TW202342737A - Systems and methods for enhanced immunotherapies - Google Patents

Abstract

The present disclosure describes systems and methods for immunotherapies. 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.

Description

Systems and methods for enhancing immunotherapy

Cancer (e.g., neoplasms, tumors) is a leading cause of death worldwide, killing approximately 10 million people each year. Cancer continues to place an increasing health, financial, and emotional burden on individuals, families, communities, and nations. Increased understanding of cancer biology (e.g., cancer immunobiology in particular) and genetic engineering have led to the development of adoptive cell therapies (e.g., cellular immunotherapy) with the goal of treating or controlling many different cancers.

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

The present disclosure provides methods and systems for treating viral infections and/or cancer. Some aspects of the present disclosure provide engineered immune cells, such as engineered natural killer (NK) cells, and methods for treating such as hematological malignancies, solid tumors, viral infections, and the like.

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

In some embodiments of any of the engineered NK cells disclosed herein, the virus includes one or more members selected from HIV, HBV, HCV, EBV, HPV, Lassa virus, influenza virus, coronavirus, and derivatives thereof things. In some embodiments of any of the engineered NK cells disclosed herein, the virus includes one or more members selected from: HBV, HCV, and derivatives thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell exhibits enhanced response to a target cell comprising an antigen (e.g., a viral antigen) compared to a control cell lacking a chimeric polypeptide receptor. toxicity. In some embodiments of any of the engineered NK cells disclosed herein, the enhanced cytotoxicity of the engineered NK cells to the target cells is at least about 10%, at least about 20%, greater than the cytotoxicity of the control cells to the target cells. 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 at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.8 times, at least about 1 times, or More. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell is configured to within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours of incubation with the target cells. or exhibit enhanced cytotoxicity to target cells within a shorter period of time. In some embodiments of any of the engineered NK cells disclosed herein, the enhanced cytotoxicity is at an effector-to-target (E:T) ratio of at least about 5, at least about 10, or at least about 20.

In some embodiments, engineered NK cells containing chimeric polypeptide receptors (e.g., for viral antigens) and control NK cells lacking chimeric polypeptide receptors have better response to target cells (e.g., expression and/or presence of viral antigens). ) may be greater than the difference (Diff-2) in cytotoxicity between engineered NK cells and control NK cells against control target cells (e.g., no expression and/or presence of viral antigen) 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 of the engineered NK cells disclosed herein, the antigen-binding moiety comprises at least a portion of an antibody that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the engineered NK cells disclosed herein, the antigen-binding portion does not comprise at least a portion of an antibody that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the engineered NK cells disclosed herein, the antigen-binding portion comprises at least a portion of a cellular protein that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the engineered NK cells disclosed herein, the cellular protein is a surface receptor. In some embodiments of any of the engineered NK cells disclosed herein, the surface receptor is a CD receptor. In some embodiments of any of the engineered NK cells disclosed herein, the CD receptor is CD4. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor contains at least two different signaling domains or at least three different signaling domains. In some embodiments of any of the engineered NK cells disclosed herein, the viral antigen is present on the surface of the target cell.

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

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

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

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

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

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell further comprises a safety switch capable of causing death of the engineered NK cell. In some embodiments of any of the engineered NK cells disclosed herein, the safety switch comprises a caspase (eg, caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR, and One or more members of B cell CD20.

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

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous immunomodulatory polypeptide. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous immunomodulatory polypeptide comprises HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, One or more members of CD46, CD55 and CD59.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell exhibits reduced expression or activity of endogenous immunomodulatory polypeptides. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous immune modulatory polypeptide comprises an immune checkpoint inhibitor or hypoimmune modulator. In some embodiments of any of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, One or more members of the TGFβ receptor and 2B4. In some embodiments of any of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any of the engineered NK cells disclosed herein, the hypoimmune modulator comprises B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86 , one or more of ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59 members.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a CD16 variant for enhanced CD16 signaling compared to control cells, wherein the CD16 variant is different from the engineered NK cell. source. In some embodiments of any of the engineered NK cells disclosed herein, the CD16 variant comprises a sequence selected from SEQ ID NO. 1-8.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit enhanced cytotoxicity against the target cells compared to control cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells induce a reduced immune response in the host cell compared to control cells.

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

In some embodiments of any of the engineered NK cells disclosed herein, the isolated stem cells include embryonic stem cells. In some embodiments of any of the engineered NK cells disclosed herein, the induced stem cells include induced pluripotent stem cells.

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

In one aspect, the present disclosure provides a method comprising administering to an object in need thereof a population of NK cells comprising engineered NK cells comprising a chimeric polypeptide receptor comprising a chimeric polypeptide receptor capable of interacting with The antigen-binding portion of a virus that specifically binds to an antigen, where the virus is not CMV.

In some embodiments of any of the methods disclosed herein, the virus includes one or more members selected from HIV, HBV, HCV, EBV, HPV, Lassa virus, influenza virus, coronavirus, and derivatives thereof. In some embodiments of any of the methods disclosed herein, the virus includes one or more members selected from: HBV, HCV, and derivatives thereof.

In some embodiments of any of the methods disclosed herein, the engineered NK cells exhibit enhanced cytotoxicity against target cells containing an antigen (eg, viral antigen) compared to control cells lacking the chimeric polypeptide receptor. In some embodiments of any of the methods disclosed herein, the engineered NK cells have enhanced cytotoxicity on the target cells that is at least about 10%, at least about 20%, at least about 30% greater than the cytotoxicity of the control cells on the target cells. %, 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 at least about 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.8 times, at least about 1 times or more. In some embodiments of any of the methods disclosed herein, the engineered NK cells are configured to respond within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, or less of incubation with the target cells. Exhibits enhanced cytotoxicity to target cells over time. In some embodiments of any of the methods disclosed herein, the enhanced cytotoxicity is at an efficacy-to-target (E:T) ratio of at least about 5, at least about 10, or at least about 20.

In some embodiments of any of the methods disclosed herein, the antigen-binding moiety comprises at least a portion of an antibody that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the methods disclosed herein, the antigen-binding moiety does not comprise at least a portion of an antibody that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the methods disclosed herein, the antigen-binding moiety comprises at least a portion of a cellular protein that exhibits specific affinity for an antigen of the virus. In some embodiments of any of the methods disclosed herein, the cellular protein is a surface receptor. In some embodiments of any of the methods disclosed herein, the surface receptor is a CD receptor. In some embodiments of any of the methods disclosed herein, the CD receptor is CD4. In some embodiments of any 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 of the methods disclosed herein, the viral antigen is present on the surface of the target cell.

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

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

In some embodiments of any of the methods disclosed herein, heterologous IL-15 or fragments thereof are secreted by engineered NK cells. Alternatively, in some embodiments of the methods disclosed herein, heterologous IL-15 or fragments thereof are membrane-bound.

In some embodiments of any of the methods disclosed herein, the engineered NK cells further comprise initiating enhanced expression of NK receptors.

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

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

In some embodiments of any of the methods disclosed herein, the engineered NK cells further comprise a heterologous cytokine. In some embodiments of any of the methods disclosed herein, the heterologous cytokine comprises one or more members selected from IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments of any of the methods disclosed herein, the heterologous cytokine is other than IL15.

In some embodiments of any of the methods disclosed herein, the engineered NK cells further comprise a heterologous immunomodulatory polypeptide. In some embodiments of any of the methods disclosed herein, the heterologous immunomodulatory polypeptide comprises HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and one or more members of CD59.

In some embodiments of any of the methods disclosed herein, the engineered NK cells exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some embodiments of any of the methods disclosed herein, the endogenous immunomodulatory polypeptide comprises an immune checkpoint inhibitor or hypoimmune modulator. In some embodiments of any of the methods disclosed herein, the immune checkpoint inhibitor comprises PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGFβ receptor and one or more members of 2B4. In some embodiments of any of the methods disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any of the methods disclosed herein, the hypoimmune modulator comprises B2M, CIITA, TAP1, TAP2, tap-associated protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1 , one or more members of MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the methods disclosed herein, the engineered NK cells comprise a CD16 variant for enhanced CD16 signaling compared to control cells, wherein the CD16 variant is heterologous to the engineered NK cell. In some embodiments of any of the methods disclosed herein, the CD16 variant comprises a sequence selected from SEQ ID NO. 1-8.

In some embodiments of any of the methods disclosed herein, the engineered NK cells exhibit enhanced cytotoxicity against the target cells compared to control cells.

In some embodiments of any of the methods disclosed herein, the engineered NK cells induce a reduced immune response in the host cell compared to control cells.

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

In some embodiments of any of the methods disclosed herein, the isolated stem cells include embryonic stem cells. In some embodiments of any of the methods disclosed herein, the induced stem cells include induced pluripotent stem cells.

In some embodiments of any of the methods disclosed herein, the method further comprises administering engineered NK cells to the object.

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

Other aspects and advantages of the disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be realized, the 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 considered illustrative in nature and not restrictive. [Incorporated 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 that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, this specification is intended to supersede and/or take precedence over any such conflicting material.

[cross reference]

This application claims the benefit of International Application No. PCT/CN2022/075418, filed on February 7, 2022, and International Application No. PCT/CN2023/071422, filed on January 9, 2023, which are incorporated herein by reference in their entirety.

While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications and substitutions will occur to those skilled in the art without departing from the teachings of the present 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 "chimeric transmembrane receptor" includes multiple chimeric transmembrane receptors.

The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which acceptable error range will depend in part on how the value is measured or determined, i.e., measured System limitations. For example, "about" may mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, "about" may 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 may mean within one order of magnitude of a value, preferably within 5 times the value, more preferably within 2 times the value. Where a specific value is recited in this application and in the claims, the term "about" shall be assumed to mean within an acceptable error range for that specific value, unless otherwise stated.

The use of alternatives (eg, "or") should be understood to refer to one, both, or any combination of the alternatives. The term "and/or" should be understood to refer to one or both of the alternatives.

The term "cell" generally refers to biological cells. Cells are generally the basic structural, functional and/or biological units of living organisms. Cells can be derived from any organism that has one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., from plant crops, fruits, vegetables, cereals, Soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopods, hornworts, liverworts , moss cells), algal cells (for example, Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens) C. Agardh, etc.), seaweeds (e.g. kelp), fungal cells (e.g. yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g. Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates Cells from animals (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans etc.) cells, etc. Sometimes, cells are not derived from a natural organism (for example, cells may be synthetically prepared, sometimes called artificial cells).

As used interchangeably herein, the terms "reprogramming," "dedifferentiation," "increasing cell potential," or "increasing developmental potential" generally refer to methods that increase cell potency or dedifferentiate cells to a less differentiated state. For example, cells with increased cellular potential have more developmental plasticity (i.e., can differentiate into more cell types) than the same cells in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a lower state of differentiation than the same cell in a non-reprogrammed state.

The term "differentiation" generally refers to the process by which non-specialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (eg, immune cells). Differentiated or differentiation-induced cells are cells that occupy more specialized ("committed") positions in the cell lineage. The term "committed" generally refers to a cell that has progressed to a point in the differentiation pathway at which, under normal circumstances, the cell would continue to differentiate into a specific cell type or subclass of cell types and would otherwise be unable to differentiate to a different cell type or reverts 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., embryoid body). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be a continuum of developmental potential, ranging from cells that are not fully or partially pluripotent (such as ectodermal stem cells), which are unable to give rise to a complete organism, to more primitive, more potent cells, which are able to give rise to intact organisms (e.g. embryonic stem cells)).

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

The term "embryonic stem cells" (ESC) generally refers to the native pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent, giving rise to all derivatives of the three major germ layers (ectoderm, endoderm and mesoderm) during development. In some cases, ESCs can be engineered to differentiate directly into committed cells (eg, NK cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (eg, HSCs), which can then be further induced to differentiate into committed cells (eg, NK cells).

The term "isolated stem cells" generally refers to any type of stem cell disclosed herein (eg, ESCs, HSCs, mesenchymal stem cells (MSCs), etc.) isolated from a multicellular organism. For example, HSCs can be isolated from mammalian bodies (eg, humans). In another example, embryonic stem cells can be isolated from embryos.

The term "isolated" generally refers to a cell or cell population that has been separated from its original environment. For example, the new environment of an isolated cell is substantially free of at least one component found in the environment in which an "unisolated" reference cell exists. Isolated cells can be cells that have been removed from some or all components found in their natural environment, such as cells isolated from tissue or biopsy samples. The term also includes cells in which at least one, some or all components have been removed from cells found in a non-naturally occurring environment, such as cells isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component (including other substances, cells or populations of cells) found in nature or grown, stored or maintained in a non-naturally occurring environment.

As used interchangeably herein, the terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., differentiate into NK cells), and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that can give rise to all blood cell types, including myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes , megakaryocytes/platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). In some cases, HSCs can be CD34+ hematopoietic cells capable of giving rise to mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells.

The term "immune cell" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells may include NK cells, T cells, monocytes, innate lymphocytes, tumor-infiltrating lymphocytes, macrophages, granulocytes, and the like.

The term "NK cells" or "natural killer cells" 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, the NK cells, phenotypically CD3- and CD56+, express at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both to the same or different extents), and optionally express CD16, However, it lacks the expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some cases, isolated subsets of CD56+ NK cells may exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and/or DNAM-1.

As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may 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 may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP and nucleotide derivatives, which confer nuclease resistance to the nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling can also be done with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Fluorescent labels for nucleotides may include, but are not limited to, luciferin, 5-carboxyluciferin (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyluciferin ( JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-rhodamine (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-sulfonate ACID (EDANS). Specific examples of fluorescently labeled nucleotides may include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [TAMRA]dCTP, available from Perkin Elmer, Foster City, Calif. JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP and [dROX]ddTTP, available FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor , Ind.'s Luciferin-15-dATP, Luciferin-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Luciferin-12-ddUTP, Luciferin-12 - UTP and luciferin-15-2′-dATP; and chromosomally labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY available from Molecular Probes, Eugene, Oreg. -TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Luciferin-12 -UTP, Luciferin-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. Nucleotides can also be labeled or labeled by chemical modification. The 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 terms "polynucleotide," "oligonucleotide," or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleosides Acids, or analogues thereof, whether in single-, double- or multi-chain form. Polynucleotides may be exogenous or endogenous to the cell. Polynucleotides can exist in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide can be DNA. The polynucleotide can be RNA. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Polynucleotides may contain one or more analogs (eg, altered backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, can be imparted before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenogeneic nucleic acids, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycin, 7 - deazo-GTP, fluorophores (e.g. rhodamine or sugar-linked luciferin), thiol-containing nucleotides, biotin-linked nucleotides, fluorobase analogs, CpG islands, Methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braidin and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (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 nucleic acids (eg, DNA such as genomic DNA and cDNA) encoding RNA transcripts and their corresponding nucleotide sequences. As used herein with respect to genomic DNA, the term includes intervening non-coding regions as well as regulatory regions, and may include the 5' and 3' ends. In some uses, the term encompasses transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain the "open reading frame" that encodes the polypeptide. In some uses of the term, a "gene" includes only the coding sequence necessary to encode a polypeptide (eg, an "open reading frame" or "coding region"). In some cases, the gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. Genes may refer to "foreign genes" or unnatural genes. Non-native genes may refer to genes that are not normally present in a host organism but are introduced into the host organism by gene transfer. Non-native genes may also refer to genes that are not in their natural location in the genome of an organism. Non-natural genes may also refer to naturally occurring nucleic acid or polypeptide sequences (eg, non-natural sequences) that contain mutations, insertions, and/or deletions.

The term "expression" generally refers to one or more processes by which polynucleotides are transcribed from a DNA template (e.g., transcribed into mRNA or other RNA transcripts) and/or the transcribed mRNA is subsequently translated into peptides, polypeptides, or protein process. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. With respect to expression, "up-regulation" 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 the wild-type state, while "down-regulation" generally refers to A reduced level of expression of a polynucleotide (eg, RNA such as mRNA) and/or polypeptide sequence relative to its expression in the wild-type state. Expression of transfected genes can occur transiently or stably in the cell. In "transient expression," the transfected gene is not transferred to daughter cells during cell division. Expression of the gene is lost over time because its expression is restricted to the transfected cells. In contrast, stable expression of the transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cells. Such a selective advantage could be resistance to a certain toxin presented to the cell.

As used interchangeably herein, the terms "peptide," "polypeptide," or "protein" generally refer to a polymer of at least two amino acid residues linked by peptide bond(s). This term does not denote a specific length of the polymer and is not intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis or is naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (eg, domains). The term also encompasses amino acid polymers that have been modified, for example by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation (eg conjugation with a labeling component). As used herein, the term "amino acid" refers generally to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

As used herein with respect to a polypeptide, the term "derivative," "variant," or "fragment" generally refers to a polypeptide that is related to a wild-type polypeptide, e.g., 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 may contain one or more amino acid changes (eg, mutations, insertions and deletions), truncations, modifications, or combinations thereof compared to the wild-type polypeptide.

As used herein with respect to a polypeptide molecule (e.g., a protein), the terms "engineered," "chimeric," or "recombinant" generally refer to the expression of the polypeptide molecule resulting from the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule. A polypeptide molecule with a heterologous amino acid sequence or an altered amino acid sequence derived from a cell or organism. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the term "engineered" or "recombinant" generally refers to polynucleotides that have heterologous nucleic acid sequences or altered nucleic acid sequences as a result of the application of genetic engineering techniques. Acid molecules. Genetic engineering technologies 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, engineered or recombinant polynucleotides (eg, genomic DNA sequences) may be modified or altered by gene editing moieties.

The term "gene editing moiety" generally refers to a moiety that can edit a nucleic acid sequence, whether exogenous or endogenous to the cell containing the nucleic acid sequence. In some embodiments, gene editing moieties regulate gene expression by editing nucleic acid sequences. In some cases, gene editing moieties can modulate gene expression by editing genomic DNA sequences. In some cases, gene editing moieties can modulate gene expression by editing the mRNA template. In some cases, editing a nucleic acid sequence can alter the underlying pattern of gene expression.

Alternatively or additionally, the gene editing moiety may function by specifically binding to a target sequence operably coupled to the gene (or a target sequence within the gene) and modulating the production of mRNA from DNA (e.g., chromosomal DNA or cDNA). , to regulate gene expression or activity. In some cases, the gene editing moiety can recruit or contain at least one transcription factor that binds to specific DNA sequences to control the rate of transcription of genetic information from DNA to mRNA. The gene-editing moiety itself can bind to DNA and regulate transcription through physical barriers, such as preventing proteins (such as RNA polymerase) and other related proteins from assembling on the DNA template. Gene editing moieties can regulate gene expression at the translational level, for example, by modulating the proteins produced from the mRNA template. In some cases, gene editing moieties can modulate gene expression by affecting the stability of mRNA transcripts.

The term "antibody" generally refers to protein-binding molecules with immunoglobulin-like functions. The term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, etc.). Derivatives, variants or fragments thereof may refer to functional derivatives or fragments that retain (eg, complete and/or partial) binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibody, diabody and single domain antibody ("sdAb"). ” or “Nanobody” or “Camelidae”). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).

The term "chimeric polypeptide receptor" generally refers to a non-natural polypeptide receptor (or heterologous receptor) containing one or more antigen-binding moieties, each antigen-binding moiety capable of binding a specific antigen. Chimeric polypeptide receptors can be monospecific (ie, capable of binding one type of specific antigen). Alternatively, the chimeric polypeptide receptor may be multispecific (ie, capable of binding two or more different types of specific antigens). Chimeric polypeptide receptors can be monovalent (ie, contain a single antigen-binding moiety). Alternatively, the chimeric polypeptide receptor may be multivalent (ie, contain multiple antigen-binding moieties). In some cases, the chimeric polypeptide receptor may comprise a T cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR).

As used interchangeably herein, the terms "antigen binding domain" or "antigen binding portion" generally refer to a construct that exhibits preferential binding to a specific target antigen. The antigen binding domain may be a polypeptide construct such as an antibody, modifications thereof, fragments thereof, or combinations thereof. The antigen binding domain can be any antibody disclosed herein or functional variant thereof. Non-limiting examples of antigen-binding domains may include murine antibodies, human antibodies, humanized antibodies, camel Ig, shark heavy chain only antibodies (VNAR), Ig NARs, chimeric antibodies, recombinant antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), microantibodies, diabodies, triabodies, tetrabodies, single domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain antibodies (VHH) only and can maintain the binding specificity of the entire antibody other antibody fragments. Alternatively or additionally, a polypeptide construct of an antigen-binding domain may comprise at least a portion of a naturally occurring complementary molecule of a particular target antigen. Naturally complementary molecules and specific target antigens may naturally form complexes (eg, in cells, on cell surfaces, etc.). In some examples, a specific target antigen can be a receptor (e.g., a cell surface receptor), and the natural complementary molecule can be a ligand that binds and/or is bound by the receptor (e.g., a cell surface receptor). ligand) and vice versa. In some examples, a specific target antigen and its natural complementary molecule can be either receptors that bind to each other (eg, cell surface receptors) or soluble ligands that bind to each other.

In some embodiments, the antigen binding domain can be derived from an antibody. In some embodiments, the antigen-binding domain may not be derived from an antibody and need not be derived from an antibody. Antigen binding domains can be derived from cellular polypeptides that exhibit binding affinity for a specific target antigen. For example, a cellular polypeptide may be at least a portion of a receptor protein capable of binding one or more target antigens (e.g., at least a portion of the extracellular domain of a transmembrane receptor protein that exhibits 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 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 acids 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 residues, at least or at most about 90 amino acid residues, at least or at most about 100 amino acid residues, at least or at most about 110 amino acid residues, at least or at most about 120 amino acid residues, at least or at most about 130 amino acid residues residues, at least or at most about 140 amino acid residues, at least or at most about 150 amino acid residues, at least or at most about 160 amino acid residues, at least or at most about 170 amino acid residues, at least or at most about 180 amino acid residues residues, at least or at most about 190 amino acid residues, at least or at most about 200 amino acid residues, at least or at most about 210 amino acid residues, at least or at most about 220 amino acid residues, at least or at most about 230 amino acid residues residues, at least or at most about 240 amino acid residues, at least or at most about 250 amino acid residues, at least or at most about 300 amino acid residues, or at least or at most about 500 amino acid residues in length.

The term "safety switch" generally refers to engineered polypeptide constructs designed to prevent potential toxicity or other adverse effects of cell therapies. When expressed in a cell, the safety switch can induce host cell death, thereby inactivating the activity of the cell in the host (eg, in vivo). Therefore, the safety switch can be the suicide part. In some cases, cells can be programmed to express the suicidal component at a certain stage of their life cycle (e.g., time-programmed). In some cases, expression of the suicide moiety in the cell may be conditional or inducible. In some examples, conditional regulation (eg, expression) of the suicide moiety may include control through small molecule-mediated post-translational initiation as well as tissue-specific and/or temporal transcriptional regulation. Therefore, the safety switch can be part of the inducible suicide. Safety switches 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, the safety switch can be activated by an exogenous molecule (e.g., a drug or prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., an engineered NK cell disclosed herein). die.

The term "immunomodulatory polypeptide" generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secreted polypeptide, cleavable polypeptide, non-immune polypeptide) capable of modulating or controlling one or more properties of immune cells (e.g., NK cells). Cleaved peptides, etc.). One or more properties of the immune cell may include differentiation of the immune cell, immune cell morphology, expression of polynucleotide or polypeptide constructs within the immune cell, or activity of the immune cell (e.g., engineered NK cells against diseased cells (e.g., cancer) cells) cytotoxic activity). The immunomodulatory polypeptide can be endogenous to the host cell. Alternatively or additionally, the immunomodulatory polypeptide may be heterologous to the host cell. In some cases, control of one or more properties of immune cells can be mediated by down-regulating expression (eg, inhibition, knockdown, or knockout) of an immunomodulatory polypeptide. Alternatively or additionally, control of one or more properties of immune cells may be mediated by up-regulation of expression of an immunomodulatory polypeptide (eg, upregulation of an endogenous gene or knock-in of a heterologous gene encoding an immunomodulatory polypeptide). In another alternative or additionally, controlling one or more properties of the immune cell may be mediated by maintaining expression of the immunomodulatory polypeptide for a longer period of time than the native or normal expression profile of the immunomodulatory polypeptide in the host cell. In some cases, the immunomodulatory polypeptide may contain a low immunomodulatory agent. In some cases, the immunomodulatory polypeptide may comprise an immune checkpoint inhibitor.

The term "hypoimmune modulator" generally refers to a polypeptide construct in a cell in which there is enhanced expression (e.g., by knock-in of a heterologous gene) or reduced expression (e.g., by knock-in of a heterologous gene) of the hypoimmune modulator in the cell. Knockout or knockdown of an endogenous gene) may help cells reduce or avoid an immune response (e.g., immune attack, such as adaptive immune rejection) from the host body upon administration to the host body. In some cases, cells (e.g., engineered NK cells disclosed herein) can be modified to exhibit enhanced expression or reduced expression of hypoimmune modulators such that the cells can be used upon a second or further infusion of the cells into the host. (i.e., receptor) can evade host immune attack. Therefore, the cell (i) will not be rejected by the host's immune system and/or (ii) will be rejected by the host's immune system at a lower rate than a control cell without enhanced expression or reduced expression of a low immunogenicity modulator. Slow rate rejection. Cells that exhibit enhanced expression or reduced expression of hypoimmunity modulators may be said to exhibit "hypoimmunity" or "immune privilege."

The term "immune checkpoint inhibitors" generally refers to a group of molecules present on the cell surface of immune cells (e.g., T cells, myeloid cells, NK cells, B cells, etc.) that can downregulate or inhibit immune cell response to The immune response of target cells, such as cancer cells (ie, anti-cancer or anti-tumor immune response), is used to modulate the immune response of the cell. The target cells may express receptors or ligands for immune checkpoint inhibitors present on the surface of the immune cells to engage with the immune checkpoint inhibitors and downregulate or inhibit the immune response of the immune cells to the target cells. Therefore, in some cases, downregulating or inhibiting the expression of immune checkpoint inhibitors in immune cells can enhance or prolong the immune response of immune cells to target cells.

The term "immune response" generally refers to a T-cell-mediated and/or B-cell-mediated immune response from the host's immune system to an object (eg, a foreign body). Examples of immune responses may include T cell responses, such as cytokine production and cellular cytotoxicity. In some cases, the immune response can be achieved indirectly through T cell priming, such as antibody production (humoral response) and priming of cytokine response cells such as macrophages.

The term "enhanced expression," "increased expression," or "upregulated expression" generally refers to the production of a relevant moiety (e.g., a polynucleotide or a polypeptide) to a level higher than normal for the relevant moiety in a host strain (e.g., a host cell). The level of expression. A normal expression level can be essentially zero (or null) or above zero. Relevant portions may comprise endogenous genes or polypeptide constructs of the host strain. Relevant moieties may comprise heterologous genes or polypeptide constructs introduced into or into a host strain. For example, a heterologous gene encoding a related polypeptide can be knocked-in (KI) into the genome of a host strain for enhanced expression of the related polypeptide in the host strain.

The term "enhanced activity," "increased activity," or "upregulated activity" generally refers to a modified portion of interest (e.g., a polynucleotide or polypeptide) that has a higher activity than that of the portion of interest in a host strain (e.g., a host cell) of normal activity levels. Normal activity levels may be essentially zero (or null) or above zero. Relevant portions may comprise polypeptide constructs of the host strain. Relevant moieties may comprise heterologous polypeptide constructs introduced into or introduced into a host strain. For example, a heterologous gene encoding a related polypeptide can be knocked-in (KI) into the genome of a host strain for enhanced activity of the related polypeptide in the host strain.

The term "reduced expression", "reduced expression" or "down-regulated expression" generally refers to the production of a relevant moiety (e.g., a polynucleotide or a polypeptide) that is less than normal for the relevant moiety in a host strain (e.g., a host cell). The level of expression. Normal expression levels are above zero. Relevant portions may comprise endogenous genes or polypeptide constructs of the host strain. In some cases, the relevant portion can be knocked out or knocked down in the host strain. In some examples, reduced expression of the relevant moiety may include complete inhibition of such expression in the host strain.

The term "reduced activity", "reduced activity" or "down-regulated activity" generally refers to a modified relevant part (e.g., polynucleotide or polypeptide) that has less activity than the relevant part in the host strain (e.g., host cell) of normal activity levels. Normal activity levels are above zero. Relevant portions may comprise endogenous genes or polypeptide constructs of the host strain. In some cases, the relevant portion can be knocked out or knocked down in the host strain. In some examples, reduced activity of a relevant moiety may include complete inhibition of such activity in the host strain.

As used interchangeably herein, the terms "subject", "individual" or "patient" generally refer to a vertebrate animal, preferably a mammal, such as a human. Mammals include, but are not limited to, rats, apes, humans, farm animals, sporting animals, and pets. Also includes tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

The term "therapy" or "treatment" generally refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefits and/or preventive benefits. For example, treatment may include administration of a system or cell population disclosed herein. Therapeutic benefit refers to any therapeutically relevant improvement or effect on one or more diseases, conditions or symptoms being treated. For prophylactic benefit, the composition may be administered to an article that is at risk of developing a particular disease, condition, or symptom, or to an article that reports one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifest.

The term "effective amount" or "therapeutically effective amount" generally refers to an amount of a composition, e.g., an amount of a composition comprising immune cells, such as lymphocytes (e.g., T lymphocytes and/or NK cells), containing the system of the present disclosure, This amount is sufficient to produce the desired activity when applied to an article in need. In the context of the present disclosure, the term "therapeutically effective" generally refers to an amount of the composition sufficient to delay the manifestation, prevent progression, alleviate, or alleviate at least one symptom of the condition treated by the methods of the present disclosure.

A. Overview

T cells are part of the adaptive immune system and can be sensitized to recognize specific threats by recognizing immune proteins (i.e., antigens) on the surface of foreign cells. In contrast, NK cells are part of the innate immune response and can respond to a variety of objects considered "non-self." Generally, unlike T cells, NK cells can attack their target cells without the need for sensitization (i.e., antigen-specific sensitization) to eliminate foreign substances.

Unmodified NK cells derived from an article (eg, HSC derived from an article) can be cultured and expanded ex vivo and then administered to a subject as a therapy to attack their target cells (eg, cancer cells). However, NK cell-based therapies may be limited due to their short half-life and/or poor proliferation ex vivo or in vivo. Additionally, unmodified NK cells may not effectively target more difficult-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 generate a limited supply of NK cells for effective adoptive immunotherapy.

Therefore, there remains a large unmet need for NK cells that are sourced and engineered to exhibit, for example, enhanced proliferation, half-life, and cytotoxic activity against target cells.

There remains a substantial unmet need for NK cells that exhibit enhanced cytotoxicity against virally infected cells or cells in the presence of viral antigens for use in the treatment of viral diseases such as, but not limited to, influenza, common cold, bronchiolitis, Acute respiratory diseases, measles, smallpox, chickenpox, hepatitis, etc. Additionally, some viruses can cause cancer (for example, human papillomavirus (HPV) can be a factor in cervical cancer). Accordingly, in some aspects, the present disclosure provides systems and methods for immunotherapy based on engineered immune cells (e.g., engineered NK cells) that exhibit enhanced targeting of virally infected cells and/or cytotoxicity, for example, for the treatment of viral diseases and/or cancer (eg, tumors).

B. Engineered immune cells

This disclosure describes systems and methods for immunotherapy. Immune cells can be engineered to exhibit enhanced half-life compared to control cells (eg, non-engineered immune cells). Immune cells can be engineered to exhibit enhanced proliferation compared to control cells. Immune cells can be engineered to effectively and specifically target diseased cells (such as cancer cells) that control cells are insufficient or unable to target. The engineered immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases in vivo. Engineered immune cells prepared ex vivo or ex vivo can be administered to an object in need to treat a disease (eg, myeloma or solid tumor). Engineered immune cells can be autologous to the object. Alternatively, the engineered immune cells may be allogeneic to the object.

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

In some cases, the stem cells disclosed herein (eg, isolated stem cells, induced stem cells) may be or derived from autologous cells. Autologous cells can be obtained from a subject who has a condition or is suspected of having the condition. Alternatively, autologous cells may be obtained from the subject before the subject is found to have the condition. In some cases, autologous cells may be allogeneic cells, such as universal stem cells that have reduced immunogenicity, reduced numbers, or do not require immunosuppressive drugs. Autologous cells can be obtained from healthy donors.

In some cases, engineered immune cells (eg, engineered NK cells) may be autologous cells. Engineered immune cells can be obtained from subjects suffering from a condition or suspected of having the condition. Alternatively, engineered immune cells can be obtained from the subject before the subject is found to have the condition. In some cases, the engineered immune cells may be allogeneic cells, such as for general allogeneic immunotherapy with reduced immunogenicity and requiring reduced amounts or no immunosuppressive drugs. Engineered immune cells can be obtained from healthy donors.

In some aspects, T cells can be engineered to exhibit enhanced half-life compared to control cells (eg, non-engineered T cells). T cells can be engineered to exhibit enhanced proliferation compared to control cells. T cells can be engineered to effectively and specifically target diseased cells (such as cancer cells) that control cells are insufficient or unable to target. The engineered T cells disclosed herein can be engineered ex vivo, in vitro, and in some cases in vivo. Engineered T cells prepared ex vivo or ex vivo can be administered to an object in need to treat a disease (eg, myeloma or solid tumor). The engineered T cells can be autologous to the subject. Alternatively, the engineered T cells may be allogeneic to the object.

In some aspects, NK cells can be engineered to exhibit enhanced half-life compared to control cells (eg, non-engineered NK cells). NK cells can be engineered to exhibit enhanced proliferation compared to control cells. NK cells can be engineered to efficiently and specifically target diseased cells (such as cancer cells) that control cells are insufficient or unable to target. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases in vivo. Engineered NK cells prepared ex vivo or in vitro can be administered to an object in need to treat a disease (eg, myeloma or solid tumor). Engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the object.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells can include cytokines that are foreign to the immune cells (eg, secreted cytokines). In some cases, the allogeneic cytokine may include allogeneic interleukin (IL) (eg, heterologous secreted IL-15). The engineered immune cells may further comprise one or both of the following: (i) a CD16 variant for enhanced CD16 signaling compared to control cells and (ii) a chimera comprising an antigen-binding moiety capable of binding to the antigen Peptide receptors. In some examples, the antigen is not CD19. Therefore, the antigen-binding moiety may not exhibit, and need not exhibit, any specific binding to CD19, but rather to an antigen other than CD19 (eg, one or more antigens).

The engineered immune cells disclosed herein (e.g., engineered NK cells) may include a heterologous receptor that is a corresponding receptor for a heterologous cytokine disclosed herein (e.g., a heterologous IL-15 receptor for heterologous IL-15). (IL-15R, such as IL-15α or IL-15β). Alternatively, the engineered immune cells may not contain and need not contain any heterologous receptors that are the corresponding receptors for the heterologous cytokine. For example, the inclusion of heterologous Engineered immune cells for IL (e.g., IL-15) lack heterologous receptors for heterologous IL (e.g., IL-15R).

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells can include cytokines that are foreign to the immune cells (eg, secreted cytokines). Allogeneic cytokines may also include allogeneic interleukins (IL) (eg, allogeneic secreted IL-15). The engineered immune cells may and need not contain a heterologous receptor (eg, heterologous IL-15R) that is the corresponding receptor for the heterologous cytokine.

The heterologous cytokines disclosed herein (eg, allogeneic IL) may be of the same species as the engineered immune cells (eg, engineered NK cells). For example, both the heterologous cytokines and the engineered immune cells can be of human origin. Alternatively, the heterologous cytokines can be of a different species than the engineered immune cells.

Heterologous cytokines disclosed herein (eg, heterologous IL) can be introduced into engineered immune cells (eg, engineered NK cells) by contacting a heterologous polynucleotide encoding the heterologous cytokine with the engineered immune cell. Heterologous polynucleotides can be integrated into the chromosomes (eg, nuclear chromosomes) of engineered immune cells. Alternatively, the heterologous polynucleotide may not be, and need not be, integrated into the chromosome of the engineered immune cell. In one example, mRNA encoding a heterologous cytokine can be introduced (or inserted) into engineered immune cells.

In some cases, the heterologous cytokine disclosed herein may be heterologous IL. The heterologous IL disclosed herein may comprise at least 1, 2, 3, 4, 5, or more different types of heterologous IL. The heterologous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of heterologous IL. Alternatively, the heterologous IL may be a single type of heterologous IL. Non-limiting examples of allogeneic IL may 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, heterologous IL can include one or more members selected from IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof. For example, the engineered immune cells (eg, engineered NK cells) disclosed herein can comprise at least a portion of heterologous human IL-15 (or the gene encoding it).

Allogeneic cytokines (eg, allogeneic IL) disclosed herein may be secreted cytokines. Alternatively, heterologous cytokines may not be and need not be secreted by engineered immune cells. In this case, for example, heterologous cytokines could be bound to the cell surface of engineered immune cells.

In some cases, the heterologous cytokines (eg, heterologous IL) disclosed herein may be secreted cytokines. Expression cassettes encoding heterologous cytokines can be introduced into engineered immune cells. The expression cassette may further encode additional heterologous polypeptides, such as heterologous receptors. Within the expression cassette, a first polynucleotide sequence encoding a heterologous cytokine and a second polynucleotide sequence encoding an additional heterologous polypeptide (e.g., a heterologous receptor) can be expressed by a polynucleotide encoding a cleavage linker joints coupled to each other. In some examples, the heterologous receptor may be the corresponding receptor for the heterologous cytokine (eg, heterologous IL-15α or IL-15β for heterologous IL-15). Alternatively, the expression cassette may not encode, and need not encode, any other heterologous polypeptide other than the heterologous cytokine.

Cleavable linkers as disclosed herein may 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 the genus Orthovirus, such as foot-and-mouth disease virus (FMDV), equine rhinitis virus type A (ERAV), and Thosea asigna virus. virus) (TaV) and porcine Titus virus-1 (PTV-1), as well as cardioviruses such as Theiler's virus (e.g. Theile's murine encephalomyelitis) and encephalomyocarditis virus. Non-limiting examples of self-cleaving 2A peptides may include "F2A", "E2A", "P2A", "T2A" and functional variants thereof.

In some cases, the heterologous cytokines disclosed herein (eg, heterologous IL) can be bound to the cell surface of engineered immune cells (eg, engineered NK cells). In some examples, engineered immune cells can be genetically modified to couple a heterologous polynucleotide sequence encoding a heterologous cytokine to a gene encoding an endogenous transmembrane protein of the engineered immune cell. In one example, the endogenous transmembrane protein may be the corresponding receptor for a heterologous cytokine (eg, heterologous IL-15α or IL-15β for heterologous IL-15). In some examples, an expression cassette encoding a heterologous fusion polypeptide comprising (i) a heterologous cytokine coupled to (ii) a heterologous receptor can be introduced into engineered immune cells. Here, the heterologous cytokine may not be, and need not be, cleavable from the heterologous receptor. Non-limiting examples of heterologous receptors may include corresponding receptors for heterologous cytokines (eg, heterologous IL-15α or IL-15β for heterologous IL-15), or different receptors, such as a common gamma chain ( _γC ) receptors or modifications thereof.

Expression cassettes disclosed herein can be integrated into the genome of engineered cells (eg, engineered NK cells) through the action of gene editing moieties disclosed herein. Alternatively, the expression cassette may not be integrated into, and need not be integrated into, the genome of the engineered cell.

The engineered immune cells (e.g., engineered NK cells) disclosed herein can exhibit expression related to heterologous cytokines (e.g., heterologous IL, such as heterologous IL-15) and/or heterologous receptors as disclosed herein ( For example, enhanced signaling of the endogenous signaling pathway of heterologous IL receptors, such as heterologous IL-15R. Enhanced signaling of the endogenous signaling pathways disclosed herein can be determined by a number of methods, including, but not limited to: (i) downstream signaling proteins (e.g., JAK3, STAT3, STAT5 for IL-15/IL-15R etc.) or (ii) downstream genes (e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc for IL-15/IL-15R) by Western blot or polymerase chain reaction (PCR) techniques , Ifng, etc.) expression.

In some cases, induced by heterologous cytokines and/or heterologous receptors (e.g., induced by heterologous cytokines and/or heterologous receptors as disclosed herein, such as IL-15/IL-15R ) Enhanced signaling of endogenous signaling pathways in engineered immune cells of the present disclosure may be characterized by an increase in phosphorylation of downstream signaling proteins by at least or at most about 0.1-fold, at least or at most about 0.2-fold compared to control cells , at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, At least or at most about 1 time, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least Or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or At most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most About 5,000 times, or at least or at most about 10,000 times.

In some cases, induced by heterologous cytokines and/or heterologous receptors (e.g., induced by heterologous cytokines and/or heterologous receptors as disclosed herein, such as IL-15/IL-15R ) Enhanced signaling of endogenous signaling pathways in the engineered immune cells of the present disclosure may be characterized by an increase in the expression of downstream genes at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most, compared to control cells At most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most About 1 time, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 7 times 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times , or at least or at most about 10,000 times.

Enhanced CD16 signaling (eg, constitutively initiated signaling of CD16) of engineered immune cells (eg, engineered NK cells) as disclosed herein can be achieved by having non-cleavable CD16 variants in the target cells. CD16 (e.g., CD16a) is a transmembrane protein expressed by immune cells (e.g., NK cells) that binds to monomeric IgG attached to target cells to prime immune cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC) . In non-engineered immune cells, the binding between CD16 and monomeric IgG induces cleavage of the CD16 protein at cleavage sites near the transmembrane domain, thereby regulating the cell surface density of CD16 during immune cell priming. Therefore, endogenous CD16 of engineered immune cells can be modified to enhance their signaling. Alternatively, enhanced signaling variants of CD16 can be artificially introduced into engineered immune cells.

In some cases, the endogenous gene encoding CD16 of an engineered immune cell can be genetically modified in its extracellular domain (e.g., F176V) through the action of the gene editing moieties disclosed herein, such that the modification is compared to native CD16. CD16 exhibits higher binding affinity to its target (e.g., monomeric IgG). In some cases, a heterologous gene encoding such modified CD16 can be introduced into the cell.

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

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

Heterologous genes disclosed herein can be integrated into the genome of engineered cells (eg, engineered NK cells) through the action of gene editing moieties disclosed herein. Alternatively, the heterologous gene may not be integrated into, and need not be integrated into, the genome of the engineered cell.

Enhanced CD16 signaling of engineered immune cells (e.g., engineered NK cells) disclosed herein can be determined by a variety of methods, including but not limited to (i) phosphorylation of downstream signaling proteins (e.g., SHP-1) by Western blotting lation, or (ii) expression of downstream genes (e.g., CD25, IFN-γ, TNF, etc.) by Western blotting or PCR techniques.

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

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

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

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

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

Engineered immune cells (e.g., engineered NK cells) can include different heterologous cytokines and one or both of: (b) reduced expression of endogenous immune modulatory polypeptides (e.g., non-B2M polypeptides) or activity, and (c) safety switch. Engineered immune cells include reduced expression or activity of endogenous immunomodulatory polypeptides (eg, non-B2M polypeptides) and one or both of: (a) different heterologous cytokines, and (c) a safety switch. Engineered immune cells include a safety switch and one or both of: (a) different heterologous cytokines, and (b) reduced expression or activity of endogenous immune modulatory polypeptides (eg, non-B2M polypeptides). Engineered immune cells contain all of (a), (b) and (c).

As disclosed herein, engineered immune cells (eg, engineered NK cells) can exhibit enhanced signaling of endogenous signaling pathways involving heterologous cytokines compared to control cells.

The expression or activity of endogenous immunomodulatory polypeptides can be reduced in engineered immune cells (eg, engineered NK cells), for example, by the action of gene editing moieties disclosed herein.

Reduced expression or activity of endogenous immunomodulatory polypeptides in engineered immune cells (e.g., engineered NK cells) disclosed herein can be determined by a variety of methods, including but not limited to (i) downstream signaling proteins (e.g., for PD1 /PDL1 signaling SHP2, Igα/β, Syk, etc.) phosphorylation or dephosphorylation; or (ii) expression of endogenous immunomodulatory polypeptides (e.g., PD1) by Western blot or PCR techniques.

In some cases, reduced expression of endogenous immune modulatory polypeptides in engineered immune cells (eg, engineered NK cells) disclosed herein can be characterized by expression of endogenous immune modulatory polypeptides (eg, PD1) compared to control cells. Reduce at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, At least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least Or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or At most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most About 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells (e.g., engineered NK cells) disclosed herein can be characterized by the decrease in activity of downstream signaling proteins (e.g., for PD1/PDL1) compared to control cells. The phosphorylation of SHP2) signaling is reduced at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold , at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, At least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least Or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or Up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times.

In some cases, reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells (e.g., engineered NK cells) disclosed herein can be characterized by lower activity of downstream target signaling proteins (e.g., for PD1/ Increases phosphorylation of Igα/β or Syk) in PDL1 signaling by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least Or up to about 0.6 times, at least or up to about 0.7 times, at least or up to about 0.8 times, at least or up to about 0.9 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 3 times, at least or At most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most About 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times. The downstream target signaling protein may be a protein that is normally inhibited by the action of the functional signaling pathway of the endogenous immunomodulatory polypeptide.

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

As disclosed herein, engineered immune cells (eg, engineered NK cells) can exhibit enhanced CD16 signaling compared to control cells.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells may include reduced activity of endogenous cytokine signaling (eg, endogenous IL signaling, eg, endogenous IL-17 signaling). Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, engineered NK cells can be treated with inhibitors of endogenous cytokine signaling (eg, small molecule inhibitors). In some cases, engineered NK cells may contain reduced expression of endogenous IL-17 or its endogenous receptor (eg, by indel or transgene mutation, by transient or permanent suppression, etc.). In some cases, engineered NK cells can include reduced expression of endogenous IL-17. In some cases, engineered NK cells can include reduced expression of endogenous IL-17R. In some cases, engineered NK cells can include reduced expression of endogenous IL-17 and endogenous IL-17R.

In some cases, the endogenous cytokine disclosed herein can be endogenous IL. The endogenous IL disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of endogenous IL. The endogenous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of endogenous IL. Alternatively, the endogenous IL may be a single type of endogenous IL. Non-limiting examples of endogenous IL may 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 may include IL-17A, IL-17F and natural mutations thereof. For example, engineered immune cells (eg, engineered NK cells) disclosed herein may exhibit reduced expression or activity of IL-17A or IL-17F.

In some cases, endogenous genes disclosed herein encoding endogenous cytokines (eg, endogenous IL, eg, IL-17) can be modified through the action of gene editing moieties disclosed herein.

The endogenous receptor may be the corresponding receptor for any cytokine disclosed herein (eg, the corresponding receptor for any IL disclosed herein). In some cases, the endogenous receptor may be the corresponding receptor for IL (eg, IL-17R for IL-7 signaling). Non-limiting examples of IL-17R may include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof. In one example, endogenous IL-17R includes IL-17RA.

In some cases, reduced expression or activity of an endogenous cytokine (e.g., endogenous IL, e.g., IL-17) or its endogenous receptor disclosed herein can be determined by a variety of methods, including, but not limited to: (i) Downstream Phosphorylation of signaling proteins (e.g. PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) by Western blotting Or the expression of downstream genes of PCT technology. In some examples, genes downstream of IL cytokines, such as IL-17, may include chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.), cytokines (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.), acute phase response molecules (e.g., SAA, CRP, lipocalin 2/24p3, etc.) and/or enzymes (e.g., metalloproteinases, e.g., MMP1, MMP3, MMP9, MMP13).

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

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

In some cases, compared to control cells that do not exhibit reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, e.g., endogenous IL-17 signaling) as disclosed herein, as disclosed herein Engineered immune cells (eg, engineered NK cells) can exhibit enhanced expression profiles of specific cellular markers (eg, NK cell markers) directed against immune cells. For example, non-limiting examples of specific cell markers that target NK cells may include CD57 or killer cell immunoglobulin-like receptors (KIR). KIR may include KIR2D and/or KIR3D. KIR2D may include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5. KIR3D may include KIR3DL1, KIR3DL2, KIR3DL3 and/or KIR3DS1.

As disclosed herein, enhanced expression profiles of specific cellular markers for targeting immune cells (eg, CD57 or KIR for NK cells) can be determined by a variety of methods, including, but not limited to, Western blotting or PCR techniques.

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

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells can include one or both of the following: (i) heterologous transcription factors (e.g., heterologous STATs), (ii) endogenous cytokine signaling (e.g., endogenous IL signaling disclosed herein) Reduced activity, and (iii) reduced expression or activity of endogenous enzymes (eg, ligases, such as CBL-B). Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

Heterologous transcription factors may include at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factors. Heterologous transcription factors can include up to 5, 4, 3 or 2 different types of transcription factors. Alternatively, the heterologous transcription factor can have a single type of transcription factor. Transcription factors may be related to immune activity, proliferation, apoptosis, and/or differentiation of engineered immune cells. In some cases, the heterologous transcription factor of engineered immune cells (eg, engineered NK cells) can be STAT. Non-limiting examples of STATs may include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof. In one example, STAT can include STAT3. In another example, STAT can include STAT5B.

In some cases, compared to control cells without reduced activity of (i) heterologous transcription factors (e.g., heterologous STATs) and (ii) endogenous cytokine signaling (e.g., endogenous IL-17 signaling) , the engineered immune cells (eg, engineered NK cells) disclosed herein can exhibit enhanced survival in the presence of tumor cells. In some cases, the engineered immune cells can survive at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times longer than control cells in the presence of tumor cells. At least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least Or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or At most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most About 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). The engineered immune cells may exhibit reduced expression or activity of specific endogenous cell markers (eg, NK cell markers, such as KIR) for the directed immune cells disclosed herein compared to control cells. In some examples, the specific endogenous cell marker is KIR. The engineered immune cells may 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 as disclosed herein (e.g., heterologous IL, such as IL-15), (c) CD16 variants for enhanced CD16 signaling compared to control cells, wherein the CD16 variant is heterologous to engineered NK cells, as herein Disclosed; (d) an immunomodulatory polypeptide as disclosed herein, wherein the immunomodulatory polypeptide is heterologous to an engineered immune cell, and (e) reduced expression or activity of an endogenous immunomodulatory polypeptide as disclosed herein.

The engineered immune cells may comprise a chimeric polypeptide receptor and one or more (e.g., 1, 2, 3, or 4) of: (b) a heterologous cytokine, (c) for enhanced CD16 signaling CD16 variants, (d) heterologous immunomodulators, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

The engineered immune cells may comprise a heterologous cytokine and one or more (e.g., 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (c) for enhanced CD16 signaling CD16 variants, (d) heterologous immunomodulators, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

The engineered immune cells may comprise a CD16 variant for enhanced CD16 signaling and one or more (eg, 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (b) Reduced expression or activity of a heterologous cytokine, (d) a heterologous immunomodulator, and (e) an endogenous immunomodulatory polypeptide.

The engineered immune cells may comprise a heterologous immunomodulator and one or more (e.g., 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (b) a heterologous cytokine, ( c) CD16 variants for enhanced CD16 signaling, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

The engineered immune cells may comprise reduced expression or activity of an endogenous immunomodulatory polypeptide and one or more (e.g., 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (b) Allogeneic cytokines, (c) CD16 variants for enhanced CD16 signaling, and (d) allogeneic immunomodulators.

As disclosed herein, reduced expression or activity of specific endogenous cell markers for targeting immune cells (e.g., KIR for NK cells) can be determined by a variety of methods, including, but not limited to, Western blotting or PCR techniques .

In some cases, expression of a specific endogenous cell marker for directed immune cells (e.g., KIR for NK cells) in an engineered immune cell of the present disclosure may be at least, or at most, lower than its expression by a control cell About 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times , at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, At least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (eg, CD94, CD96, TGFβ receptor, SHIP2, etc.). In some cases, the engineered immune cells may exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGFβ receptor, and ( iv) Endogenous SHIP (eg SHIP2).

In some cases, the engineered immune cells may exhibit reduced expression or activity of endogenous CD94 and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: (ii) endogenous CD96, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some cases, the engineered immune cells may exhibit reduced expression or activity of endogenous CD96 and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: (i) Endogenous CD94, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some cases, the engineered immune cells may exhibit reduced expression or activity of endogenous TGFβ receptors and also exhibit reduced expression of one or more (eg, 1, 2, or all) of the following or Activity: (i) endogenous CD94, (ii) endogenous CD96 and (iv) endogenous SHIP (eg SHIP2).

In some cases, the engineered immune cells may exhibit reduced expression or activity of endogenous SHIP (eg, SHIP2) and also exhibit reduced expression of one or more (eg, 1, 2, or all) of the following Expression or activity of: (i) endogenous CD94, (ii) endogenous CD96 and (iii) endogenous TGFβ receptor.

In some cases, the reduced expression or activity of immune checkpoint inhibitors (e.g., CD94, CD96, TGFβ receptor, SHIP2, etc.) in engineered immune cells (e.g., engineered NK cells) of the present disclosure can be compared with control cells. Its expression is at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or At most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most About 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous CD94 in an engineered immune cell (eg, an engineered NK cell) of the present disclosure may be at least, or at most about 0.1-fold, at least or less than its expression by a control cell. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous CD96 in an engineered immune cell (eg, an engineered NK cell) of the present disclosure may be at least, or at most about 0.1-fold, at least or less than its expression by a control cell. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous TGFβ receptors in engineered immune cells (e.g., engineered NK cells) of the present disclosure may be at least or up to about 0.1-fold lower than its expression by control cells. At least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least Or up to about 0.9 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 4 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or At most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most About 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous SHIP (eg, SHIP2) in an engineered immune cell (eg, an engineered NK cell) of the present disclosure may be at least or at most about 0.1 lower than its expression by a control cell. times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times , at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, At least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least Or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or Up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some cases, the endogenous immunomodulatory polypeptide contains one or more hypoimmune modulators. In some cases, the engineered immune cells exhibit reduced expression or activity from one or more hypoimmune modulators of: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB or (vi) endogenous NKG2DL. Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, as disclosed herein, endogenous low-immunity modulators (e.g., CD80, CD86, The reduced expression or activity of ICOSL, CD40L, MICA, MICB, NKG2DL, etc.) may be at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least less than the expression thereof by control cells. Or up to about 0.4 times, at least or up to about 0.5 times, at least or up to about 0.6 times, at least or up to about 0.7 times, at least or up to about 0.8 times, at least or up to about 0.9 times, at least or up to about 1 times, at least or At most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most About 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous CD80 in engineered immune cells (e.g., engineered NK cells) of the present disclosure may be at least, or at most about 0.1-fold, at least or less than its expression by control cells. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous CD86 in an engineered immune cell (eg, an engineered NK cell) of the present disclosure may be at least, or at most, about 0.1-fold, at least, or less than its expression by a control cell. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous ICOSL in an engineered immune cell (eg, an engineered NK cell) of the present disclosure may be at least, or at most, about 0.1-fold, at least, or less than its expression by a control cell. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of the endogenous immunomodulatory modulator CD40L in engineered immune cells (e.g., engineered NK cells) of the present disclosure may be at least, or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times , at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, At least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least Or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times.

In some cases, the reduced expression or activity of endogenous MICA or MICB in an engineered immune cell (e.g., an engineered NK cell) of the present disclosure may be at least or up to about 0.1-fold lower than its expression by a control cell. At least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least Or up to about 0.9 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 4 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or At most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most About 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, the reduced expression or activity of endogenous NKG2DL in an engineered immune cell (e.g., an engineered NK cell) of the present disclosure may be at least, or at most about 0.1-fold, at least or less than its expression by a control cell. At most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most About 0.9 times, at least or at most about 1 time, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times , at least or at most about 5,000 times, or at least or at most about 10,000 times.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some cases, the endogenous immunomodulatory polypeptide includes a hypoimmune modulator (eg, ICAM1). The engineered immune cells 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., as disclosed herein) , heterologous IL, such as IL-15), and (c) CD16 variants for enhanced CD16 signaling compared to control cells.

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

In some cases, the engineered immune cells (e.g., engineered NK cells) comprise a heterologous cytokine disclosed herein (e.g., a heterologous IL, such as IL-15) and one or both of: (a) as disclosed herein Chimeric polypeptide receptors, and (c) CD16 variants for enhanced D16 signaling.

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

In some cases, as disclosed herein, reduced expression or activity of endogenous ICAM1 in engineered immune cells (e.g., engineered NK cells) of the present disclosure can be compared with control cells as determined by Western blot or PCT techniques. Its expression is at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or At most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most About 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some cases, the endogenous immunomodulatory polypeptide includes a hypoimmune modulator (eg, ICAM1). Engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, as disclosed herein, the reduced expression or activity of endogenous ICAMl in engineered immune cells (eg, engineered NK cells) of the present disclosure may be lower than its expression by control cells.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). The engineered immune cells can comprise an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some cases, immunomodulatory polypeptides include hypoimmune modulators. The hypoimmune modulator may be PDL2. Alternatively or additionally, the hypoimmune modulator may be TGF-β.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). The engineered immune cells can comprise an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some cases, immunomodulatory polypeptides include hypoimmune modulators. The hypoimmune modulator may comprise one or more members of: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous Source CD59. Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

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

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

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

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

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

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). Engineered immune cells may include heterologous cytokines other than IL-15 (eg, heterologous IL) as disclosed herein. In some examples, the allogeneic cytokine includes IL-21 or a variant thereof. Engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). As disclosed herein, the engineered immune cells can comprise a chimeric polypeptide receptor comprising an antigen-binding moiety capable of binding to an antigen. The antigen may be an antigen of a virus. In some cases, the virus includes one or more members selected from HIV, HBV, HCV, EBV, HPV, Lassa virus, influenza virus, coronavirus, and derivatives thereof. In some instances. The virus is not cytomegalovirus (CMV).

In some cases, an antigen of a virus may be a fragment of a viral protein of the virus presented by a target cell (eg, an immune cell). In some cases, viral infection induces host cell apoptosis as a mechanism for viral spread after the host cells have been used to replicate the virus. Therefore, without wishing to be bound by theory, targeting of viral antigens by engineered immune cells (e.g., engineered NK cells expressing CARs to viral antigens) as disclosed herein may allow engineered immune cells to interact with other virally infected cells. cells are in close proximity, thereby inducing cell death in such virus-infected cells.

In some cases, certain viruses may cause or increase the risk of certain types of cancer, and therefore engineered immune cells or uses thereof as disclosed herein may be used to prevent or treat such cancers. For example, Epstein-Barr virus (EBV) is a herpes virus that can be spread through saliva, and EBV can increase the risk of Burkitt lymphoma, some types of Hodgkin and non-Hodgkin lymphoma, and stomach cancer . Hepatitis B virus (HBV) can be transmitted through infected blood, semen and other body fluids, and HBV can increase the risk of liver cancer. Hepatitis C virus (HCV) can be spread through infected blood, and HCV can increase the risk of liver cancer and non-Hodgkin lymphoma. Human immunodeficiency virus (HIV) can be transmitted through infected semen, vaginal fluid, 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 cancer-causing viruses. Other oncogenic viruses cause cancer. HIV-related cancers include Kaposi's sarcoma, non-Hodgkin's lymphoma and Hodgkin's lymphoma, cervical cancer, and cancers of the anus, liver, mouth, throat and lungs. Human herpesvirus 8 (HHV-8) can increase the risk of Kaposi's sarcoma in people with weakened immune systems. Human papillomavirus (HPV) increases the risk of cancers in men and women, including anal, cervical, penile, throat, vaginal and vulvar cancers. Human T-cell leukemia virus type 1 (HTLV-1) can be transmitted through infected semen, vaginal fluid, blood, and breast milk and can increase the risk of T-cell leukemia/lymphoma in adults.

In some cases, viruses as disclosed herein can be DNA viruses or RNA viruses. For example, the virus may be a double-stranded DNA virus, a single-stranded DNA virus, a double-stranded RNA virus, a sense single-stranded RNA virus, an antisense single-stranded RNA virus, a single-stranded RNA retrovirus (retrovirus), or a double-stranded DNA reverse transcription virus. Virus. Examples of DNA viruses may include, but are not limited to, cytomegalovirus, herpes simplex virus, Epstein-Barr virus, simian virus 40, bovine papilloma virus, adeno-associated virus, adenovirus, vaccinia virus, and baculovirus. Examples of RNA viruses may include, but are not limited to, coronavirus, Semliki forest virus, Sindbis virus, Poko virus, rabies virus, influenza virus, SV5, respiratory syncytial virus, Venezuelan equine encephalitis virus, Kunjin virus, Sendai virus, vesicular virus Stomatitis viruses and retroviruses.

Various specific viruses include, but are not limited to, Papovaviridae, Adenoviridae, Herpesviridae, Herpesvirales, Ascoviridae, Bottlevirus Ampullaviridae, Asfarviridae, Baculoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hypertrophic sialadenitis Hytrosaviridae, Iridoviridae, Lipothrixviridae, Nimaviridae, Poxviridae, Tectiviridae, Corticoviridae , Sulfolobus, Caudovirales, Covering Phageidae, Multilayered Phages, Ligamenvirales, Vialoviridae, Bicaudaviridae, Rhabdoviridae (Clavaviridae), Parvoviridae, Spheroviridae, Trichoviridae, Turriviridae, Ascovirus, Baculovirus, Hypertrophiviridae, Iridoviridae, Polydnaviruses, Mimiviridae, Marseillevirus, Megavirus, Mavirus virophage, Sputnik virophage, Threadviridae, Algal DNAviridae (Phycodnaviridae), pleolipoviruses, Plasmaviridae, Pandoraviridae, Dinodnavirus, Rhizidiovirus, Salt end Salterprovirus, Sphaerolipoviridae, Anelloviridae, Bidnaviridae, Circoviridae, Geminiviridae, 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, fungal single negative strand RNA 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 type 1, monkey and Feline immunodeficiency virus, Spumavirus, simian foamy virus, Orthoretrovirinae, Spumaretrovirinae, Metaviridae, Pseudoviridae , Retroviridae, Hepadnaviridae and Caulimoviridae.

In one aspect, the present disclosure provides an engineered immune cell (eg, engineered NK cell). As disclosed herein, the engineered immune cells can comprise a chimeric polypeptide receptor comprising an antigen-binding moiety capable of binding to an antigen. In some cases, the antigen can be secreted by the target cell and bound to the surface of the target cell (eg, to the plasma membrane). In some cases, the antigen targeted by the chimeric polypeptide receptor may be an antibody produced by target cells (eg, B cells) of the subject (eg, a patient suffering from or suspected of having an autoimmune disease). Antibodies can be autoantibodies. In some examples, B cells can secrete antibodies and/or release the same antibodies against the object's own cells, thereby exhibiting local concentrations of antibodies in or adjacent to the B cells. Antibodies can be autoantibodies that target other cells of the same object to affect various diseases and/or debilitating conditions. Accordingly, by targeting and binding to such antibodies via the action of chimeric polypeptide receptors, the engineered immune cells disclosed herein can specifically target B cells (e.g., malignant or diseased B cells) to, for example, reduce or inhibit Activity of B cells in a subject or induction of B cell death. Without wishing to be bound by theory, administration of engineered immune cells (eg, engineered NK cells) to an object can target and inhibit or deplete the object's self-attacking B cells for the treatment of autoimmune diseases.

In some cases, the antigen-binding domain of the chimeric polypeptide receptor can be an antibody or variant thereof that is configured to bind to a target immune cell (e.g., a self-attacking B cell) secreted by or present in the target One or more antibodies on immune cells. In some cases, the antigen-binding domain of the chimeric polypeptide receptor may be an antigen (and therefore not an antibody) that is recognized and secreted by or present on the target immune cell (e.g., a self-attacking B cell) bound by the antibody. In either case, the antigen-binding portion of the chimeric polypeptide receptor may be referred to as the "antibody-binding portion."

In some cases, the antigen-binding domain (i.e., the antibody-binding domain) of the chimeric polypeptide receptor can be designed such that the antigen-binding domain does not induce any other biological effects on the object (eg, other cells of the object). For example, the antigen-binding domain may be a peptide sequence derived from a target protein of an autoantibody of the B cells of a subject suffering from 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 does not (i) exhibit any function of the original target protein or (e.g., interact with the object) Unintended interactions with other cells or proteins) (ii) Any biological effect on the article other than serving as a target for autoantibodies.

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. 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 amino acid residues, at least or at most about 11 amino acid residues, at least or at most about 12 amino acid residues, at least or at most about 13 amino acid residues, at least or at most about 14 amino acid residues, at least or at most about 15 amino acid residues 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 amino acid residues, at least or at most about 25 amino acid residues, at least or at most about 30 amino acid residues, at least or at most about 35 amino acid residues, at least or at most about 40 amino acid residues, at least or at most about 45 amino acid residues amino acid residues or at least or up to about 50 amino acid residues.

In some cases, the viral antigen may be at least a portion of a viral protein. Viral proteins can be surface proteins, glycoproteins, envelope proteins, membrane proteins, nucleocapsid proteins, gene polymerases, proteases, etc. For example, examples of viral glycoproteins include hemagglutinin 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 proteins Gp1-Gp2 (e.g., for Ebola virus), E dimer (e.g., for dengue virus), E1 and E2 (e.g., for chikungunya virus )wait.

In some cases, the antigen-binding domain or antigen-binding portion of a heterologous receptor (eg, a chimeric polypeptide receptor) may comprise an antibody or fragment thereof (eg, scFv) that exhibits specific binding to a viral antigen.

In some cases, the antigen-binding domain or antigen-binding portion of a heterologous receptor (e.g., a chimeric polypeptide receptor) can comprise at least a portion of a cellular protein that exhibits specific affinity for a target antigen (e.g., an antigen of a virus) . In some cases, the cellular proteins are not viral proteins. For example, cellular proteins are not derived from viral proteins. In some cases, the cellular protein is endogenous (eg, encoded by the cell's native genome). In some cases, cellular proteins are surface receptors. 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 variant thereof. In some cases, the surface receptor can be CXCR4, CCR5, G protein-coupled receptor, Niemann-Pick disease C1 receptor, integrin receptor (e.g., αvβ1, αvβ3, αvβ6, and αvβ8), sialic acid, immune Globulin superfamily receptor (eg, JAM-A) or any variant thereof.

In some cases, the control cells may be cells, which may be immune cells, such as NK cells, for comparison purposes. In some cases, the control cells may be cells that do not contain the heterologous cytokine (eg, IL-15). In some cases, the control cells may be cells that do not contain a CD16 variant for enhanced CD16 signaling. In some cases, the control cell may be a cell containing a chimeric polypeptide receptor that contains an antigen-binding portion capable of binding to the antigen. In some cases, the control cells may be cells containing heterologous IL-15R. In some cases, the control cells may be cells that do not contain a membrane-bound heterologous cytokine (eg, IL-15). In some cases, a control cell may be a cell that does not exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide. In some cases, a control cell may be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (eg, IL-17) or its receptor (eg, IL-17R). In some cases, control cells may be cells that do not contain heterologous transcription factors (eg, STATs). In some cases, control cells may be cells that do not exhibit reduced expression or activity of specific endogenous cell markers for directed immune cells (eg, NK cell markers, such as KIR). In some cases, the control cells may be cells that do not contain the heterologous immunomodulatory polypeptide. In some cases, the control cells may be cells that do not exhibit reduced expression or activity of one or more of: endogenous CD94, endogenous CD96, endogenous TGFβ receptor, or endogenous SHIP2. In some cases, a control cell may 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 may be a cell that does not exhibit reduced expression or activity of ICAM1. In some cases, the control cells may be cells that do not contain heterologous PDL2 or heterologous TGFβ. In some cases, the control cells may be cells that do not contain one or more of: allogeneic CCL21, allogeneic IL-10, allogeneic CD46, allogeneic CD55, or allogeneic CD59. In some cases, the control cells may be cells that do not contain heterologous IL-21. In some cases, control cells may be cells that are not derived from a cell line. In some cases, control cells may be cells that are not derived from isolated ESCs. In some cases, control cells may be cells that are not derived from iPSCs.

C. Other aspects of engineered immune cells

In some embodiments of any of the engineered immune cells disclosed herein (eg, engineered NK cells), the engineered immune cell can comprise a heterologous cytokine disclosed herein (eg, heterologous IL, such as IL-15). In some cases, the engineered immune cells (eg, engineered NK cells) comprise a heterologous receptor that is a corresponding receptor for a heterologous cytokine (eg, heterologous IL-15R), as disclosed herein. Accordingly, engineered immune cells (e.g., engineered NK cells) may exhibit induction by heterologous cytokines and/or heterologous receptors disclosed herein (e.g., induced by heterologous cytokines and/or heterologous receptors such as IL -15/IL-15R-induced) enhanced signaling of the intrinsic signaling pathway.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells may exhibit reduced expression or activity of endogenous CD38 compared to control cells. Such engineered immune cells can be used to treat subjects who have or are suspected of having leukocyte cancers such as multiple myeloma (MM).

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the expression or activity of endogenous CD38 of the engineered immune cell may not be modified and need not be modified. Such engineered immune cells can be used to treat subjects who have or are suspected of having diseases other than multiple myeloma (eg, cancer, tumors).

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise heterologous IL-15 or fragments thereof, and the heterologous IL-15 or fragments thereof can be composed of Engineered immune cell secretion.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise heterologous IL-15 or fragments thereof, and the heterologous IL-15 or fragments thereof can bind onto the cell surface membrane of engineered immune cells.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise at least one chimeric polypeptide receptor comprising an antigen-binding portion capable of binding to an antigen, such as provided in this disclosure. In some examples, engineered immune cells can contain multiple different chimeric polypeptide receptors to specifically bind multiple different antigens. In some examples, engineered immune cells can include at least one chimeric polypeptide receptor that includes multiple antigen-binding moieties to specifically bind multiple different antigens.

In some embodiments of any of the engineered immune cells disclosed herein (eg, engineered NK cells), the engineered immune cell can contain a safety switch capable of affecting the death of the engineered immune cell. Engineered immune cells can contain genes encoding safety switches (e.g., integrated into the genome of the immune cell) through the action of gene editing moieties, as disclosed herein. In some cases, if adverse events occur or when adaptive immunotherapy is no longer needed, prodrugs can be introduced into the engineered immune cells (e.g., administered to an article containing the engineered immune cells) and can be switched via a safety switch molecule to activate prodrugs to kill object immune cells. In some cases, the safety switch may include a caspase (eg, caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR, B cell CD20, and functional variants thereof one or more members of. In some cases, safety switches can be activated by initiators (e.g., small molecules or proteins, such as antibodies) for post-translational, temporal, and/or site-specific regulation of death (or exhaustion) of engineered immune cells. . Non-limiting examples of safety switches and their initiators may 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 additionally, modified epidermal growth factor receptor (EGFR) containing an epitope recognized by an antibody (e.g., anti-EGFR Ab, e.g., cetuximab) can be used to deplete engineering when the subject cells are exposed to the antibody. oxidize immune cells. In some cases, the engineered immune cells (e.g., engineered NK cells) disclosed herein may comprise caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR and the safety switch protein CD20.3 in B cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can comprise a heterologous immune receptor polypeptide. The immunomodulatory polypeptide may comprise one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells may exhibit reduced expression or activity of endogenous immunomodulatory polypeptides, as disclosed herein. Endogenous immunomodulatory polypeptides include immune checkpoint inhibitors or hypoimmune modulators (or both).

In some cases, the immune checkpoint inhibitors disclosed herein can include selected from PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGFβ receptor, and 2B4 one or more members. In some cases, the immune checkpoint inhibitor may include SHIP2.

In some cases, the hypoimmune modulators disclosed herein may comprise B2M, CIITA, TAP1, TAP2, tap-associated protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB , one or more members of ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can comprise a CD16 variant for enhanced CD16 signaling compared to control cells, Among them, the CD16 variant is heterologous to the engineered immune cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can exhibit enhanced cytotoxicity against target cells compared to control cells. In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can be effector cells. In some embodiments, the control cells are wild-type NK cells. In some cases, the control cells are immune cells (eg, NK cells) lacking one or more engineering aspects provided herein. In some embodiments, the target cell may present or express at least a portion of an antigen of the virus. In some embodiments, the target cells may present or express the virus. In some embodiments, the target cells may present or express viral proteins. In some embodiments, the enhanced cytotoxicity is at an 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 cells disclosed herein may exhibit cytotoxicity to a target cell or population of target cells (e.g., in vitro, ex vivo, or in vivo), as determined, for example, by tracking changes in the number of target cell populations. It is at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.5 times more cytotoxic than control cells. 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times , at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, At least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, as determined, for example, by tracking changes in the number of target cell populations (e.g., via fluorescence-initiated cell sorting or FACS) , compared to control cells, the engineered immune cells can perform better 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 Exhibits enhanced cytotoxicity against target cells in a shorter period of time.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can be induced from isolated immune cells (e.g., isolated T cells in vitro and /or a decrease in the immune response of B cells, or immune cells of the host after the engineered immune cells are administered to the host. In some cases, such as by, for example, measuring (i) changes in the number of an initial population of engineered immune cells following exposure to isolated immune cells, or (ii) changes in the number of isolated immune cells following exposure to an initial population of engineered immune cells. The engineered immune cells disclosed herein can reduce the immune response from the isolated immune cells by at least or up to about 0.1-fold compared to the immune response from the isolated immune cells when exposed to control cells, as determined by changes in cytokine release. , at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, At least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least Or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or At most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most About 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can be compared to control cells after exposure to isolated immune cells (e.g., isolated T cells in vitro). and/or B cells, or immune cells of the host) exhibit enhanced half-life when the engineered immune cells are administered to the host. In some cases, the half-life of engineered immune cells upon exposure to isolated immune cells (e.g., in vitro or in vivo) can be compared to The half-life of the control cells is at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least Or up to about 0.7 times, at least or up to about 0.8 times, at least or up to about 0.9 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or At most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most About 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve enhanced function or pathological conditions in body tissue compared to control cells. In some cases, treatment with engineered immune cells can achieve at least or at most about 0.1-fold, at least or at most about 0.2-fold enhancement in the function or pathological condition of the body tissue of the object compared to control cells or vehicles (i.e., no cells) , at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, At least or at most about 1 time, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least Or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or At most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most About 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve delayed degradation of the function or pathological condition of the body tissue compared to control cells. In some cases, treatment with engineered immune cells can achieve a delay in degradation of a functional or pathological condition of body tissue of an object by at least or at most about 0.1-fold, at least or at most about 0.1-fold compared to control cells or vehicles (i.e., no cells). 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times , at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, At least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least Or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the body tissue can include selected from the group consisting of blood, plasma, serum, urine, perilymph, feces, saliva, semen, amniotic fluid, Cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomitus, bones, heart, thymus, arteries, blood vessels, lungs, muscles, stomach, intestines, liver, pancreas, spleen, kidneys, gallbladder, thyroid, adrenal glands, One or more members of the breast, ovary, prostate, testicle, skin, fat, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can induce an immune response against a target cell. The target may be, for example, diseased cells, cancer cells, tumor cells, etc.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the heterologous gene can be operably associated with constitutive, inducible, temporal, tissue-specific, and/or cellular Type-specific promoter coupling (e.g., knock-in). Non-limiting examples of promoters of interest may include CMV, EFla, PGK, CAG, and UBC. Non-limiting examples of insertion sites may include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tap-related protein, 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 of the engineered immune cells (e.g., engineered NK cells) disclosed herein, may exhibit reduced expression or activity of one or more of the following endogenous genes to enhance the performance of the engineered immune cells in the tumor microenvironment (i.e., tumor microenvironment genes or "TME"). In some cases, reduced expression or activity of TME can enhance the immune activity of engineered immune cells against target cells. In some cases, TME genes can be immune checkpoint inhibitors. Non-limiting examples of TMEs may include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGFβ receptor, 2B4, and SHIP2.

In some embodiments of any of the engineered immune cells disclosed herein (e.g., engineered NK cells), one or more heterologous genes (e.g., knock-in) may be expressed for enhanced function, e.g.: CD137, CD80, CD86 , DAP10 (e.g., with or without point mutations).

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

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more heterologous genes (e.g., knock-ins) may be expressed for, e.g., hypoimmunity: HLA-E, CD47 , CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments, any of the engineered immune cells disclosed herein (eg, engineered NK cells) may express one or more heterologous genes (eg, knock-in): CD3, CD4, CD80, 41BBL, and CD131.

D. chimeric antigen receptor

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

The chimeric polypeptide receptor may comprise 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 the TCR extracellular domain, (iii) at least a portion of the TCR transmembrane domain, and (iv) at least a portion of the TCR intracellular domain.

In some cases, the endogenous T cell receptor (TCR) of engineered immune cells (eg, engineered NK cells) of the present disclosure can be inactivated. In some examples, the function of the endogenous TCR of engineered immune cells can be inhibited by inhibitors. In some examples, genes encoding subunits of the endogenous TCR can be inactivated (eg, edited by the action of gene editing moieties as disclosed herein) such that the endogenous TCR is inactivated. The gene encoding an endogenous TCR subunit may be one or more of the following: TCRα, TCRβ, CD3ε, CD3δ, CD3γ, and CD3ζ.

Chimeric polypeptide receptors may include chimeric antigen receptors (CARs). 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 functional signaling structure derived from a stimulatory molecule The cytoplasmic signaling domain of the domain (also referred to herein as the "intracellular or intrinsic signaling domain"). In some cases, the stimulatory molecule may be a zeta chain associated with a T cell receptor complex. In some cases, the intracellular signaling domain also includes one or more functional signaling domains derived from at least one costimulatory molecule. In some cases, costimulatory molecules may include 4-1BB (i.e., CD137), CD27, and/or CD28. In one aspect, the CAR includes an optional leader sequence at the amino terminus (N-terminus) 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 derived from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. cutting.

The 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 specific for a particular antigen (e.g., an antibody or an antigen-binding fragment thereof, such as a scFv, a Fab fragment, a VHH domain, or the VH of a heavy chain antibody only) domain), a transmembrane domain derived from a self-adjusting immune receptor (e.g., a transmembrane domain of a CD28 receptor), and a signaling domain derived from a self-adjusting immune receptor (e.g., one or more (e.g., Three) ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ). Second-generation CARs are developed by adding co-stimulatory receptors (e.g., derived from co-stimulatory receptors that interact with T cell receptors such as CD28, CD137/4-1BB, and CD134/OX40) to the intracellular signaling domain portion of the CAR. The stimulatory domain is used to modify first-generation CARs, which eliminates the need for cofactors (such as IL-2) to be administered with first-generation CARs. Third-generation CARs add multiple costimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB). Fourth-generation CARs work by adding priming cytokines (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more costimulatory domains and the CD3ζ ITAM domain). ) or modify a second or third generation CAR under the control of a CAR-inducible promoter (e.g., NFAT/IL-2 minimal promoter). In some cases, the CAR may be a new generation CAR system that is different from the first, second, third or fourth generation CAR systems disclosed herein.

The hinge domain (e.g., the linker between the extracellular antigen-binding domain and the transmembrane domain) of the CAR in engineered immune cells (e.g., engineered NK cells) disclosed herein may comprise 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 , the full length or at least a portion of the native or modified transmembrane region of a T cell receptor polypeptide.

The transmembrane domain of the CAR in engineered immune cells (eg, engineered NK cells) disclosed herein may comprise 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 The entire length or at least a portion of the native or modified transmembrane region of a cell receptor polypeptide.

The hinge domain and transmembrane domain of the CARs disclosed herein (eg, for engineering immune cells, such as engineered NK cells) can be derived from the same protein (eg, CD8). Alternatively, the hinge domain and transmembrane domain of the CAR disclosed herein can be derived from different proteins.

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

The signaling domain (e.g., the signaling peptide of the intracellular signaling domain) of the CAR in the engineered immune cells (e.g., engineered NK cells) disclosed herein may comprise CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB ), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D or any combination thereof.

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

In some cases, the engineered immune cells (eg, engineered NK cells) disclosed herein comprise a chimeric polypeptide receptor (eg, CAR) comprising at least the CD8 transmembrane domain and one or more of: Two: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some cases, the engineered cells (e.g., engineered NK cells) disclosed herein comprise a chimeric polypeptide receptor (e.g., TFP or CAR) comprising at least (i) a CD8 transmembrane domain, (ii) 2B4 signaling domain and (iii) DAP10 signaling domain. The 2B4 signaling domain can be flanked by a CD8 transmembrane domain and a DAP10 signaling domain. Alternatively, the DAP10 signaling domain can be flanked by a CD8 transmembrane domain and a 2B4 signaling domain. In some cases, the chimeric polypeptide receptors disclosed herein can further comprise additional signaling domains derived from CD3ζ.

The antigen (ie, target antigen) of the antigen-binding portion of the chimeric polypeptide receptor (eg, TFP or CAR) disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.

Non-limiting examples of antigens (i.e., target antigens) of the antigen-binding portion of chimeric polypeptide receptors (e.g., TFP or CAR) disclosed herein may include ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, oncinoembryonic 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, antigens from cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP 2), epithelial glycoprotein 40 (EGP-40), epithelial cell adhesion molecule ( EpCAM), EGFRvIII, receptor tyrosine protein kinase 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), kappa light chain, kinase domain receptor (KDR), kappa, Lewis A (CA19.9), Lewis Y (LeY), L1 cells Adhesion molecule (L1-CAM), LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1), MICA/B, mucin 1 (Muc-1), mucin 16 (Muc-16), interstitial Cortisol (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, carcinoembryonic 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 antigens (eg, those derived from viruses, bacteria, fungi, parasites, and protozoa capable of causing disease). In some examples, the pathogen antigen is derived from HIV, HBV, HCV, EBV, HPV, Lassa virus, influenza virus, or coronavirus.

Other examples of antigens for the antigen-binding portion of chimeric polypeptide receptors as disclosed herein may include: 1-40-beta-amyloid, 4-1BB, 5AC, 5T4, promoter 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-lymph Tumor cells, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eosinophil chemotactic factor-1), CCR4 , CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD25 (IL-2 receptor alpha chain), CD27, CD274, CD28, CD3, CD3ε, 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, agglutination factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus Glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli Shigella toxin type 1, E. coli Shigella toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, E. coli, respiratory syncytium Viral F protein, FAP, fibrin II beta chain, fibronectin extra domain B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptors, ganglioside GD2, GD2, GD3 ganglioside Lipids, glypican 3, GMCSF receptor alpha 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 amyloid beta, 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 α7β7, integrin αIIbβ3, integrin αvβ3, interferon α/β receptor, gamma interferon-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), neuronal apoptosis-regulating proteinase 1, NGF, N-hydroxyacetylneuraminic acid, NOGO-A, Notch receptor, NRP1, acupoint Rabbit (Oryctolagus cuniculus), OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, sodium phosphate cotransporter, phospholipid serine, platelet-derived growth factor receptor beta, prostate cancer cells, pseudomonas aeruginosa cystis, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine 1-phosphate, gold Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, tenascin C, TFPI, TGF-β1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA 16.88, tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

Other examples of antigens for the antigen-binding portion of the chimeric polypeptide receptors disclosed herein may include: 707-AP, biotinylated molecules, 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, carcinoembryonic antigen (h5T4 ), OS-9, P peptide, P15, P53, PRAME, PSA, PCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survival Protein (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.

Other examples of antigens of the antigen-binding portions of chimeric polypeptide receptors as disclosed herein may include antibodies, fragments thereof, or variants thereof. Such antibodies may be natural antibodies (eg, naturally secreted by the immune cells of the object (eg, B cells)), synthetic antibodies, or modified antibodies. In some cases, the antigen of the antigen-binding portion of the chimeric polypeptide receptor disclosed herein may comprise an Fc domain of an antibody from the group consisting of: 20-(74)-(74) (milatizumab (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab), 8H9, A33, AB -16B5, abavolumab, abciximab, abituzumab, zlintuzumab), actoxumab, adalimumab, ADC-1013 , ADCT-301, ADCT-402, adelimumab, aducanumab, afitumumab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F , AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, atimumab valerate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, Leisin -Anetumab Ravtansine, anifrolumab, anrukinzumab, APN301, APN311, avolizumab, APX003/SIM-BD0801 (sevacizumab), APX005M, acitumomab, ARX788, ascrinvacumab, asezolizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also known as tocilizumab), atomumab, avelumab, B-701, bapineuzumab, basiliximab, baviliximab, BAY1129980, BAY1187982 , betumolumab, begelomab, belimumab, benralizumab, becilizumab, besilosomab, betalutin (177Lu-tetraxetan -tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biximumab, bimagrumab, bimekizumab, mobivacizumab (bivatuzumab mertansine), BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, Vibu brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab (canakinumab), cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, carolizumab pentidetide, carolizumab ( carlumab), catumaxomab (catumaxomab), CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cilizumab, ceto Citatuzumab, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, Citatuzumab bogatox, Totumumab, clazakizumab, cliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine , conatumumab, concizumab, Cotara (iodine I-131, delotuzumab biotin), cR6261, crizumab, DA-3111 (trastuzumab Biosimilars), daclizumab, daclizumab, daratumumab, dapirolizumab pegol, daratumumab, daratumumab ( Daratumumab Enhanze, daratumumab), Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuximab, depatuxizumab (depatuxizumab mafodotin), derlotuximab biotin (derlotuximabbiotin), Detumomab, DI-B4, denutuximab, delitavumab, DKN-01, DMOT4039A, atotuzumab, trazituzumab, DS-1123, DS-8895, Dugo Tocilizumab, dupilumab, durvalumab, duseltuzumab, emeleximab, eculizumab, ebalizumab, edelizumab, efalizumab, elfin Columab, edilutumab, egmantumumab, elotuzumab, exituzumab, imotuzumab, emituzumab, inatuzumab, fumatin, entuximab, enotuzumab, enokizumab, enoticumab, entuximab, etoluzumab cetuximab, ipilizumab, erlizumab, ertuzumab, daclizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, frantuzumab, ff Latuximab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulatuzumab, flutuximab, galiximab, ganizumab, gantezumab, plus vitumomab Anti, gemtuzumab, 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, etalizumab, tisartan, ivolumab, idelizumab, IGN002, IGN523, igovumab, IMAB362, IMAB362 (claudiximab), imalumab , IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 ipilumab), IMMU-114, ImmuTune IMP701 antagonist antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin , infliximab, intumomab, intetumumab, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumab, ituximab, istelolumab, atetuzumab monoclonal antibody, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, Lebozumab monoclonal antibody, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivirumab, lolotumumab methacholine, lucalumab, Lulizumab, lumelumab, lutuzumab, 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, miratuzumab, miratuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK -4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, mogamulizumab, motuzumab, motuximab, moromonab Monoclonal Antibodies-CD3, tacolatox, narcolumab, namilumab, estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, novolumab, nolfitatumumab , NOV-10, obinutuximab, obinumab, ocalizumab, oculizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, entuzumab Anti-, entuximab, opimumab, octuximab, oregovomab, octrexumab, octuximab, octretuzumab, OX002/MEN1309, octuximab, ozanib Tilizumab, oxalizumab, pakizumab, palivizumab, panitumumab, panolizumab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, pateclizumab anti, PAT-SC1, PAT-SM6, pembrolizumab, pembrolizumab, palatizumab, pertuzumab, pectizumab, PF-05082566 (utomilumab), PF-06647263, PF- 06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, prituximab, prituzumab, PRO 140, Proxinium, PSMA ADC, ranibizumab monoclonal antibody, ractomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilomab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovizumab, 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, silotuzumab monoclonal antibody, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (fortuximab and motuximab), Sym015, TAB08, tabatumumab, takizumab, tetrahydromethadone, takizumab, talizumab, tanizumab, talizumab, taplitumomab paptox, tarextumab, TB-403 , tefibazumab, Teleukin, telimomab aritox, tenatumumab, teneximab, tepiluzumab, tepilumab, tecilumab, tetumolumab TG-1303, TGN1412, Thorium -227-Epratuzumab conjugate, tinitizumab, tigaltuzumab, tiralizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosartuzumab, Tositumumab, tocilumab, trastuzumab, trastuzumab, trastuzumab amidanshinine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042 , TTI-200.7, trastuzumab celmoleukin, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustenitumab, vadatuximab talilin, vandotux VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, Zalutumumab, zanotumumab, zatuximab, ziptumomab, and zolimumab.

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

In some cases, the engineered immune cells (e.g., engineered NK cells) disclosed herein can comprise a chimeric polypeptide receptor (e.g., TFP or CAR) that contains an antigen-binding protein capable of specifically binding to a target cell antigen. domain, and the engineered immune cells may exhibit reduced expression or activity of the endogenous gene encoding the same antigen of the chimeric polypeptide receptor. Thus, populations of engineered immune cells can avoid targeting and killing each other, for example, when administered to an object in need.

In some cases, the engineered immune cells (e.g., engineered NK cells) disclosed herein can comprise a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds The domain may be capable of binding specifically and preferentially to CD38. In some cases, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38. In some cases, engineered immune cells containing chimeric peptide receptors for CD38 may be able to target and effect death (or degradation) of plasma cells.

In some cases, the engineered immune cells (e.g., engineered NK cells) disclosed herein can comprise a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds The domain may be capable of binding specifically and preferentially to CD38. In some examples, the engineered immune cells are engineered NK cells derived from isolated ESCs or induced stem cells (eg, iPSCs). In some cases, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38.

E. stem cells

Any of the engineered immune cells (eg, engineered NK cells) disclosed herein can be derived from isolated stem cells (eg, ESCs) or induced stem cells (iPSCs). Isolated stem cells or induced stem cells can be modified (eg, genetically modified) to produce engineered immune cells.

In some cases, the pluripotency of stem cells (eg, ESCs or iPSCs) can be determined in part by assessing the pluripotency characteristics of the cells. Pluripotency characteristics may include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential; (iii) 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 expression of CD50; (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of teratomas composed of all three somatic lineages; Formation of embryoid bodies composed of cells of three somatic lineages.

In some cases, stem cells (eg, ESCs or iPSCs) can be genetically modified to produce (eg, induced to differentiate into) CD34+ hematopoietic stem cells. Stem cells can be genetically modified to express any of the heterologous polypeptides disclosed herein (eg, cytokines, receptors, etc.) before, after, or during induced hematopoietic stem cell differentiation. Stem cells can be genetically modified before, after, or during induced hematopoietic stem cell differentiation to reduce the expression or activity of any of the endogenous genes or polypeptides (eg, cytokines, receptors, etc.) as disclosed herein. In some cases, such genetically modified CD34+ hematopoietic stem cells are or are derived from any of the engineered immune cells of the present disclosure.

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

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

In some examples, in 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 Genetically modified CD34+ hematopoietic stem cells are cultured in the presence of Flt3L (e.g., about 20 ng/mL) and Flt3L (e.g., about 10 ng/mL) to differentiate into CD45+ NK cells.

In some cases, CD45+ NK cells can be expanded in culture using the gas permeable rapid expansion (G-Rex) platform, for example, in media containing IL-2, mbIL-21 aAPC.

In some cases, iPSC-derived NK cells disclosed herein can be cultured with one or more heterologous cytokines including II-2, IL-15, or IL-21. In some cases, iPSC-derived NK cells disclosed herein can be cultured with one or more heterologous cytokines selected from II-2, IL-15, and IL-21 (eg, for cell expansion). In some cases, iPSC-derived NK cells disclosed herein can be combined with two or more heterologous cytokines selected from 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) are cultured together simultaneously or sequentially in any order. In some cases, iPSC-derived NK cells disclosed herein can be cultured with all of II-2, IL-15, and IL-21 simultaneously or sequentially in any order.

F. Gene editing or genetic material delivery

The gene editing part disclosed herein may include CRISPR-related polypeptides (Cas), zinc finger nucleases (ZFN), zinc finger-related gene regulatory polypeptides, transcription initiation factor-like effector nucleases (TALENs), transcription initiation factor-like effector-related genes Regulatory polypeptides, meganucleases, natural master transcription factors, epigenetic modification enzymes, recombinases, flippases, transposases, RNA binding proteins (RBPs), Argonaute proteins, any derivatives thereof, any variants thereof, or Any fragment. In some embodiments, the actuator moiety includes a Cas protein and the system further includes a guide RNA (gRNA) complexed with the Cas protein. In some embodiments, the actuator moiety contains an RBP complexed with the gRNA, which is capable of forming a complex with the Cas protein. In some embodiments, the gRNA comprises a targeting fragment that exhibits at least 80% sequence identity to the target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

In some cases, suitable gene editing moieties include 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, and type III CRISPR-associated (Cas) polypeptides. (Cas) peptide, type IV CRISPR-associated (Cas) peptide, type V CRISPR-associated (Cas) peptide, and type VI CRISPR-associated (Cas) peptide; zinc finger nuclease (ZFN); transcription initiation factor-like effector nuclease (TALEN) ); meganuclease; RNA-binding protein (RBP); CRISPR-related RNA-binding protein; recombinase; flippase; transposase; Argonaute (Ago) proteins (such as 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 forms thereof.

In some cases, the gene editing moiety disclosed herein can be fused with an additional functional moiety (e.g., to form a fusion moiety), and non-limiting examples of the functions of the additional functional moiety can include: methyltransferase activity, demethylation Base enzyme activity, dismutase activity, alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, unwinding Enzyme activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenosyllation Activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, Hydrolase activity, lyase activity, isomerase activity, synthase activity, synthase activity and demyristation activity. For example, the fusion protein can be a fusion of a Cas protein and an effector or repressor functional portion.

Alternatively or additionally, gene editing (eg knock-in) or delivery of heterologous genetic material may be achieved. Other viral and non-viral based gene transfer methods can be used to introduce nucleic acids into host cells (eg, 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 into a host organism). Viral vector delivery systems can include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells. Non-viral vector delivery systems may include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes.

Systems based on RNA or DNA viruses can be used to target specific cells and deliver the viral payload to the cell's nucleus. Viral vectors can be used to treat cells in vitro, and modified cells can optionally be administered (ex vivo). Alternatively, the viral vector can be administered directly (in vivo) to the object. Virus-based systems may include retroviral, lentiviral, adenoviral, adeno-associated virus, and herpes simplex virus vectors for gene transfer. Utilizing retroviral, lentiviral, and adeno-associated viral gene transfer methods, integration into the host genome can occur, which may result in long-term expression of the inserted transgene.

Non-viral delivery methods of nucleic acids may include lipofection, nucleofection, microinjection, gene guns, virions, liposomes, immunoliposomes, polycationic or lipid-nucleic acid conjugates, naked DNA, artificial virions, and Enhanced absorption of DNA reagents. Both cationic and neutral lipids of lipofection can be recognized using efficient receptors suitable for polynucleotides.

Alternatively or additionally, antisense oligonucleotides can be used to inhibit or silence target gene expression. Non-limiting examples of antisense oligonucleotides may include short hairpin RNA (shRNA), microRNA (miRNA), and small interfering RNA (siRNA).

G. combination therapy

The engineered immune cells (eg, engineered NK cells) of the present disclosure can be combined with combination therapeutic agents to treat an object in need thereof. In some cases, the engineered immune cells can be administered to the article before, simultaneously with, or after the combination therapeutic agent is administered to the article.

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

In some cases, the engineered immune cells may comprise a heterologous cytokine disclosed herein (e.g., IL-15) and one or both of: (ii) a CD16 variant for enhanced CD16 signaling, and (iii) ) A chimeric polypeptide receptor comprising an antigen-binding portion.

In some cases, the engineered immune cells may comprise a CD16 variant for enhanced CD16 signaling and one or both of: (i) a heterologous cytokine (e.g., IL-15), and (iii) Antigen-binding portion of chimeric peptide receptors.

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

Non-limiting examples of combination therapeutic agents may include: cytotoxic agents, chemotherapeutic agents, growth inhibitors, agents for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer. , for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., pembrolizumab), platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (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, TRAIL/Apo2, other biologically active agents, and organic chemicals. ).

The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. Non-limiting examples of cytotoxic agents may include: radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu), chemotherapeutic agents (e.g., methotrexate , doxorubicin), vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or others 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 chemotherapeutic agents may include: alkylating agents, such as thiotiguat and CYTOXAN® cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and guapsulfan; nitrogen Propidines, such as benzodopa, carboquinone, meteredopa and uredopa; ethyleneimines and methylmelamines, including hexammelamine, triethylenemelamine, triethylenephosphatamide, triethylene Ethylthiophosphoramide and trimethylolmelamine; Acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan , CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; sponge polyketenes; CC-1065 including its adolesin, caszelasin, and bizoletin New synthetic analogues); podophyllotoxin; podophylphyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; becarmycin (including synthetic analogs substances, KW-2189 and CB1-TM1); argyrosin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide , estramustine, ifosfamide, nitrogen mustard, nitrogen mustard hydrochloride, melphalan, nitrogen mustard, mustard cholesterol, prednimustine, trophosphamide, uracil mustard; nitrite Ureas such as carmustine, chlorotrimethrin, fomustine, lomustine, nimustine and ramustine; antibiotics such as enediyne antibiotics; dynemicin, Including danithromycin A; esperamycin; and new tumor suppressor protein chromophores and related pigment proteins enediyne antibiotic chromophores), aclacinomysin, actinomycin, and Aspergillus Authramycin, diazoserine, bleomycin, actinomycin C, carabicin, carcinogen, carcinogen, chromomycin, actinomycin D, daunorubicin, ditomycin Bixin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholine-doxorubicin, 2-pyrrolino- Doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, masiromycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin , olivinemycin, pelomycin, potfiromycin (potfiromycin), puromycin, triferric doxorubicin, rhodobicin, streptozotocin, streptozotocin, tuberculin, ebonylin Mesac, zistin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, methotrexate, pteropterin, and trimetrexate Sand; purine analogs such as fludarabine, 6-mercaptopurine, thioimidine, thioguanine; pyrimidine analogs such as amcitabine, azacitidine, 6-azuridine, carmofur, arabinosporine Glycosides, dideoxyuridine, deoxyfluridine, enocitabine, floxuridine; androgens such as caprotestosterone, drostandrosterone propionate, cyclothiandrostenol, meandrosteane, testolactone; anti- Adrenal hormones such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folic acid; acetyl acetone; aminophosphatide glycosides; aminoacetate; enyluracil; dapsic acid; acetaminophen Mustine; bisantrene; edatraxate; deoxyamine; demecosin; diazinon; efafronate; elaplatin acetate; epothilone; etaglucoside; gallium nitrate; hydroxyl Urea; lentinan; lonidainine; maytansinoid alkaloids such as maytansine and antomycin; mitoguanone; mitoxantrone; mopantenol; nitramide; pentostatin; hematoxylin; pirarupin Bisin; Oxanthrone; 2-Ethylhydrazine; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); Propyrimidine; Rhizopuscin; Sizoran; Spiral Germanium; Bovine Sulfonic acids; triazinones; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, rhodamine A, and guanidine); urea; vincaine Depsin (ELDISINE®, FILDESIN®); dacarbazine; manlutin; midopanool; mitonideol; tepobromide; cytosine; arabinoside (“Ara-C”); thioteguat; Taxoids, such as taxanes, including TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™, an albumin-engineered nanoparticle formulation of paclitaxel without Cremophor (American Pharmaceutical Partners, Schaumberg , Ill.) and TAXOTERE® docetaxel (Rhône-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analog Drugs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin ; Chlorophyll; Vinorelbine®; Norvizone; Idatrexed; Daunorubicin; Aminopterin; Ibandronic acid; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); retinoids such as tretinoin; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above such as CHOP ( Cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin) . Additional chemotherapeutic agents include cytotoxic agents used as antibody drug conjugates, such as maytansinoid alkaloids (eg DMl) and the auristatins such as MMAE and MMAF.

Examples of chemotherapeutic agents may also include "antihormonal agents" or "endocrine therapeutic agents," which act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are typically in a systemic form, or systemic therapy . They may be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4- Hydroxytamoxifen, trovoxifene, raloxifene, LY117018, onapristone, and FARESTON® toremifene; antiprogestins; estrogen receptor downregulators (ERDs); have the ability to suppress or shut down ovarian function drugs such as luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD leuprolide acetate, goserelin acetate, buserelin acetate and triptorelin; 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 4(5)-imidazole, aminoglutethimide , MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fazhidazole, RIVISOR® vorozole, FEMARA® letrozole and ARIMIDEX® anastrozole. Additionally, the definition of this chemotherapeutic agent includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate phosphonates, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; and trosatabine (1,3-dioxola alkyl nucleoside cytosine analogues); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in mucosal cell proliferation, such as PKC-α, Raf, H-Ras and epidermal growth factor receptor (EGFR); vaccines, such as THERATOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; xylene Lapatinib sulfonate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Examples of chemotherapeutic agents may also include antibodies, such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab ( Bexxar, Corixia) and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Other humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: avolizumab, asezolizumab, atlizumab, bapizumab, bivaltuzumab, mesartan, Cantumumab mesartan, cedilizumab, certolizumab (certolizumab pegol), cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab (efalizumab), epratizumab monoclonal antibody, ercilizumab, feMzumab, fentuzumab, gemtuzumab ozogamicin, intuzumab, ipilimumab, labezumab, lintuzumab anti, matuzumab, mepolizumab, movitizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, pa Rilizumab, pacolizumab, pecfusituzumab, pectuzumab, pecilizumab, ralivizumab, ranibizumab, risulizumab, risulizumab, rovizumab, rupuzumab, Silotolizumab, ciprilizumab, sertolizumab, takizumab tetraracetam, tadolizumab, talizumab, tilefibizumab, tocilizumab Anti-, tocilizumab, trastuzumab, interleukin-12, trastuzumab, umavizumab, ucilizumab, velcilizumab, and anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), a recombinant full-length IgG1λ antibody genetically modified to recognize interleukin 12 p40 protein specifically for human sequences.

Examples of chemotherapeutic agents may also include "tyrosine kinase inhibitors," such as EGFR targeting agents (e.g., small molecules, antibodies, etc.); small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724, 714, oral selective inhibitors of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits HER2 and Cells overexpressing both EGFR; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, e.g. Carnetinib (CI-1033; Pharmacia); Raf-1 inhibitors, such as ISIS-5132, an antisense agent that inhibits Raf-1 signaling, available from ISIS Pharmaceuticals; non-HER-targeted TK inhibitors, such as Imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-target tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors Agents, 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; pyridinepyrimidine; pyrimidopyrimidine; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[ 2,3-d]pyrimidine; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing nitrothiophene moiety Acid; PD-0183805 (Warner-Lamber); antisense molecules (e.g., molecules that bind to HER-encoding nucleic acids); quinoxaline (U.S. Patent No. 5,804,396); tryphostin (U.S. Patent 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 (GLEEVEC®); 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 Rapa Mycins (sirolimus, RAPAMUNE®).

Examples of chemotherapeutic agents may also include: dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurine Alcohol, amifostine, arsenic trioxide, aspartase, BCG activity, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, alfa Faibetin, erlotinib, filgrastim, histrelin acetate, itumomab, interferon alpha-2a, interferon alpha-2b, lenalidomide, levamisole, mesna , methoxsalen, nandrolone, narabebine, norfilumab, opreleukin, pamidronate, pegase, pegaspargase, pegfilgrastim, pemetrexed disodium , plucamycin, porphyrom sodium, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, retinoic acid, ATRA, valrubicin, zoledronic acid salts and zoledronic acid and its pharmaceutically acceptable salts.

Examples of chemotherapeutic agents may also include hydrocortisone, hydrocortisone acetate, cortisone acetate, hydrocortisone pivalate, triamcinolone acetonide, triamcinolone acetonide, mometasone, amcinonide, budesonide Acetonide, desonide, fluocinolone acetonide, fluocinonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocorterone, hydrocortisone 17-butyrate, hydrogenated Cortisone 17 valerate, beclomethasone propionate, betamethasone valerate, betamethasone propionate, prednisolate, clobetasol 17-butyrate, clobetasol 17-propionate esters, flubutolone caproate, flubutolone pivalate, and flubutylene acetate: immunoselective anti-inflammatory peptides (ImSAID), such as phenylalanine-glutamine-glycine (FEG ) and its D-isomer (feG) (IMULAN BioTherapeutics, LLC); antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, Leflunomide minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA) ®), certolizumab polyethylene glycol (CIMZIA®), golimumab (SIMPONI®), interleukin 1 (IL-1) blockers such as anakinra (KINERET®), T cell costimulation blockers such as abatacept (ORENCIA®), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers, such as rotalizumab; beta7 integrin blockers, such as rhuMAb beta7; IgE pathway blockers, such as anti-M1 elicitors; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa/β2 blockers, such as antilymphotoxin alpha (LTa); other investigational reagents, such as thioplatinum, PS-341, phenyl butyrate, ET-18-OCH3, or Farmagen Transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, picrol, epigallocatechin gallate, theaflavins, flavanols , proanthocyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapadone; lapachol; colchicine; birch acids; acetylcamptothecin, scopolectin and 9-aminocamptothecin); podophyllotoxins; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (e.g. BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate salts (AREDIA®), tilodronate (SKELID®) or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE® vaccine; fenfos, COX -2 inhibitors (such as celecoxib or etoricoxib), proteosome inhibitors (such as PS341); CCI-779; tipamib (R11577); olafenib, ABT510; Bcl-2 inhibitors, For example, orimethane sodium (GENASENSE®); pixantrone; farnesyl transferase inhibitors, such as lonafamib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and two of the above one or more combinations.

The term "growth inhibitory agent" generally refers to a compound or composition that inhibits the growth and/or proliferation of cells (eg, cells whose growth depends on expression of PD-L1) in vitro or in vivo. The growth inhibitor can be one that significantly reduces the percentage of cells in S phase. Non-limiting examples of growth inhibitors include agents that block cell cycle progression (outside of S phase), such as agents that induce G1 arrest and M phase arrest. Classic M phase blockers include vinca (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-lyxopyranosyl-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6, 8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthylenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that block G1 also spill over into S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and Cytarabine (Ara-C). Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from taxane. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer) is derived from European yew and is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly of tubulin dimers and stabilize microtubules by preventing depolymerization, which results in inhibition of mitosis in cells.

H. Instructions

Engineered immune cells (eg, engineered NK cells) of the present disclosure can be used (eg, administered) to treat an object in need thereof. The object may suffer or may be suspected of suffering from a condition such as a disease (eg, cancer, tumor, tissue degeneration, fibrosis, etc.). Cells can be obtained from an object (eg, stem cells or committed adult cells), and such cells can be cultured ex vivo and genetically modified to produce any of the object engineered immune cells (eg, any engineered NK cells) disclosed herein. The engineered immune cells can then be administered to the object for adaptive immunotherapy.

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 can be used. An object is treated (eg, administered) with 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 of a population of engineered immune cells (eg, engineered NK cells) of the present disclosure.

A composition (eg, unit dosage form) comprising engineered immune cells as provided herein can be administered to an article. Can be at least or at most about 1 × 10 ⁴ cells per kilogram of body weight (cells/kg), at least or at most about 2 × 10 ⁴ cells/kg, at least or at most about 3 × 10 ⁴ cells/kg, at least or at most About 4×10 ⁴ cells/kg, at least or at most about 5×10 ⁴ cells/kg, at least or at most about 6×10 ⁴ cells/kg, at least or at most about 7×10 ⁴ cells/kg, at least Or at most about 8×10 ⁴ cells/kg, at least or at most about 9×10 ⁴ cells/kg, at least or at most about 1×10 ⁵ cells/kg, at least or at most about 2×10 ⁵ cells/kg , at least or at most about 3×10 ⁵ cells/kg, at least or at most about 4×10 ⁵ cells/kg, at least or at most about 5×10 ⁵ cells/kg, at least or at most about 6×10 ⁵ cells /kg, at least or at most about 7×10 ⁵ cells/kg, at least or at most about 8×10 ⁵ cells/kg, at least or at most about 9×10 ⁵ cells/kg, at least or at most about 1×10 ⁶ cells/kg, at least or at most about 2×10 ⁶ cells/kg, at least or at most about 3×10 ⁶ cells/kg, at least or at most about 4×10 ⁶ cells/kg, at least or at most about 5× 10 ⁶ cells/kg, at least or at most about 6×10 ⁶ cells/kg, at least or at most about 7×10 ⁶ cells/kg, at least or at most about 8×10 6 cells/kg, at least or at most about 7× ^{10 6} cells/kg 9×10 ⁶ cells/kg, at least or at most about 1×10 ⁷ cells/kg, at least or at most about 2×10 ⁷ cells/kg, at least or at most about 3×10 ⁷ cells/kg, at least or At most about 4×10 ⁷ cells/kg, at least or at most about 5×10 ⁷ cells/kg, at least or at most about 6×10 ⁷ cells/kg, at least or at most about 7×10 ⁷ cells/kg, At least or at most about 8×10 ⁷ cells/kg, at least or at most about 9×10 ⁷ cells/kg, at least or at most about 1×10 ⁸ cells/kg, at least or at most about 2×10 ⁸ cells/kg kg, at least or at most about 3×10 ⁸ cells/kg, at least or at most about 4×10 ⁸ cells/kg, at least or at most about 5×10 ⁸ cells/kg, at least or at most about 6×10 ⁸ cells/kg cells/kg, at least or at most about 7×10 ⁸ cells/kg, at least or at most about 8×10 ⁸ cells/kg, at least or at most about 9×10 ⁸ cells/kg, at least or at most about 1×10 8 cells/kg ⁹ cells/kg, at least or at most about 2×10 ⁹ cells/kg, at least or at most about 3×10 ⁹ cells/kg, at least or at most about 4×10 ⁹ cells/kg, at least or at most about 5 ×10 ⁹ cells/kg, at least or at most about 6 × 10 ⁹ cells/kg, at least or at most about 7 × 10 ⁹ cells/kg, at least or at most about 8 × 10 ⁹ cells/kg, at least or at most About 9×10 ⁹ cells/kg, at least or at most about 1×10 ¹⁰ cells/kg, at least or at most about 2×10 ¹⁰ cells/kg, at least or at most about 3×10 ¹⁰ cells/kg, at least Or at most about 4×10 ¹⁰ cells/kg, at least or at most about 5×10 ¹⁰ cells/kg, at least or at most about 6×10 ¹⁰ cells/kg, at least or at most about 7×10 ¹⁰ cells/kg , at least or at most about 8×10 ¹⁰ cells/kg, at least or at most about 9×10 ¹⁰ cells/kg, or at least or at most about 1×10 ¹¹ cells/kg at a total concentration or dose of administering the engineered immunity to the object cells. The dose of engineered immune cells provided herein can be administered to the article in a single unit dosage form or in multiple unit dosage forms (eg, at least 2, at least 3, at least 4, or at least 5 unit dosage forms).

In some cases, the engineered immunity provided herein may be administered to the article by a route selected from the group consisting of subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intravenous administration, intranasal administration, intralymphatic injection, and oral administration. composition of cells.

In one aspect, the present disclosure provides a method comprising: (a) obtaining a cell from a subject; and (b) producing from the cell any of the engineered immune cells disclosed herein (eg, engineered NK cells). In some cases, the cells obtained from the object are ESCs. In some cases, cells obtained from an object (eg, fibroblasts, such as adult skin fibroblasts) are modified and converted into iPSCs.

In one aspect, the present disclosure provides a method comprising administering to an object in need thereof a population of NK cells comprising any of the engineered immune cells (eg, engineered NK cells) disclosed herein. In some cases, the method may further comprise administering to the object a combination therapeutic agent (eg, a chemotherapeutic agent, an anti-CD20 antibody, etc.).

In one aspect, the present disclosure provides a method comprising applying any of the compositions disclosed herein to an object in need thereof. In some cases, a composition can comprise (i) any one of the engineered immune cells (eg, engineered NK cells) as disclosed herein, and (ii) a combination therapeutic agent (eg, chemotherapeutic agents, anti-CD20 antibodies, etc.).

Any of the methods disclosed herein may be used to treat target cells, target tissues, target conditions, or target diseases of an object.

Target diseases may be viral, bacterial, and/or parasitic infections; inflammatory and/or autoimmune diseases; or neoplasms, such as cancers and/or tumors.

The target cells may be diseased cells. Diseased cells may have altered metabolic, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. Diseased cells may be cells from a diseased object. Exemplary diseases may include hematological disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, etc.

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

Target cells can be stem cells or progenitor cells. Target cells may include stem cells (eg, adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (eg, cardiac progenitor cells, neural progenitor cells, etc.). Target cells may include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, and the like. Clonal cells can contain the cells' descendants. Target cells can contain target nucleic acids. The target cells can be in living organisms. The target cells can be genetically modified cells. The target cell can be a host cell.

The target cells may be totipotent stem cells, however, in some embodiments of the present disclosure, the term "cell" may be used but may not refer to totipotent stem cells. The target cell may be a plant cell, but in some embodiments of the present disclosure, the term "cell" may be used but may not refer to a plant cell. Target cells can be pluripotent cells. For example, the target cell may be a multipotent 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 cells. The target cells may be able to develop into an entire organism. The target cells may or may not be able to develop into the entire organism. Target cells can be whole organisms.

Target cells can be primary cells. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15, or more times. Cells can be single-celled organisms. Cells can be grown in culture.

The target cells may be diseased cells. Diseased cells may have altered metabolic, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. Diseased cells may be cells from a diseased object. Exemplary diseases may include hematological disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, etc.

If the target cells are primary cells, they can be harvested from the individual by any method. For example, leukocytes can be harvested by apheresis, leukapheresis, density gradient separation, and the like. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Harvested cells can be dispersed or suspended using appropriate solutions. Such solutions may typically be balanced salt solutions (e.g., physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal calf serum or other naturally occurring factors and low concentrations of acceptable Buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately, or they can be stored (eg by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, medium buffer (eg 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 that may be target cells include, but are not limited to: lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells Cells, cytokine-induced killer (CIK) cells (see e.g. US20080241194 for details); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils/superfine neutrophils) ), monocytes/macrophages, red blood cells (reticulocytes), mast cells, platelets/megakaryocytes, dendritic cells; cells from the endocrine system, including the thyroid (thyroid epithelial cells, parafollicular cells), thyroid Cells of the parathyroid glands (parathyroid chief cells, eosinophils), adrenal glands (chromaffin cells), and pineal glands (pineal cells); cells of the nervous system, including glial cells (astrocytes, micronerves) glial cells), magnocellular neurosecretory cells, stellate cells, Boettcher cells, and pituitary gland (gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, prolactinocytes); cells of the respiratory system, including lung cells (Type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including the stomach (gastric chief cells, parietal cells) , goblet cells, Panett cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells) , cartilage/bone/muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); chondrocytes, including chondrocytes, chondrocytes; skin cells, including trophic cells Cells (Trichocytes), keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, intraglomerular mesangial cells/extraglomerular kidney Mesangial cells, proximal renal tubule brush border cells, macula densa cells; reproductive system cells, including sperm, supporting cells, Leydig cells, eggs; and other cells, including adipocytes, fibroblasts, tenocytes, and epidermis Keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of fingernails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, epidermal hair stem cells, epidermal hair root sheath cells, Hairy root sheath cells of Huxley layer, hairy root sheath cells of Henle layer, outer hairy root sheath cells, hair matrix cells (stem cells), moist layer barrier epithelial cells, (cornea, tongue, oral cavity, esophagus, anal canal, distal urethra Surface epithelial cells of the stratified squamous epithelium (of the distal end and vagina), basal cells (stem cells) of the epithelium (of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina), urothelial cells (lining the bladder and within the urethra), exocrine secretory epithelial cells, salivary gland mucous cells (polysaccharide-rich secretions), salivary gland serous cells (glycoprotein-rich secretions), Von Ebner gland cells of the tongue (taste bud wash), mammary gland cells ( Milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland clear cells (small molecule secretion), apical Sudomotor cells (odorous secretions, sensitive to sex hormones), Moll cell glands in the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebaceous secretions), Bowman's gland cells in the nasal cavity (olfactory epithelial wash) , Brunner gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secret semen components, including fructose for sperm swimming), prostate cells (secret semen components), bulbourethral gland cells (mucus secretions), Bartholin gland cells ( Vaginal lubricant secretions), Littre gland cells (mucus secretions), endometrial cells (carbohydrate secretions), isolated goblet cells of the respiratory and digestive tracts (mucus secretions), gastric mucosal cells (mucus secretions) , gastric gland enzyme-producing cells (pepsinogen secretion), gastric gland enzyme secretion cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), small intestinal Panet cells (lysozyme secretion), Type II pneumocytes of the lung (surfactant secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, somatotroph cells, prolactin cells, thyrotropin cells, gonadotrophic cells, corticotroph cells , intermediate pituitary cells, large cell neurosecretory cells, intestinal and respiratory cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells , testicular Ley dig cells, ovarian theca cells, luteal cells of ruptured follicles, granulosa cells, theca luteal cells, juxtamloglomerular cells (renin secretion), renal macula densa cells, metabolic and storage cells, barrier Functional cells (lungs, intestines, exocrine glands and genitourinary tract), kidneys, type I pneumocytes (air spaces lining the lungs), pancreatic duct cells (cardiac cells), non-striated ductal cells (sweat glands, salivary glands, mammary glands, etc. ), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining the closed lumen, ciliated cells with propulsive function, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and Immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bones), dendritic cells ( Lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, blood and immune system (various types) Suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitor cells, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory Detector cells, autonomic neuron cells, sensory organ and peripheral neuron support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia/ Oocytes, sperm cells, spermatocytes, spermatogonia (spermocyte stem cells), spermatozoa, trophic cells, follicle cells, supporting cells (in the testes), thymic epithelial cells, Leydig cells and interstitial kidney cells.

Of particular concern are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include cancer cells including: acanthoma, acinar cell carcinoma, acoustic neuroma, acral melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia , acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, ameloma, adenocarcinoma, adenoid cystic Carcinoma, adenoma, odontogenic adenomatoid tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastoma fibroma, anal cancer , anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratoid rhabdomyomas, basal cell Carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini's duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, cloth Renner's tumor, bronchial neoplasm, bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Giant lymph node hyperplasia, embryonal tumors of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, enchondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphoma Cellular 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, cutaneous sarcoma protuberans, dermoid cyst, proliferative small round cell tumor, diffuse large B-cell lymphoma, embryonal proliferative neuroepithelial tumor, embryonal carcinoma, intimal sinus tumor, uterus Endometrial cancer, endometrium uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroid leukemia, esophageal cancer, nasal glia tumor, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, Paget's disease of the breast, fallopian tube cancer, parasitic fetus, fibroma, fibrosarcoma , follicular lymphoma, follicular thyroid carcinoma, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gastrointestinal Tract stromal tumor, germ cell tumor, genital tumor, choriocarcinoma of pregnancy, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, glioma of the brain, glomus tumor, glucemia Tumor, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart disease, hemangioblastoma, hemangioendothelioma, angiosarcoma, hematological malignancies, hepatocellular carcinoma , hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, pancreatic islets cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatkin's tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, nevus maligna melanoma neoplasm, 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 rhabdomyomas, malignant salamanderoma, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinum Germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult Primary metastatic squamous carcinoma, metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, oral cancer, mucinous neoplasms, multiple endocrine neoplasms, multiple myeloma, multiple myeloma , mycosis, mycosis, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, tumor, neuroma, Neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumors, Oligoastrocytoma, oligodendrogliomas, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, epithelial ovarian cancer, ovary Germ cell tumors, ovarian tumors of low malignant potential, Paget's disease of the breast, prostate tumors, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paranasal glioma, paranasal sinus cancer, parathyroid Adenocarcinoma, penile carcinoma, hemangioepitheloid cell tumor, pharyngeal carcinoma, pheochromocytoma, moderately differentiated spinal cord parenchyma, fibroblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, polyblastoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary Peritoneal cancer, primary neuroectodermal tumor, prostate cancer, Pseudomonas peritonei, rectal cancer, renal cell carcinoma, respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma , Richter transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary tumors, seminomas, serous tumors, Sertoli-Leydig cell tumors, sex cord stromal tumors, Cezalay syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small bowel cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumors , spinal cord tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T Large-cell lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic cancer, thymoma, thyroid Carcinoma, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urethral cancer, urethral cancer, genitourinary tract tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulva carcinoma, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a population of cancer cells, such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, such as lymphoma. The antigen may be a tumor-associated antigen.

In some cases, target cells (eg, B cells) disclosed herein are associated or suspected of being associated with an autoimmune disease. A subject treated with any of the engineered immune cells (eg, engineered NK cells) of the present disclosure may have, or may be suspected of having, an autoimmune disease.

Non-limiting examples of autoimmune diseases may include: acute diffuse encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, anaphylaxis Rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant 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 and neuronal neuropathy, Barrot's disease, Behcet's disease, bullous pemphigoid, Cardiomyopathy, giant lymphadenopathy, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal myelitis (CRMO), Churg-Strauss Syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, idiopathic mixed Cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dresler syndrome, endometriosis disease, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Pulmonary hemorrhage-nephritis syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Balinese syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypoglobulin anaemia, hyperglobulinaemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing diseases, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus ( Type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen planus sclerosis, xylem 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), Mullen's ulcer, Mucha -Habermann's disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism , PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome disease, cycloplanitis (peripheral uveitis), pemphigus, peripheral neuropathy, venous encephalomyelitis, pernicious anemia, POEMS syndrome, polyinflammatory nodosa, autologous types I, II, and III Immune polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis , psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red blood cell dysplasia, Raynaud's 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 and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susak syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse spinal cord ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatosis, vitiligo, Waldenström's macroglobulinemia (WM), and Wegener's granulomatosis (granulomas with multiple Vasculitis (GPA)).

In some cases, the autoimmune disease includes one or more members selected from the group consisting of: rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiplex syndrome Sclerosis, scleroderma, Addison's disease, bullous pemphigus, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypercytosis Proteinemia, 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 transplant rejection (e.g., for tissue transplants, such as kidney transplants). In examples, the autoimmune disease may be type 1 diabetes, lupus, or rheumatoid arthritis.

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

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 of the engineered immune cells (e.g., engineered NK cells) disclosed herein can be administered to an object in need to treat BCL, the engineered immune cells comprising one or more of the following: (i) as described herein Disclosed are chimeric polypeptide receptors comprising an antigen-binding domain capable of binding CD19, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) as disclosed herein for use in CD16 variants with enhanced CD16 signaling.

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

In some cases, the target cells form tumors (i.e., solid tumors). Tumors treated with the methods herein may result in stable tumor growth (e.g., one or more tumors that increase in size by no more than 1%, 5%, 10%, 15%, or 20%, and/or do not metastasize). In some cases, the tumors remain stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some cases, the tumors remain stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some cases, the tumors remain stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some cases, the size of the 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, tumors are completely eliminated or reduced below detection levels. In some cases, the subject remains tumor-free (eg, in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after treatment. In some cases, the object remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after treatment. In some cases, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after treatment. [Example]

Example 1 : Engineering NK Cell:

Table 1 shows examples of engineered NK cells with or without genetic modifications, along with possible functions and therapeutic indications. Examples of treatment indications may include acute myeloid leukemia (AML), multiple myeloma (MM), myelodysplastic syndrome (MDS), B-cell leukemias, T-cell leukemias, solid tumors, and blood cancers. Table 1 No. genetic modification Function Treatment indications 1 without Unengineered sNK AML,MM,MDS 2 aCD19-CAR + IL15 + hnCD16 Target specificity + persistence + ADCC B-cell leukemia +/- anti-CD20 3 aCD19-CAR + IL15 + hnCD16 + i-caspase 9 Target specificity + persistence + ADCC + safety switch B-cell leukemia +/- anti-CD20 4 hnCD16+IL15 ADCC + persistence Solid tumors + anti-PD-1 5 hnCD16-CD64 + IL15 ADCC + persistence Solid tumors + anti-PD-1 6 hnCD16 + IL15 + CD38-KO ADCC + persistence Multiple myeloma-/+ anti-CD38 7 aBCMA-CAR+ hnCD16 + IL15 + CD38-KO Target specificity + persistence + ADCC Multiple myeloma-/+ anti-CD38 8 CAR + IL15 + hnCD16 + TME gene Target specificity + persistence + ADCC + enhanced activity Blood cancer, solid tumors 9 CAR + IL15 + hnCD16 + TME gene + low immunity gene (for re-dosing) Target specificity + persistence + ADCC + enhanced activity + low immunity (for re-dosing) Blood cancer, solid tumors 10 CAR + IL15 + hnCD16 + TME gene + low immunity gene (for re-dosing) + i-caspase 9 Target specificity + persistence + ADCC + enhanced activity + low immunity (re-dosing) + safety switch Blood cancer, solid tumors

Example 2 : Engineered resistance - antibody NK cells

Engineering NK Cell:

To improve adaptive immunotherapy, NK cells can be engineered to express chimeric receptor polypeptides (e.g., TFP or CAR) that contain autoantibodies that engage immune cells (e.g., B cells) The binding extracellular domain. The subject may have, or may be suspected of having, an autoimmune disease indicated by increased self-attack activity of B cells. Thus, a chimeric receptor polypeptide can comprise (i) an antibody that binds an autoantibody or (ii) an antigen for an autoantibody, thereby allowing engineered NK cells to find B cells, target B cells, and induce B cell death.

Goodpasture Syndrome:

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

Pemphigus:

In another example, the subject has or is suspected of having pemphigus, and the subject's B cells express autoantibodies to a cellular structural protein, such as desmoglein. Thus, engineered NK cells (e.g., from HSCs, ESCs, or iPSCs) are generated to express chimeric receptor polypeptides with antigens derived from targeting cellular structural proteins (e.g., desmoglein) Binding domain (i.e., antibody binding domain). A population of such engineered NK cells is administered to an object as a treatment for Goodpasture syndrome.

Example 3 : enhanced CD16 Signaling

To improve adaptive immunotherapy, NK cells can be engineered to exhibit enhanced CD16 signaling.

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

MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCET KLLLQRPGLQLYFSFYMGSKTLRGRNTSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWF HV SFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

Engineering NK Cell:

NK-92 cells are engineered to exhibit enhanced CD16 signaling. Engineered NK-92 cells are modified to express CD64/CD16A fusion protein (ie, hnCD16) (SEQ ID NO. 1).

The resulting hnCD16 NK-92 was 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 primed cell sorting (FACS) cells, as shown in Figure 1A. Wild-type (WT) NK-92 cells were used as control.

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

Enhanced targeting:

hnCD16 NK-92 cells were cultured alone (unstimulated, control) or in the presence of K562 cells capable of priming NK cells (K562) or phorbol 12-myristate 13-acetate (PMA) to turn on CD16 and induce its cleavage. Data show that hnCD16 NK-92 cells are highly resistant to priming-induced CD16a cleavage compared with peripheral blood (PB) NK cells as controls (Fig. 1B). For example, for hnCD16 NK92 cells, treatment with PMA slightly reduced the percentage of CD16+ cells from 92% to 85%, while the same treatment reduced the percentage of CD16+ cells from 96% to 25% (Figure 1B). The persistence of hnCD16 in hnCD16 NK-92 cells was also confirmed by using anti-CD64 antibody (Fig. 1C). Additionally, it was observed that hnCD16 NK-92 cells did not downregulate endogenous CD16 expression when stimulated (e.g., K652 or PMA) (Figures 1D and 1E).

Enhanced antibody combination therapy

Target cells (Raji cells) were treated with (i) hnCD16 NK-92 cells and (ii) anti-CD20 antibody or hIgG as control. Data show that compared with controls (e.g., wild-type NK-92 cells with anti-CD20 antibody, or hnCD16 NK92 cells with hIgG), hnCD16 NK-92 cells combined with anti-CD20 antibodies induce increased death of target cells ( For example, death mediated at least in part by ADCC) (Figure 1F and 1G).

Example 4 : NK Enhanced cell survival and persistence

To improve the durability of adaptive immunotherapy, NK cells can be engineered to contain at least (i) heterologous transcription factors (e.g., STATs) and (ii) endogenous cytokine receptors (e.g., endogenous IL receptors, such as IL- Reduced expression or activity of 17R). Having the combination of (i) and (ii) in engineered NK cells can synergistically improve the persistence of engineered NK cells compared to having only one or neither of (i) and (ii).

Engineering NK Cell:

NK cells are generated from isolated ESCs or iPSCs. NK cells are engineered to express heterologous STATs (eg, STAT3 and/or STAT5B). Genes encoding heterologous STATs are incorporated into the genome of NK cells by viral transduction or by the action of gene editing moieties disclosed herein. NK cells have also been engineered to exhibit reduced expression or activity of endogenous IL-17R (ie, STAT3 ⁺ IL-17R ^- NK cells). NK cells with reduced expression or activity of one of (i) heterologous STAT and (ii) IL-17R or unengineered NK cells were used as controls.

In vitro survival and persistence:

Engineered STAT3 ⁺ IL-17R ^- NK cells can be cultured in vitro in the absence of exogenous cytokines to assess the viability and growth (or proliferation ability) of engineered STAT3 ⁺ IL-17R ^- NK cells. NK cells were cultured in culture medium without the addition of exogenous cytokines for 3-6 weeks. Engineered STAT3 ⁺ IL-17R ^- NK cells exhibited significantly higher NK cell numbers compared with control cells, indicating enhanced survival and persistence of engineered STAT3 ⁺ IL-17R ^- NK cells in vitro.

In vivo pharmacokinetics (PK) :

Engineered STAT3 ⁺ IL-17R ^- NK cells can be administered in NCG mice with 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 accommodate xenograft models. Engineered STAT3 ⁺ IL-17R ⁻ NK cells and control cells were administered to respective Raji xenograft model mice via intravenous (IV) tail vein injection, at a dose of approximately 1 × ¹⁰ cells per animal. From approximately 7 days to approximately 28 days after infusion, mice injected with engineered STAT3 ⁺ IL-17R ^- NK cells showed higher NK cell concentrations in peripheral blood, indicating that engineered STAT3 ⁺ IL-17R ^- NK cells Enhanced in vivo survival and persistence.

Example 5 : Engineered resistance CD19NK cells

NK-92 cells were engineered to express anti-CD19 CAR and then cultured in the presence of CD19+ Raji cells to evaluate the targeting of Raji cells by the engineered anti-CD19 NK cells. Wild-type (WT) NK-92 cells were used as control. Anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against Raji cells (as determined by reduced numbers of viable Raji cells) compared to controls (Figures 2A and 2B). Furthermore, when cultured in the presence of Raji cells, anti-CD19 CAR NK cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) compared to controls (Figures 2C and 2D). Furthermore, when cultured in the presence of Raji cells, anti-CD19 CAR NK cells exhibited enhanced cytokine production (e.g., IFN-γ and/or TNF-α production) compared to controls (Figures 2E-2G).

Example 6 ：engineered hIL15NK cells

Engineering NK Cell:

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 fusion-1 or "fus1") is designed to have a linker between hIL-15 and hIL15R, the linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8 or more repeats) "GGGGS" (SEQ ID NO. 9), such as "GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS" (SEQ ID NO. 10). The second variant (i.e., hIL15-IL15Ra fusion-2 or "fus2") is designed to have a linker between hIL-15 and hIL15R, the linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8 or more repeats) "GGGGS" (SEQ ID NO. 9) and one or more (for example, at least 1, 2, 3, 4, 5, 6, 7, 8 or more repeats) "EGKSSGSGSESKST" (SEQ ID NO. 11), such as "EGKSSGSGSESKSTEGKSSSGSGSESKSTGGGGS" (SEQ ID NO. 12). NK-92 cells knocking in any of the hIL-15-hIL15R fusion polypeptide variants were positive for hIL-15 (Fig. 3A).

Additionally, engineered NK-92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling compared to control NK-92 cells engineered to express a secreted form of IL-15 Exhibits longer persistence. Western blot analysis showed that compared with secretory IL-15 (IL15), in NK-92 cells expressing hIL15-IL15Ra fusion-1 (fus1) or hIL15-IL15Ra fusion-2 (fus2), IL-15-stimulated Phosphorylation of STAT5 was increased (Fig. 3B).

hIL15-IL15Ra fusion-1 sequence (SEQ ID NO.13): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS GGGGSGGGGSGGGGSGGGGSGGGG SGGGGS GITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEALPVTWGTSS RDEDLENCSHHL

hIL15-IL15Ra fusion -2 sequence (SEQ ID NO.14) : MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS EGKSSGSGSESKSTEGKSSGS GSESKSTGGGGS GITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPV TWGTSSRDEDLENCSHHL

Example 7 : Engineered antiviral antigens ( For example, against -EBV antigen ) CAR-NK cells

NK cells can be engineered to express heterologous receptors that exhibit specific binding to viral antigens, such as at least a portion of a viral protein. For example, the heterologous receptor may be a chimeric antigen receptor (CAR) containing an antigen-binding domain (or antigen-binding portion) directed to a viral antigen. The antigen-binding domain may comprise at least a portion of an antibody that exhibits specific binding to a viral antigen (e.g., a human Fab that specifically recognizes a polypeptide in the extramembranous domain of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) At least a portion of a fragment (eg, a scFv derived from a HLEA Fab)) or derived therefrom. Alternatively or additionally, the antigen-binding domain may comprise at least a portion of a natural complementary molecule of a senescence marker (eg, at least a portion of the intracellular TNFR-associated factor (TRAF) to target LMP1 as an EBV viral antigen) or be composed of its derivatives.

Engineering NK Cell:

As shown in Figure 4A, NK cells (eg, NK-92 cells) are engineered to target viral (eg, EBV) gene products (eg, LMP1). As shown in Figure 4A, the anti-EBV CAR structure can comprise an anti-EBV scFv (e.g., derived Anti-LMP1 scFv from HLEA-Fab). Anti-EBV CAR structures can also include 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 domain (ICD) (e.g., 4-1BB ICD, CD3ζ ICD).

CD8a leader (SEQ ID NO. 15): MALPPVTALLLPLALLLHAARP

VH (SEQ ID NO. 16): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLRMNSLRAEDTAVYYCARAWGYSSYYFDYWGQGTLVTVSP

Linker (SEQ ID NO. 17): GGGGSGGGGSGGGGS

VL (SEQ ID NO. 18): ELQMTQSPSSSLSASVGDRVAITCRASQSISSHLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK

CD8a hinge (SEQ ID NO. 19): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

CD8a TM (SEQ ID NO. 20): DIYIWAPLAGTCGVLLLSLVITLYC

CD28 TM (SEQ ID NO. 21): FWVLVVVGGVLACYSLLVTVAFIIFWV

CD28 cos (SEQ ID NO. 22): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

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

CD3ζ ICD (SEQ ID NO. 24): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

A plasmid with an anti-EBV CAR construct (e.g., anti-LMP1 CAR or LMP1-CAR, as used interchangeably herein) is packaged into a lentivirus and used to transduce NK cells (e.g., NK92 cells). As shown in Figure 4B (see CAR1 and CAR2), FACS analysis showed that NK cells transduced with the anti-LMP1 CAR construct (LMP1-CAR NK92 cells) expressed LMP-1 CAR on the surface of the NK cells.

Engineering NK cells target EBV positive (EBV+) In vitro killing of target cells:

Target cells labeled with carboxyfluorescein succinimidyl ester (CSFE) (e.g., Nalm6 cells, B95.8, Lymphoblastoid cell line (LCL)) with engineered NK cells (e.g., LMP1-CAR NK92 cells). Nalm6 cells were used as a control cell population that did not contain EBV viral antigens, while B95.8 and LCL cells were used as EBV-expressing or producing cells. After co-culture (eg, 6 hours of co-culture), the cytotoxicity of LMP1-CAR NK cells against target cells is measured via flow cytometry. As shown in Figure 5B, LMP-1 CAR NK cells exhibited stronger cytotoxicity against B95.8 cells than wild-type NK cells (NK92 wild type (WT)). After co-culture (e.g., 6 hours) at an E:T ratio of 20, LMP1-CAR NK cells exhibit at least about 10% greater cytotoxicity against B95.8 target cells than wild-type NK cells (e.g., as killed by as measured by the percentage of dead target cells). Conversely, the difference in cytotoxicity between LMP1-CAR NK cells and wild-type NK cells against Nalm6 cells is less than about 10% (e.g., less than about 6%), indicating viral antigen targeting to target cells that induce the expression or presence of viral antigens (e.g., B95.8 cells) death plays a role. Additionally, as assessed by flow cytometry, there was no significant difference in the response between wild-type NK cells (Nk92 WT) and LMP1-targeted NK cells (LMP1-CAR NK92) with LMP1-expressing target cells (e.g., LCL, B95.8) and After co-culture at a 1:1 ratio (e.g., 5 days of co-culture), almost all target cells were killed, while target cells survived in the absence of LMP1-targeting NK cells (see Figure 5C).

Without wishing to be bound by theory, 5 days of co-culture of wild-type NK cells (NK92-wt) and target cells may be long enough for wild-type NK to induce cytotoxicity in the absence of anti-LMP1 CAR (see Figure 5C). Without wishing to be bound by theory, when anti-LMP1 CAR-expressing NK cells are co-cultured with 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.) , such NK cells may exhibit higher cytotoxicity to target cells (e.g., LCL, B95) than wild-type NK cells.

Example 8 : Engineered antiviral antigens ( For example, against -HIV antigen ) CAR-NK cells

Engineering NK Cell:

NK cells can be engineered to express heterologous receptors that exhibit specific binding to viral antigens, such as at least a portion of a viral protein. For example, the heterologous receptor may be a chimeric antigen receptor (CAR) containing an antigen-binding domain (or antigen-binding portion) directed to a viral antigen. The antigen-binding domain may comprise or be derived from at least a portion of an antibody that exhibits specific binding to a viral antigen, such as a viral glycoprotein (eg, at least a portion of a human anti-HIB-1 gp120 recombinant antibody, such as a VRC clone). Alternatively or additionally, the antigen-binding domain may comprise or be derived from at least a portion of a natural complementary molecule of the viral protein (eg, at least a portion of CD4 to target gp120 as an HIV viral antigen).

Engineering NK Cell:

As shown in Figure 6A, NK cells (eg, NK-92 cells) are engineered to target viral (eg, HIV) proteins (eg, gp120). As shown in Figure 6A, anti-HIV CAR constructs can contain CD4 extracellular (EC) fragments or anti-gp120 scFv, both of which specifically target gp120. A CAR may also include 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 an intracellular domain (ICD) (e.g., 4-1BB ICD, CD3ζ ICD).

CD8a leader (SEQ ID NO. 15): MALPPVTALLLPLALLLHAARP

CD8a hinge (SEQ ID NO. 19): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

CD8a TM (SEQ ID NO. 20): DIYIWAPLAGTCGVLLLSLVITLYC

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

CD3ζ ICD (SEQ ID NO. 24): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD4 EC (SEQ ID NO. 25): KVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAF QKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMMWQCLLSDSGQVLLESNIKVLPTWSTPVQP

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

Validation of engineered NK cells. As shown in Figure 6B, FACS analysis showed that CD4 EC expression was higher in engineered NK92 cells (CD4 CAR NK92) compared with control wild-type NK cells (e.g., NK92, non-engineered NK cells), indicating that CD4 CAR expression. As shown in Figure 6C, compared with control wild-type NK cells (e.g., NK92, non-engineered NK cells), 4-1BB mRNA levels were significantly higher in engineered NK cells (e.g., CD4 CAR NK92, anti-gp120 scFv CAR- NK92), indicating expression of CD4 CAR or anti-gp120 scFv CAR. Furthermore, CD4 CAR NK92 with anti-CD4 (e.g., 10ug/ml) demonstrated higher NK than CD4-CAR NK92 alone (8.33%) as assessed by plate-immobilized antigen stimulation assay and flow cytometry Surface expression of the initiation marker CD69 (50.09%) (Fig. 6D). Control NK cells (anti-SS1 CAR NK), which did not show specific binding to gp120, treated with PMA and ionomycin (P/I) were used as positive controls.

Engineering NK cells target HIV positive (HIV+) In vitro killing of target cells:

Addition of engineered NK cells to target cells labeled with carboxyfluorescein succinimide ester (CSFE) at various effector to target cell (E:T) ratios (e.g., 5, 10, 15, 20, 25) (e.g., CD4 CAR NK92, anti-gp120 scFv CAR-NK92). The target cells are a control population of cells that do not contain HIV viral antigen, or the target cells are cells that express or produce HIV (eg, target cells presenting gp120). After co-culture (eg, 6 hours of co-culture), the cytotoxicity of CD4 CAR NK92 or anti-gp120 scFv CAR-NK92 cells on target cells is measured via flow cytometry. CD4 CAR NK92 and anti-gp120 scFv CAR-NK92 cells can exhibit greater cytotoxicity than wild-type NK cells against gp120-presenting target cells, indicating that engineered NK cells (e.g., CD4 CAR NK92, anti-gp120 scFv CAR- NK92) ability to target HIV+ target cells.

Example 9 : Engineered antiviral antigens ( For example, against -HBV antigen ) CAR NK cells

NK cells can be engineered to express heterologous receptors that exhibit specific binding to viral antigens, such as at least a portion of a viral protein. For example, the heterologous receptor may be a chimeric antigen receptor (CAR) containing an antigen-binding domain (or antigen-binding portion) directed to a viral antigen. The antigen-binding domain may comprise or consist of at least a portion of an antibody that exhibits specific binding to a viral antigen, such as a viral surface antigen (e.g., at least a portion of an anti-hepatitis B surface antigen (anti-HBsAg) scFv, such as the F124 clone) derivative. Alternatively or additionally, the antigen-binding domain may comprise at least a portion of a native complementary molecule of the viral protein (eg, at least a portion of the hepatitis B surface antigen-binding protein (SBP) in HepG2 cells for targeting HBV viruses as antigen (HBsAg) or derived therefrom.

Engineering NK Cell:

As shown in Figure 7, NK cells (eg, NK-92 cells) are engineered to target viral (eg, HBV) proteins (eg, HBsAg). As shown in Figure 4, an anti-HBV CAR structure can comprise a linker-containing anti-HBV scFv (e.g., anti-HBsAg scFv), a variable fragment heavy chain (VH), and a variable fragment light chain (VL). Anti-HBV CAR structures can also include a signal peptide (e.g., CD8a, CD8a leader), a hinge (e.g., CD8a hinge), a transmembrane domain (TM) (e.g., CD8aTM, CD28TM), a CD28 costimulatory domain ( CD28 cos), intracellular domain (ICD) (e.g., 4-1BB ICD, CD3ζ ICD).

CD8a leader (SEQ ID NO. 15): MALPPVTALLLPLALLLHAARP

VH-1 (SEQ ID NO. 27): EVQLQESGPGLVKPSQTLSLTCAVSGSSITYGYHWNWIRQFPGNKLEWIGYISYDGSVLYNPSLENRVTITRDTSKNQFFLKLSSVTAEDTAKYYCASGFDHWGQGTTLTVSS

Linker (SEQ ID NO. 17): GGGGSGGGGSGGGGS

VL-1 (SEQ ID NO. 28): DVVMTQSPLSLPVTLGEPASISCRSSQSLVHSYGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYYCSQNTHVPYTFGGGTKLEIKR

VH-2 (SEQ ID NO. 29): QVQLVQSGGDLIQPGGSLRLSCEASGFTFNRNWMHWVRQAPGMGLVWVSRVNSDGTSSTYADSVKGQFTISRDNAKNTLFLQMNSLRVDDTAVYYCARETWYRCDYWGQGTVVTVSSASTKGPSVTSGQAGQ

VL-2 (SEQ ID NO. 30): ELQMTQSPSILSASVGDRVTITCRASQSLNSWVAWYQQAPGRPPTLLISKTSSLESGVPSRFRGGGSGAEFTLTITSLQPDDAASYYCQQYETYPYTFGQGTKVDIK

CD8a hinge (SEQ ID NO. 19): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

CD8a TM (SEQ ID NO. 20): DIYIWAPLAGTCGVLLLSLVITLYC

CD28 TM (SEQ ID NO. 21): FWVLVVVGGVLACYSLLVTVAFIIFWV

CD28 cos (SEQ ID NO. 22): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

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

CD3ζ ICD (SEQ ID NO. 24): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Engineering NK cells target HBV positive (HBV+) In vitro killing of target cells:

Addition of engineered NK cells to target cells labeled with carboxyfluorescein succinimide ester (CSFE) at various effector to target cell (E:T) ratios (e.g., 5, 10, 15, 20, 25) (eg, anti-HBsAg scFv CAR-NK92). The target cells are a control cell population that does not contain HBV viral antigen or cells that express or produce HIV (eg, target cells presenting HBsAg). After co-culture (eg, 6 hours of co-culture), the cytotoxicity of anti-HBsAg scFv CAR-NK92 cells against target cells is measured via flow cytometry. HBsAg scFv CAR-NK92 cells can exhibit greater cytotoxicity than wild-type NK cells against HBsAg-presenting target cells, demonstrating the ability of engineered NK cells (e.g., HBsAg scFv CAR-NK92) to target HBV+ target cells.

Example 10 : Engineering NK In vivo killing of virus-infected cells by cells

anti- -LMP1 CAR-NK In vivo anti-tumor efficacy of cells:

To evaluate the in vivo antitumor activity of anti-LMP1 CAR-NK cells, a xenograft model was used. On day 0, 5 × 10 ⁶ SUNE1-LMP1 cells were subcutaneously injected into 6-week-old male BALB/c nude mice. ^{Approximately} 10 days after tumor cell inoculation, when the tumor load reached approximately 100 mm, mice were randomly assigned into three different groups (N = 5/group). On days 10, 13, and 16, 5 × 10 ⁶ NK cells/100 μL were injected percutaneously into the tumors of the animals. Group A received anti-LMP1 CAR-NK cells, Group B received control NK cells, and Group C received normal saline. Tumor growth was monitored by caliper measurements and tumor volume was calculated using the following formula: 1/2 × length × (width). In this model, anti-LMP1 CAR-NK cells can substantially inhibit tumor growth, while control NK cells may not.

CD4CAR-NK Cellular in vivo resistance -HIV active:

Bone marrow-liver-thymus (BLT) humanized mice were challenged with 20,000 half tissue culture infectious dose (TCID50) of HIV via intraperitoneal injection. Three weeks after HIV challenge, all infected mice were administered low-dose antiretroviral therapy (ART) consisting of 1 mg kg−1 EFdA and 25 mg kg−1 dolutegravir by intraperitoneal injection every other day for 4 weeks. (Dolutegravir) composition. At the time of ART initiation, HIV-infected mice were divided into three groups. Treated mice were infused with CD4 CAR NK cells. Control mice were infused with wild-type NK cells or left untreated. Mice were euthanized 7 weeks post-infection and tissues collected for analysis. Infusion of CD4 CAR-NK cells reduces the frequency of HIV-infected cells in tissues.

While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The invention is not intended to be limited to the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustrations of embodiments herein are not intended to be construed in a limiting sense. Many variations, modifications and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, constructions, or relative proportions set forth herein depending on the various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice the invention. Therefore, it is contemplated that the present invention also covers 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.

The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description, in which illustrative embodiments utilizing the principles of the invention are set forth, and the accompanying drawings, also referred to herein as (for "Attachment" and "Figure"):

Figures 1A-1G illustrate engineered NK cells containing CD16 variants for enhanced CD16 signaling;

Figures 2A-2G illustrate engineered NK cells containing chimeric antigen receptors for CD19; and

Figures 3A and 3B show engineered T cells containing heterologous human IL-15.

Figures 4A and 4B show engineered NK cells for targeting latent membrane protein 1 (LMP1), a marker of Epstein-Barr virus (EBV) infection.

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

Figures 6A-6D illustrate engineered NK cells for targeting CD4 or gp120 as markers of human immunodeficiency virus (HIV) infection.

Figure 7 shows engineered NK cells for targeting hepatitis B surface antigen (HBsAg).

TW202342737A_112104137_SEQL.xml

Claims (52)

An engineered NK cell containing: A chimeric polypeptide receptor comprising an antigen-binding portion 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 includes one or more members selected from the group consisting of HIV, HBV, HCV, EBV, HPV, Lassa virus, influenza virus, coronavirus and derivatives thereof. The engineered NK cell of any one of the preceding claims, wherein the virus includes one or more members selected from the group consisting of HBV, HCV and derivatives thereof. The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell exhibits enhanced cytotoxicity against target cells containing the antigen compared to control cells 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 at least greater than the cytotoxicity of the control cell against the target cell. About 0.1 times, at least about 0.2 times, at least about 0.3 times, at least about 0.4 times, at least about 0.5 times, at least about 0.6 times, at least about 0.8 times, at least about 1 times or more. The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is configured to within about 24 hours, within about 18 hours, within about 12 hours, about The enhanced cytotoxicity is demonstrated against the target cells within 8 hours or less. The engineered NK cell of any one of the preceding claims, wherein the enhanced cytotoxicity is at an 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 said antigen-binding portion comprises at least a portion of an antibody that exhibits specific affinity for said antigen of said virus. The engineered NK cell of any one of the preceding claims, wherein said antigen-binding portion does not comprise at least a portion of an antibody that exhibits specific affinity for said antigen of said virus. The engineered NK cell of any one of the preceding claims, wherein said antigen-binding portion comprises at least a portion of a cellular protein that exhibits specific affinity for said antigen of said 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 said antigen of said virus is present on the surface of the target cell. The engineered NK cell according to any one of the preceding claims, further comprising heterologous IL-15 or a fragment thereof. The engineered NK cell of any one of the preceding claims, further comprising a receptor comprising 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 isolated stem cells or induced stem cells. The engineered NK cell of claims 1 to 18, wherein the engineered NK cell exhibits reduced expression or activity of endogenous CD38. The engineered NK cell of claims 1 to 18, wherein the expression or activity of endogenous CD38 of the engineered NK cell is not modified. The engineered NK cell of claims 1 to 20, wherein the heterologous IL-15 or fragment thereof is secreted by the engineered NK cell. The engineered NK cell of claims 1 to 20, wherein the heterologous IL-15 or fragment thereof is membrane-bound. The engineered NK cell of any one of the preceding claims, further comprising initiating enhanced expression of NK receptors. The engineered NK cell according to any one of the preceding claims, further comprising an additional chimeric polypeptide receptor, the additional chimeric polypeptide receptor comprising an antigen-binding portion capable of binding to an antigen. The engineered NK cell of any one of the preceding claims, wherein the antigen-binding portion of the chimeric polypeptide receptor is a multispecific binding portion capable of specifically binding to two or more different antigens. 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, κ, 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 an NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. The engineered NK cell according to any one of the preceding claims, further comprising a safety switch capable of causing death of the engineered NK cell. The engineered NK cell of any one of the preceding claims, wherein the safety switch comprises a caspase (such as caspase 3, 7 or 9), thymidine kinase, cytosine deaminase, modified EGFR and one or more members of B cell CD20. The engineered NK cell according to 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 according to any one of the preceding claims, further comprising a heterologous immune modulating polypeptide. The engineered NK cell according to any one of the preceding claims, wherein the heterologous immunomodulatory polypeptide comprises HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10 , one or more members of 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 endogenous immunomodulatory polypeptides. The engineered NK cell of any one of the preceding claims, wherein the endogenous immunomodulatory polypeptide comprises an immune checkpoint inhibitor or a low-immunity modulator. The engineered NK cell according to any one of the preceding claims, wherein the immune checkpoint inhibitor comprises PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT , one or more members of TGFβ 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 according to any one of the preceding claims, wherein the low-immunity modulator comprises B2M, CIITA, TAP1, TAP2, tap-related protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL , one or more members of CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, 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 compared to control cells, wherein the CD16 variant is consistent with the Engineered NK Cells Allogeneic. The engineered NK cell of any one of the preceding claims, wherein the CD16 variant comprises a sequence selected from SEQ ID NO. 1-8. The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell induces a reduced immune response in the host cell compared to control cells. The engineered NK cell according to 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 cells comprise embryonic stem cells. The engineered NK cell according to any one of the preceding claims, wherein the induced stem cells comprise induced pluripotent stem cells. A method that includes: Get cells from an object; and An engineered NK cell according to any of the preceding claims is produced from said cells. The method of any one of the preceding claims, further comprising administering the engineered NK cells to the object. A method that includes: A population of NK cells comprising engineered NK cells according to any one of the preceding claims is administered to an object in need thereof. The method of any one of the preceding claims, further comprising administering to the object a separate therapeutic agent. A method as claimed in any one of the preceding claims, wherein said separate therapeutic agent is a chemotherapeutic agent. The method of any one of the preceding claims, wherein the administration of the engineered NK cells treats cancer or reduces the risk of cancer in the subject. The method of any one of the preceding claims, wherein the administration of the engineered NK cells treats a viral infection of the subject.