 variant that (i) exhibits higher binding affinity to its target (e.g., monomeric IgG) and (ii) is non-cleavable can be introduced to the cell (i.e., hnCD16) . In some examples, the heterologous CD16 variant can be a modified CD16 comprising, for example, F176V and S197P, as disclosed herein. In some examples, the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.

A heterologous gene as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein. Alternatively, the heterologous gene may not and need not be integrated into the genome of the engineered cell.

The enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., SHP-1) via Western blotting or (ii) expression of a downstream gene (e.g., CD25, IFN-gamma, TNF, etc. ) via Western blotting or PCR techniques.

In some cases, the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be greater than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.

In some cases, enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

In some cases, enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

In some cases, the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be more prolonged (e.g., a longer duration of time of activated CD16 signaling) than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.

The engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) . The engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) . Alternatively, the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .

In some cases, the engineered NK cell can be treated with inhibitors (e.g., small molecule inhibitors) of the endogenous cytokine signaling. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL (e.g., endogenous IL-17) or endogenous receptor thereof (e.g., via indel or transgene mutation, via transient or permanent suppression, etc. ) . In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17R. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17 and endogenous IL-17R.

In some cases, the endogenous cytokine as disclosed herein can be an endogenous IL. An endogenous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of endogenous ILs. An endogenous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of endogenous ILs. Alternatively, the endogenous IL can be a single type of endogenous IL. Non-limiting examples of the endogenous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. In some examples, the endogenous IL can be IL-17. Non-limiting examples of endogenous Il-17 can include IL-17A, IL-17F, and natural mutations thereof. For example, the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F.

In some cases, an endogenous gene encoding the endogenous cytokine (e.g., an endogenous IL, such as IL-17) as disclosed herein can be modified via action of a gene editing moiety as disclosed herein.

The endogenous receptor can be a respective receptor of any cytokine as disclosed herein (e.g., a respective receptor of any IL as disclosed herein) . In some cases, the endogenous receptor can be a respective receptor of IL (e.g., IL-17R for IL-7 signaling) . Non-limiting examples of IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof. In an example, the endogenous IL-17R comprises IL-17RA.

In some cases, the reduced expression or activity of the endogenous cytokine (e.g., an endogenous IL, such as IL-17) or endogenous receptor thereof as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) expression of a downstream gene via Western blotting or PCT techniques. In some examples, a downstream gene of IL cytokine, such as IL-17, can include a chemokine (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc. ) , a cytokine (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc. ) , an acute phase response molecule (e.g., SAA, CRP, lipocalin 2/24p3, etc. ) , and/or an enzyme (e.g., a metalloproteinase, such as MMP1, MMP3, MMP9, MMP13) .

In some cases, reduced expression or activity of the endogenous cytokine (e.g., the endogenous IL, such as IL-17) or endogenous receptor thereof in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be characterized by a decrease in phosphorylation of a downstream signaling protein of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

In some cases, reduced expression or activity of the endogenous cytokine (e.g., the endogenous IL, such as IL-17) or endogenous receptor thereof in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be characterized by a decrease in the expression of a downstream gene of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein. For example, non-limiting examples of a specific cell marker for committed NK cells can include CD57 or killer immunoglobulin-like receptors (KIR) . KIR can comprise KIR2D and/or KIR3D. KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5. KIR3D can comprise KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.

The enhanced expression profile of the specific cell marker for the committed immune cell (e.g., CD57 or KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.

In some cases, the expression of the specific cell marker for a committed immune cell (e.g., CD57 or KIR or NK cells) in the engineered immune cell of the present disclosure can be greater than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) comprising one or more members (e.g., two or more members) selected from the group consisting of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can exhibit enhanced survival in the presence of tumor cells as compared to a control cell lacking at least one of the one or more members. In some case, the engineered immune cell can, in the presence of tumor cells, survive longer than the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit reduced expression or activity of one or more immune checkpoint inhibitors (e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc. ) . The one or more immune checkpoint inhibitors can be endogenous to the engineered immune cell. In an example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous PD1. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous CTLA-4. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous TIM-3. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous KIR2D. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous CD94. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous NKG2A. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous TIGIT. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous TGF beta receptor. In another example, the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous 2B4.

In some cases, the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96, TGF beta receptor, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit reduced expression or activity of one or more hypo-immunity regulators (e.g., one or more endogenous immune regulator polypeptides) , as disclosed herein. The one or more hypo-immunity regulators can be selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit reduced expression or activity of one or more endogenous polypeptides (e.g., endogenous immune regulating polypeptide) . The one or more endogenous polypeptides can comprise one or more members of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN2, PTPN6, and/or CD70. Alternatively or in addition to, the one or more endogenous polypeptides can comprise one or more members of NLRC5, RFXANK, RFXAP, CD80, CD7, TAP2, TAP1, TAPBP. For example, the reduced expression or activity of such one or more endogenous polypeptides in the engineered NK cell can enhance one or more characteristics of the engineered NK cell (e.g., persistence or viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, etc. ) . For example, the reduced expression or activity of such one or more endogenous polypeptides in the engineered NK cell can enhance persistence of the engineered cell in a sub-optimal environment. In another example, the reduced expression or activity of such one or more endogenous polypeptides in the engineered NK cell can enhance resistance against immune rejection (e.g., ADCC cytotoxicity of the engineered NK cell) .

In some cases, the expression or activity of the hypo-immunity regulator (e.g., the endogenous hypo-immunity regulator) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than the same in a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by Western blotting or PCT techniques, as disclosed herein.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can exhibit reduced expression or activity of endogenous CD38 as compared to a control cell. Such engineered immune cell may be used to treat a subject who has or is suspected of having white blood cell cancer, such as multiple myeloma (MM) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, expression or activity of endogenous CD38 of the engineered immune cell may not and need not be modified. Such engineered immune cell may be used to treat a subject who has or is suspected of having a disease (e.g., cancer, tumor) that is not multiple myeloma.

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) can comprise the chimeric polypeptide receptor (or at least one chimeric polypeptide receptor) comprising an antigen binding moiety capable of binding to an antigen, as provided in the present disclosure. In some examples, the engineered immune cell can comprise a plurality of different chimeric polypeptide receptors to specifically bind a plurality of different antigens. In some examples, the engineered immune cell can comprise at least one chimeric polypeptide receptor that comprises a plurality of antigen binding moieties to specifically bind a plurality of different antigens.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can comprise a safety switch capable of effecting death of the engineered immune cell. The engineered immune cell can comprise a gene encoding the safety switch (e.g., integrated into the genome of the immune cell) , via action of the gene editing moiety, as disclosed herein. In some cases, a prodrug can be introduced to the engineered immune cell (e.g., administered to a subject comprising the engineered immune cell) in the event of an adverse event or when the adaptive immunotherapy is no longer necessary, and the prodrug can be activated by the safety switch molecule to kill the subject immune cell. In some cases, the safety switch can comprise one or more members selected from the group consisting of caspase (e.g.,

caspase

3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and functional variants thereof. In some cases, the safety switch can be activated via an activator (e.g., a small molecule or a protein, such as an antibody) for post-translational, temporal, and/or site-specific regulation of death (or depletion) of the subject engineered immune cell. Non-limiting examples of a safety switch and its activator can include Caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV) ; and cytosine deaminase (CD) and 5-fluorocytosine (5-FC) . Alternatively or in addition to, modified epidermal growth factor receptor (EGFR) containing epitope recognized by an antibody (e.g., anti-EGFR Ab, such as cetuximab) can be used to deplete the engineered immune cells when the subject cells are exposed to the antibody. In some cases, the engineered immune cells (e.g., the engineered NK cells) as disclosed herein can comprise a safety switch protein selected from the group consisting of caspase 9 (caspase 3 or 7) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.3

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can exhibit enhanced persistence or survival (e.g., in an environment substantially free of exogenous cytokine, such as IL-2 or IL-5; or in an environment with sub-optimal dose of such exogenous cytokine; in vitro environment; in vivo environment, etc. ) as compared to a control cell. In some cases, the engineered immune cell as disclosed herein can exhibit enhanced persistence or survival (e.g., in vitro, ex vivo, or in vivo) that is greater than that of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by, e.g., tracking a change in the number of the engineered immune cells (e.g., the engineered NK cells) over time.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can exhibit enhanced proliferation or growth rate (e.g., in an environment substantially free of exogenous cytokine, such as IL-2 or IL-5; or in an environment with sub-optimal dose of such exogenous cytokine; in vitro environment; in vivo environment, etc. ) as compared to a control cell. In some cases, the engineered immune cell as disclosed herein can exhibit enhanced proliferation or growth rate (e.g., in vitro, ex vivo, or in vivo) that is greater than that of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by, e.g., tracking a change in the number of the engineered immune cells (e.g., the engineered NK cells) over time.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can exhibit enhanced cytotoxicity against a target cell as compared to a control cell. In some cases, the engineered immune cell as disclosed herein can exhibit cytotoxicity (e.g., in vitro, ex vivo, or in vivo) against a target cell or a target population of cells that is greater than that of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by, e.g., tracking a change in the number of the target population of cells.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can induce reduced immune response from separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host) as compared to a control cell. In some cases, the engineered immune cell as disclosed herein can reduce the immune response from the separate immune cells by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to an immune response from the separate immune cells when exposed to a control cell, as ascertained by, e.g., measuring (i) a change in the number of the initial population of the engineered immune cells upon exposure to the separate immune cells or (ii) a change in cytokine release of the separate immune cells upon exposure to the initial population of the engineered immune cells.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can exhibit enhanced half-life upon exposure to separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host) as compared to a control cell. In some cases, upon exposure to the separate immune cells (e.g., in vitro or in vivo) , the half-life of the engineered immune cells can be greater than that of the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by monitoring the number of the engineered immune cells over time (e.g., via FACS) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can effect enhanced function or pathological condition of a bodily tissue of a subject as compared to a control cell. In some cases, treatment with the engineered immune cell can effect enhanced function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell or vehicle (i.e., no cells) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject as compared to a control cell. In some cases, treatment with the engineered immune cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell or vehicle (i.e., no cells) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the bodily tissue can comprise one or more members selected from the group consisting of blood, plasma, serum, urine, perilymph fluid, feces, saliva, semen, amniotic fluid, cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bone, heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine, liver, pancreas, spleen, kidney, gall bladder, thyroid gland, adrenal gland, mammary gland, ovary, prostate gland, testicle, skin, adipose, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can induce immune response towards a target cell. The target can be, for example, a diseased cell, a cancer cell, a tumor cell, etc.

C. Additional aspects of the engineered immune cells

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, a heterologous gene can be operatively coupled to (e.g., for knock-in) a constitutive, inducible, temporal, tissue-specific, and/or cell type-specific promoter. Non-limiting examples of a promoter of interest can include CMV, EF1a, PGK, CAG, and UBC. Non-limiting examples of an insertion site can include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit reduced expression or activity of one or more of the following endogenous genes for enhancing function of the engineered immune cell in a tumor microenvironment (i.e., tumor microenvironment gene or “TME” ) . In some cases, having reduced expression or activity of a TME can enhance the engineered immune cell’s immune activity against a target cell. In some cases, a TME gene may be an immune checkpoint inhibitor. Non-limiting examples of the TME can include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGF beta receptor, 2B4, and SHIP2.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) for, e.g., enhanced function: CD137, CD80, CD86, DAP10 (e.g., with or without point mutation) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit reduced expression or activity of one or more of the following endogenous genes for, e.g., hypo-immunity: B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) .

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.

In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) : CD3, CD4, CD80, 41BBL, and CD131.

D. Chimeric antigen receptor

The engineered immune cell (e.g., the engineered NK cell) of the present disclosure can comprise a chimeric polypeptide receptor as disclosed herein (e.g., at least 1, 2, 3, 4, 5, or more different types of chimeric polypeptide receptors) . The engineered immune cell can be engineered to express a chimeric polypeptide receptor transiently or permanently. In some cases, a recombinant chimeric polypeptide receptor can be delivered to the engineered immune cell via, e.g., a liposome, and be incorporated into the engineered immune cell via membrane fusion. In some cases, a heterologous polynucleotide construct (e.g., DNA or RNA) encoding the chimeric polypeptide receptor can be delivered to the engineered immune cell. The heterologous polynucleotide construct (i.e., a gene) encoding the heterologous polynucleotide construct can be incorporated into the chromosome of the engineered immune cell (i.e., chromosomal gene) or, alternatively, may not or need not be integrated into the chromosome of the engineered immune cell as disclosed herein.

A chimeric polypeptide receptor can comprises a T cell receptor fusion protein (TFP) . The term “T cell receptor fusion protein” or “TFP” generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of TCR extracellular domain, (iii) at least a portion of TCR transmembrane domain, and (iv) at least a portion of TCR intracellular domain.

In some cases, an endogenous T cell receptor (TCR) of the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be inactivated. In some examples, a function of the endogenous TCR of the engineered immune cell can be inhibited by an inhibitor. In some examples, a gene encoding a subunit of the endogenous TCR can be inactivated (e.g., edited via action of the gene editing moiety as disclosed herein) such that the endogenous TCR is inactivated. The gene encoding the subunit of endogenous TCR can be one or more of: TCRα, TCRβ, CD3ε, CD3δ, CD3γ, and CD3ζ.

A chimeric polypeptide receptor can comprises a chimeric antigen receptor (CAR) . The term “chimeric antigen receptor” or “CAR” generally refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular or intrinsic signaling domain” ) comprising a functional signaling domain derived from a stimulatory molecule. In some cases, the stimulatory molecule may be the zeta chain associated with the T cell receptor complex. In some cases, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In some cases, the costimulatory molecule may comprise 4-1BB (i.e., CD137) , CD27, and/or CD28. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

A CAR may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. First-generation CARs (e.g., CD19R or CD19CAR) include an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody) , a transmembrane domain derived from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor) , and a signaling domain derived from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ζ receptor or FcεRIγ) . Second-generation CARs modify the first-generation CAR by addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137/4-1BB, and CD134/OX40) , which abrogates the need for administration of a co-factor (e.g., IL-2) alongside a first-generation CAR. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB) . Fourth-generation CARs modify second-or third-generation CARs by the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3ζ ITAM domain) or under the control of a CAR-induced promoter (e.g., the NFAT/IL-2 minimal promoter) . In some cases, a CAR may be a new generation CAR system that is different than the first-, second-, third-, or fourth-generation CAR system as disclosed herein.

A hinge domain (e.g., the linker between the extracellular antigen binding domain and the transmembrane domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.

A transmembrane domain of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.

The hinge domain and the transmembrane domain of a CAR as disclosed herein (e.g., for the engineered immune cell, such as the engineered NK cell) can be derived from the same protein (e.g., CD8) . Alternatively, the hinge domain and the transmembrane domain of the CAR as disclosed herein can be derived from different proteins.

A signaling domain of a CAR can comprise at least or up to about 1 signaling domain, at least or up to about 2 signaling domains, at least or up to about 3 signaling domains, at least or up to about 4 signaling domains, at least or up to about 5 signaling domains, at least or up to about 6 signaling domains, at least or up to about 7 signaling domains, at least or up to about 8 signaling domains, at least or up to about 9 signaling domains, or at least or up to about 10 signaling domains.

A signaling domain (e.g., a signaling peptide of the intracellular signaling domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.

Alternatively or in addition to (i.e., a co-stimulatory domain) , the signaling domain CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein comprises the chimeric polypeptide receptor (e.g., CAR) that comprises at least CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some cases, the engineered cell (e.g., the engineered NK cell) as disclosed herein comprises the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises at least (i) CD8 transmembrane domain, (ii) 2B4 signaling domain, and (iii) DAP10 signaling domain. The 2B4 signaling domain can be flanked by the CD8 transmembrane domain and the DAP10 signaling domain. Alternatively, the DAP10 signaling domain can be flanked by the CD8 transmembrane domain and the 2B4 signaling domain. In some cases, the chimeric polypeptide receptor as disclosed herein can further comprise yet an additional signaling domain derived from CD3ζ.

An antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) as disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.

Non-limiting examples of an antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) as disclosed herein can include ADGRE2, carbonic anhydrase IX (CA1X) , CCRI, CCR4, carcinoembryonic antigen (CEA) , CD3ζ, CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD133, CD138, CD269 (BCMA) , CD S, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , EGFRvIII, receptor tyrosine-protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-a, Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , gp100, human Epidermal Growth Factor Receptor 2 (HER-2) , human telomerase reverse transcriptase (hTERT) , ICAM-1, Integrin B7, Interleukin-13 receptor subunit alpha-2 (IL-13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Kappa, Lewis A (CA19.9) , Lewis Y (LeY) , L1 cell adhesion molecule (L1-CAM) , LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1) , MICA/B, Mucin 1 (Muc-1) , Mucin 16 (Muc-16) , Mesothelin (MSLN) , NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, oncofetal antigen (h5T4) , PRAIVIE, prostate stem cell antigen (PSCA) , PRAME prostate-specific membrane antigen (PSMA) , ROR1, tumor-associated glycoprotein 72 (TAG-72) , TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2) , Wilms tumor protein (WT-1) , and various pathogen antigen (e.g., pathogen antigens derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases) . In some examples, a pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus.

Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 1-40-β-amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1) , B7-H3, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphoma cell, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX) , cardiac myosin, CCL11 (eotaxin-1) , CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin) , CD15, CD152, CD154 (CD40L) , CD19, CD2, CD20, CD200, CD22, CD221, CD25 (α chain of IL-2receptor) , CD27, CD274, CD28, CD3, CD3 epsilon, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type-2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor α-chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54) , IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-17, IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, influenza A virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7 β7, integrin αIIbβ3, integrin αvβ3, interferon α/β receptor, interferon gamma-induced protein, ITGA2, ITGB2 (CD18) , KIR2D, Lewis-Y antigen, LFA-1 (CD11a) , LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L) , LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen) , neural apoptosis-regulated proteinase 1, NGF, N-glycolylneuraminic acid, NOGO-A, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R α, phosphate-sodium co-transporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostatic carcinoma cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, Rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGF-β 1, TGF-β 2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, TRAIL-R4 (DCR2) , tumor antigen CTAA16.88, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75) , VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2) , abl-bcr alb-b4 (b3a2) , adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, b-Catenin, bcr-abl, bcr-abl p190 (e1a2) , bcr-abl p210 (b2a2) , bcr-abl p210 (b3a2) , BING-4, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6) , HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR/FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic enzyme, Mammaglobin-A, MART-1/Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, Myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, oncofetal antigen (h5T4) , OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, Tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain.

Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an antibody, a fragment thereof, or a variant thereof. Such antibody can be a natural antibody (e.g., naturally secreted by a subject’s immune cell, such as B cells) , a synthetic antibody, or a modified antibody. In some cases, the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an Fc domain of an antibody from the group comprising 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab) , APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab) , atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab) , bevacizumab, BEVZ92 (bevacizumab biosimilar) , bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab) , CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine I-131 derlotuximab biotin) , cR6261, crenezumab, DA-3111 (trastuzumab biosimilar) , dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab) , Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 Antibody-Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab) , imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan) , IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab) , MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab) , PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab) , Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to an antigen comprising one or more members selected from the group comprising BCMA, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100. Non-limiting examples of the NKG2D ligand comprises one or more members selected from the group comprising of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain capable of specifically binding an antigen of a target cell, and the engineered immune cell can exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor. As such, a population of the engineered immune cells can avoid targeting and killing each other, e.g., upon administration to a subject in need thereof.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38. In some cases, the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38. In some cases, the subject engineered immune cells comprising the chimeric polypeptide receptor against CD38 can be capable of targeting and effecting death (or degradation) of plasma cells.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38. In some examples, the engineered immune cell is an engineered NK cell that is derived from an isolated ESC or an induced stem cell (e.g., iPSC) . In some cases, the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.

E. Stem cells

Any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can be derived from an isolated stem cell (e.g., an ESC) or an induced stem cell (iPSC) . The isolated stem cell or the induced stem cell can be modified (e.g., genetically modified) to generate the engineered immune cell.

In some cases, pluripotency of stem cells (e.g., ESCs or iPSCs) can be determined, in part, by assessing pluripotency characteristics of the cells. Pluripotency characteristics can include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only) , SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm) ; (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.

In some cases, stem cells (e.g., ESCs or iPSCs) can be genetically modified to generate (e.g., induce differentiation into) CD34+ hematopoietic stem cells. The stem cells can be genetically modified to express any one of the heterologous polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation. The stem cells can be genetically modified to reduce expression or activity of any one of the endogenous genes or polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation. In some cases, such genetically modified CD34+hematopoietic stem cell is or is a source of any one of the engineered immune cell of the present disclosure.

In some examples, stem cells as disclosed herein can be cultured in APEL media with ROCKi (Y-27632) (e.g., at about 10 micromolar (μM) ) , SCF (e.g., at about 40 nanograms per milliner (ng/mL) of media) , VEGF (e.g., at about 20 ng/mL of media) , and BMP-4 (e.g., at about 20 ng/mL of media) to differentiate into CD34+ hematopoietic stem cells.

In some cases, the CD34+ hematopoietic stem cells (e.g., genetically modified with one or more features of any one of the engineered immune cell of the present disclosure) can be induced to differentiate in to a committed immune cell, such as T cells or NK cells. As such, in some cases, the induced differentiation process generates any one of the engineered NK cell of the present disclosure.

In some examples, genetically modified CD34+ hematopoietic stem cells are cultured in the presence of IL-3 (e.g., about 5 ng/mL) , IL-7 (e.g., about 20 ng/mL) , IL-15 (e.g., about 10 ng/mL) , SCF (e.g., about 20 ng/mL) , and Flt3L (e.g., about 10 ng/mL) to differentiate into CD45+ NK cells.

In some cases, the CD45+ NK cells can be expanded in culture, e.g., in a media comprising IL-2, mbIL-21 aAPC using Gas Permeable Rapid Expansion (G-Rex) platform.

In some cases, iPSC-derived NK cells as disclosed herein can be cultured with one or more heterologous cytokines comprising Il-2, IL-15, or IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with (e.g., for cell expansion) one or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with two or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-2 and IL-21, or IL-15 and IL-21) , either simultaneously or sequentially in any order. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with all of Il-2, IL-15, and IL-21, either simultaneous or sequentially in any order.

F. Gene editing or genetic material delivery

The gene editing moiety as disclosed herein can comprise a CRISPR-associated polypeptide (Cas) , zinc finger nuclease (ZFN) , zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN) , transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP) , an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof. In some embodiments, the actuator moiety comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the actuator moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein. In some embodiments, the gRNA comprises a targeting segment which exhibits at least 80%sequence identity to a target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

In some cases, a suitable gene editing moiety comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN) ; transcription activator-like effector nucleases (TALEN) ; meganucleases; RNA-binding proteins (RBP) ; CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo) , archaeal Argonaute (aAgo) , and eukaryotic Argonaute (eAgo) ) ; any derivative thereof, any variant thereof; and any fragment thereof.

Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD) , Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12) , Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA) , Cse2 (CasB) , Cse3 (CasE) , Cse4 (CasC) , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

In some cases, the gene editing moiety as disclosed herein can be fused with an additional functional moiety (e.g., to form a fusion moiety) , and non-limiting examples of a function of the additional functional moiety can include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity. For example, a fusion protein can be a fusion in a Cas protein and an effector or repressor functional moiety.

Alternatively or in addition to, gene editing (e.g., knock in) or delivery of heterologous genetic material can be achieved other viral and non-viral based gene transfer methods can be used to introduce nucleic acids in host cells (e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein) . Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism) . Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g. a transcript of a vector described herein) , naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.

RNA or DNA viral based systems can be used to target specific cells and traffick the viral payload to the nucleus of the cell. Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo) . Alternatively, viral vectors can be administered directly (in vivo) to the subject. Viral based systems can include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, which can result in long term expression of the inserted transgene.

Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.

Alternatively or in addition to, antisense oligonucleotides can be utilized to suppress or silence a target gene expression. Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA) , microRNA (miRNA) , and small interfering RNA (siRNA) .

G. Co-therapy

The engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be combined with a co-therapeutic agent to treat a subject in need thereof. In some cases, the engineered immune cell can be administered to the subject prior to, concurrent with, or subsequent to administration of the co-therapeutic agent to the subject.

In one aspect, the present disclosure provides a composition comprising (a) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (b) a co-therapeutic agent (i.e., a separate therapeutic agent) (e.g., an antibody, such as anti-CD20 antibody or anti-PD1 antibody) . In some cases, the engineered immune cell can comprise one or more of: (i) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein, (ii) a CD16 variant for enhanced CD16 signaling as disclosed herein, and (iii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclose herein. In some examples, the co-therapeutic agent comprises an anti-CD20 antibody.

In some cases, the engineered immune cell can comprise the heterologous cytokine (e.g., IL-15) as disclosed herein and one or both of: (ii) the CD16 variant for enhanced CD16 signaling and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.

In some cases, the engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.

In some cases, the engineered immune cell can comprise the chimeric polypeptide receptor comprising the antigen binding moiety and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (ii) the CD16 variant for enhanced CD16 signaling.

Non-limiting examples of a co-therapeutic agent can include cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., Pembrolizumab) platelet derived growth factor inhibitors (e.g., GLEEVEC ^TM (imatinib mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-β, BlyS, APRIL, BCMA receptor (s) , TRAIL/Apo2, other bioactive and organic chemical agents, and the like.

The term “cytotoxic agent” generally refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. Non-limiting examples of a cytotoxic agent can include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu) , chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide) , doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.

Non-limiting examples of a chemotherapeutic agent can include alkylating agents such as thiotepa and

cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; delta-9-tetrahydrocannabinol (dronabinol,

) ; beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan

CPT-11 (irinotecan,

) , acetylcamptothecin, scopolectin, and 9-aminocamptothecin) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ; podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8) ; dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1) ; eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores) , aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,

doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aidophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;

polysaccharide complex (JHS Natural Products, Eugene, Oreg. ) ; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2′, 2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine

dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; thiotepa; taxoids, for example taxanes including

paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J. ) , ABRAXANE ^TM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill. ) , and

docetaxel (

Rorer, Antony, France) ; chloranbucil; gemcitabine

6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine

platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine

oxaliplatin; leucovovin; vinorelbine

novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoids such as retinoic acid; capecitabine

pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN ^TM) combined with 5-FU and leucovorin. Additional chemotherapeutic agents include the cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (DM1, for example) and the auristatins MMAE and MMAF, for example.

Examples of a chemotherapeutic agent can also include “anti-hormonal agents” or “endocrine therapeutics” that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (including

tamoxifen) ,

raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and

toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs) ; agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as

and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide,

megestrol acetate,

exemestane, formestanie, fadrozole,

vorozole,

letrozole, and

anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example,

or

) ,

etidronate, NE-58095,

zoledronic acid/zoledronate,

alendronate,

pamidronate,

tiludronate, or

risedronate; as well as troxacitabine (a1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR) ; vaccines such as

vaccine and gene therapy vaccines, for example,

vaccine,

vaccine, and

vaccine;

topoisomerase 1 inhibitor;

rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016) ; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Examples of a chemotherapeutic agent can also include antibodies such as alemtuzumab (Campath) , bevacizumab (

Genentech) ; cetuximab (

Imclone) ; panitumumab (

Amgen) , rituximab (

Genentech/Biogen Idec) , pertuzumab (

2C4, Genentech) , trastuzumab (

Genentech) , tositumomab (Bexxar, Corixia) , and the antibody drug conjugate, gemtuzumab ozogamicin (

Wyeth) . Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgG1λ antibody genetically modified to recognize interleukin-12 p40 protein.

Examples of a chemotherapeutic agent can also include “tyrosine kinase inhibitors” such as an EGFR-targeting agent (e.g., small molecule, antibody, etc. ) ; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724, 714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI) ; dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline) , an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis) ; pan-HER inhibitors such as canertinib (CI-1033; Pharmacia) ; Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (

available from Glaxo SmithKline) ; multi-targeted tyrosine kinase inhibitors such as sunitinib (

available from Pfizer) ; VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG) ; MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia) ; quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2, 3-d] pyrimidines; curcumin (diferuloyl methane, 4, 5-bis (4-fluoroanilino) phthalimide) ; tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber) ; antisense molecules (e.g., those that bind to HER-encoding nucleic acid) ; quinoxalines (U.S. Pat. No. 5,804,396) ; tryphostins (U.S. Pat. No. 5,804,396) ; ZD6474 (Astra Zeneca) ; PTK-787 (Novartis/Schering AG) ; pan-HER inhibitors such as CI-1033 (Pfizer) ; Affinitac (ISIS 3521; Isis/Lilly) ; imatinib mesylate

PKI 166 (Novartis) ; GW2016 (Glaxo SmithKline) ; CI-1033 (Pfizer) ; EKB-569 (Wyeth) ; Semaxinib (Pfizer) ; ZD6474 (AstraZeneca) ; PTK-787 (Novartis/Schering AG) ; INC-1C11 (Imclone) ; and rapamycin (sirolimus,

) .

Examples of a chemotherapeutic agent can also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

Examples of a chemotherapeutic agent can also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate: immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC) ; anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A) , D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept

infliximab

adalimumab

certolizumab pegol

golimumab

Interleukin 1 (IL-1) blockers such as anakinra

T-cell costimulation blockers such as abatacept

Interleukin 6 (IL-6) blockers such as tocilizumab

Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa/β2 blockers such as Anti-lymphotoxin alpha (LTa) ; miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or famesyl transferase inhibitors (L-739749, L-744832) ; polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol,

) ; beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin) ; podophyllotoxin; tegafur

bexarotene

bisphosphonates such as clodronate (for example,

or

) , etidronate

NE-58095, zoledronic acid/zoledronate

alendronate

pamidronate

tiludronate

or risedronate

and epidermal growth factor receptor (EGF-R) ; vaccines such as

vaccine; perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib) , proteosome inhibitor (e.g., PS341) ; CCI-779; tipifamib (R11577) ; orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium

pixantrone; famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASAR ^TM) ; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

The term “growth inhibitory agent” generally refers to a compound or composition which inhibits growth and/or proliferation of a cell (e.g., a cell whose growth is dependent on PD-L1 expression) either in vitro or in vivo. The growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Non-limiting examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) , such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ( (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy-α-L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8-(hydroxyacetyl) -1-methoxy-5, 12-naphthacenedione) , epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (

Rhone-Poulenc Rorer) , derived from the European yew, is a semisynthetic analogue of paclitaxel (

Bristol-Myers Squibb) . Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

H. Methods of production and use

In some embodiments, the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be generated from an isolated stem cell (e.g., isolated ESCs, iPSCs, etc. ) . The one or more members of the group consisting of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) , as disclosed herein, can be introduced during any stage (or cellular state) between (and including) (a) the isolated stem cell and (b) the differentiated immune cell state thereof (e.g., a terminally differentiated immune cell state, such as a terminally differentiated NK cell state) .

In some cases, the engineered NK cell can be derived from iPSCs, and the one or more members of the group consisting of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) can be introduced to the cell at (A) the iPSC state, (B) the hematopoietic stem cell state, and/or (C) the NK cell state. The one or more members can be introduced to the cell once during one of (A) , (B) , and (C) . Alternatively, the one or more members can be introduced to the cell multiple times during two or all of (A) , (B) , and (C) .

In some cases, the engineered NK cell can be derived from iPSCs, and two or more members of the group consisting of (#1) , (#2) , (#3) , (#4) , (#5) , (#6) , (#7) , (#8) , (#9) , and (#10) can be introduced to the cell at (A) the iPSC state, (B) the hematopoietic stem cell state, and/or (C) the NK cell state. The two or more members can be introduced to the cell at the same state selected from the group comprising (A) , (B) , and/or (C) . Alternatively, the two or more members can be introduced to the cell at different states selected from the group comprising (A) , (B) , and/or (C) . For example, the iPSCs can be (A) engineered to exhibit the heterologous CD16 variant for enhanced CD16 signaling at the iPSC state, (B) subsequently differentiated into hematopoietic stem cells, and (C) engineered to exhibit the enhanced expression and/or activity level of an activating NK receptor during and/or subsequent to being differentiated into NK cells.

The engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be used (e.g., administered) to treat a subject in need thereof. The subject can have or can be suspected of having a condition, such as a disease (e.g., cancer, tumor, tissue degeneration, fibrosis, etc. ) . A cell (e.g., a stem cell or a committed adult cell) can be obtained from the subject, and such cell can be cultured ex vivo and genetically modified to generate any subject engineered immune cell (e.g., any engineered NK cell) as disclosed herein. Subsequently, the engineered immune cell can be administered to the subject for adaptive immunotherapy.

The subject can be treated (e.g., administered with) a population of engineered immune cells (e.g., engineered NK cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses.

In one aspect, the present disclosure provides a method comprising (a) obtaining a cell from a subject; and (b) generating, from the cell, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. In some cases, the cell obtained from the subject is ESC. In some cases, the cell (e.g., a fibroblast, such as an adult skin fibroblast) obtained from the subject is modified and transformed into an iPSC.

In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. In some cases, the method can further comprise administering to the subject a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .

In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof any one of the composition disclosed herein. In some cases, the composition can comprise (i) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (ii) a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .

Any one of the methods disclosed herein can be utilized to treat a target cell, a target tissue, a target condition, or a target disease of a subject.

A target disease can be a viral, bacterial, and/or parasitic infection; inflammatory and/or autoimmune disease; or neoplasm such as a cancer and/or tumor.

A target cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.

A variety of target cells can be killed using any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. A target cell can include a wide variety of cell types. A target cell can be in vitro. A target cell can be in vivo. A target cell can be ex vivo. A target cell can be an isolated cell. A target cell can be a cell inside of an organism. A target cell can be an organism. A target cell can be a cell in a cell culture. A target cell can be one of a collection of cells. A target cell can be a mammalian cell or derived from a mammalian cell. A target cell can be a rodent cell or derived from a rodent cell. A target cell can be a human cell or derived from a human cell. A target cell can be a prokaryotic cell or derived from a prokaryotic cell. A target cell can be a bacterial cell or can be derived from a bacterial cell. A target cell can be an archaeal cell or derived from an archaeal cell. A target cell can be a eukaryotic cell or derived from a eukaryotic cell. A target cell can be a pluripotent stem cell. A target cell can be a plant cell or derived from a plant cell. A target cell can be an animal cell or derived from an animal cell. A target cell can be an invertebrate cell or derived from an invertebrate cell. A target cell can be a vertebrate cell or derived from a vertebrate cell. A target cell can be a microbe cell or derived from a microbe cell. A target cell can be a fungi cell or derived from a fungi cell. A target cell can be from a specific organ or tissue.

A target cell can be a stem cell or progenitor cell. Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc. ) . Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Clonal cells can comprise the progeny of a cell. A target cell can comprise a target nucleic acid. A target cell can be in a living organism. A target cell can be a genetically modified cell. A target cell can be a host cell.

A target cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell. A target cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell. A target cell can be a pluripotent cell. For example, a target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell. A target cell may be able to develop into a whole organism. A target cell may or may not be able to develop into a whole organism. A target cell may be a whole organism.

A target cell can be a primary cell. For example, cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. Cells can be unicellular organisms. Cells can be grown in culture.

A target cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.

If the target cells are primary cells, they may be harvested from an individual by any method. For example, leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution can generally be a balanced salt solution, (e.g. normal saline, phosphate-buffered saline (PBS) , Hank's balanced salt solution, etc. ) , conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration. Buffers can include HEPES, phosphate buffers, lactate buffers, etc. Cells may be used immediately, or they may be stored (e.g., by freezing) . Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10%DMSO, 50%serum, 40%buffered medium) , and/or some other such common solution used to preserve cells at freezing temperatures.

Non-limiting examples of cells which can be target cells include, but are not limited to, lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell) , Natural killer cell, cytokine induced killer (CIK) cells (see e.g. US20080241194) ; myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil) , Monocyte/Macrophage, Red blood cell (Reticulocyte) , Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell) , parathyroid (Parathyroid chief cell, Oxyphil cell) , adrenal (Chromaffin cell) , pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia) , Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph) ; cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte) , Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell) , Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell) , Cartilage/bone/muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast) ; cartilage cells, including Chondroblast, Chondrocyte; skin cells, including Trichocyte, Keratinocyte, Melanocyte (Nevus cell) ; muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell/Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell) , Epidermal basal cell (stem cell) , Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell) , Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell) , Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts) , Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion) , Salivary gland serous cell (glycoprotein enzyme-rich secretion) , Von Ebner's gland cell in tongue (washes taste buds) , Mammary gland cell (milk secretion) , Lacrimal gland cell (tear secretion) , Ceruminous gland cell in ear (wax secretion) , Eccrine sweat gland dark cell (glycoprotein secretion) , Eccrine sweat gland clear cell (small molecule secretion) . Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive) , Gland of Moll cell in eyelid (specialized sweat gland) , Sebaceous gland cell (lipid-rich sebum secretion) , Bowman's gland cell in nose (washes olfactory epithelium) , Brunner's gland cell in duodenum (enzymes and alkaline mucus) , Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm) , Prostate gland cell (secretes seminal fluid components) , Bulbourethral gland cell (mucus secretion) , Bartholin's gland cell (vaginal lubricant secretion) , Gland of Littre cell (mucus secretion) , Uterus endometrium cell (carbohydrate secretion) , Isolated goblet cell of respiratory and digestive tracts (mucus secretion) , Stomach lining mucous cell (mucus secretion) , Gastric gland zymogenic cell (pepsinogen secretion) , Gastric gland oxyntic cell (hydrochloric acid secretion) , Pancreatic acinar cell (bicarbonate and digestive enzyme secretion) , Paneth cell of small intestine (lysozyme secretion) , Type II pneumocyte of lung (surfactant secretion) , Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of ruptured ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion) , Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract) , Kidney, Type I pneumocyte (lining air space of lung) , Pancreatic duct cell (centroacinar cell) , Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc. ) , Duct cell (of seminal vesicle, prostate gland, etc. ) , Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell) , Megakaryocyte (platelet precursor) , Monocyte, Connective tissue macrophage (various types) , Epidermal Langerhans cell, Osteoclast (in bone) , Dendritic cell (in lymphoid tissues) , Microglial cell (in central nervous system) , Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types) , Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte) , Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis) , Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.

Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells include cells of cancers including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms'tumor, and combinations thereof. In some embodiments, the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of a hematopoietic lineage, such as a lymphoma. The antigen can be a tumor associated antigen.

In some cases, the target cell (e.g., B cells) as disclosed herein is associated or is suspected of being associated with an autoimmune disease. The subject being treated with any one of the engineered immune cell (e.g., engineered NK cell) of the present disclosure can have or can be suspected of having an autoimmune disease.

Non-limiting examples of an autoimmune disease can include acute disseminated encephalomyelitis (ADEM) , acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplantation rejection, anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome (APS) , autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED) , autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP) , autoimmune thyroid disease, autoimmune urticaria, axonal &neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) , chronic recurrent multifocal ostomyelitis (CRMO) , Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica) , discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis) , glomerulonephritis, goodpasture's syndrome, granulomatosis with polyangiitis (GPA) , Graves'disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP) , IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes (type 1) , interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD) , lupus (SLE) , lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD) , monoclonal gammopathy of undetermined significance (MGUS) , Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's ) , neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus) , paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH) , Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis) , pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm &testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE) , Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP) , Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD) , uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia (WM) , and Wegener's granulomatosis (Granulomatosis with Polyangiitis (GPA) ) .

In some cases, the autoimmune disease comprises one or more members selected from the group comprising rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis, multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves'Disease, Wegener's Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplants such as renal transplant) . In examples, the autoimmune disease can be type 1 diabetes, lupus, or rheumatoid arthritis.

In some cases the target disease is acute myeloid leukemia (AML) . For example, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to an antigen (e.g., CD33) as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat AML.

In some cases, the target disease is non-Hodgkin’s lymphoma (NHL) .

In some cases, the target disease is chronic lymphocytic leukemia (CLL) .

In some cases, the target disease is B-cell leukemia (BCL) . For example, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD19 as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat BCL.

In some cases, the target disease is non-small-cell lung carcinoma (NSCLC) .

In some cases, the target cells form a tumor (i.e., a solid tumor) . A tumor treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20%in size, and/or do not metastasize) . In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some cases, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more. In some cases, the tumor is completely eliminated, or reduced below a level of detection. In some cases, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some cases, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some cases, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

EXAMPLES

Example 1: Enhanced expression and/or activity level of an activating NK receptor

For improved cell therapy (e.g., stem cell therapy, adaptive immunotherapy, etc. ) , cells of interest can be engineered with enhanced or introduced expression level of activating NK receptor. The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .

For example, iPSCs can be engineered to generate engineered NK cells exhibiting enhanced expression and/or activity level of an activating NK receptor, e.g., NKG2D, NKP30, NKP44, NKP46, DNAM1, NKP80 or a variant thereof (e.g., see TABLE 1) . As illustrated in FIG. 1, FIG. 13, and FIG. 14, a heterologous polypeptide selected from the group comprising 120, 130, 140, 150, 160, 170, 180, 190, and 200, each heterologous polypeptide encoding at least one activating NK receptor, can be in contact with an iPSC or a differentiated cell derivative thereof, to generate the engineered NK cells. The heterologous polypeptide 110 is a control vector that does not encode any activating NK receptor. Such engineered NK cells can exhibit enhanced cytotoxicity against cancer cells, e.g., for pan-cancer treatment. In some cases, the heterologous polypeptide selected from the group comprising 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 can further encode a heterologous cytokine and/or a heterologous receptor thereof (e.g., IL15RF) . In some cases, the heterologous polypeptide selected from the group comprising 120, 130, 140, 150, 160, 170, 180, 190, and 200 can further encode a heterologous CD16 variant for enhanced CD16 signaling.

Table 1

In some examples, any one of the heterologous polypeptide selected from the group comprising 120, 130, 140, 150, 160, 170, 180, 190, and 200 can come in contact (e.g., via transfection or viral transduction) with the iPSCs prior to inducing differentiation of the iPSCs (e.g., into hematopoietic stem cells) . As shown in FIG. 14, iPSCs can be engineered to express NK receptors.

In some examples, any one of the heterologous polypeptide selected from the group comprising 120, 130, 140, 150, 160, 170, 180, 190, and 200 can come in contact (e.g., via transfection or viral transduction) with hematopoietic stem cells derived from the iPSCs.

In some examples, any one of the heterologous polypeptide selected from the group comprising 120, 130, 140, 150, 160, 170, 180, 190, and 200 can come in contact (e.g., via transfection or viral transduction) with NK cells derived from the iPSCs.

Example 2: An engineered immune cell with (A) enhanced expression and/or activity level of DAP and (B) enhanced expression and/or activity level of an activating NK receptor.

For improved cell therapy (e.g., stem cell therapy, adaptive immunotherapy, etc. ) , cells of interest can be engineered with enhanced or introduced expression level of a plurality of polypeptides. The plurality of polypeptides can be different polypeptides. The plurality of polypeptides can be complimentary to one another, e.g., in terms of expression level, activity level, etc. The plurality of polypeptides may directly interact (e.g., directly bind) with each other. Alternatively, the plurality of polypeptides may not directly interact with each other. The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .

For example, in an engineered immune cell (e.g., an engineered NK cell) as disclosed herein, the presence of (A) enhanced expression and/or activity level of DAP (e.g., DAP10) can facilitate or further amplify (B) enhanced expression and/or activity level of the activating NK receptor (e.g., NKG2D) . Thus, (A) can have a synergistic effect on (B) , or vice versa, in the engineered immune cell.

Referring to FIG. 2A, 293T cells were modified with one of the

heterologous polynucleotides

210, 220, and 230. The heterologous polynucleotide 230 encodes a heterologous NKG2D without a heterologous DAP10. The heterologous polynucleotide 220 encodes a heterologous DAP10 without a heterologous NKG2D. The heterologous polynucleotide 210 encodes both the heterologous NKG2D and the heterologous DAP10, to examine the synergy effect of NKG2D and DAP10. Referring to FIGs. 2B and 2C, co-expression of the heterologous NKG2D and the heterologous DAP10 showed enhanced expression of membrane-bound NKG2D, relative to (i) control 293T cells without any heterologous polynucleotide modification, (ii) 293T cells modified with the heterologous NKG2D alone, and (ii) 293T cells modified with the heterologous DAP10 alone. Thus, induced expression of heterologous activating NK receptor (e.g., NKG2D) in cells (e.g., 293T cells, iPSC cells, NK cells) can be dependent on induced expression of an additional heterologous protein, such as DAP (e.g., DAP-10) .

Example 3: Engineered NK cells for acute myeloid leukemia (AML) therapy.

For improved cell therapy (e.g., stem cell therapy, adaptive immunotherapy, etc. ) , cells of interest can be engineered with a plurality of modifications. The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .

For example., an engineered NK cell can be prepared to exhibit at least three modifications: (A) a chimeric polypeptide receptor (e.g., a chimeric antigen receptor (CAR) , an engineered T cell receptor (TCR) , etc. ) comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not a ligand of the activating NK receptor (e.g., not a ligand of NKG2D; (B) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a ligand of the activating NK receptor (e.g., NKG2D ligand) ; and (C) reduced expression and/or activity (e.g., via genetic knockout) of an endogenous gene, such as CD33, CD70, and/or TIM3. The features (A) , (B) , and (C) can be introduced in any order during the development of the engineered NK cell (e.g., from iPSCs) .

For example, the engineered NK cells can be prepared to express a heterologous CAR encoded by any one of the heterologous polynucleotides 310 (ani-CLL-1 CAR) , 320 (anti-CD33 CAR) , 330 (anti-CD70 CAR) , 340 (anti-CD123 CAR) , and/or 350 (anti-TIM3 CAR) (FIG. 3A) . The engineered NK cells can also be modified to exhibit enhanced CD16 signaling by inserting the CD16 variant 360 (CD16-IL15RF fusion protein) or 365 (CD16-CD64-IL15RF fusion protein) into the cell’s endogenous CD16 gene (FIG. 3B) . The engineered NK cells can also be modified to exhibit knock out of one or more immune regulating proteins, such as 370 (CD33) , 380 (CD70) , and/or 390 (TIM3) (FIG. 3C) . By testing different and various combinations as disclosed herein and the effect thereof on the engineered NK cell’s activity (e.g., persistence or viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, etc. ) , different optimal combinations can be identified for different applications, such as AML therapy.

Example 4: Engineered CAR NK cells

For improved cell therapy (e.g., adaptive immunotherapy, etc. ) , cells of interest can be engineered to comprise a heterologous chimeric polypeptide (e.g., a chimeric antigen receptor) comprising an antigen binding moiety against a specific antibody of a target cell (e.g., a cancer or tumor cell) , to exhibit enhanced cytotoxicity against such target cell. The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. For example, for NK cells derived from stem cells, one or more genetic modifications as disclosed herein can be introduced at (A) the stem cell state (e.g., iPSC state) , (B) the hematopoietic stem cell state, and/or (C) the NK cell state. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .

For example, NK cells expressing a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to CLL01 was generated. Schematic of anti-CLL-1 CAR structure design is shown in FIG. 4A, wherein TM is short for Transmembrane domain, and SCFV is short for single chain variable fragment. As shown in FIG. 4B, CLL1-CAR NK92 cells demonstrated targeted cytotoxicity against HL60 tumor cells that high express CLL-1, as compared to wild type (WT) NK92 cells that lack the anti-CLL-1 CAR (E/T (Effector/Target) equals 1: 1; 1: 5, or 1: 10) . As shown in FIG. 4C, upon contacting the JL60 tumor cells, NK92 cells comprising anti-CLL-1 CAR exhibited a higher expression level of CD107a, as compared to the NK92 cells lacking the anti-CCL-CAR. NK cells are involved in innate immunity against targets, such as tumors and viral infections. Lysosome-associated membrane protein-1 (LAMP-1 or CD107a, as used interchangeably herein) can be a marker for degranulation of NK cells, and enhanced expression level of CD107a in NK cells can be an indication of cytotoxic activity determination of the NK cell (e.g., the anti-CLL-1 CAR NK cell as disclosed herein) .

Example 5: Engineered NK cells from iPSCs

iPSCs can be engineered to generate engineered NK cells exhibiting enhanced expression and/or activity level of an activating NK receptor, e.g., NKG2D, NKP30, NKP44, NKP46, or a variant thereof. As shown in FIG. 5A, engineered iPSCs were differentiated into engineered NK cells (super NK cells) , where there was no difference in hCD56+ cells versus wildtype (ANB) (e.g., iPSC derived NK cells without genetic modifications) . FACS fluoresces intensity showed that the engineered NK cells overexpressed an activating NK receptor (FIG. 5B) .

Example 6: iPSC derived engineered NK exhibit cytotoxicity in vitro

iPSCs engineered with an activating NK receptor (e.g., NKG2D, NKP30) were differentiated to engineered NK cells (super NK) . Super NK cells were incubated with solid tumor cells (e.g., Calu-3) at various NK to solid tumor cells ratio (e.g., 10: 1, 1: 1) . Super NK cells showed superior cytotoxicity to solid tumor cells, compared to NK cells differentiated from

iPSC cells (FIG. 6A) . Serial cytotoxicity analysis with super NK cells incubated with solid tumor cells (e.g., THP1 or SKOV3) at 1: 1 ratio showed the ability of super NK cells to maintain long-term cytotoxicity to the solid tumor cells (FIG. 6B) . iPSCs was engineered with more than one activating NK receptors (e.g., NKG2D and NKP30) and differentiated to NK cells (super NK v4.0) . As shown in FIG. 6C, super NK v4.0 showed superior killing capability incubated with solid tumor cells (e.g., THP1 or MOLM13) compared to NK cells derived from

iPSCs, while maintaining high expression of the NK receptors (FIG. 6D) .

Example 7: iPSC derived engineered NK exhibit cytotoxicity in vivo

Mice were inoculated with tumor cells labeled with luciferase (e.g., THP1 or SKOV3) only or together with engineered NK cells expressing NK receptors (e.g., NKG2D and NKP30) . Tumor burden, calculated through the luminescence intensity, revealed that engineered NK cells expressing NK receptors (super NK v4.0) exhibited more cytotoxicity compared to NK cells derived from

iPSCs (FIG. 7 and 8D) . The mice with super NK v4.0 had weight change post injection (FIG. 8A) but had higher percent survival compared to mice inoculated with tumor cells only (FIG. 8B) . The mice with high levels of super NK v4.0 also maintained hCD56+ cells, as measured by pharmacokinetics in peripheral blood (FIG. 8C) .

Example 8: Gene knockout to enhance NK function

For improved cell therapy (e.g., adaptive immunotherapy, etc. ) , genes involved in inhibiting NK cell activity can be knocked down to enhance NK function. The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. For example, for NK cells derived from stem cells, one or more genetic modifications as disclosed herein can be introduced at (A) the stem cell state (e.g., iPSC state) , (B) the hematopoietic stem cell state, and/or (C) the NK cell state. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .

TGFb-R2 was knocked out in NK cells (e.g., with sgRNAs targeting TGFb-R2) and were incubated with GFP labeled tumor cells (e.g., THP1 cells) , with different TGF-b dosage (e.g., 10 ng/ml and 50 ng/ml) at various times (e.g., at 24 hours and 72 hours) . As shown in FIG. 9A and 9B, knocking out TGFb-R2 rescued NK function under TGF-b treatment, enhancing NK cytotoxicity function. Knocking out TGFb-R2 (FIG. 10A) in NK cells also rescued NKG2D and NKP30 expression from TGF-b treatment, as analyzed by FACS (FIG. 10B) .

CBNK was incubated with wild type K562 and HLA-E K562 cells at different effector cell to target cell ratios (e.g., E/T ratio at 4: 1, 1: 1, and 0.5: 1) . Cytotoxicity assay showed that HLAE inhibits NK cytotoxicity function (FIG. 11A) . As shown in FIG. 11B, HLA-E K562 cells decreased the percentage of CD107a+ cells, presenting the percentage of activated NK cells. Upon knockout of NKG2A (e.g. with sgRNAs with Cas9 protein) in CBNK (FIG. 11C) , the NKG2A knocked out cells were incubated with wild type K562 and HLA-E K562. As shown in FIG. 11D, NKG2A knock out rescued NK function during targeting of the HLA-E cells, assessed via cytotoxicity assay.

SOCS2 was knocked out in iPSCs. As shown in FIG. 12A, engineered iPSCs were differentiated into engineered NK cells (TD, representing various SOCS2 knock outs) , where there was no difference in hCD56+ cells versus wildtype (ANB) (e.g., iPSC derived NK cells without genetic modifications) , as quantified via FACS fluoresces intensity (FIG. 12B) . When cultured in vitro in presence of allogeneic peripheral blood cells, to mimic in vivo conditions, with IL-2 or IL-15 supplementation, engineered SOCS2 knockout NK cells showed greater persistence compared to unmodified NK cells (FIG. 12C) .

EMBODIMENTS

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1: An engineered NK cell, comprising:

(1) enhanced expression or activity level of an activating NK receptor as compared to a control NK cell; and/or

(2) one or more members selected from the group consisting of:

(vi) a safety switch capable of effecting death of the engineered NK cell.

optionally wherein:

(a) the engineered NK cell comprises (i) the heterologous CD16 variant; and/or

(b) the engineered NK cell comprises (ii) the reduced expression and/or activity level of the endogenous CD38; and/or

(c) the engineered NK cell comprises (iii) the reduced expression of the one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70; and/or

(d) the engineered NK cell comprises (iv) the reduced expression and/or activity of the immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4; and/or

(e) the engineered NK cell comprises (v) the reduced expression and/or activity of the hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and/or

(f) the engineered NK cell comprises (vi) the safety switch; and/or

(g) the engineered NK cell comprises two or more members selected from the group consisting of (i) , (ii) , (iii) , (iv) , and (v) ; and/or

(h) wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking (1) the enhanced expression or activity level of the activating NK receptor and/or (2) the one or more members selected from the group consisting of (i) - (iv) ; and/or

(i) wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking (1) the enhanced expression or activity level of the activating NK receptor and/or (2) the one or more members selected from the group consisting of (i) - (iv) ; and/or

(j) further comprising a heterologous interleukin (IL) cytokine and/or receptor for enhanced interleukin signaling as compared to the control NK cell; and/or

(k) further comprising a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not a NKG2D ligand.

Embodiment 2. An engineered NK cell, comprising:

enhanced expression or activity level of an activating NK receptor as compared to a control NK cell,

wherein:

the engineered NK cell exhibits at least about 0.1-fold or at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to the control NK cell; and/or

the enhanced expression or activity level of the activating NK receptor on the engineered NK cell is at least about 0.1-fold or at least about 0.5-fold greater than that in the control NK cell,

optionally wherein:

(a) the activating NK receptor does not comprise a heterologous intracellular signaling domain; and/or

(b) the activating NK receptor does not comprise a heterologous transmembrane domain; and/or

(c) the activating NK receptor does not comprise a heterologous antigen binding domain.

Embodiment 3. An engineered NK cell, comprising:

enhanced expression or activity level of an activating NK receptor as compared to a control NK cell, wherein the activating NK receptor is configured to bind an antigen of NKG2D, NKP30, and/or NKP44, NKP46, NKP80, and/or DNAM1 and wherein the activating NK receptor does not comprise a heterologous intracellular signaling domain.

Embodiment 4. The engineered NK cell of any one of the preceding Embodiments, wherein:

(a) the activating NK receptor is endogenous to the engineered NK cell;

(b) the engineered NK cell comprises a heterologous polynucleotide sequence encoding the activating NK receptor;

(c) the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the enhanced expression or activity level of the activating NK receptor; and/or

(d) the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking the enhanced expression or activity level of the activating NK receptor.

Embodiment 5. An engineered NK cell, comprising:

a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is selected from the group consisting of CD70, CLEC12A, and TIM-3, CD9, CD26, TRAIL-R4 (DCR2) , and NKG2D ligand,

wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the chimeric polypeptide receptor,

optionally wherein:

(a) the antigen is selected from the group consisting of CD70, CLEC12A, and TIM-3; and/or

(b) the antigen is CD70 or TIM-3; and/or

(c) the engineered NK cell exhibits at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the cytotoxicity against the target cell as compared to the control NK cell; and/or

(d) the engineered NK cell exhibits at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the proliferation as compared to the control NK cell.

Embodiment 6. An engineered NK cell, comprising

(1) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is CD38 and/or BCMA; and

(2) one or more members selected from the group consisting of:

(iv) a safety switch capable of effecting death of the engineered NK cell,

wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the one or more members,

optionally wherein:

(a) the engineered NK cell comprises (i) the enhanced expression or activity level of the activating NK receptor; and/or

(b) the engineered NK cell comprises (ii) the reduced expression and/or activity of the immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4; and/or

(c) the engineered NK cell comprises (iii) the reduced expression and/or activity of rgw hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and/or

(d) the engineered NK cell comprises (iv) the safety switch; and/or

(e) the engineered NK cell comprises two or more members selected from the group consisting of (i) , (ii) , (iii) , and (iv) .

Embodiment 7. An engineered NK cell, comprising:

(1) reduced expression or activity level of a suppressor of cytokine (SOCS) protein; and

(2) one or more members selected from the group consisting of:

(vii) a safety switch capable of effecting death of the engineered NK cell; and

Embodiment 8. The engineered NK cell of any one of the preceding Embodiments, wherein:

(a) the NK cell comprises (i) the enhanced expression or activity level of endogenous activating NK receptor; and/or

(b) the engineered NK cell comprises (ii) the heterologous interleukin (IL) cytokine and/or receptor; and/or

(c) the engineered NK cell comprises (iii) the heterologous CD16 variant; and/or

(d) the engineered NK cell comprises (iv) the reduced expression and/or activity level of the endogenous CD38; and/or

(e) the engineered NK cell comprises (v) the reduced expression and/or activity of the immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4; and/or

(f) the engineered NK cell comprises (vi) the reduced expression and/or activity of the hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59;

(g) the engineered NK cell comprises (vii) the safety switch; and/or

(h) the engineered NK cell comprises (viii) the chimeric polypeptide receptor; and/or

(i) the engineered NK cell comprises two or more members selected from the group consisting of (i) , (ii) , (iii) , (iv) , (v) , (vi) , (vii) , and (viii) ; and/or

(j) the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking reduced expression or activity level of the SOCS protein; and/or

(k) the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking reduced expression or activity level of the SOCS protein; and/or

(l) the SOCS protein is an endogenous protein; and/or

(m) the SOCS protein is selected from the group consisting of CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7; and/or

(n) the SOCS protein is SOCS2; and/or

(o) further comprising an enhanced expression or activity level of a dead-associated protein (DAP) , optionally wherein the DAP is DAP-10 or DAP-12; and/or

(p) the activating NK receptor comprises NKG2D, NKP30, NKP44, NKP46, NKP80, DNAM1, a modification thereof, a functional variant thereof, or a combination thereof; and/or

(q) the activating NK receptor comprises NKG2D or a modification thereof; and/or

(r) the activating NK receptor comprises NKP30 or a modification thereof; and/or

(s) the activating NK receptor comprises NKP44 or a modification thereof; and/or

(t) the activating NK receptor comprises NKP46 or a modification thereof; and/or

(u) the activating NK receptor comprises NKP80 or a modification thereof; and/or

(v) the activating NK receptor comprises DNAM1 or a modification thereof; and/or

(w) the activating NK receptor is heterologous to the engineered immune cell; and/or

(x) the activating NK receptor is a chimeric polypeptide receptor comprising at least an extracellular portion of NKG2D, NKP30, NKP44, or NKP46, NKP80, or DNAM1; and/or

(y) the activating NK receptor is endogenous to the engineered immune cell; and/or

(z) the heterologous IL cytokine and/or receptor comprises IL-15, IL-15R, and/or a fragment thereof; and/or

(aa) the antigen is not a NKP30 ligand, a NKP44 ligand, and/or a NKP46 ligand, NKP80 ligand, and/or DNAM1 ligand; and/or

(bb) the antigen comprises BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10; and/or

(cc) the engineered NK cell exhibits enhanced cytotoxic activity against a population of target cells as compared to the control NK cell; and/or

(dd) the enhanced cytotoxic activity against the population of target cells is ascertained by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in a size of the population of target cells as compared to the control NK cell; and/or

(ee) the engineered NK cell exhibits reduction in tumor size as compared to as compared to the control NK cell; and/or

(ff) the tumor size is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%as compared to the control NK cell; and/or

(gg) the engineered NK cell induces reduced immune response in a host cell as compared to the control NK cell; and/or

(hh) the engineered NK cell is for use in treating a subject in need thereof; and/or

(ii) the engineered NK cell is allogeneic to the subject; and/or

(jj) the engineered NK cell is autologous to the subject; and/or

(kk) the engineered NK cell is derived from an isolated stem cell or an induced stem cell; and/or

(ll) the engineered NK cell is derived from a cord blood natural killer cell (CBNK) or a peripheral blood natural killer cell (PBNK) ; and/or

(mm) the engineered NK cell is for use in a method for inducing death of a target cell, optionally wherein the target cell is a cancer cell or a tumor cell; and/or

(nn) the engineered NK cell is for use in a method for treating a subject in need thereof, wherein the subject has or is suspected of having a condition, optionally wherein:

the condition is cancer or tumor; and/or

the engineered NK cell is either autologous or allogeneic to the subject; and/or

(oo) the engineered NK cell is for the manufacture of medicament for inducing death of a target cell, optionally wherein the target cell is a cancer cell or a tumor cell; and/or

(pp) the engineered NK cell is for the manufacture of medicament for treating a subject in need thereof, wherein the subject has or is suspected of having a condition, optionally wherein the condition is cancer or tumor; and/or

(qq) the engineered NK cell is either autologous or allogeneic to the subject.

Embodiment 9. A composition comprising the engineered NK cell of any one of the Embodiments provided herein,

optionally wherein the composition further comprises a separate therapeutic agent, further optionally wherein the separate therapeutic agent is a chemotherapeutic agent.

Embodiment 10. A method comprising:

obtaining a cell from a subject; and

generating, from the cell, the engineered NK cell of any one of the Embodiments provided herein.

Embodiment 11. A method comprising:

contacting the target cell with the engineered NK cell of any one of the Embodiments provided herein,

optionally wherein the contacting is sufficient to induce the killing of the target cell.

Embodiment 12. A method of treating a subject in need thereof, comprising administering the engineered NK cell of any any one of the Embodiments provided herein,

optionally wherein the method further comprises administering a separate therapeutic agent, further optionally wherein the separate therapeutic agent is a chemotherapeutic agent.

Embodiment 13. A method of converting a stem cell to the engineered NK cell of any one of the Embodiments provided herein, comprising:

contacting in vitro the stem cell with a transgenic nucleic acid encoding the activating NK receptor, wherein the stem cell is an isolated stem cell or an induced stem cell, for conversion of the stem cell to the engineered NK cell,

wherein, upon the contacting, the engineered NK cell exhibits enhanced expression or activity level of the activating NK receptor as compared to a control NK cell.

It shall be understood that different aspects of the invention can be appreciated individually, collectively, or in combination with each other. Various aspects of the invention described herein may be applied to any of the particular applications disclosed herein. The compositions of matter including the engineered immune cells of the present disclosure may be utilized in the method section including methods of use and production disclosed herein, or vice versa.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An engineered NK cell, comprising:

(1) enhanced expression or activity level of an activating NK receptor as compared to a control NK cell; and

(2) one or more members selected from the group consisting of:

(i) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a NKG2D ligand;

(ii) reduced expression and/or activity level of an endogenous CD38 as compared to the control NK cell;

(iii) a reduced expression of one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70;

(iv) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(v) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and

(vi) a safety switch capable of effecting death of the engineered NK cell.
The engineered NK cell of claim 1, comprising two or more members selected from the group consisting of (i) , (ii) , (iii) , (iv) , and (v) .
The engineered NK cell of any one of the preceding claims, comprising (i) .
The engineered NK cell of any one of the preceding claims, comprising (ii) .
The engineered NK cell of any one of the preceding claims, comprising (iii) .
The engineered NK cell of any one of the preceding claims, comprising (iv) .
The engineered NK cell of any one of the preceding claims, comprising (v) .
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking (1) and/or (2) .
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking (1) and/or (2) .
The engineered NK cell of any one of the preceding claims, further comprising a heterologous interleukin (IL) cytokine and/or receptor for enhanced interleukin signaling as compared to the control NK cell.
The engineered NK cell of any one of the preceding claims, further comprising a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not a NKG2D ligand.
An engineered NK cell, comprising:

enhanced expression or activity level of an activating NK receptor as compared to a control NK cell, wherein the activating NK receptor is configured to bind an antigen of NKG2D, NKP30, NKP44, NKP46, NKP80, and/or DNAM1 and wherein the activating NK receptor does not comprise a heterologous intracellular signaling domain.
The engineered NK cell of claim 12, wherein the activating NK receptor is endogenous to the engineered NK cell.
The engineered NK cell of claim 12, wherein the engineered NK cell comprises a heterologous polynucleotide sequence encoding the activating NK receptor.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the enhanced expression or activity level of the activating NK receptor.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking the enhanced expression or activity level of the activating NK receptor.
An engineered NK cell, comprising:

a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is selected from the group consisting of CD70, CLEC12A, and TIM-3, CD9, CD26, TRAIL-R4 (DCR2) , and NKG2D ligand,

wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the chimeric polypeptide receptor.
The engineered NK cell of any one of the preceding claims, wherein the antigen is selected from the group consisting of CD70, CLEC12A, and TIM-3.
The engineered NK cell of any one of the preceding claims, wherein the antigen is CD70 or TIM-3.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the cytotoxicity against the target cell as compared to the control NK cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the proliferation as compared to the control NK cell.
An engineered NK cell, comprising:

(1) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is CD38 and/or BCMA; and

(2) one or more members selected from the group consisting of:

(i) enhanced expression or activity level of an activating NK receptor as compared to a control NK cell;

(ii) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(iii) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59; and

(iv) a safety switch capable of effecting death of the engineered NK cell,

wherein the engineered NK cell exhibits at least about 0.5-fold increase in cytotoxicity against a target cell, as compared to a control NK cell lacking the one or more members.
The engineered NK cell of any one of the preceding claims, comprising two or more members selected from the group consisting of (i) , (ii) , (iii) , and (iv) .
The engineered NK cell of any one of the preceding claims, comprising (i) .
The engineered NK cell of any one of the preceding claims, comprising (ii) .
The engineered NK cell of any one of the preceding claims, comprising (iii) .
The engineered NK cell of any one of the preceding claims, comprising (iv) .
An engineered NK cell, comprising:

(1) reduced expression or activity level of a suppressor of cytokine (SOCS) protein; and

(2) one or more members selected from the group consisting of:

(i) enhanced expression or activity level of endogenous activating NK receptor as compared to a control NK cell;

(ii) a heterologous interleukin (IL) cytokine and/or receptor for enhanced interleukin signaling as compared to the control NK cell;

(iii) a heterologous CD16 variant for enhanced CD16 signaling as compared to the control NK cell, wherein the heterologous CD16 variant does not bind a NKG2D ligand;

(iv) reduced expression and/or activity level of an endogenous CD38 as compared to the control NK cell;

(v) reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4;

(vi) reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59;

(vii) a safety switch capable of effecting death of the engineered NK cell; and

(viii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not a NKG2D ligand.
The engineered NK cell of any one of the preceding claims, comprising two or more members selected from the group consisting of (i) , (ii) , (iii) , (iv) , (v) , (vi) , and (viii) .
The engineered NK cell of any one of the preceding claims, comprising (i) .
The engineered NK cell of any one of the preceding claims, comprising (ii) .
The engineered NK cell of any one of the preceding claims, comprising (iii) .
The engineered NK cell of any one of the preceding claims, comprising (iv) .
The engineered NK cell of any one of the preceding claims, comprising (v) .
The engineered NK cell of any one of the preceding claims, comprising (vi) .
The engineered NK cell of any one of the preceding claims, comprising (vii) .
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in cytotoxicity against a target cell, as compared to a control NK cell lacking reduced expression or activity level of the SOCS protein.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proliferation, as compared to a control NK cell lacking reduced expression or activity level of the SOCS protein.
The engineered NK cell of any one of the preceding claims, wherein the SOCS protein is an endogenous protein.
The engineered NK cell of any one of the preceding claims, wherein the SOCS protein is selected from the group consisting of CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7.
The engineered NK cell of any one of the preceding claims, wherein the SOCS protein is SOCS2.
The engineered NK cell of any one of the preceding claims, further comprising an enhanced expression or activity level of a dead-associated protein (DAP) .
The engineered NK cell of claim 32, wherein the DAP is DAP-10.
The engineered NK cell of claim 32, wherein the DAP is DAP-12.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises NKG2D, NKP30, NKP44, NKP46, NKP80, DNAM1, a modification thereof, a functional variant thereof, or a combination thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises NKG2D or a modification thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises NKP30 or a modification thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises NKP44 or a modification thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises NKP46 or a modification thereof.
The engineered NK cell of any of the preceding claims, wherein the activating NK receptor comprises NKP80 or a modification thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor comprises DNAM1 or a modification thereof.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor is heterologous to the engineered immune cell.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor is a chimeric polypeptide receptor comprising at least an extracellular portion of NKG2D, NKP30, NKP44, or NKP46, NKP80, or DNAM1.
The engineered NK cell of any one of the preceding claims, wherein the activating NK receptor is endogenous to the engineered immune cell.
The engineered NK cell of any one of the preceding claims, wherein the heterologous IL cytokine and/or receptor comprises IL-15, IL-15R, and/or a fragment thereof.
The engineered NK cell of any one of the preceding claims, wherein the antigen is not a NKP30 ligand, a NKP44 ligand, and/or a NKP46 ligand, NKP80 ligand, and/or DNAM1 ligand.
The engineered NK cell of any one of the preceding claims, wherein the antigen comprises BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits enhanced cytotoxic activity against a population of target cells as compared to the control NK cell.
The engineered NK cell of claim 58, wherein the enhanced cytotoxic activity against the population of target cells is ascertained by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in a size of the population of target cells as compared to the control NK cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits reduction in tumor size as compared to as compared to the control NK cell.
The engineered NK cell of claim 60, wherein the tumor size is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%as compared to the control NK cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell induces reduced immune response in a host cell as compared to the control NK cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is for use in treating a subject in need thereof.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is allogeneic to the subject.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is autologous to the subject.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is derived from a cord blood natural killer cell (CBNK) or a peripheral blood natural killer cell (PBNK) .
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is for use in a method for inducing death of a target cell, optionally wherein the target cell is a cancer cell or a tumor cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is for use in a method for treating a subject in need thereof, wherein the subject has or is suspected of having a condition.
The engineered NK cell of any one of the preceding claims, wherein the condition is cancer or tumor.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is either autologous or allogeneic to the subject.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is for the manufacture of medicament for inducing death of a target cell, optionally wherein the target cell is a cancer cell or a tumor cell.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is for the manufacture of medicament for treating a subject in need thereof, wherein the subject has or is suspected of having a condition.
The engineered NK cell of any one of the preceding claims, wherein the condition is cancer or tumor.
A composition comprising the engineered NK cell of any one of the preceding claims.
The composition of claim 75, further comprising a separate therapeutic agent.
The composition of claim 76, wherein the separate therapeutic agent is a chemotherapeutic agent.
A method comprising:

obtaining a cell from a subject; and

generating, from the cell, the engineered NK cell of any one of the preceding claims.
A method of killing a target cell, comprising:

contacting the target cell with the engineered NK cell of any one of the preceding claims.
The method of claim 79, wherein the contacting is sufficient to induce the killing of the target cell.
A method of treating a subject in need thereof, comprising administering the engineered NK cell of any one of the preceding claims.
The method of claim 81, further comprising administering a separate therapeutic agent.
The method of claim 82, wherein the separate therapeutic agent is a chemotherapeutic agent.
A method of converting a stem cell to an engineered NK cell, comprising:

contacting in vitro the stem cell with a transgenic nucleic acid encoding an activating NK receptor, wherein the stem cell is an isolated stem cell or an induced stem cell, for conversion of the stem cell to the engineered NK cell,

wherein, upon the contacting, the engineered NK cell exhibits enhanced expression or activity level of the activating NK receptor as compared to a control NK cell.