WO2023147776A1

WO2023147776A1 - Systems and methods for enhanced immunotherapies

Info

Publication number: WO2023147776A1
Application number: PCT/CN2023/074518
Authority: WO
Inventors: Jing Xu; Lu Liu; Yanan YUE; Luhan Yang; Yangbin Gao
Original assignee: Hangzhou Qihan Biotechnology Co., Ltd.
Priority date: 2022-02-07
Filing date: 2023-02-06
Publication date: 2023-08-10
Also published as: TW202342737A

Abstract

Provided are systems and methods for immunotherapy. 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 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.

Description

SYSTEMS AND METHODS FOR ENHANCED IMMUNOTHERAPIES

CROSS-REFERENCE

This application claims the benefit International Application No. PCT/CN2022/075418, filed February 07, 2022, and International Application No. PCT/CN2023/071422, filed January 09, 2023, which are incorporated herein by reference in their entirety.

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.

In addition, increased understanding of diseases related to viral infections has also encouraged the development of adoptive cell therapies (e.g., cellular immunotherapy) to treat such diseases.

SUMMARY

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

In one aspect, the present disclosure provides an engineered NK cell, comprising: a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to an antigen of a virus, wherein the virus is not CMV. In some embodiments, the engineered NK cell is derived from an isolated stem cell or an induced stem cell.

In some embodiments of any one of the engineered NK cells disclosed herein, the virus comprises one or more members selected from the group consisting of HIV, HBV, HCV, EBV, HPV, Lasse Virus, Influenza Virus, Coronavirus, and a derivative thereof. In some embodiments of any one of the engineered NK cells disclosed herein, the virus comprises one or more members selected from the group consisting of HBV, HCV, and a derivative thereof.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell exhibits enhanced cytotoxicity against a target cell comprising the antigen (e.g., a viral antigen) , as compared to a control cell lacking the chimeric polypeptide receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the enhanced cytotoxicity of the engineered NK cell against the target cell is greater than that of the control cell by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, at least about 200%, or more, or by at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.8-fold, at least about 1-fold, or more. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell is configured to exhibit the enhanced cytotoxicity against the target cell within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, or less of incubation with the target cell. In some embodiments of any one of the engineered NK cells disclosed herein, the enhanced cytotoxicity is at effector to target (E: T) ratio of at least about 5, at least about 10, or at least about 20.

In some embodiments, the difference of cytotoxicity of target cells (e.g., expressing and/or presenting viral antigen (s) ) between the engineered NK cells comprising the chimeric polypeptide receptor (e.g., against the viral antigen (s) ) and control NK cells lacking the chimeric polypeptide receptor (Diff-1) may be greater than the difference of cytotoxicity of control target cells (e.g., not expressing and/or presenting viral antigen) between the engineered NK cells and the control NK cells (Diff-2) , by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more.

In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding moiety comprises at least a portion of an antibody exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding moiety does not comprise at least a portion of an antibody exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding moiety comprises at least a portion of a cellular protein exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the engineered NK cells disclosed herein, the cellular protein is a surface receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the surface receptor is a CD receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the CD receptor is CD4. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least two different signaling domains or at least three different signaling domains. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen of the virus is presented on a surface of a target cell.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous IL-15 or a fragment thereof. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a receptor comprising a heterologous IL-15R or a fragment thereof.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell exhibits reduced expression or activity of endogenous CD38. Alternatively, in some embodiments of any one of the engineered NK cells disclosed herein, expression or activity of endogenous CD38 of the engineered NK cell is not modified.

In some embodiments of any one of the engineered NK cells disclosed herein, the heterologous IL-15 or the fragment thereof is secreted by the engineered NK cell. Alternatively, in some embodiments of any one of the engineered NK cells disclosed herein, the heterologous IL-15 or the fragment thereof is membrane-bound.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises an enhanced expression of an activating NK receptor.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises an additional chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding moiety of the chimeric polypeptide receptor is a multispecific binding moiety capable of specifically binding to two or more antigens that are different. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen comprises one or more members selected from the group consisting of: BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a safety switch capable of effecting death of the engineered NK cell. In some embodiments of any one of the engineered NK cells disclosed herein, the safety switch comprises one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous cytokine. In some embodiments of any one of the engineered NK cells disclosed herein, the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments of any one of the engineered NK cells disclosed herein, the heterologous cytokine is not IL15.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous immune regulator polypeptide. In some embodiments of any one of the engineered NK cells disclosed herein, the heterologous immune regulator polypeptide comprises one or more members selected from the group consisting of 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 NK cells disclosed herein, the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide. In some embodiments of any one of the engineered NK cells disclosed herein, the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator. In some embodiments of any one of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4. In some embodiments of any one of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any one of the engineered NK cells disclosed herein, the hypo-immunity regulator comprises one or more members 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 of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell. In some embodiments of any one of the engineered NK cells disclosed herein, the CD16 variant comprises a sequence selected from the group consisting of: SEQ ID NOs. 1-8.

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

In some embodiments of any one of the engineered NK cells disclosed herein, the isolated stem cell comprises an embryonic stem cell. In some embodiments of any one of the engineered NK cells disclosed herein, the induced stem cell comprises an induced pluripotent stem cell.

In one aspect, the present disclosure provides a method comprising: (1) obtaining a cell from a subject; and (2) generating, from the cell an engineered NK cell that comprises a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to an antigen of a virus, wherein the virus is not CMV. In some embodiments of any one of the methods disclosed herein, the engineered NK cell is derived from an isolated stem cell or an induced stem cell.

In one aspect, the present disclosure provides a method comprising: administering to a subject in need thereof a population of NK cells comprising an engineered NK cell comprising a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to an antigen of a virus, wherein the virus is not CMV.

In some embodiments of any one of the methods disclosed herein, the virus comprises one or more members selected from the group consisting of HIV, HBV, HCV, EBV, HPV, Lasse Virus, Influenza Virus, Coronavirus, and a derivative thereof. In some embodiments of any one of the methods disclosed herein, the virus comprises one or more members selected from the group consisting of HBV, HCV, and a derivative thereof.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell exhibits enhanced cytotoxicity against a target cell comprising the antigen (e.g., a viral antigen) , as compared to a control cell lacking the chimeric polypeptide receptor. In some embodiments of any one of the methods disclosed herein, the enhanced cytotoxicity of the engineered NK cell against the target cell is greater than that of the control cell by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, at least about 200%, or more, or by at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.8-fold, at least about 1-fold, or more. In some embodiments of any one of the methods disclosed herein, the engineered NK cell is configured to exhibit the enhanced cytotoxicity against the target cell within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, or less of incubation with the target cell. In some embodiments of any one of the methods disclosed herein, the enhanced cytotoxicity is at effector to target (E: T) ratio of at least about 5, at least about 10, or at least about 20.

In some embodiments of any one of the methods disclosed herein, the antigen binding moiety comprises at least a portion of an antibody exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the methods disclosed herein, the antigen binding moiety does not comprise at least a portion of an antibody exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the methods disclosed herein, the antigen binding moiety comprises at least a portion of a cellular protein exhibiting specific affinity to the antigen of the virus. In some embodiments of any one of the methods disclosed herein, the cellular protein is a surface receptor. In some embodiments of any one of the methods disclosed herein, the surface receptor is a CD receptor. In some embodiments of any one of the methods disclosed herein, the CD receptor is CD4. In some embodiments of any one of the methods disclosed herein, the chimeric polypeptide receptor comprises at least two different signaling domains or at least three different signaling domains. In some embodiments of any one of the methods disclosed herein, the antigen of the virus is presented on a surface of a target cell.

In some embodiments of any one the methods disclosed herein, the engineered NK cell further comprises a heterologous IL-15 or a fragment thereof. In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises a receptor comprising a heterologous IL-15R or a fragment thereof.

In some embodiments of any one of the methods herein, the engineered NK cell exhibits reduced expression or activity of endogenous CD38. Alternatively, in some embodiments of any one of the methods disclosed herein, expression or activity of endogenous CD38 of the engineered NK cell is not modified.

In some embodiments of any one of the methods disclosed herein, the heterologous IL-15 or the fragment thereof is secreted by the engineered NK cell. Alternatively, in some embodiments of the methods disclosed herein, the heterologous IL-15 or the fragment thereof is membrane-bound.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises an enhanced expression of an activating NK receptor.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises an additional chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some embodiments of any one of the methods disclosed herein, the antigen binding moiety of the chimeric polypeptide receptor is a multispecific binding moiety capable of specifically binding to two or more antigens that are different. In some embodiments of any one of the methods disclosed herein, the antigen comprises one or more members selected from the group consisting of: BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100. In some embodiments of any one of the methods disclosed herein, the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises a safety switch capable of effecting death of the engineered NK cell. In some embodiments of any one of the methods disclosed herein, the safety switch comprises one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises a heterologous cytokine. In some embodiments of any one of the methods disclosed herein, the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments of any one of the methods disclosed herein, the heterologous cytokine is not IL15.

In some embodiments of any one of the methods disclosed herein, the engineered NK cell further comprises a heterologous immune regulator polypeptide. In some embodiments of any one of the methods disclosed herein, the heterologous immune regulator polypeptide comprises one or more members selected from the group consisting of 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 methods disclosed herein, the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide. In some embodiments of any one of the methods disclosed herein, the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator. In some embodiments of any one of the methods disclosed herein, the immune checkpoint inhibitor comprises one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4. In some embodiments of any one of the methods disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any one of the methods disclosed herein, the hypo-immunity regulator comprises one or more members 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 of any one of the methods disclosed herein, the engineered NK cell comprises a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell. In some embodiments of any one of the methods disclosed herein, the CD16 variant comprises a sequence selected from the group consisting of: SEQ ID NOs. 1-8.

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

In some embodiments of any one of the methods disclosed herein, the isolated stem cell comprises an embryonic stem cell. In some embodiments of any one of the methods disclosed herein, the induced stem cell comprises an induced pluripotent stem cell.

In some embodiments of any one of the methods disclosed herein, the method further comprises administering the engineered NK cell to the subject.

In some embodiments of any one of the methods disclosed herein, the method further comprises administering to the subject a separate therapeutic agent. In some embodiments of any one of the methods disclosed herein, the separate therapeutic agent is a chemotherapeutic agent. In some embodiments of any one of the methods disclosed herein, the administration of the engineered NK cells treats or reduces a risk of a cancer in a subject. In some embodiments of any one of the methods disclosed herein, the administration of the engineered NK cell treats a viral infection in the subject.

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:

FIGs. 1A-1G illustrate engineered NK cells comprising a CD16 variant for enhanced CD16 signaling;

FIGs. 2A-2G illustrate engineered NK cells comprising a chimeric antigen receptor against CD19; and

FIGs. 3A and 3B illustrate engineered T cells comprising heterologous human IL-15.

FIGs. 4A and 4B illustrate engineered NK cells for targeting latent membrane protein 1 (LMP1) as a marker for Epstein-Barr virus (EBV) infection.

FIGs. 5A-5C illustrate cytotoxicity of engineered NK cells against LMP1-expressing target cells.

FIGs. 6A-6D illustrate engineered NK cells for targeting CD4 or gp120 as marker (s) for human immunodeficiency virus (HIV) infection.

FIG. 7 illustrate engineered NK cells for targeting Hepatitis B surface antigen (HBsAg) .

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 (or a heterologous 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” or “antigen binding moiety, ” as used interchangeably herein, 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. Alternatively or in addition to, the polypeptide construct of the antigen binding domain can comprise at least a portion of a natural complementary molecule of the specific target antigen. The natural complementary molecule and the specific target antigen may naturally form a complex (e.g., in a cell, on a cell surface, etc. ) . In some examples, the specific target antigen can be a receptor (e.g., a cell surface receptor) , and the natural complementary molecule can be a ligand that binds to and/or is bound by the receptor (e.g., a ligand of the cell surface receptor) , or vice versa. In some examples, the specific target antigen and its natural complementary molecule can both be receptors (e.g., cell surface receptors) that bind to one another, or can both be soluble ligands that binds to one another.

In some embodiments, the antigen binding domain may be derived from an antibody. In some embodiments, the antigen binding domain may not and need not be derived from an antibody. The antigen binding domain can be derived from a cellular polypeptide exhibiting binding affinity to a specific target antigen. For example, the cellular polypeptide can be at least a portion of a receptor protein capable of binding to one or more target antigens (e.g., at least a portion of an extracellular domain of a transmembrane receptor protein exhibiting specific binding to an antigen derived from a virus) .

In some embodiments, the antigen binding domain can be a polypeptide construct, and the polypeptide construct can have a length of at least or up to about 10 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 110 amino acid residues, at least or up to about 120 amino acid residues, at least or up to about 130 amino acid residues, at least or up to about 140 amino acid residues, at least or up to about 150 amino acid residues, at least or up to about 160 amino acid residues, at least or up to about 170 amino acid residues, at least or up to about 180 amino acid residues, at least or up to about 190 amino acid residues, at least or up to about 200 amino acid residues, at least or up to about 210 amino acid residues, at least or up to about 220 amino acid residues, at least or up to about 230 amino acid residues, at least or up to about 240 amino acid residues, at least or up to about 250 amino acid residues, at least or up to about 300 amino acid residues, or at least or up to about 500 amino acid residues.

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 “immune regulator polypeptide” generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secretory polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc. ) capable of regulating or controlling one or more attributes of an immune cell, such as a NK cell. One or more attributes of an immune cell can comprise differentiation of the immune cell, immune cell morphology, expression of a polynucleotide or polypeptide construct within the immune cell, or activity of the immune cell (e.g., cytotoxic activity of an engineered NK cell against a diseased cell, such as a cancer cell) . An immune regulator polypeptide can be endogenous to a host cell. Alternatively or in addition to, an immune regulator polypeptide can be heterologous to a hots cell. In some cases, controlling the one or more attributes of the immune cell can be mediated by downregulating expression of the immune regulator polypeptide (e.g., suppression, knock-down or knock-out) . Alternatively or in addition to, controlling the one or more attributes of the immune cell can be mediated by upregulating expression of the immune regulator polypeptide (e.g., upregulation of an endogenous gene or knock-in of a heterologous gene encoding the immune regulator polypeptide) . Yet in another alternative or additionally, controlling the one or more attributes of the immune cell can be mediated by maintaining expression of the immune regulator polypeptide for time period that is longer than a natural or normal expression profile of the immune regulator polypeptide in a host cell. In some cases, an immune regulator polypeptide can comprise a hypo-immunity regulator. In some case, an immune regulator polypeptide can comprise an immune checkpoint inhibitor.

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 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. ”

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.

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 can 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 NK cells sourced and engineered to exhibit, for example, enhanced proliferation, half-life, and cytotoxic activity against target cells.

There also remains a significant unmet need for NK cells to exhibit enhanced cytotoxicity against virally infected cells or cells that are presenting viral antigens, for treatment of viral disease such as, but not limited to, influenza, common cold, bronchiolitis, acute respiratory disease, measles, smallpox, chickenpox, hepatitis, etc. In addition, some viruses can lead to cancer (e.g., human papillomavirus (HPV) can be one of the factors causing cervical cancer) . Thus, in some aspects, the present disclosure provides systems and methods for immunotherapies based on engineered immune cells (e.g., engineered NK cells) exhibiting enhanced targeting and/or cytotoxicity against virally infected cells, e.g., for treatment of viral diseases and/or cancer (e.g., tumor) .

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 a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell. In some cases, the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) . The engineered immune cell can further comprise one or both of: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell and (ii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some examples, the antigen is not CD19. Thus, the antigen binding moiety may not and need not exhibit any specific binding to CD19, but rather a specific binding to an antigen (e.g., one or more antigens) that is not CD19.

The engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise a heterologous receptor that is a respective receptor of the heterologous cytokine as disclosed herein (e.g., heterologous IL-15 receptor (IL-15R, such as IL-15α or IL-15β) for heterologous IL-15) . Alternatively, the engineered immune cell may not and need not comprise any heterologous receptor that is a respective receptor of the heterologous cytokine. For example, the engineered immune cell comprising a heterologous IL (e.g., IL-15) lacks a heterologous receptor of the heterologous IL (e.g., IL-15R) .

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell. The heterologous cytokine can further comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) . The engineered immune cell may and need not comprise a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) .

The heterologous cytokine (e.g., the heterologous IL) 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 the engineered immune cell can be of human origin. Alternatively, the heterologous cytokine can be of a different species than that of the engineered immune cell.

A heterologous cytokine (e.g., the heterologous IL) 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 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 heterologous cytokine as disclosed herein can be a heterologous IL. A heterologous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous ILs. A heterologous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of heterologous ILs. Alternatively, the heterologous IL can be a single type of heterologous IL. Non-limiting examples of the heterologous 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 heterologous 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 human IL-15 (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.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a heterologous cytokine as disclosed herein, wherein the heterologous cytokine is bound to a membrane (e.g., plasma membrane) of the engineered immune cell. In some cases, the heterologous cytokine can comprise a heterologous IL as disclosed herein (e.g., a heterologous IL-15) . The engineered immune cell can further comprise one, two, or all of: (a) a different heterologous cytokine (e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell) , (b) reduced expression or activity of an endogenous immune regulator polypeptide, and (c) a safety switch. In some examples, the endogenous immune regulator polypeptide is not B2M. Thus, the endogenous immune regulator can be, for example, a polypeptide other than B2M.

The engineered immune cell (e.g., an engineered NK cell) can comprise the different heterologous cytokine and one or both of (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and (c) the safety switch. The engineered immune cell comprise the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and one or both of (a) the different heterologous cytokine and (c) the safety switch. The engineered immune cell comprise the safety switch and one or both of (a) the different heterologous cytokine and (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) . The engineered immune cell comprise all of (a) , (b) , and (c) .

The engineered immune cell (e.g., the engineered NK cell) can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine as compared to a control cell, as disclosed herein.

The expression or activity of the endogenous immune regulator polypeptide can be reduced in the engineered immune cell (e.g., the engineered NK cell) , for example, via action of a gene editing moiety as disclosed herein.

The reduced expression or activity of the endogenous immune regulator polypeptide in 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 or dephosphorylation of a downstream signaling protein (e.g., SHP2, Igα/β, Syk, etc. for PD1/PDL1 signaling) or (ii) expression of the endogenous immune regulator polypeptide (e.g., PD1) via Western blotting or PCR techniques.

In some cases, reduced expression of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by a decrease in the expression of the endogenous immune regulator polypeptide (e.g., PD1) 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 activity of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by a decrease in phosphorylation of a downstream signaling protein (e.g., SHP2 for PD1/PDL1 signaling) 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 activity of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by an increase in phosphorylation of a downstream target signaling protein (e.g., Igα/β or Syk for PD1/PDL1 signaling) 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. The downstream target signaling protein may be a protein that is normally inhibited by action of a functional signaling pathway of the endogenous immune regulator polypeptide.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a CD16 variant as disclosed herein for enhanced CD16 signaling in the engineered NK cell. In some cases, the CD16 variant (e.g., a heterologous CD16 variant) can comprise at least a portion of CD16 and at least a portion of CD64 (e.g., hnCD16 as disclosed herein) . The engineered immune cell can further comprise reduced expression or activity of an endogenous immune regulator polypeptide as compared to a control cell, as disclosed herein.

The engineered immune cell (e.g., the engineered NK cell) can exhibit enhanced CD16 signaling as compared to a control cell, as disclosed herein.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell 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-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 one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise one or both of: (i) a heterologous transcription factor (e.g., a heterologous STAT) , (ii) reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling as disclosed herein) , and (iii) reduced expression or activity of endogenous enzyme (e.g., a ligase, such as CBL-B) . 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) .

The heterologous transcription factor can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factor. The heterologous transcription factor can comprise at most 5, 4, 3, or 2 different types of transcription factor. Alternatively, the heterologous transcription factor can have a single type of transcription factor. The transcription factor can be involved in the engineered immune cell’s immune activity, proliferation, apoptosis, and/or differentiation. In some cases, the heterologous transcription factor for the engineered immune cell (e.g., the engineered NK cell) can be STAT. Non-limiting examples of STAT can include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof. In an example, STAT can comprise STAT3. In another example, STAT can comprise STAT5B.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced survival in the presence of tumor cells as compared to a control cell without (i) the heterologous transcription factor (e.g., the heterologous STAT) or (ii) the reduced activity of endogenous cytokine signaling (e.g., endogenous IL-17 signaling) . 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 one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell as disclosed herein (e.g., a NK cell marker, such as KIR) as compared to a control cell. In some examples, the specific endogenous cell marker is KIR. The engineered immune cell can further comprise one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell, as disclosed herein, (d) an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell, and (e) reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.

The engineered immune cell can comprise the chimeric polypeptide receptor and one or more of (e.g., 1, 2, 3, or 4 of) : (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.

The engineered immune cell can comprise the heterologous cytokine and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.

The engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.

The engineered immune cell can comprise the heterologous immune regulator and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.

The engineered immune cell can comprise the reduced expression or activity of an endogenous immune regulator polypeptide and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (d) the heterologous immune regulator.

The reduced expression or activity of the specific endogenous cell marker for the committed immune cell (e.g., 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 endogenous cell marker for a committed immune cell (e.g., KIR or NK cells) in the engineered immune 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 one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) . In some cases, the engineered immune cell can exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .

In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous CD94 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .

In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous CD96 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .

In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous TGF beta receptor and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iv) endogenous SHIP (e.g., SHIP2) .

In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous SHIP (e.g., SHIP2) and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iii) endogenous TGF beta receptor.

In some cases, the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96, TGF beta receptor, SHIP2, 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 cases, the reduced expression or activity of the endogenous CD94 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 cases, the reduced expression or activity of the endogenous CD96 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 cases, the reduced expression or activity of the endogenous TGF beta receptor 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 cases, the reduced expression or activity of the endogenous SHIP (e.g., SHIP2) 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 one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise one or more hypo-immunity regulators. In some cases, the engineered immune cell exhibits reduced expression or activity of one or more hypo-immunity regulators from: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL. 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 reduced expression or activity of the endogenous hypo-immunity regulator (e.g., CD80, CD86, ICOSL, CD40L, MICA, MICB, NKG2DL, 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, as ascertained by Western blotting or PCT techniques, as disclosed herein.

In some cases, the reduced expression or activity of the endogenous CD80 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 cases, the reduced expression or activity of the endogenous CD86 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 cases, the reduced expression or activity of the endogenous ICOSL 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 cases, the reduced expression or activity of the endogenous hypo-immunity regulator CD40L 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 cases, the reduced expression or activity of the endogenous MICA or MICB 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 cases, the reduced expression or activity of the endogenous NKG2DL 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 one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) . The engineered immune cell further comprises one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, and (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell.

In some cases, the engineered immune cell (e.g., the engineered NK cell) comprises a chimeric polypeptide receptor as disclosed herein and one or both of: (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.

In some cases, the engineered immune cell (e.g., the engineered NK cell) comprise the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.

In some cases, the engineered immune cell (e.g., the engineered NK cell) comprises the CD16 variant for enhanced CD16 signaling and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein.

In some cases, the reduced expression or activity of the endogenous ICAM1 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, as ascertained by Western blotting or PCT techniques, as disclosed herein.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) . The engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .

In some cases, the reduced expression or activity of the endogenous ICAM1 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, as disclosed herein.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell. In some cases, the immune regulator polypeptide comprises a hypo-immunity regulator. The hypo-immunity regulator can be PDL2. Alternatively or in addition to, the hypo-immunity regulator can be TGF-beta.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell. In some cases, the immune regulator polypeptide comprises a hypo-immunity regulator. The hypo-immunity regulator can comprise one or more members from: (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59. 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 immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CCL21 and one or more of (e.g., 1, 2, 3, or all of) : (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous IL-10 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD46 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iv) a heterologous CD55, and (v) a heterologous CD59.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD55 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (v) a heterologous CD59.

In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD59 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (iv) a heterologous CD55.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL) , as disclosed herein that is not IL-15. In some examples, the heterologous cytokine comprises IL-21 or variants thereof. The engineered immune cell can be derived from an induced stem cell (e.g., iPSC) .

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein. The antigen can be an antigen of a virus. In some cases, the virus comprises one or more members selected from the group comprising HIV, HBV, HCV, EBV, HPV, Lasse Virus, Influenza Virus, Coronavirus, and a derivative thereof. In some examples, the virus is not Cytomegalovirus (CMV) .

In some cases, the antigen of the virus can be a fragment of a viral protein of the virus that is presented by a target cell (e.g., an immune cell) . In some cases, viral infection induces apoptosis of the host cell after the host cell is used to replicate the virus, as a mechanism of virus spread. Thus, without wishing to be bound by theory, targeting of an antigen of a virus by the engineered immune cells as disclosed herein (e.g., engineered NK cell expressing a CAR against the virus antigen) can allow the engineered immune to be in close proximity to other virally infected cells, thereby inducing death of such virally infected cells.

In some cases, certain viruses can cause or increase the risk of certain types of cancers, and thus the engineered immune cells as disclosed herein or uses thereof can be utilized for preventing or treating such cancers. For example, Epstein-Barr virus (EBV) is a herpes virus that can spread through saliva, and EBV can increase the risk of Burkitt lymphoma, some types of Hodgkin’s and non-Hodgkin’s lymphoma, and stomach cancer. Hepatitis B virus (HBV) can spread through infected blood, semen, and other body fluids, and HBV can increase the risk of liver cancer. Hepatitis C virus (HCV) can spread through infected blood, and HCV can increase the risk of liver cancer and non-Hodgkin’s lymphoma. Human immunodeficiency virus (HIV) can spread through infected semen, vaginal fluids, blood, and breast milk, and HIV can increase the risk of cancer by damaging the immune system, which reduces the body’s defenses against other oncoviruses, thereby enabling other oncoviruses to cause cancer. HIV-associated cancers can include Kaposi sarcoma, non-Hodgkin’s and Hodgkin’s lymphoma, cervical cancer, and cancers of the anus, liver, mouth, throat, and lung. Human herpes virus 8 (HHV-8) can increase the risk of Kaposi sarcoma in people who have a weakened immune system. Human papillomavirus (HPV) can increase the risk of cancer in men and women, including anal, cervical, penile, throat, vaginal, and vulvar cancer. Human T-cell leukemia virus type (HTLV-1) can spread through infected semen, vaginal fluids, blood and breast milk, and it can increase the risk of adult T-cell leukemia/lymphoma.

In some embodiments, the virus as disclosed herein can be a DNA virus or a RNA virus. The virus may be, for example, a double stranded DNA virus, a single stranded DNA virus, a double stranded RNA virus, a positive sense single stranded RNA virus, a negative sense single stranded RNA virus, a single stranded RNA-reverse transcribing virus (retrovirus) or a double stranded DNA reverse transcribing virus. Examples of DNA viruses cam include, but are not limited to, cytomegalo virus, Herpex Simplex, Epstein-Barr virus, Simian virus 40, Bovine papillomavirus, Adeno-associated virus, Adenovirus, Vaccinia virus, and Baculo virus. Examples of RNA viruses can include, but are not limited to, Coronavirus, Semliki Forest virus, Sindbis virus, Poko virus, Rabies virus, Influenza virus, SV5, Respiratory Syncytial virus, Venezuela equine encephalitis virus, Kunjin virus, Sendai virus, Vesicular stomatitisvirus, and Retroviruses.

Various specific viruses include, but are not limited to, Papovaviridae, Adenoviridae, Herpesviridae, Herpesvirales, Ascoviridae, Ampullaviridae, Asfarviridae, Baculoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Lipothrixviridae, Nimaviridae, Poxviridae, Tectiviridae, Corticoviridae, Sulfolobus, Caudovirales, Corticoviridae, Tectiviridaea, Ligamenvirales, Ampullaviridae, Bicaudaviridae, Clavaviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Turriviridae, Ascovirus, Baculovirus, Hytrosaviridae, Iridoviridae, Polydnaviruses, Mimiviridae, Marseillevirus, Megavirus, Mavirus virophage, Sputnik virophage, Nimaviridae, Phycodnaviridae , pleolipoviruses, Plasmaviridae, Pandoraviridae, Dinodnavirus, Rhizidiovirus, Salterprovirus, Sphaerolipoviridae, Anelloviridae, Bidnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, Spiraviridae, Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Quadriviridae, Reoviridae, Totiviridae, Nidovirales, Picornavirales, Tymovirales, Mononegavirales, Bornaviridae, Filoviridae, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae, Anphevirus, Arlivirus, Chengtivirus, Crustavirus, Wastrivirus, Bunyavirales, Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, Arenaviridae, Ophioviridae, Orthomyxoviridae, Deltavirus, Taastrup virus, Alpharetrovirus, Avian leukosis virus; Rous sarcoma virus, Betaretrovirus , Mouse mammary tumor virus, Gammaretrovirus, Murine leukemia virus, Feline leukemia virus, Bovine leukemia virus, Human T-lymphotropic virus, Epsilonretrovirus, Walleye dermal sarcoma virus, Lentivirus , Human immunodeficiency virus 1, Simian and Feline immunodeficiency viruses, Spumavirus, Simian foamy virus, Orthoretrovirinae, Spumaretrovirinae, Metaviridae, Pseudoviridae, Retroviridae, Hepadnaviridae, and Caulimoviridae.

In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein. In some cases, the antigen can be secreted by a target cell or bound to a surface (e.g., on a plasma membrane) of the target cell. In some cases, the antigen that is targeted by the chimeric polypeptide receptor can be an antibody that is produced by the target cell (e.g., B cells) of a subject (e.g., a subject who has or is suspected of having an autoimmune disease) . The antibodies can be autoantibodies. In some examples, B cells can secrete antibodies and/or release the same antibodies against the subject’s own cells, thereby exhibiting a localized concentration of the antibodies near or adjacent to the B cells. The antibodies can be auto-antibodies that target other cells of the same subject, to effect various diseases and/or debilitating conditions. Thus, by targeting and binding to such antibodies via action of the chimeric polypeptide receptor, the engineered immune cell disclosed herein can specifically target the B cells (e.g., malignant or diseased B cells) to, e.g., reduce or inhibit activity of the B cells or induce death of the B cells in the subject. Without wishing to be bound by theory, administering the subject engineered immune cells (e.g., engineered NK cells) can target and suppress or deplete self-attacking B cells in the subject, for treatment of an autoimmune disease.

In some cases, the antigen binding domain of the chimeric polypeptide receptor can be an antibody or a variant thereof that is configured to bind one or more antibodies that are secreted by or presented on the target immune cells (e.g., self-attacking B cells) . In some cases, the antigen binding domain of the chimeric polypeptide receptor can be an antigen (thus not an antibody) that is recognized and bound by the antibodies that are secreted by or presented on the target immune cells (e.g., self-attacking B cells) . In either case, the antigen binding moiety of the chimeric polypeptide receptor may be referred to as an “antibody binding moiety. ”

In some cases, the antigen binding domain (i.e., antibody binding domain) of the chimeric polypeptide receptor can be designed such that the antigen binding domain does not induce any other biological effect to the subject (e.g., to other cells of the subject) . For example, the antigen binding domain can be a peptide sequence derived from a target protein of an autoantibody of a B cell of a subject who has an autoimmune disease. While the peptide sequence is designed to promote high specificity and binding affinity between the peptide and the autoantibody, the peptide sequence is also designed such that it would not (i) exhibit any function of the original target protein or (e.g., unintended interaction with other cells or proteins of the subject) (ii) elicit any biological effect on the subject other than being used as a target for the autoantibody.

In some cases, the antigen binding domain (i.e., antibody binding domain) of the chimeric polypeptide receptor can comprise at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 6 amino acid residues, at least or up to about 7 amino acid residues, at least or up to about 8 amino acid residues, at least or up to about 9 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 11 amino acid residues, at least or up to about 12 amino acid residues, at least or up to about 13 amino acid residues, at least or up to about 14 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 16 amino acid residues, at least or up to about 17 amino acid residues, at least or up to about 18 amino acid residues, at least or up to about 19 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 45 amino acid residues, or at least or up to about 50 amino acid residues.

In some cases, the antigen of the virus can be at least a portion of a viral protein. The viral protein can be a surface protein, a glycoprotein, an envelope protein, a membrane protein, a nucleocapsid protein, a gene polymerase, a protease, etc. For example, examples of viral glycoproteins include haemaglutinin and neuraminidase (e.g., for influenza virus) , spike glycoprotein (e.g., for SARS-CoV) , E1 and E2 (e.g., for HCV) , gp120, gp160, and gp41 (e.g., for HIV) , spike protein Gp1-Gp2 (e.g., for Ebola virus) , E dimer (e.g., for Dengue virus) , E1 and E2 (e.g., for Chikungunya virus) , etc.

In some cases, the antigen binding domain or antigen binding moiety of a heterologous receptor (e.g., a chimeric polypeptide receptor) can comprise an antibody or a fragment thereof (e.g., scFv) that exhibits specific binding to a viral antigen.

In some cases, the antigen binding domain or antigen binding moiety of a heterologous receptor (e.g., a chimeric polypeptide receptor) can comprise at least a portion of a cellular protein exhibiting specific affinity to target antigens (e.g., antigen of a virus) . In some cases, the cellular protein is not a viral protein. For example, the cellular protein is not derived from a viral protein. In some cases, the cellular protein is an endogenous protein (e.g., encoded by the native gnome of the cell) . In some cases, the cellular protein is a surface receptor. For example, the surface receptor can be a CD receptor. In some cases, the CD receptor can be CD4, CD8, CD34, CD31, CD117, CD45, CD11b, CD15, CD24, CD114, CD182, CD14, CD114, CD11a, CD91, CD16, CD3, CD25, CD19, CD20, CD38, CD22, CD61, CD16, CD30, CD38, or any variations thereof. In some cases, the surface receptor can be CXCR4, CCR5, G-protein coupled receptors, Niemann-Pick C1 receptor, integrin receptors (e.g., αvβ1, αvβ3, αvβ6, and αvβ8) , sialic acids, immunoglobulin superfamily receptors (e.g., JAM-A) , or any variations thereof.

In some cases, a control cell can be a cell can be an immune cell, such as a NK cell, used for comparison purposes. In some cases, a control cell can be a cell that does not comprise a heterologous cytokine (e.g., IL-15) . In some cases, a control cell can be a cell that does not comprise a CD16 variant for enhanced CD16 signaling. In some cases, a control cell can be a cell that a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some cases, a control cell can be a cell that comprises a heterologous IL-15R. In some cases, a control cell can be a cell that does not comprise a membrane bound heterologous cytokine (e.g., IL-15) . In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous immune regulator polypeptide. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (e.g., IL-17) or a receptor thereof (e.g., IL-17R) . In some cases, a control cell can be a cell that does not comprise a heterologous transcription factor (e.g., STAT) . In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell (e.g., a NK cell marker, such as KIR) . In some cases, a control cell can be a cell that does not comprise a heterologous immune regulator polypeptide. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD94, endogenous CD96, endogenous TGF beta receptor, or endogenous SHIP2. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD80, endogenous CD86, endogenous ICOSL, endogenous CD40L, endogenous MICA or MICB, or endogenous NKG2DL. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of ICAM1. In some cases, a control cell can be a cell that does not comprise a heterologous PDL2 or heterologous TGF beta. In some cases, a control cell can be a cell that does not comprise one or more of: heterologous CCL21, heterologous IL-10, heterologous CD46, heterologous CD55, or heterologous CD59. In some cases, a control cell can be a cell that does not comprise heterologous IL-21. In some cases, a control cell can be a cell that is not derived from a cell line. In some cases, a control cell can be a cell that is not derived from an isolated ESC. In some cases, a control cell can be a cell that is not derived from an iPSC.

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, the engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein. In some cases, the engineered immune cell (e.g., the engineered NK cell) comprises a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) , as disclosed herein. As such, the engineered immune cell (e.g., the engineered NK cell) can exhibit enhanced signaling of the endogenous signaling pathway 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 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 of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can comprise a heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be secreted by the engineered immune cell.

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 heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be bound to a cell surface membrane of the engineered immune cell.

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 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.

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 heterologous immune receptor polypeptide. The immune regulator polypeptide can comprise one or more members selected from the group consisting of 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, the engineered immune cell can exhibits reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. The endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator (or both) .

In some cases, the immune checkpoint inhibitor as disclosed herein can comprise one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4. In some cases, the immune checkpoint inhibitor can comprise SHIP2.

In some cases, the hypo-immunity regulator as disclosed herein can comprise one or more members 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 of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell can comprise a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered immune cell.

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 embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, the engineered immune cell is an effector cell. In some embodiments, the control cell is a wildtype NK cell. In some embodiments, the control cell is an immune cell (e.g., NK cell) lacking one or more of the engineered aspects provided herein. In some embodiments, the target cell can present or express at least a portion of an antigen of a virus. In some embodiments, the target cell can present or express the virus. In some embodiments, the target cell can present or express a protein of the virus. In some embodiments, the enhanced cytotoxicity is at effector cell to target cell (E: T) ratio of at least about 0.2, at least about 0.5, at least about 1, at least about 5, at least about 10, at least about 20, or at least about 30. 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 exhibit enhanced cytotoxicity against a target cell as compared to a control cell within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, within about 4 hours, within about 2 hours, or less of incubation with target cell as ascertained by, e.g., tracking a change in the number of the target population of cells (e.g., via Fluorescence-Activated Cell Sorting or 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 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.

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, HCV, 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, 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, he 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, Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD) , Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csx12) , Cas10, CaslOd, Cas10, CaslOd, CasF, CasG, CasH, Cpfl, Csyl, Csy2, Csy3, Csel (CasA) , Cse2 (CasB) , Cse3 (CasE) , Cse4 (CasC) , Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4, Cul966, Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, Cas13Y, Cas14 (e.g., Cas14 variants, such as Cas14a, Cas14b, Cas14c, etc. ) 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 andcyclosphosphamide; 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 topotecanCPT-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; vindesinedacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; thiotepa; taxoids, for example taxanes includingpaclitaxel (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; vinblastineplatinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristineoxaliplatin; leucovovin; vinorelbinenovantrone; 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 (includingtamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene; 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 asand 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, andanastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, or) , etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, orrisedronate; 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 asvaccine and gene therapy vaccines, for example, vaccine, vaccine, andvaccine; 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 etanerceptinfliximabadalimumabcertolizumab pegolgolimumabInterleukin 1 (IL-1) blockers such as anakinraT-cell costimulation blockers such as abataceptInterleukin 6 (IL-6) blockers such as tocilizumabInterleukin 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 bexarotenebisphosphonates such as clodronate (for example, or) , etidronateNE-58095, zoledronic acid/zoledronate alendronatepamidronatetiludronateor risedronateand 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 sodiumpixantrone; 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 use

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.

The subject can be administered with a composition (e.g., a unit dosage form) comprising the engineered immune cell as provided herein. A subject can be administered with the engineered immune cell at a total concentration or dose of at least or up to about 1 x10⁴ cells/kilogram of body weight (cells/kg) , at least or up to about 2 x10⁴ cells/kg, at least or up to about 3 x10⁴ cells/kg, at least or up to about 4 x10⁴ cells/kg, at least or up to about 5 x10⁴ cells/kg, at least or up to about 6 x10⁴ cells/kg, at least or up to about 7 x10⁴ cells/kg, at least or up to about 8 x10⁴ cells/kg, at least or up to about 9 x10⁴ cells/kg, at least or up to about 1 x10⁵ cells/kg, at least or up to about 2 x10⁵ cells/kg, at least or up to about 3 x10⁵ cells/kg, at least or up to about 4 x10⁵ cells/kg, at least or up to about 5 x10⁵ cells/kg, at least or up to about 6 x10⁵ cells/kg, at least or up to about 7 x10⁵ cells/kg, at least or up to about 8 x10⁵ cells/kg, at least or up to about 9 x10⁵ cells/kg, at least or up to about 1 x10⁶ cells/kg, at least or up to about 2 x10⁶ cells/kg, at least or up to about 3 x10⁶ cells/kg, at least or up to about 4 x10⁶ cells/kg, at least or up to about 5 x10⁶ cells/kg, at least or up to about 6 x10⁶ cells/kg, at least or up to about 7 x10⁶ cells/kg, at least or up to about 8 x10⁶ cells/kg, at least or up to about 9 x10⁶ cells/kg, at least or up to about 1 x10⁷ cells/kg, at least or up to about 2 x10⁷ cells/kg, at least or up to about 3 x10⁷ cells/kg, at least or up to about 4 x10⁷ cells/kg, at least or up to about 5 x10⁷ cells/kg, at least or up to about 6 x10⁷ cells/kg, at least or up to about 7 x10⁷ cells/kg, at least or up to about 8 x10⁷ cells/kg, at least or up to about 9 x10⁷ cells/kg, at least or up to about 1 x10⁸ cells/kg, at least or up to about 2 x10⁸ cells/kg, at least or up to about 3 x10⁸ cells/kg, at least or up to about 4 x10⁸ cells/kg, at least or up to about 5 x10⁸ cells/kg, at least or up to about 6 x10⁸ cells/kg, at least or up to about 7 x10⁸ cells/kg, at least or up to about 8 x10⁸ cells/kg, at least or up to about 9 x10⁸ cells/kg, at least or up to about 1 x10⁹ cells/kg, at least or up to about 2 x10⁹ cells/kg, at least or up to about 3 x10⁹ cells/kg, at least or up to about 4 x10⁹ cells/kg, at least or up to about 5 x10⁹ cells/kg, at least or up to about 6 x10⁹ cells/kg, at least or up to about 7 x10⁹ cells/kg, at least or up to about 8 x10⁹ cells/kg, at least or up to about 9 x10⁹ cells/kg, at least or up to about 1 x10¹⁰ cells/kg, at least or up to about 2 x10¹⁰ cells/kg, at least or up to about 3 x10¹⁰ cells/kg, at least or up to about 4 x10¹⁰ cells/kg, at least or up to about 5 x10¹⁰ cells/kg, at least or up to about 6 x10¹⁰ cells/kg, at least or up to about 7 x10¹⁰ cells/kg, at least or up to about 8 x10¹⁰ cells/kg, at least or up to about 9 x10¹⁰ cells/kg, or at least or up to about 1 x10¹¹ cells/kg. The dose of the engineered immune cell provided herein can be administered to the subject in a single unit dosage form, or a plurality of unit dosage forms (e.g., at least 2, at least 3, at least 4, or at least 5 unit dosage forms) .

In some cases, a composition comprising the engineered immune cell provided herein can be administered to the subject by a route selected from subcutaneous injection, intramuscular injection, intradermal injection, percutaneous administration, intravenous administration, intranasal administration, intralymphatic injection, and oral administration.

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: Engineered NK cells

Table 1 illustrates examples of engineered NK cells with or without genetic modifications, along with possible functions, and therapeutic indications. Examples of therapeutic indications can include acute myeloid leukemia (AML) , multiple myeloma (MM) , Myelodysplastic syndrome (MDS) , B cell leukemia, T cell leukemia, solid tumor, and blood cancer.

TABLE 1

Example 2: Engineered anti-antibody NK Cells

Engineered NK cells:

For improved adaptive immunotherapy, NK cells can be engineered to express a chimeric receptor polypeptide (e.g., a TFP or a CAR) that comprises an extracellular domain that engages in binding with an autoantibody of an immune cell (e.g., B cell) of s subject. The subject can have or can be suspected of having an autoimmune disease, indicated by a heightened auto-attacking activity of B cells. Thus, the chimeric receptor polypeptide can comprise (i) an antibody that binds the autoantibody or (ii) an antigen of the autoantibody, thereby allowing the engineered NK cell to find, target, and induce death of the B cells.

Goodpasture's syndrome:

In an example, the subject has or is suspected of having goodpasture's syndrome, and B cells of the subject express autoantibodies against a basement membrane protein of a tissue (e.g., lung, kidney, etc. ) , such as alpha-3 subunit of type IV collagen, Thus, engineered NK cells are generated (e.g., from HSCs, ESCs, or iPSCs) to express a chimeric receptor polypeptide having an antigen binding domain (i.e., antibody binding domain) that is derived from the targeted basement membrane protein (e.g., alpha-3 subunit of type IV collagen) . A population of such engineered NK cells are administered to the subject as a treatment for goodpasture’s syndrome.

Pemphigus:

In another example, the subject has or is suspected of having pepphigus, and B cells of the subject express autoantibodies against a cell structure protein, such as desmoglein, Thus, engineered NK cells are generated (e.g., from HSCs, ESCs, or iPSCs) to express a chimeric receptor polypeptide having an antigen binding domain (i.e., antibody binding domain) that is derived from the targeted cell structural protein (e.g., desmoglein) . A population of such engineered NK cells are administered to the subject as a treatment for goodpasture’s syndrome.

Example 3: Enhanced CD16 signaling

For improved adaptive immunotherapy, NK cells can be engineered to exhibit enhanced CD16 signaling.

hnCD16 amino acid sequence (SEQ ID NO. 1) :

Engineered NK cells:

NK-92 cells were engineered to exhibit enhanced CD16 signaling. The engineered NK-92 cells were modified to express CD64/CD16A fusion protein (i.e., hnCD16) (SEQ ID NO. 1) .

The resulting hnCD16 NK-92 cells were validated by identifying enhanced expression of both CD16 (e.g., via anti-CD16-PE antibody) and CD64 (e.g., via anti-CD64-APC/AF700 antibody) using fluorescence-activated cell sorting (FACS) , as shown in FIG. 1A. Wild-type (WT) NK-92 cells were used as control.

The hnCD16 construct sequence can comprise “FHVS” (SEQ ID NO. 2) . The hnCD16 construct sequence can comprise “WFHVS” (SEQ ID NO. 3) . The hnCD16 construct sequence can comprise “FHVSF” (SEQ ID NO. 4) . The hnCD16 construct sequence can comprise “WFHVSF” (SEQ ID NO. 5) . The hnCD16 construct sequence can comprise “VWFHVSFC” (SEQ ID NO. 6) . The hnCD16 construct sequence can comprise “PVWFHVSFCL” (SEQ ID NO. 7) . The hnCD16 construct sequence can comprise “TPVWFHVSFCLV” (SEQ ID NO. 8) .

Enhanced targeting:

The hnCD16 NK-92 cells were cultured alone (unstimulated, control) or in the presence of K562 cells capable of activating NK cells (K562) or phorbol 12-myristate 13-acetate (PMA) to activate CD16 and induce cleavage thereof. Data revealed that the hnCD16 NK-92 cells were highly resistant to the activation-induced cleavage of CD16a, as compared to peripheral blood (PB) NK cells as a control (FIG. 1B) . For example, treatment with PMA marginally reduced the percentage of CD16+ cells from 92%to 85%for the hnCD16 NK-92 cells, whereas the same treatment reduced the percentage of CD16+ cells from 96%to 25% (FIG. 1B) . Persistency of hnCD16 in the hnCD16 NK-92 cells was also confirmed by using anait-CD64 antibody (FIG. 1C) . Also, it was observed that hnCD16 NK-92 cells did not downregulate endogenous CD16 expression upon stimulation (e.g., K652 or PMA) (FIGs. 1D and 1E) .

Enhanced co-antibody therapy

The target cells (Raji cells) were treated with (i) the hnCD16 NK-92 cells and (ii) either anti-CD20 antibody or hIgG as a control. Data revealed that the hnCD16 NK-92 cells in combination with anti-CD20 antibody induced enhanced death (e.g., at least partially ADCC-mediated death) of the target cells as compared to controls (e.g., wildtype NK92 cells with anti-CD20 antibody, or the hnCD16 NK-92 cells with hIgG) (FIGs. 1F and 1G) .

Example 4: Enhanced survival and persistency of NK Cells

For enhanced persistence of adaptive immunotherapy, NK cells can be engineered to comprise at least (i) a heterologous transcription factor (e.g., STAT) and (ii) reduced expression or activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) . Having the combination of (i) and (ii) in the engineered NK cell can synergistically improve persistence of the engineered NK cells as compared to having either one of (i) and (ii) alone, or none.

Engineered NK cells:

NK cells are generated from isolated ESCs or iPSCs. The NK cells are engineered to express a heterologous STAT (e.g., STAT3 and/or STAT5B) . A gene encoding the heterologous STAT is incorporated into the NK cell’s genome via either viral transduction or via action of a gene editing moiety as disclosed herein. The NK cells are also engineered to exhibit reduced expression or activity of endogenous IL-17R (i.e., STAT3⁺IL-17R^-NK cells) . NK cells with either one of (i) the heterologous STAT and (ii) reduced expression or activity of IL-17R, or non-engineered NK cells are used as a control.

In vitro survival and persistency:

The engineered STAT3⁺IL-17R^-NK cells can be cultured in vitro to assess viability and growth (or proliferative capacity) of the engineered STAT3⁺IL-17R^-NK cells in absence of an exogenous cytokine. The NK cells are cultured in culture medium without the addition of exogenous cytokines for 3-6 weeks. The engineered STAT3⁺IL-17R^-NK cells exhibit a significantly higher number of NK cells as compared to the control cells, indicating the enhanced survival and persistency of the engineered STAT3⁺IL-17R^-NK cells in vitro.

In vivo pharmacokinetics (PK) :

The engineered STAT3⁺IL-17R^-NK cells can be administered in NCG mice having a Raji xenograft model. NCG mice are triple immunodeficient and lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function to host the xenograft model. The engineered STAT3⁺IL-17R^-NK cells and the control cells are each administered into the respective Raji xenograft model mice via intravenous (IV) tail vein injection, at a dose of about 1 × 10⁶ cells per animal. Mice injected with the engineered STAT3⁺IL-17R^-NK cells exhibit higher NK cell concentrations in the peripheral blood from about 7 days to about 28 days post-infusion, demonstrating the enhanced survival and persistency of the engineered STAT3⁺IL-17R^-NK cells in vivo.

Example 5: Engineered anti-CD19 NK Cells

NK-92 cells were engineered to express anti-CD19 CAR, then cultured in the presence of CD19+ Raji cells to assess targeting of the Raji cells by the engineered anti-CD19 NK cells. Wild type (WT) NK-92 cells were used as control. The anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against the Raji cells (as ascertained by a reduced number of alive Raji cells) as compared to the control (FIGs. 2A and 2B) . In addition, when cultured in the presence of the Raji cells, the anti-CD19 CAR NK cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) as compared to the control (FIGs. 2C and 2D) . Furthermore, when cultured in the presence of the Raji cells, the anti-CD19 CAR NK cells exhibited enhanced cytokine production (e.g., IFN-gamma and/or TNF-alpha production) as compared to the control (FIGs. 2E-2G) .

Example 6: Engineered hIL15 NK Cells

Engineered NK cells:

NK-92 cells were engineered with (i) hIL-15 knock in or (ii) hIL-15-hIL15R fusion polypeptide knock in. Two variants of the hIL-15-hIL15R fusion polypeptide were tested. The first variant (i.e., hIL15-IL15Ra fused-1 or “fus1” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 9) , e.g., “GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS” (SEQ ID NO. 10) . The second variant (i.e., hIL15-IL15Ra fused-2 or “fus2” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 9) and one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “EGKSSGSGSESKST” (SEQ ID NO. 11) , e.g., “EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS” (SEQ ID NO. 12) . NK-92 cells with either of the hIL-15-hIL15R fusion polypeptide variant knocked-in were positive for hIL-15 (FIG. 3A) .

In addition, the engineered NK-92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling exhibited longer persistency as compared to control NK-92 cells engineered express secretory form of IL-15. Western blotting analysis revealed increased phosphorylation of IL-15-stimulated STAT5 in the NK-92 cells expressing either hIL15-IL15Ra fused-1 (fus1) or hIL15-IL15Ra fused-2 (fus2) , as compared to the secretory IL-15 (IL15) (FIG. 3B) .

hIL15-IL15Ra fused-1 sequence (SEQ ID NO. 13) :

hIL15-IL15Ra fused-2 sequence (SEQ ID NO. 14) :

Example 7: Engineered anti-viral antigen (e.g., anti-EBV antigen) CAR-NK Cells

NK cells can be engineered to express a heterologous receptor exhibiting specific binding against a viral antigen, such as at least a portion of a viral protein. The heterologous receptor can be, for example, a chimeric antigen receptor (CAR) comprising an antigen binding domain (or antigen binding moiety) against the viral antigen. The antigen binding domain can comprise or be derived from at least a portion of an antibody that exhibits specific binding against the viral antigen (e.g., at least a portion of a human Fab fragment (e.g., a scFv derived from HLEA-Fab) that specifically recognizes a polypeptide in the extramembrane domain of an Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) ) . Alternatively or in addition to, the antigen binding domain can comprise or be derived from at least a portion of a natural complementary molecule of the senescence marker (e.g., at least a portion of intracellular TNFR-associated factors (TRAFs) , to target LMP1 as a EBV viral antigen) .

Engineered NK cells:

As shown in FIG. 4A, NK cells (e.g., NK-92 cells) were engineered to target viral (e.g., EBV) gene products (e.g., LMP1) . As shown in FIG. 4A, anti-EBV CAR structure can comprise of an anti-EBV scFv (e.g., anti-LMP1 scFv derived from HLEA-Fab) comprising a linker (e.g., (G4S) 3) , a variable fragment heavy chain (VH) , and a variable fragment light chain (VL) . The anti-EBV CAR structure can also comprise of a signal peptide (e.g., CD8a, CD8a leader) , a hinge (e.g., CD8a hinge) , a transmembrane domain (TM) (e.g., CD8a TM, CD28 TM) , a CD28 costimulatory domain (CD28 cos) , and/or intracellular domains (ICD) (e.g., 4-1BB ICD, CD3ζICD) .

CD8a leader (SEQ ID NO. 15) :

VH (SEQ ID NO. 16) :

Linker (SEQ ID NO. 17) :

VL (SEQ ID NO. 18) :

CD8a hinge (SEQ ID NO. 19) :

CD8a TM (SEQ ID NO. 20) :

CD28 TM (SEQ ID NO. 21) :

CD28 cos (SEQ ID NO. 22) :

4-1BB ICD (SEQ ID NO. 23) :

CD3ζ ICD (SEQ ID NO. 24) :

Plasmid with anti-EBV CAR structure (e.g., anti-LMP1 CAR or LMP1-CAR, as used interchangeably herein) was packaged into lentivirus and NK cells (e.g., NK92 cells) were transduced with the lentivirus. FACS analysis revealed that NK cells transduced with anti-LMP1 CAR constructs (LMP1-CAR NK92 cells) expressed the LMP-1 CAR on the surface of the NK cells, as shown in FIG. 4B (see CAR1 and CAR2) .

In vitro killing of engineered NK cells against EBV positive (EBV+) target cell:

Engineered NK cells (e.g., LMP1-CAR NK92 cells) were added to the target cells (e.g., Nalm6 cells, B95.8, lymphoblastoid cell line (LCL) ) labeled with carboxyfluorescein succinimidyl ester (CSFE) in various effector cells to target cells (E: T) ratio (e.g., 1, 5, and 20) . Nalm6 cells were used as a control cell population that does not comprise EBV viral antigens, while B95.8 and LCL cells were used as EBV-expressing or producing cells. After coculturing (e.g., 6 hours of coculturing) , cytotoxicity by LMP1-CAR NK cells against the target cells was measured via flow cytometry. As shown in FIG. 5B, LMP-1 CAR NK cells exhibited greater cytotoxicity against B95.8 cells than by wildtype NK cells (NK92 wildtype (WT) ) . After coculturing (e.g., 6 hours) at E: T ratio of 20, the LMP1-CAR NK cells exhibited a greater cytotoxicity against the B95.8 target cells (e.g., as measured by the percentage of target cells killed) than the wildtype NK cells, by at least about 10%. On the contrary, a difference of cytotoxicity against Nalm6 cells between the LMP1-CAR NK cells and the wildtype NK cells was less than about 10% (e.g., less than about 6%) , suggesting a role of viral antigen targeting in inducing death of target cells (e.g., B95.8 cells) that express or present the viral antigen. In addition, after coculturing (e.g., five days of coculturing) wildtype NK cells (Nk92 WT) and LMP1 targeting NK cells (LMP1-CAR NK92) with LMP1-expressing target cells (e.g., LCL, B95.8) at 1: 1 ratio, nearly all of the target cells were killed, whereas the target cells survived in absence of the LMP1 targeting NK cells, as evaluated by flow cytometry (see FIG. 5C) .

Without wishing to be bound by theory, 5 days of co-culture between wildtype NK cells (NK92-wt) and the target cells may have been long enough for the wildtype NK cells to induce cytotoxicity in absence of the anti-LMP1 CAR (see FIG. 5C) . Without wishing to be bound by theory, the anti-LMP1 CAR expressing NK cells may exhibit a higher cytotoxicity against the target cells (e.g., LCL, B95) than wildtype NK cells when such NK cells are co-cultured with the target cells for less than 5 days (e.g., less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than about 12 hours, etc. ) .

Example 8: Engineered anti-viral antigen (e.g., anti-HIV antigen) CAR-NK cells

Engineered NK cells:

NK cells can be engineered to express a heterologous receptor exhibiting specific binding against a viral antigen, such as at least a portion of a viral protein. The heterologous receptor can be, for example, a chimeric antigen receptor (CAR) comprising an antigen binding domain (or antigen binding moiety) against the viral antigen. The antigen binding domain can comprise or be derived from at least a portion of an antibody that exhibits specific binding against the viral antigen such as a viral glycoprotein (e.g., at least a portion of a human anti-HIB-1 gp120 recombinant antibody, such as VRC clones) . Alternatively or in addition to, the antigen binding domain can comprise or be derived from at least a portion of a natural complementary molecule of the viral protein (e.g., at least a portion of CD4 to target gp120 as a HIV viral antigen) .

Engineered NK cells:

As shown in FIG. 6A, NK cells (e.g., NK-92 cells) were engineered to target viral (e.g., HIV) protein (e.g., gp120) . As shown in FIG. 6A, anti-HIV CAR structure can comprise of an CD4 extracellular (EC) fragment or anti-gp120 scFv, both of which can specifically target gp120. The CAR can also comprise of a signal peptide (e.g., CD8a, CD8a leader) , a hinge (e.g., CD8a hinge) , a transmembrane domain (TM) (e.g., CD8a TM, CD28 TM) , and intracellular domains (ICD) (e.g., 4-1BB ICD, CD3ζ ICD) .

CD8a leader (SEQ ID NO. 15) :

CD8a hinge (SEQ ID NO. 19) :

CD8a TM (SEQ ID NO. 20) :

4-1BB ICD (SEQ ID NO. 23) :

CD3ζ ICD (SEQ ID NO. 24) :

CD4 EC (SEQ ID NO. 25) :

Anti-gp120 scFv (SEQ ID NO. 26) :

The engineered NK cells were validated. As shown in FIG. 6B, FACS analysis revealed greater expression of CD4 EC in engineered NK92 cells (CD4 CAR NK92) compared to control wildtype NK cells (e.g., NK92, non-engineered NK cells) , suggesting expression of the CD4 CAR. As shown in FIG. 6C, mRNA levels of 4-1BB were higher in engineered NK cells (e.g., CD4 CAR NK92, anti-gp120 scFv CAR-NK92) compared to control wildtype NK cells (e.g., NK92, non-engineered NK cells) , suggesting expression of the CD4 CAR or the anti-gp120 scFv CAR. Furthermore, as assessed by plate-immobilized antigen stimulation assay and flow cytometry, CD4 CAR NK92 with anti-CD4 (e.g., 10ug/ml) exhibited greater surface expression of NK activation marker CD69 (50.09%) than CD4-CAR NK92 alone (8.33%) (FIG. 6D) . Control NK cells that do not exhibit specific binding to gp120 (anti-SS1 CAR NK) treated with PMA and Ionomycin (P/I) was used as a positive control.

In vitro killing of engineered NK cells against HIV positive (HIV+) target cell:

Engineered NK cells (e.g., CD4 CAR NK92, anti-gp120 scFv CAR-NK92) are added to the target cells labeled with carboxyfluorescein succinimidyl ester (CSFE) in various effector cells to target cells (E: T) ratio (e.g., 5, 10, 15, 20, 25) . Target cells are a control cell population that does not comprise HIV viral antigens or the target cell are cells that are HIV-expressing or producing cells (e.g., gp120-presenting target cells) . After coculturing (e.g., 6 hours of coculturing) , cytotoxicity by CD4 CAR NK92 or anti-gp120 scFv CAR-NK92 cells against the target cells is measured via flow cytometry. CD4 CAR NK92 and anti-gp120 scFv CAR-NK92 cells may exhibit greater cytotoxicity against gp120-presenting target cells than by wildtype NK cells, indicating ability of engineered NK cells (e.g., CD4 CAR NK92, anti-gp120 scFv CAR-NK92) against HIV+ target cells.

Example 9: Engineered anti-viral antigen (e.g., anti-HBV antigen) CAR NK cells

NK cells can be engineered to express a heterologous receptor exhibiting specific binding against a viral antigen, such as at least a portion of a viral protein. The heterologous receptor can be, for example, a chimeric antigen receptor (CAR) comprising an antigen binding domain (or antigen binding moiety) against the viral antigen. The antigen binding domain can comprise or be derived from at least a portion of an antibody that exhibits specific binding against the viral antigen such as a viral surface antigen (e.g., at least a portion of anti-Hepatitis B surface antigen (anti-HBsAg) scFv, such as F124 clone) . Alternatively or in addition to, the antigen binding domain can comprise or be derived from at least a portion of a natural complementary molecule of the viral protein (e.g., at least a portion of a hepatitis B surface antigen binding protein (SBP) in HepG2 cells, for targeting HBsAg as a HBV viral antigen) .

Engineered NK cells:

As shown in FIG. 7, NK cells (e.g., NK-92 cells) were engineered to target viral (e.g., HBV) protein (e.g., HBsAg) . As shown in FIG. 4, anti-HBV CAR structure can comprise an anti-HBV scFv (e.g., anti-HBsAg scFv) comprising a linker, a variable fragment heavy chain (VH) , and a variable fragment light chain (VL) . The anti-HBV CAR structure can also comprise of a signal peptide (e.g., CD8a, CD8a leader) , a hinge (e.g., CD8a hinge) , a transmembrane domain (TM) (e.g., CD8a TM, CD28 TM) , a CD28 costimulatory domain (CD28 cos) , intracellular domains (ICD) (e.g., 4-1BB ICD, CD3ζ ICD) .

CD8a leader (SEQ ID NO. 15) :

VH-1 (SEQ ID NO. 27) :

Linker (SEQ ID NO. 17) :

VL-1 (SEQ ID NO. 28) :

VH-2 (SEQ ID NO. 29) :

VL-2 (SEQ ID NO. 30) :

CD8a hinge (SEQ ID NO. 19) :

CD8a TM (SEQ ID NO. 20) :

CD28 TM (SEQ ID NO. 21) :

CD28 cos (SEQ ID NO. 22) :

4-1BB ICD (SEQ ID NO. 23) :

CD3ζ ICD (SEQ ID NO. 24) :

In vitro killing of engineered NK cells against HBV positive (HBV+) target cell:

Engineered NK cells (e.g., anti-HBsAg scFv CAR-NK92) are added to the target cells labeled with carboxyfluorescein succinimidyl ester (CSFE) in various effector cells to target cells (E: T) ratio (e.g., 5, 10, 15, 20, 25) . Target cells are a control cell population that does not comprise HBV viral antigens or cells that are HIV-expressing or producing cells (e.g., HBsAg-presenting target cells) . After coculturing (e.g., 6 hours of coculturing) , cytotoxicity by anti-HBsAg scFv CAR-NK92 cells against the target cells is measured via flow cytometry. HBsAg scFv CAR-NK92 cells may exhibit greater cytotoxicity against HBsAg-presenting target cells than by wildtype NK cells, indicating ability of engineered NK cells (e.g., HBsAg scFv CAR-NK92) against HBV+ target cells.

Example 10: In vivo killing of virally infected cells by engineered NK cells

Anti-tumor efficacy of anti-LMP1 CAR-NK cells in vivo:

To evaluate the in vivo antitumor activity of anti-LMP1 CAR-NK cells, a xenograft model is used. On day 0, 6-week-old male BALB/c nude mice are subcutaneously injected with 5×10⁶ SUNE1-LMP1 cells. When the tumor burden reaches about 100mm³ approximately 10 days after tumor cell inoculation, the mice are randomly assigned to three different groups (N =5/group) . The animals are percutaneously intratumorally injected with 5 × 10⁶ NK cells/100μL on day 10, 13, and 16. Group A receives anti-LMP1 CAR-NK cells, Group B receives control NK cells and Group C receives normal saline. Tumor growth is monitored by calliper measurement, and tumor volume is calculated using the formula: 1/2 × length × (width) . In this model, the anti-LMP1 CAR-NK cells may substantially inhibit the growth of tumors, while control NK cells may not.

Anti-HIV activity of CD4 CAR-NK cells in vivo:

Bone marrow-liver-thymus (BLT) humanized mice are challenged with 20,000 median tissue culture infectious dose (TCID50) HIV via intraperitoneal injection. At 3 weeks post-HIV challenge, all infected mice are given low-dose antiretroviral therapy (ART) consisting of 1 mg kg-1 EFdA and 25 mg kg-1 Dolutegravir every other day by intraperitoneal injection for 4 weeks. At the time of ART initiation, HIV-infected mice are allocated into three groups. Treated mice are infused with CD4 CAR NK cells. Control mice are infused with wildtype NK cells or untreated. The mice are euthanized, and tissues are collected for analysis 7 weeks post-infection. The infusion of CD4 CAR-NK cells may reduce the frequency of HIV-infected cells in tissues.

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:

a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to an antigen of a virus, wherein the virus is not CMV.
The engineered NK cell of claim 1, wherein the virus comprises one or more members selected from the group consisting of HIV, HBV, HCV, EBV, HPV, Lasse Virus, Influenza Virus, Coronavirus, and a derivative thereof.
The engineered NK cell of any one of the preceding claims, wherein the virus comprises one or more members selected from the group consisting of HBV, HCV, and a derivative thereof.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits enhanced cytotoxicity against a target cell comprising the antigen, as compared to a control cell lacking the chimeric polypeptide receptor.
The engineered NK cell of any one of the preceding claims, wherein the enhanced cytotoxicity of the engineered NK cell against the target cell is greater than that of the control cell by at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.8-fold, at least about 1-fold, or more.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is configured to exhibit the enhanced cytotoxicity against the target cell within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, or less of incubation with the target cell.
The engineered NK cell of any one of the preceding claims, wherein the enhanced cytotoxicity is at effector to target (E: T) ratio of at least about 5, at least about 10, or at least about 20.
The engineered NK cell of any one of the preceding claims, wherein the antigen binding moiety comprises at least a portion of an antibody exhibiting specific affinity to the antigen of the virus.
The engineered NK cell of any one of the preceding claims, wherein the antigen binding moiety does not comprise at least a portion of an antibody exhibiting specific affinity to the antigen of the virus.
The engineered NK cell of any one of the preceding claims, wherein the antigen binding moiety comprises at least a portion of a cellular protein exhibiting specific affinity to the antigen of the virus.
The engineered NK cell of any one of the preceding claims, wherein the cellular protein is a surface receptor.
The engineered NK cell of any one of the preceding claims, wherein the surface receptor is a CD receptor.
The engineered NK cell of any one of the preceding claims, wherein the CD receptor is CD4.
The engineered NK cell of any one of the preceding claims, wherein the chimeric polypeptide receptor comprises at least two different signaling domains or at least three different signaling domains.
The engineered NK cell of any one of the preceding claims, wherein the antigen of the virus is presented on a surface of a target cell.
The engineered NK cell of any one of the preceding claims, further comprising a heterologous IL-15 or a fragment thereof.
The engineered NK cell of any one of the preceding claims, further comprising a receptor comprising a heterologous IL-15R or a fragment thereof.
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 claims 1-18, wherein the engineered NK cell exhibits reduced expression or activity of endogenous CD38.
The engineered NK cell of claims 1-18, wherein expression or activity of endogenous CD38 of the engineered NK cell is not modified.
The engineered NK cell of claims 1-20, wherein the heterologous IL-15 or the fragment thereof is secreted by the engineered NK cell.
The engineered NK cell of claims 1-20, wherein the heterologous IL-15 or the fragment thereof is membrane-bound.
The engineered NK cell of any one of the preceding claims, further comprising an enhanced expression of an activating NK receptor.
The engineered NK cell of any one of the preceding claims, further comprising an additional chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen.
The engineered NK cell of any one of the preceding claims, wherein the antigen binding moiety of the chimeric polypeptide receptor is a multispecific binding moiety capable of specifically binding to two or more antigens that are different.
The engineered NK cell of any one of the preceding claims, wherein the antigen comprises one or more members selected from the group consisting of: BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
The engineered NK cell of any one of the preceding claims, wherein the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.
The engineered NK cell of any one of the preceding claims, further comprising a safety switch capable of effecting death of the engineered NK cell.
The engineered NK cell of any one of the preceding claims, wherein the safety switch comprises one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.
The engineered NK cell of any one of the preceding claims, further comprising a heterologous cytokine.
The engineered NK cell of any one of the preceding claims, wherein the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21.
The engineered NK cell of any one of the preceding claims, wherein the heterologous cytokine is not IL15.
The engineered NK cell of any one of the preceding claims, further comprising a heterologous immune regulator polypeptide.
The engineered NK cell of any one of the preceding claims, wherein the heterologous immune regulator polypeptide comprises one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide.
The engineered NK cell of any one of the preceding claims, wherein the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator.
The engineered NK cell of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
The engineered NK cell of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises SHIP2.
The engineered NK cell of any one of the preceding claims, wherein the hypo-immunity regulator comprises one or more members 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.
The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell comprises a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell.
The engineered NK cell of any one of the preceding claims, wherein the CD16 variant comprises a sequence selected from the group consisting of: SEQ ID NOs. 1-8.
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 a control cell.
The engineered NK cell of any one of the preceding claims, wherein the host cell is an immune cell.
The engineered NK cell of any one of the preceding claims, wherein the isolated stem cell comprises an embryonic stem cell.
The engineered NK cell of any one of the preceding claims, wherein the induced stem cell comprises an induced pluripotent stem cell.
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.
The method of any one of the preceding claims, further comprising administering the engineered NK cell to the subject.
A method comprising:

administering to a subject in need thereof a population of NK cells comprising the engineered NK cell of any one of the preceding claims.
The method of any one of the preceding claims, further comprising administering to the subject a separate therapeutic agent.
The method of any one of the preceding claims, wherein the separate therapeutic agent is a chemotherapeutic agent.
The method of any one of the preceding claims, wherein administration of the engineered NK cell treats or reduces a risk of a cancer in the subject.
The method of any one of the preceding claims, wherein administration of the engineered NK cell treats a viral infection in the subject.