WO2023093763A1 - Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules - Google Patents

Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules Download PDF

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WO2023093763A1
WO2023093763A1 PCT/CN2022/133745 CN2022133745W WO2023093763A1 WO 2023093763 A1 WO2023093763 A1 WO 2023093763A1 CN 2022133745 W CN2022133745 W CN 2022133745W WO 2023093763 A1 WO2023093763 A1 WO 2023093763A1
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cell
engineered
heterologous
fold
receptor
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PCT/CN2022/133745
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English (en)
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Jing Xu
Yangbin Gao
Luhan Yang
Huashun LI
Yaqi Lv
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Hangzhou Qihan Biotechnology Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/16143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Cancer e.g., neoplasm, tumor
  • cancer is a leading cause of death worldwide, accounting for about 10 million deaths annually. Cancer continues to bring increasing health, economic, and emotional burden on individuals, families, communities, and countries. Increase understanding of cancer biology (e.g., specifically cancer immune biology) and genetic engineering has encouraged development of adoptive cell therapies (e.g., cellular immunotherapy) , with a goal to treat or control a number of different cancers.
  • adoptive cell therapies e.g., cellular immunotherapy
  • the present disclosure provides methods and systems for treating cancer.
  • Some aspects of the present disclosure provide engineered immune cells (e.g., engineered natural killer (NK) cells) and methods of use thereof for treatment of cancer, such as, e.g., as hematologic malignancies or solid tumors.
  • engineered immune cells e.g., engineered natural killer (NK) cells
  • NK natural killer
  • the present disclosure provides an engineered NK cell configured to secrete a heterologous adaptor into an extracellular environment
  • the heterologous adaptor comprises (i) a coupling domain for coupling to the engineered NK cell and (ii) an antigen binding domain exhibiting specific binding to an antigen of a target cell, wherein, upon secretion of the heterologous adaptor to the extracellular environment by the engineered NK cell, (a) the coupling domain of the heterologous adaptor couples to the engineered NK cell and (b) the antigen binding domain of the heterologous adaptor binds to the antigen of the target cell, such that the engineered NK cell specifically targets the target cell, and wherein the engineered NK cell exhibits enhanced cytotoxicity against the target cell, as compared to a control NK cell without the heterologous adaptor.
  • the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the cytotoxicity against a target cell, as compared to the control NK cell.
  • the heterologous adaptor is an antibody or a modification thereof.
  • the heterologous adaptor is an engineered antibody having a size that is at most about 80 kilodalton (kDa) , at most about 60 kDa, or less.
  • the heterologous adaptor lacks one or more members selected from the group consisting of C L , C H 1, C H 1, and C L.
  • the heterologous adaptor is scFv-Fc.
  • the coupling domain comprises IgG, IgE, IgA, IgM, a modification thereof, or a combination thereof.
  • the coupling domain couples to an extracellular portion of a surface receptor of the engineered NK cell.
  • the surface receptor comprises at least a portion of a Fc receptor.
  • the Fc receptor comprises Fc ⁇ RI (or CD64) , Fc ⁇ RII (or CD32) , Fc ⁇ RIII (or CD16) , Fc ⁇ RI, Fc ⁇ RI (or CD89) , Fc ⁇ R, Fc ⁇ / ⁇ R (or CD351) , a modification thereof, or a combination thereof.
  • the Fc receptor comprises a fusion polypeptide comprising at least a portion of CD16 and at least a portion of CD64.
  • the surface receptor is a heterologous receptor.
  • the engineered NK cell is sufficient to kill the target cell.
  • the engineered NK cell is configured to secrete the heterologous adaptor substantially constitutively.
  • an engineered NK cell comprising: a heterologous receptor; and a heterologous adaptor comprising (i) a coupling domain for coupling to the heterologous receptor and (ii) an antigen binding domain exhibiting specific binding to an antigen of a target cell, wherein, upon expression of the heterologous receptor and the heterologous adaptor by the engineered NK cell, (a) the coupling domain of the heterologous adaptor couples to the heterologous receptor and (b) the antigen binding domain of the heterologous adaptor binds to the antigen of the target cell, such that the engineered NK cell specifically targets the target cell.
  • the engineered NK cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the cytotoxicity against a target cell, as compared to a control NK cell lacking the heterologous receptor and/or the heterologous adaptor.
  • the coupling of the heterologous adaptor and the heterologous receptor forms a complex adjacent to an extracellular surface of the engineered NK cell, which complex is capable of targeting the antigen.
  • the coupling domain couples to an extracellular domain of the heterologous receptor.
  • the heterologous adaptor is an antibody or a modification thereof.
  • the heterologous adaptor is an engineered antibody having a size that is at most about 80 kilodalton (kDa) , at most about 60 kDa, or less.
  • the heterologous adaptor lacks one or more members selected from the group consisting of C L , C H 1, C H 1, and C L.
  • the heterologous adaptor is scFv-Fc.
  • the coupling domain comprises IgG, IgE, IgA, IgM, a modification thereof, or a combination thereof.
  • the heterologous receptor comprises at least a portion of a Fc receptor.
  • the Fc receptor comprises Fc ⁇ RI (or CD64) , Fc ⁇ RII (or CD32) , Fc ⁇ RIII (or CD16) , Fc ⁇ RI, Fc ⁇ RI (or CD89) , Fc ⁇ R, Fc ⁇ / ⁇ R (or CD351) , a modification thereof, or a combination thereof.
  • the Fc receptor comprises a fusion polypeptide comprising at least a portion of CD16 and at least a portion of CD64.
  • the present disclosure provides an engineered immune cell, comprising: a heterologous receptor; and a heterologous adaptor comprising (i) a coupling domain for coupling to an extracellular domain of the heterologous receptor and (ii) an antigen binding domain exhibiting specific binding to an antigen of a target cell, wherein, upon expression of the heterologous receptor and secretion of the heterologous adaptor by the engineered immune cell, (a) the coupling domain of the heterologous adaptor couples to the extracellular domain of the heterologous receptor and (b) the antigen binding domain of the heterologous adaptor binds to the antigen of the target cell, such that the engineered immune cell specifically targets the target cell.
  • the engineered immune cell is a NK cell.
  • the engineered immune cell exhibits at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in the cytotoxicity against a target cell, as compared to a control immune cell lacking the heterologous receptor and/or the heterologous adaptor.
  • the heterologous adaptor is an antibody or a modification thereof.
  • the heterologous adaptor is an engineered antibody having a size that is at most about 80 kilodalton (kDa) , at most about 60 kDa, or less.
  • the heterologous adaptor lacks one or more members selected from the group consisting of C L , C H 1, C H 1, and C L.
  • the heterologous adaptor is scFv-Fc.
  • the coupling domain comprises IgG, IgE, IgA, IgM, a modification thereof, or a combination thereof.
  • the heterologous receptor comprises at least a portion of a Fc receptor.
  • the Fc receptor comprises Fc ⁇ RI (or CD64) , Fc ⁇ RII (or CD32) , Fc ⁇ RIII (or CD16) , Fc ⁇ RI, Fc ⁇ RI (or CD89) , Fc ⁇ R, Fc ⁇ / ⁇ R (or CD351) , a modification thereof, or a combination thereof.
  • the Fc receptor comprises a fusion protein comprising at least a portion of CD16 and at least a portion of CD64.
  • the coupling domain comprises (1) a first coupling domain for coupling to a first surface receptor or heterologous receptor and (2) a second coupling domain for coupling to a second surface receptor or heterologous receptor, wherein the first surface receptor or heterologous receptor is different from the second surface receptor or heterologous receptor.
  • any one of the engineered immune cell or the engineered NK cell as disclosed herein (1) the first surface receptor or heterologous receptor is presented on a first immune cell, and (2) the second surface receptor or heterologous receptor is presented on a second immune cell, wherein the first immune cell and the second immune cell are different.
  • the first immune cell is an NK cell
  • the second immune cell is a T cell.
  • the antigen binding moiety is flanked by the first coupling domain and the second coupling domain.
  • any one of the engineered immune cell or the engineered NK cell as disclosed herein (a) the coupling of the heterologous adaptor to the heterologous receptor of the NK cell and (b) the binding of the heterologous adaptor to the antigen are necessary but are not individually sufficient for the engineered NK cell to kill the target cell.
  • the engineered immune cell or the engineered NK cell further comprises a heterologous polynucleotide sequence encoding the heterologous adaptor and/or the heterologous receptor.
  • the heterologous polynucleotide sequence is genomically integrated.
  • the engineered immune cell or the engineered NK cell exhibits reduced expression and/or activity level of an endogenous gene encoding the antigen as compared to the control NK cell.
  • the engineered immune cell or the engineered NK cell exhibits reduced expression and/or activity level of an endogenous gene encoding CD38 as compared to the control NK cell.
  • the engineered immune cell or the engineered NK cell further comprises a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to the antigen and/or a different antigen.
  • the different antigen is on the target cell. In some embodiments of any one of the engineered immune cell or the engineered NK cell as disclosed herein, the different antigen is on a different target cell.
  • the engineered immune cell or the engineered NK cell further comprises a heterologous cytokine and/or a heterologous cytokine receptor thereof for enhanced cytokine signaling, as compared to that without the heterologous cytokine and/or the heterologous cytokine receptor.
  • the engineered immune cell or the engineered NK cell exhibits reduced expression and/or activity of an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • the engineered immune cell or the engineered NK cell exhibits reduced expression and/or activity of a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • a hypo-immunity regulator selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL
  • the engineered immune cell or the engineered NK cell further comprises a safety switch capable of effecting death of the engineered NK cell.
  • the enhanced cytotoxicity against the target cell is ascertained in vitro, ex vivo, or in vivo.
  • the enhanced cytotoxicity against the target cell is ascertained by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%decrease in a size of a population of the target cell, as compared to a cytotoxicity level of the control immune cell or the control NK cell.
  • the enhanced cytotoxicity against the target cell is ascertained by a reduction in tumor size, as compared to that by the control immune cell or the control NK cell.
  • the tumor size is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as compared to the control immune cell or the control NK cell.
  • the engineered immune cell or the engineered NK cell induces reduced immune response in a host cell, as compared to the control immune cell ore the control NK cell.
  • the engineered immune cell or the engineered NK cell is for use in treating a subject in need thereof. In some embodiments of any one of the engineered immune cell or the engineered NK cell as disclosed herein, the engineered immune cell or the engineered NK cell is allogeneic to the subject. In some embodiments of any one of the engineered immune cell or the engineered NK cell as disclosed herein, the engineered immune cell or the engineered NK cell is autologous to the subject.
  • the engineered immune cell or the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the present disclosure provides a composition comprising any one of the engineered immune cell or the engineered NK cell disclosed herein.
  • the composition further comprises a separate therapeutic agent.
  • the separate therapeutic agent is a chemotherapeutic agent.
  • the present disclosure provides a method comprising (a) obtaining a cell from a subject; and (b) generating, from the cell, any one of the engineered immune cell or the engineered NK cell disclosed herein.
  • the present disclosure provides a method of killing a target cell, the method comprising: contacting the target cell with any one of the engineered immune cell or the engineered NK cell disclosed herein, wherein the contacting is sufficient to induce killing of the target cell.
  • the present disclosure provides a method of treating a subject in need thereof, comprising administering any one of the engineered immune cell or the engineered NK disclosed herein to the subject.
  • the method further comprises administering a separate therapeutic agent.
  • the separate therapeutic agent is a chemotherapeutic agent.
  • the engineered NK cell exhibits enhanced cytotoxicity against a target cell as compared to a control cell. In some embodiments of any one of the compositions disclosed herein, the engineered NK cell induces reduced immune response in a host cell as compared to a control cell.
  • the host cell is an immune cell.
  • FIG. 1 schematically illustrates an example of an engineered immune cell for antibody-dependent cellular cytotoxicity.
  • FIG. 2 schematically illustrates an example of an engineered NK cell for antibody-dependent cellular cytotoxicity.
  • FIG. 3 illustrates a nucleic acid vector encoding a heterologous adaptor (e.g., full length) for promoting antibody-dependent cellular cytotoxicity.
  • a heterologous adaptor e.g., full length
  • FIG. 4 shows secretion of a heterologous adaptor by mammalian cells.
  • FIG. 5A shows a heterologous adaptor-mediated ADCC function of cord blood NK cells against target cells.
  • FIG. 5B shows cytotoxicity against target cells by a combination of NK cells and a medium comprising antibodies secreted by mammalian cells.
  • FIG. 6 shows secretion of a heterologous adaptor by induced pluripotent stem cells.
  • FIG. 7 shows binding of a target cell by the heterologous adaptor secreted by induced pluripotent stem cells.
  • FIG. 8 shows binding of a target cell by the heterologous adaptor secreted by induced pluripotent stem cell-derived embryoid bodies.
  • FIG. 9 shows a heterologous adaptor-mediated ADCC function of induced pluripotent stem cell-derived embryoid bodies against target cells.
  • FIG. 10 shows successful differentiation of induced pluripotent stem cell to NK cells.
  • FIG. 11 shows binding of a target cell by the heterologous adaptor secreted by engineered NK cells.
  • FIG. 12 shows a heterologous adaptor-mediated ADCC function of engineered NK cells against target cells.
  • FIG. 13 shows binding of a target cell by the heterologous adaptor secreted by NK-92 cells.
  • FIG. 14 shows mRNA levels of the heterologous adaptor secreted by NK-92 cells.
  • FIG. 15 illustrates a nucleic acid vector encoding a scFv-Fc heterologous adaptor for promoting antibody-dependent cellular cytotoxicity.
  • a chimeric transmembrane receptor includes a plurality of chimeric transmembrane receptors.
  • the term “about” or “approximately” generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • a cell generally refers to a biological cell.
  • a cell can be the basic structural, functional and/or biological unit of a living organism.
  • a cell can originate from any organism having one or more cells.
  • Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses) , an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana,
  • seaweeds e.g., kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., fruit fly, cnidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell) .
  • reprogramming generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state.
  • a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state.
  • a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • differentiated generally refers to a process by which an unspecialized ( “uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ( “committed” ) position within the lineage of a cell.
  • the term “committed” generally refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • pluripotent generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) .
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .
  • iPSCs induced pluripotent stem cells
  • differentiated cells e.g., differentiated adult, neonatal, or fetal cells
  • iPSCs reprogrammed stem cells
  • the iPSCs produced do not refer to cells as they are found in nature.
  • iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells.
  • NK natural killer
  • iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • tissue-specific stem cells e.g., hematopoietic stem cells (HSCs)
  • HSCs hematopoietic stem cells
  • ESCs generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.
  • ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) .
  • ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • isolated stem cells generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism.
  • HSCs can be isolated from a mammal’s body, such as a human body.
  • an embryonic stem cells can be isolated from an embryo.
  • isolated generally refers to a cell or a population of cells, which has been separated from its original environment.
  • a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist.
  • An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample.
  • the term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
  • hematopoietic stem and progenitor cells generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • hematoblasts multipotent hematopoietic stem cells
  • HSCs Hematopoietic stem and progenitor cells
  • myeloid monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages T cells, B cells, NK cells
  • HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.
  • immune cell generally refers to a differentiated hematopoietic cell.
  • Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.
  • NK cell or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) .
  • NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) .
  • nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives can include, for example, [ ⁇ S] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots.
  • Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) .
  • FAM 5-carboxyfluorescein
  • JE 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein
  • fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif.
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be biotin-dNTP.
  • 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) .
  • polynucleotide oligonucleotide, ” or “nucleic acid, ” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • a polynucleotide can be exogenous or endogenous to a cell.
  • a polynucleotide can exist in a cell-free environment.
  • a polynucleotide can be a gene or fragment thereof.
  • a polynucleotide can be DNA.
  • a polynucleotide can be RNA.
  • a polynucleotide can have any three dimensional structure, and can perform any function, known or unknown.
  • a polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.
  • analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g., rhodamine or flurescein linked to the sugar) , thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • 5-bromouracil peptide nucleic acid
  • xeno nucleic acid morpholinos
  • locked nucleic acids glycol nucleic acids
  • threose nucleic acids dideoxyn
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript.
  • genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′and 3′ends.
  • the term encompasses the transcribed sequences, including 5′and 3′untranslated regions (5′-UTR and 3′-UTR) , exons and introns.
  • the transcribed region will contain “open reading frames” that encode polypeptides.
  • a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide.
  • genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters.
  • a gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism.
  • a gene can refer to an “exogenous gene” or a non-native gene.
  • a non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer.
  • a non-native gene can also refer to a gene not in its natural location in the genome of an organism.
  • a non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .
  • expression generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides can be collectively referred to as “gene product. ” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.
  • Up-regulated, with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state.
  • Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time.
  • stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell.
  • a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
  • amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) .
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids, ” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues.
  • Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • Amino acid analogues can refer to amino acid derivatives.
  • amino acid includes both D-amino acids and L-amino acids.
  • derivative, ” “variant, ” or “fragment, ” as used herein with reference to a polypeptide generally refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.
  • engineered, ” “chimeric, ” or “recombinant, ” as used herein with respect to a polypeptide molecule generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule.
  • Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • an engineered or recombinant polynucleotide e.g.,
  • gene editing moiety generally refers to a moiety which can edit a nucleic acid sequence, whether exogenous or endogenous to a cell comprising the nucleic acid sequence.
  • a gene editing moiety regulates expression of a gene by editing a nucleic acid sequence.
  • a gene editing moiety can regulate expression of a gene by editing genomic DNA sequence.
  • a gene editing moiety can regulate expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.
  • a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operatively coupled to the gene (or a target sequence within the gene) , and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA.
  • a gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA.
  • a gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template.
  • a gene editing moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template.
  • a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript.
  • antibody generally refers to a proteinaceous binding molecule with immunoglobulin-like functions.
  • the term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) , as well as derivatives, variants, and fragments thereof.
  • Antibodies include, but are not limited to, immunoglobulins (Ig's ) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc. ) , immunoglobulin that comprises at least two heavy (H) chains and two light (L) chains connected by disulfide, etc.
  • Each H chain can comprise a heavy chain variable region (VH) and a heavy chain constant region.
  • Each L chain can comprise a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further characterized by having complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR) .
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL can contain three CDRs and four FRs, and the variable regions of the V and H chains contain a binding domain that interacts with the antigen.
  • a derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody.
  • Antigen-binding fragments include Fab, Fab′, F (ab′) 2, variable fragment (Fv) , single chain variable fragment (scFv) , minibodies, diabodies, and single-domain antibodies ( “sdAb” or “nanobodies” or “camelids” ) .
  • the term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies) .
  • Non-limiting examples of the antibodies can include, but are not limited to, monospecific or monovalent antibodies (e.g., CH-Fc/VL-Fc, Fc/Fab-Fc, HC-FC/LC-Fc, etc. ) , IgG-like formats with appendages of scFv and/or scFab (e.g., scFv-Fc, scFv-Fc/scFv-Fc, Fab-Fc, Fab-Fc/Fab-Fc, Fab-Fc/scFab-Fc, Fv-/Fv-CrossMab IgG, Fab-CrossMab IgG, etc.
  • monospecific or monovalent antibodies e.g., CH-Fc/VL-Fc, Fc/Fab-Fc, HC-FC/LC-Fc, etc.
  • IgG-like formats with appendages of scFv and/or scFab e.g
  • IgG formats with correct LC associations e.g., IgG, CrossMab IgG, common LC-IgG, Ortho-Fab IgG, Four-in-One Cross-Mab IgG, etc. .
  • chimeric polypeptide receptor generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each antigen binding moiety capable of binding to a specific antigen.
  • a chimeric polypeptide receptor can be monospecific (i.e., capable of binding to one type of specific antigen) .
  • a chimeric polypeptide receptor can be multi-specific (i.e., capable of binding to two or more different types of specific antigens) .
  • a chimeric polypeptide receptor can be monovalent (i.e., comprising a single antigen binding moiety) .
  • a chimeric polypeptide receptor can be multivalent (i.e., comprising a plurality of antigen binding moieties) .
  • a chimeric polypeptide receptor can comprise a T-cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR) .
  • TCR T-cell receptor
  • TFP T-cell receptor
  • an antigen binding domain generally refers to a construct exhibiting preferential binding to a specific target antigen.
  • An antigen binding domain can be a polypeptide construct, such as an antibody, modification thereof, fragment thereof, or a combination thereof.
  • the antigen binding domain can be any antibody as disclosed herein, or a functional variant thereof.
  • Non-limiting examples of an antigen binding domain can include a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy-chain-only antibody (VNAR) , Ig NAR, a chimeric antibody, a recombinant antibody, or antibody fragment thereof.
  • Non-limiting examples of antibody fragment include Fab, Fab′, F (ab) ′2, F(ab) ′3, Fv, single chain antigen binding fragment (scFv) , (scFv) 2, disulfide stabilized Fv (dsFv) , minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody) , recombinant heavy-chain-only antibody (VHH) , and other antibody fragments that maintain the binding specificity of the whole antibody.
  • safety switch generally refers to an engineered polypeptide construct designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. When expressed in a cell, the safety switch can induce death of the host cell, thereby inactivating activity of the cell in a host (e.g., in a subject’s body) .
  • the safety switch can be a suicide moiety.
  • the cell can be programmed to express the suicide moiety at certain stage of its life-cycle (e.g., time-programmed) . In some cases, expression of the suicide moiety in a cell can be conditional or inducible.
  • conditional regulation (e.g., expression) of a suicide moiety can include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation.
  • the safety switch can be an inducible suicide moiety.
  • a safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion.
  • a safety switch can be activated by an exogenous molecule (e.g., a drug or a prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., engineered NK cell as disclosed herein) .
  • an exogenous molecule e.g., a drug or a prodrug
  • apoptosis and/or cell death of a cell e.g., engineered NK cell as disclosed herein
  • hypo-immunity regulator generally refers to a polypeptide construct in a cell, wherein either enhanced expression (e.g., via knock-in of a heterologous gene) or reduced expression (e.g., via knock-out or knock-down of an endogenous gene) of the hypo-immunity regulator in the cell can help the cell to reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration to the host’s body.
  • immune response e.g., immune attack, such as adaptive immune rejection
  • cells e.g., engineered NK cells as disclosed herein
  • the hypo-immunity regulator can be modified to exhibit either enhanced expression or reduced expression of the hypo-immunity regulator, such that the cells can evade the host immune attack upon second or further infusion of the cells into the host (i.e., recipient) .
  • the cells (i) would not be rejected by the host’s immune system and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator.
  • a cell exhibiting the enhanced expression or reduced expression of the hypo-immunity regulator can be referred to as exhibiting “hypo-immunity” or being “immune-privileged. ”
  • immune checkpoint inhibitor generally refers to a group of molecules presented on a cell surface of an immune cell (e.g., T cells, myeloid cells, NK cells, B cells, etc. ) that can modulate immune response of the cell by down-regulating or inhibiting the immune response of the immune cell against a target cell, such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) .
  • a target cell such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) .
  • the target cell can express a receptor or a ligand of the immune checkpoint inhibitor presented on the surface of the immune cell, to engage with the immune checkpoint inhibitor and down-regulate or inhibit the immune response of the immune cells against the target cell.
  • down-regulating or inhibiting expression of the immune checkpoint inhibitor in the immune cell can, in some cases, enhance or prolong the immune response of the immune cell against a target cell.
  • immune response generally refers to T cell mediated and/or B cell mediated immune responses from a host’s immune system to an object (e.g., a foreign object) .
  • An example of an immune response include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • an immune response can be indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, such as macrophages.
  • the term “enhanced expression, ” “increased expression, ” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of expression can be substantially zero (or null) or higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain.
  • a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.
  • the term “enhanced activity, ” “increased activity, ” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of activity can be substantially zero (or null) or higher than zero.
  • the moiety of interest can comprise a polypeptide construct of the host strain.
  • the moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain.
  • a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.
  • reduced expression, ” “decreased expression, ” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of expression is higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can be knocked-out or knocked-down in the host strain.
  • reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.
  • reduced activity, ” “decreased activity, ” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of activity is higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can be knocked-out or knocked-down in the host strain.
  • reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.
  • subject generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • treatment generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a treatment can comprise administering a system or cell population disclosed herein.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • an effective amount or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof.
  • lymphocytes e.g., T lymphocytes and/or NK cells
  • therapeutically effective generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.
  • T cells are part of the adaptive immune system and can be primed to recognize a specific threat by recognizing immune proteins (i.e., antigens) on a foreign cell surface.
  • immune proteins i.e., antigens
  • NK cells are part of the innate immune response and can respond to a broad range of objects that consider to be “non-self. ”
  • NK cells can attack their target cells without sensitization (i.e., antigen-specific priming) to eliminate foreign substances.
  • Unmodified NK cells derived from a subject can be cultured and expanded ex vivo, then administered to the subject as a treatment to attack their target cells, e.g., cancer cells.
  • target cells e.g., cancer cells.
  • NK cell-based therapies can be limited due to short half-life and/or poor proliferation of NK cells ex vivo or in vivo.
  • unmodified NK cells can be ineffective in targeting harder-to-treat cancers, such as myeloma or solid tumors.
  • ex vivo production of NK cells based on blood-derived stem cells e.g., HSCs
  • immune cells e.g., NK cells, T cells, etc.
  • NK cells e.g., T cells, etc.
  • Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell) .
  • Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered Immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered Immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.
  • engineered immune cells e.g., engineered NK cells
  • engineered immune cells disclosed herein can be derived from an isolated stem cell (e.g., isolated ESCs) .
  • engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSCs) .
  • the stem cell disclosed herein can be an autologous cell or derived from the autologous cell.
  • the autologous cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the autologous cell can be obtained from the subject before the subject is found to have the condition.
  • the autologous cell can be an allogeneic cell, e.g., a universal stem cell with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs.
  • the autologous cell can be obtained from a healthy donor.
  • the engineered immune cell (e.g., engineered NK cell) can be an autologous cell.
  • the engineered immune cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the engineered immune cell can be obtained from the subject before the subject is found to have the condition.
  • the engineered immune cell can be an allogeneic cell, e.g., for a universal allogenic immunotherapy with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs.
  • the engineered immune cell can be obtained from a healthy donor.
  • T cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered T cell) .
  • T cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • T cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered T cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered T cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered T cells can be autologous to the subject. Alternatively, the engineered T cells can be allogeneic to the subject.
  • NK cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered NK cell) .
  • NK cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered NK cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) comprising a heterologous adaptor.
  • the heterologous adaptor can comprise (i) (i) a coupling domain for coupling to the engineered immune cell (e.g., to a surface of the engineered immune cell, such as an extracellular portion of a receptor protein on the surface of the engineered immune cell) and (ii) an antigen binding domain exhibiting specific binding to an antigen of a target cell.
  • the engineered immune cell can secrete the heterologous adaptor, such that the heterologous adaptor can couple to the engineered immune cell via the coupling domain.
  • the coupling between the coupling domain and the engineered immune cell may be non-covalent (e.g., via hydrogen bond (s) ) .
  • the heterologous adaptor can couple to the receptor protein within the cell, and the resulting complex (e.g., non-covalent complex) comprising the heterologous adaptor and the receptor protein can be moved to the membrane of the engineered immune cell, such that the heterologous adaptor coupled to the receptor protein is presented adjacent to the surface of the engineered immune cell.
  • the engineered immune cell can thus target (or bind) the target cell, via action of the heterologous adaptor, to exhibit enhanced cytotoxicity against the target cell or to kill the target cell.
  • the coupling as provided herein can comprise a non-covalent binding between two moieties (e.g., between the coupling domain and the extracellular portion of the receptor protein of the engineered immune cell or another engineered immune cell) .
  • the heterologous adaptor is an antibody, a fragment thereof, or a modification thereof, as disclosed herein.
  • the coupling domain of the heterologous adaptor can comprise at least a portion of an immunoglobulin, such as, for example, IgG, IgE, IgA, IgM, a modification thereof, or a combination thereof.
  • the heterologous adaptor may be derived from an IgG antibody, but without being a full length IgG antibody.
  • the heterologous adaptor may be a fully length IgG antibody.
  • the heterologous adaptor can be or can be an engineered variant of (i) a humanized antibody (e.g., via insertion of relevant complementarity-determining region (CDR) segments into a human antibody scaffold, such as, for example, the CDR segments derived from obinutuzumab, veltuzumab, ocrelizumab, etc. for CD20 targeting) , (ii) a fully human antibody (e.g., ofatumumab, mAb 1.5.3, etc.
  • a humanized antibody e.g., via insertion of relevant complementarity-determining region (CDR) segments into a human antibody scaffold, such as, for example, the CDR segments derived from obinutuzumab, veltuzumab, ocrelizumab, etc. for CD20 targeting
  • CDR complementarity-determining region
  • a chimeric antibody e.g., via splicing a non-human variable region into a human constant region, such as, for example, rituximab for CD20 targeting.
  • the coupling domain can comprise a polypeptide sequence having a length of at least or up to about 2 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 110 amino acid residues, at least or up to about 120 amino acid residues, at least or up to
  • the heterologous adaptor can be a polypeptide, and the polypeptide can have a size of at least or up to about 5 kilodalton (kDa) , at least or up to about 10 kDa, at least or up to about 15 kDa, at least or up to about 20 kDa, at least or up to about 30 kDa, at least or up to about 40 kDa, at least or up to about 50 kDa, at least or up to about 60 kDa, at least or up to about 70 kDa, at least or up to about 80 kDa, at least or up to about 90 kDa, at least or up to about 100 kDa, at least or up to about 110 kDa, at least or up to about 120 kDa, at least or up to about 130 kDa, at least or up to about 140 kDa, at least or up to about 150 kDa, at least or up to about 160 kDa,
  • the heterologous adaptor can comprise (i) a variable heavy chain (e.g., VH, VHH, etc. ) and optionally a variable light chain (e.g., VL) that is coupled to (e.g., fused directly or indirectly to) (ii) a fragment but not the entirety of the crystallisable fragment (Fc) of an antibody.
  • the heterologous adaptor can be a derivative of an antibody that is smaller than the antibody, e.g., lacking one or more members selected from the group consisting of C L , C H 1, C H 1, C L , C H 2, and C H 3.
  • the heterologous adaptor can be a derivative of an antibody that is smaller than the antibody, e.g., lacking one or more members (e.g., one member, two members, three members, or all four members) of the following: C L , C H 1, C H 1, and C L .
  • the heterologous adaptor can lack at least C L .
  • the heterologous adaptor can lack at least C H 1.
  • the heterologous adaptor can lack at least C H 1.
  • the heterologous adaptor can lack at least C L .
  • the size of the heterologous adaptor can be smaller than a size of an antibody (e.g., a naturally occurring antibody, such as IgG) by at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, or at least or up to about 70%.
  • an antibody e.g., a naturally occurring antibody, such as IgG
  • the receptor protein can be a heterologous receptor, such that the heterologous adaptor and the heterologous receptor can form a complex (e.g., a non-covalent complex) adjacent to an extracellular surface of the engineered immune cell as disclosed herein.
  • the heterologous receptor can comprise at least a portion of a Fc receptor.
  • the Fc receptor can include Fc ⁇ RI (or CD64) , Fc ⁇ RII (or CD32) , Fc ⁇ RIII (or CD16) , Fc ⁇ RI, Fc ⁇ RI (or CD89) , Fc ⁇ R, Fc ⁇ / ⁇ R (or CD351) , a modification thereof, or a combination thereof.
  • the heterologous receptor can comprise at least a portion of Fc ⁇ RI (or CD64) .
  • the heterologous receptor can comprise at least a portion of Fc ⁇ RII (or CD32) .
  • the heterologous receptor can comprise at least a portion of Fc ⁇ RIII (or CD16) .
  • the heterologous receptor can comprise at least a portion of Fc ⁇ RI.
  • the heterologous receptor can comprise at least a portion of Fc ⁇ RI (or CD89) .
  • the heterologous receptor can comprise at least a portion of Fc ⁇ R.
  • the heterologous receptor can comprise at least a portion of Fc ⁇ / ⁇ R (or CD351) .
  • (A) the coupling of the heterologous adaptor to the receptor protein of the engineered immune cell (e.g., the engineered NK cell) and (B) the binding of the heterologous adaptor to the antigen of the target cell are necessary but are not individually sufficient for the engineered immune cell to kill the target cell.
  • (A) the coupling of the heterologous adaptor to the heterologous receptor of the engineered immune cell (e.g., the engineered NK cell) and (B) the binding of the heterologous adaptor to the antigen of the target cell are necessary but are not individually sufficient for the engineered immune cell to kill the target cell.
  • formation of the complex comprising the heterologous adaptor and the heterologous receptor may not and need not require avidin-biotin binding. In some embodiments, formation of the complex comprising the heterologous adaptor and the heterologous receptor may utilize avidin-biotin binding, or a functional variant thereof.
  • the heterologous receptor (or the surface receptor) as disclosed herein can comprise CD16, CD64, a modification thereof, or a combination thereof.
  • the heterologous receptor can be any heterologous CD16 variant for enhanced CD16 signaling, as disclosed herein.
  • the heterologous receptor can be a chimeric protein (e.g., a CD16-CD64 fusion protein, a CD16-CD64-IL15RF fusion protein, etc., as disclosed herein) .
  • he heterologous receptor can comprise at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%of the Fc receptor as disclosed herein.
  • the heterologous receptor can comprise at most about 100%, at most about 99%, 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, or less of the Fc receptor as disclosed herein.
  • the receptor protein can be an endogenous receptor (e.g., an endogenous surface receptor) .
  • the endogenous receptor can be a Fc receptor, as disclosed herein, that is endogenously expressed (e.g., expressed from an endogenous gene of the engineered immune cell) .
  • the coupling domain of the heterologous adaptor can couple to the engineered immune cell (e.g., the engineered NK cell) and (b) the antigen binding domain of the heterologous adaptor can bind to the antigen of the target cell, such that the engineered immune cell can specifically target the target cell, e.g., to kill the target cell.
  • the engineered immune cell e.g., the engineered NK cell
  • the antigen binding domain of the heterologous adaptor can bind to the antigen of the target cell, such that the engineered immune cell can specifically target the target cell, e.g., to kill the target cell.
  • the heterologous adaptor can enhance target specificity (e.g., target cell specificity) of the engineered immune cell to the target cell by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more, as compared to that in a control immune cell without the heterologous adaptor.
  • target specificity e.g., target cell specificity
  • the enhanced target specificity can be at most about 100%, at most about 99%, 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 2%, at most about 1%, or less, as compared to that in the control immune cell.
  • the enhanced target specificity can be at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more, as compared to that in the control immune cell.
  • the enhanced target specificity can be at most about 500-fold, at most about 200-fold, at most about 100-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 25-fold, at most about 20-fold, at most about 15-fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.5-fold, at most about 0.2-fold, at most about 0.1-fold, or less, as compared to that in the control immune cell.
  • the heterologous adaptor can recruit the engineered immune cell (e.g., the engineered NK cell) as disclosed herein and/or other immune cells (e.g., other NK cells of the host) towards the target cell, e.g., to induce killing of the target cell by the engineered immune cell and/or other immune cells.
  • the engineered immune cell and/or other immune cells otherwise, in absence of the heterologous adaptor, would exhibit little or no targeting specificity to the target cell.
  • the heterologous adaptor can be an antibody or a modification thereof, such that the heterologous adaptor can promote antibody dependent cell-mediated cytotoxicity (ADCC) against the target cell.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can be sufficient to induce killing of the target cell (or a population of the target cell, such as a tumor) .
  • the engineered NK cell comprising and capable of secreting the heterologous adaptor can induce killing of a population of target cells (e.g., a population of cancer or tumor cells) in an environment (e.g., in vitro or in vivo environment) that is substantially free (e.g., completely free) of a respective recombinant variant of the heterologous adaptor (e.g., a recombinant antibody, such as a recombinant monoclonal antibody) that is not secreted by the engineered NK cell.
  • a population of target cells e.g., a population of cancer or tumor cells
  • an environment e.g., in vitro or in vivo environment
  • a respective recombinant variant of the heterologous adaptor e.g., a recombinant antibody, such as a recombinant monoclonal antibody
  • administering can be sufficient to substantially treat the cancer or tumor without the need to additionally administer (e.g., simultaneously or sequentially) a non-cellular composition comprising the respective recombinant variant of the heterologous adaptor (e.g., the recombinant antibody) .
  • a non-cellular composition comprising the respective recombinant variant of the heterologous adaptor (e.g., the recombinant antibody) .
  • the engineered immune cell (e.g., the engineered NK cell) comprising and capable of secreting the heterologous adaptor as disclosed herein can induce killing of a population of target cells (e.g., cancer/tumor cells) with reduced amount of the recombinant antibody in the non-cellular composition.
  • target cells e.g., cancer/tumor cells
  • the engineered immune cell comprising and capable of secreting the heterologous adaptor can reduce the amount of the recombinant antibody in the non-cellular composition by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, as compared to a control therapy without any engineered immune cell capable of secreting the heterologous adaptor.
  • a therapy (e.g., a cancer therapy) using the engineered immune cell secreting the heterologous adaptor, as disclosed herein, may not need a co-therapy with a non-cellular composition comprising a recombinant antibody, e.g., to treat cancer.
  • administering antibodies or variants thereof to a subject in need thereof by administering engineered immune cells (e.g., engineered NK cells) that secret such antibodies (or variants) can reduce an amount of such antibodies needed to yield a therapeutic effect (e.g., cancer therapy) , as compared to direct administration of such antibodies.
  • engineered immune cells e.g., engineered NK cells
  • a therapeutic effect e.g., cancer therapy
  • a total amount (e.g., number) of the engineered immune cell (e.g., engineered NK cell) comprising the heterologous adaptor (e.g., an antibody or a modification thereof) and optionally the heterologous receptor, as disclosed herein, that is sufficient to yield a therapeutic effect in a subject may be less than
  • a total amount (e.g., number) of a respective recombinant variant of the heterologous adaptor (e.g., a recombinant antibody) that is sufficient to yield a comparable therapeutic effect in a control subject by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about
  • the total amount (e.g., number) of the engineered immune cell comprising the heterologous adaptor (e.g., the antibody or a modification thereof) and optionally the heterologous receptor, as disclosed herein, that is sufficient to yield the therapeutic effect in a subject may be less than (B) the total amount (e.g., number) of the respective recombinant variant of the heterologous adaptor that is sufficient to yield the comparable therapeutic effect in the control subject, by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at
  • a total amount (e.g., number) of the heterologous adaptor (e.g., an antibody or a modification thereof) secreted by a population of the engineered immune cell (e.g., a population of the engineered NK cell) , as disclosed herein, that is sufficient to yield a therapeutic effect in a subject (e.g., that is sufficient to kill a population of cancer or tumor cells in the subject) may be less than (B) a total amount (e.g., number) of a respective recombinant variant of the heterologous adaptor (e.g., a recombinant antibody) that is sufficient to yield a comparable therapeutic effect in a control subject, by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%,
  • the total amount (e.g., number) of the heterologous adaptor secreted by the population of the engineered immune cell, as disclosed herein, that is sufficient to yield the therapeutic effect in the subject may be less than (B) the total amount (e.g., number) of the respective recombinant variant of the heterologous adaptor that is sufficient to yield the comparable therapeutic effect in the control subject, by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-
  • a half-life of the engineered immune cells capable of secreting the heterologous adaptor may be longer than (B) a half-life of a respective recombinant variant of the heterologous adaptor (e.g., a recombinant antibody) (e.g., for direct administration to the subject) , by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 120%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or
  • the half-life of the engineered immune cells secreting the heterologous adaptor may be longer than (B) the half-life of the respective recombinant variant of the heterologous adaptor by at least about 0.1-fold, atf least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more.
  • the half-life of the engineered immune cells secreting the heterologous adaptor may be at least about 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, or more.
  • the engineered immune cell can comprise the heterologous receptor and the heterologous adaptor (e.g., a heterologous antibody) , as disclosed herein, and the heterologous receptor and the heterologous adaptor can be designed such that (A) a binding affinity between the heterologous receptor and the heterologous adaptor can be greater than (B) a binding affinity between an Fc receptor of an endogenous immune cell (e.g., endogenous NK cell) of a subject (e.g., a human subject) and a respective recombinant variant of the heterologous adaptor (e.g., a recombinant antibody) , by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at
  • the binding affinity between the heterologous receptor and the heterologous adaptor can be greater than (B) the binding affinity between the Fc receptor of the endogenous immune cell (e.g., the endogenous NK cell) of the subject and the respective recombinant variant of the heterologous adaptor, by at least about 0.1-fold, at least about 0.2-fold, at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or more.
  • administration of the engineered immune cell e.g., the engineered NK cell
  • the heterologous adaptor e.g., a heterologous antibody
  • administration of the engineered immune cell can reduce (or eliminate) one or more side effects of monoclonal antibody administration therapies, e.g., non-specific targeting, organ-specific adverse events (e.g., cardiotoxicity) , fever, chills, headache, nausea, vomiting, diarrhea, rashes, etc.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can constitutively express the heterologous adaptor (and/or the heterologous receptor) , to constitutively secrete the heterologous adaptor (and/or the heterologous receptor) .
  • a gene encoding the heterologous adaptor (and/or the heterologous receptor) can be operatively coupled to a constitutive promoter (e.g., SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems) .
  • a constitutive promoter e.g., SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can conditionally express the heterologous adaptor (and/or the heterologous receptor) , to conditionally secrete the heterologous adaptor (and/or the heterologous receptor) .
  • a gene encoding the heterologous adaptor (and/or the heterologous receptor) can be operatively coupled to a promoter specific for a cell state (or type) of the engineered immune cell.
  • a gene can be operatively coupled to an immune cell promoter.
  • the immune cell promoters can include CD8 cell-specific promoters, CD4 cell-specific promoters, neutrophil-specific promoters, and NK-specific promoters.
  • an NK-specific promoter can be a promoter that regulates expression of at least one of NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, etc.
  • the gene encoding the heterologous adaptor (and/or the heterologous receptor) can be operatively coupled to a heterologous promoter, wherein the heterologous promoter is activatable upon an external stimulus (e.g., light, magnetic field, extracellular ligand, etc. ) .
  • the heterologous promoter can be operatively coupled to a chimeric antigen receptor (CAR) signaling of the cell, such that the activated CAR activates the promoter upon binding a target antigen, to thereby activate expression of the heterologous adaptor (and/or the heterologous receptor) .
  • the heterologous promoter can be a small molecule-response promoter, such as a tetracycline response promoter (TRE) .
  • TRE tetracycline response promoter
  • the gene encoding the heterologous adaptor as disclosed herein can be genomically integrated in the engineered immune cell. In some embodiments, the gene encoding the heterologous adaptor as disclosed herein may not be genomically integrated. For example, such gene can be in a heterologous plasmid within the engineered immune cell.
  • the gene encoding the heterologous receptor as disclosed herein can be genomically integrated in the engineered immune cell. In some embodiments, the gene encoding the heterologous receptor as disclosed herein may not be genomically integrated. For example, such gene can be in a heterologous plasmid within the engineered immune cell.
  • the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise a first polynucleotide sequence encoding the heterologous adaptor and a second polynucleotide sequence encoding the heterologous receptor.
  • the first polynucleotide sequence and the second polynucleotide sequence can be part of the same nucleic acid molecule (e.g., part of the same expression cassette) . In such cases, the first polynucleotide sequence and the second polynucleotide sequence can be under the control of the same promoter.
  • the first polynucleotide sequence and the second polynucleotide sequence can be under the control of different promoters.
  • the polynucleotide sequence and the second polynucleotide sequence can be each in a different nucleic acid molecule (e.g., in different expression cassettes) .
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell as disclosed herein can comprise a single engineered immune cell comprising both the heterologous adaptor and the heterologous receptor.
  • the engineered immune cell as disclosed herein can comprise a first engineered immune cell (e.g., a first engineered NK cell) and a second engineered immune cell (e.g., a second engineered NK cell) .
  • the first engineered immune cell can be configured to express and secrete the heterologous as disclosed herein, but may not be configured to express the heterologous receptor.
  • the second engineered immune cell can be configured to express the heterologous receptor, but may not be configured to express the heterologous adaptor.
  • both the first engineered immune cell and the second engineered immune cell can be necessary but may not be individually sufficient to yield a therapeutic effect in a subject (e.g., to treat cancer or tumor in the subject) .
  • the heterologous adaptor as disclosed herein can comprise at least or up to 1, at least or up to 2, at least or up to 3, at least or up to 4, or at least or up to 5 different coupling domains.
  • the heterologous adaptor can comprise a plurality of different coupling domains, and the plurality of different coupling domains can be coupled to the same cell (e.g., the same engineered immune cell as disclosed herein) or can be coupled to different cells.
  • a first coupling domain to couple to the engineered NK cell as disclosed herein and a second coupling domain to couple to a different immune cell (e.g., T cell) .
  • the first coupling domain can be flanked by the second coupling domain and the antigen binding moiety.
  • the antigen binding moiety can be flanked by the first coupling domain and the second coupling domain.
  • the heterologous adaptor as disclosed herein can comprise at least or up to 1, at least or up to 2, at least or up to 3, at least or up to 4, or at least or up to 5 different antigen binding moieties.
  • the heterologous adaptor can comprise a plurality of different antigen binding moieties for targeting a plurality of different antigens.
  • the plurality of different antigens can be presented on a same target cell (e.g., same cancer cell) or different target cells (e.g., a cancerous target cell, and a non-cancerous immune cell) .
  • the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit reduced expression and/or activity level of an endogenous variant of the antigen, as compared to a control immune cell.
  • the engineered immune cell can exhibit reduced expression and/or activity level of an endogenous gene encoding the antigen, as compared to the control immune cell.
  • the heterologous receptor (or the surface receptor) can comprise at least a portion of CD16, or a modification thereof, to recognize and bind to the coupling domain of the heterologous adaptor (e.g., the secreted heterologous adaptor) .
  • CD16 signaling of the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can be enhanced to potentiate one or more activities of the engineered immune cell (e.g., viability, proliferation, survival, cytotoxicity against a target cell such as a cancer cell, etc. ) .
  • the CD16-based heterologous receptor may be designed such that it is constitutively activated to constitutively induce enhanced CD signaling and one or more downstream activities of the engineered immune cell.
  • the coupling between the heterologous adaptor and the CD16-based heterologous receptor can serve as an anchor to allow the engineered immune cell to identify, target, and bind the target cell more efficiently, faster, and/or longer.
  • CD16 signaling e.g., constitutively activated signaling of CD16
  • engineered immune cell e.g., engineered NK cell
  • Enhanced CD16 signaling (e.g., constitutively activated signaling of CD16) of the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be achieved by having non-cleavable CD16 variant in the subject cell.
  • CD16 e.g., CD16a
  • immune cells e.g., NK cells
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the binding between CD16 and the monomeric IgG can induce cleavage of the CD16 protein at a cleavage site near the transmembrane domain, to regulates the cell surface density of CD16 upon immune cell activation.
  • the endogenous CD16 of the engineered immune cell can be modified to enhance its signaling.
  • an enhanced signaling variant of CD16 can be artificially introduced to the engineered immune cell.
  • the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified in its ectodomain (e.g., F176V) via action of a gene editing moiety as disclosed herein, such that the modified CD16 exhibits higher binding affinity to its target (e.g., monomeric IgG) as compared to a natural CD16.
  • a heterologous gene encoding such modified CD16 can be introduced to the cell.
  • the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified via action of a gene editing moiety as disclosed herein, such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling.
  • the cleavage site e.g., position 195-198 in the membrane-proximal region (position 189-212) of CD16 can be modified or eliminated (e.g., CD16 S197P variant as a non-cleavable CD16 variant) .
  • a heterologous gene encoding such modified CD16 can be introduced to the cell.
  • a heterologous gene encoding a heterologous CD16 variant that (i) exhibits higher binding affinity to its target (e.g., monomeric IgG) and (ii) is non-cleavable can be introduced to the cell (i.e., hnCD16) .
  • the heterologous CD16 variant can be a modified CD16 comprising, for example, F176V and S197P, as disclosed herein.
  • the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • a heterologous gene as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein.
  • the heterologous gene may not and need not be integrated into the genome of the engineered cell.
  • the enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., SHP-1) via Western blotting or (ii) expression of a downstream gene (e.g., CD25, IFN-gamma, TNF, etc. ) via Western blotting or PCR techniques.
  • a downstream signaling protein e.g., SHP-1
  • a downstream gene e.g., CD25, IFN-gamma, TNF, etc.
  • the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be greater than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9- fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up or
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the CD16 signaling of the engineered immune cell can be more prolonged (e.g., a longer duration of time of activated CD16 signaling) than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can further comprise or exhibit one or more members of the following: (i) a chimeric polypeptide receptor (e.g., a chimeric antigen receptor (CAR) , an engineered T cell receptor (TCR) , etc.
  • a chimeric polypeptide receptor e.g., a chimeric antigen receptor (CAR)
  • TCR engineered T cell receptor
  • an immune checkpoint inhibitor e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc.
  • hypo-immunity regulator e.g., B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, etc.
  • a hypo-immunity regulator e.g., B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10
  • a safety switch capable of effecting death of the engineered NK cell (e.g., upon a stimulus, such as exposure to an activating moiety, such as light or antibody) ; and/or (vi) a heterologous cytokine (e.g., interleukin (IL) ) and/or a heterologous receptor thereof for enhanced cytokine signaling as compared to the control NK cell.
  • a heterologous cytokine e.g., interleukin (IL)
  • IL interleukin
  • the engineered immune cell can comprise one or more members, two or members, three or more members, four or more members, or all of the members selected from the group consisting of (i) , (ii) , (iii) , (iv) , (v) , and (vi) ( “ (i) - (vi) ” ) as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise/exhibit the chimeric polypeptide receptor (e.g., CAR, engineered TCR, etc. ) comprising the antigen binding moiety capable of binding to the antigen (e.g., an antigen of a target cell) .
  • the chimeric polypeptide receptor e.g., CAR, engineered TCR, etc.
  • the chimeric polypeptide receptor e.g., CAR, engineered TCR, etc.
  • the antigen binding moiety capable of binding to the antigen e.g., an antigen of a target cell
  • A a target antigen of the antigen binding moiety of the chimeric polypeptide receptor and
  • B a target antigen of the antigen binding moiety of the heterologous adaptor as disclosed herein may be the same.
  • the target antigen of the antigen binding moiety of the chimeric polypeptide receptor and (B) the target antigen of the antigen binding moiety of the heterologous adaptor as disclosed herein may be different.
  • the different target antigens may be of the same target cell, or of different target cell types (e.g., an immune cell and a cancer cell, two different cancer cell types, etc. ) .
  • the antigen of the antigen binding moiety of the chimeric polypeptide receptor and/or the antigen binding moiety of the heterologous adaptor as disclosed herein can BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise (or exhibit) the heterologous cytokine (e.g., IL) and/or the heterologous receptor thereof for enhanced signaling of the cytokine in the engineered immune cell, as compared to that in a control NK cell.
  • the heterologous cytokine e.g., IL
  • the heterologous cytokine (e.g., the heterologous IL) and/or the heterologous receptor thereof, as disclosed herein, can be of the same species as that of the engineered immune cell (e.g., the engineered NK cell) .
  • both the heterologous cytokine (and/or the heterologous receptor thereof) and the engineered immune cell can be of human origin.
  • the heterologous cytokine (and/or the heterologous receptor thereof) can be of a different species than that of the engineered immune cell.
  • a heterologous cytokine e.g., the heterologous IL and/or the heterologous receptor thereof, as disclosed herein, can be introduced to the engineered immune cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine and/or the heterologous receptor thereof to the engineered immune cell.
  • the heterologous polynucleotide can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) .
  • the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell.
  • a mRNA encoding a heterologous cytokine can be introduced (or inserted into) the engineered immune cell.
  • the cytokine as disclosed herein can be IL.
  • An IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of ILs.
  • An IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of ILs.
  • the IL can be a single type of IL.
  • the IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.
  • the IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof.
  • the engineered immune cell e.g., an engineered NK cell
  • the engineered immune cell can comprise at least a portion of heterologous variant of any one of the IL as disclosed herein, such as human IL-15 (or a gene encoding thereof) .
  • the engineered immune cell e.g., an engineered NK cell
  • the engineered immune cell can comprise at least a portion of heterologous variant of a receptor of any one of the IL as disclosed herein, such as human IL-15 receptor (IL-15R) (or a gene encoding thereof) .
  • IL-15R human IL-15 receptor
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine.
  • the heterologous cytokine may not and need not be secreted by the engineered immune cell.
  • the heterologous cytokine can be bound to a cell surface of the engineered immune cell.
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine.
  • An expression cassette encoding the heterologous cytokine can be introduced to the engineered immune cell.
  • the expression cassette can further encode an additional heterologous polypeptide, e.g., a heterologous receptor.
  • a first polynucleotide sequence encoding the heterologous cytokine and a second polynucleotide sequence encoding the additional heterologous polypeptide (e.g., the heterologous receptor) can be coupled to each other via a polynucleotide linker encoding a cleavage linker.
  • the heterologous receptor can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) .
  • the expression cassette may not and need not encode any additional heterologous polypeptide other than the heterologous cytokine.
  • a cleavable linker as disclosed herein can comprise a self-cleaving peptide, such as a self-cleaving 2A peptide.
  • Self-cleaving peptides can be found in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , Thosea asigna virus (TaV) and porcine tescho virus-1 (PTV-I) , and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses.
  • Non-limiting examples of the self-cleaving 2A peptide can include “F2A” , “E2A” , “P2A” , “T2A” , and functional variants thereof.
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be bound to a cell surface the engineered immune cell (e.g., the engineered NK cell) .
  • the engineered immune cell can be genetically modified such that a heterologous polynucleotide sequence encoding the heterologous cytokine is coupled to a gene encoding an endogenous transmembrane protein of the engineered immune cell.
  • the endogenous transmembrane protein can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) .
  • an expression cassette encoding a heterologous fusion polypeptide comprising (i) the heterologous cytokine that is coupled to (ii) a heterologous receptor can be introduced to the engineered immune cell.
  • the heterologous cytokine may not and need not be cleavable from the heterologous receptor.
  • Non-limiting examples of the heterologous receptor can include a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) , or a different receptor such as a common gamma chain ( ⁇ C ) receptor or a modification thereof.
  • An expression cassette as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein.
  • the expression cassette may not and need not be integrated into the genome of the engineered cell.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine (e.g., the heterologous IL, such as the heterologous IL-15) and/or the heterologous receptor (e.g., the heterologous IL receptor, such as the heterologous IL-15R) as disclosed herein.
  • the enhanced signaling of the endogenous signaling pathway as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., JAK3, STAT3, STAT5, etc.
  • a downstream gene e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng, etc. for IL-15/IL-15R
  • PCR polymerase chain reaction
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or
  • the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) .
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the engineered NK cell can be treated with inhibitors (e.g., small molecule inhibitors) of the endogenous cytokine signaling.
  • the engineered NK cell can comprise reduced expression of endogenous IL (e.g., endogenous IL-17) or endogenous receptor thereof (e.g., via indel or transgene mutation, via transient or permanent suppression, etc. ) .
  • the engineered NK cell can comprise reduced expression of endogenous IL-17.
  • the engineered NK cell can comprise reduced expression of endogenous IL-17R.
  • the engineered NK cell can comprise reduced expression of endogenous IL-17 and endogenous IL-17R.
  • the endogenous cytokine as disclosed herein can be an endogenous IL.
  • An endogenous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of endogenous ILs.
  • An endogenous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of endogenous ILs.
  • the endogenous IL can be a single type of endogenous IL.
  • Non-limiting examples of the endogenous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.
  • the endogenous IL can be IL-17.
  • Non-limiting examples of endogenous Il-17 can include IL-17A, IL-17F, and natural mutations thereof.
  • the engineered immune cell e.g., an engineered NK cell
  • the engineered immune cell as disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F.
  • an endogenous gene encoding the endogenous cytokine e.g., an endogenous IL, such as IL-17
  • an endogenous cytokine e.g., an endogenous IL, such as IL-17
  • a gene editing moiety as disclosed herein.
  • the endogenous receptor can be a respective receptor of any cytokine as disclosed herein (e.g., a respective receptor of any IL as disclosed herein) .
  • the endogenous receptor can be a respective receptor of IL (e.g., IL-17R for IL-7 signaling) .
  • IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof.
  • the endogenous IL-17R comprises IL-17RA.
  • the reduced expression or activity of the endogenous cytokine e.g., an endogenous IL, such as IL-17
  • endogenous receptor thereof as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) expression of a downstream gene via Western blotting or PCT techniques.
  • a downstream signaling protein e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc.
  • a downstream gene of IL cytokine can include a chemokine (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc. ) , a cytokine (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc. ) , an acute phase response molecule (e.g., SAA, CRP, lipocalin 2/24p3, etc. ) , and/or an enzyme (e.g., a metalloproteinase, such as MMP1, MMP3, MMP9, MMP13) .
  • a chemokine e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.
  • a cytokine e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in phosphorylation of a downstream signaling protein of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in the expression of a downstream gene of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein.
  • a specific cell marker for committed NK cells can include CD57 or killer immunoglobulin-like receptors (KIR) .
  • KIR can comprise KIR2D and/or KIR3D.
  • KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5.
  • KIR3D can comprise KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.
  • the enhanced expression profile of the specific cell marker for the committed immune cell (e.g., CD57 or KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
  • the expression of the specific cell marker for a committed immune cell (e.g., CD57 or KIR or NK cells) in the engineered immune cell of the present disclosure can be greater than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced survival in the presence of tumor cells as compared to a control cell lacking at least the heterologous adaptor and/or the heterologous receptor.
  • the engineered immune cell can, in the presence of tumor cells, survive longer than the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit reduced expression or activity of one or more immune checkpoint inhibitors (e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc. ) .
  • the one or more immune checkpoint inhibitors can be endogenous to the engineered immune cell.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous CTLA-4.
  • the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous TIM-3.
  • the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous KIR2D.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell (e.g., the engineered NK cell) can exhibit reduced expression and/or activity of endogenous NKG2A.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit reduced expression and/or activity of endogenous TIGIT.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit reduced expression and/or activity of endogenous TGF beta receptor.
  • the engineered immune cell e.g., the engineered NK cell
  • the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96, TGF beta receptor, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit reduced expression or activity of one or more hypo-immunity regulators (e.g., one or more endogenous immune regulator polypeptides) , as disclosed herein.
  • one or more hypo-immunity regulators e.g., one or more endogenous immune regulator polypeptides
  • the one or more hypo-immunity regulators can be selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the expression or activity of the hypo-immunity regulator (e.g., the endogenous hypo-immunity regulator) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than the same in a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD38 as compared to a control cell.
  • Such engineered immune cell may be used to treat a subject who has or is suspected of having white blood cell cancer, such as multiple myeloma (MM) .
  • MM multiple myeloma
  • any one of the engineered immune cell e.g., the engineered NK cell
  • expression or activity of endogenous CD38 of the engineered immune cell may not and need not be modified.
  • Such engineered immune cell may be used to treat a subject who has or is suspected of having a disease (e.g., cancer, tumor) that is not multiple myeloma.
  • the engineered immune cell (e.g., the engineered NK cell) can comprise the chimeric polypeptide receptor (or at least one chimeric polypeptide receptor) comprising an antigen binding moiety capable of binding to an antigen, as provided in the present disclosure.
  • the engineered immune cell can comprise a plurality of different chimeric polypeptide receptors to specifically bind a plurality of different antigens.
  • the engineered immune cell can comprise at least one chimeric polypeptide receptor that comprises a plurality of antigen binding moieties to specifically bind a plurality of different antigens.
  • the engineered immune cell can comprise a safety switch capable of effecting death of the engineered immune cell.
  • the engineered immune cell can comprise a gene encoding the safety switch (e.g., integrated into the genome of the immune cell) , via action of the gene editing moiety, as disclosed herein.
  • a prodrug can be introduced to the engineered immune cell (e.g., administered to a subject comprising the engineered immune cell) in the event of an adverse event or when the adaptive immunotherapy is no longer necessary, and the prodrug can be activated by the safety switch molecule to kill the subject immune cell.
  • the safety switch can comprise one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and functional variants thereof.
  • the safety switch can be activated via an activator (e.g., a small molecule or a protein, such as an antibody) for post-translational, temporal, and/or site-specific regulation of death (or depletion) of the subject engineered immune cell.
  • an activator e.g., a small molecule or a protein, such as an antibody
  • Non-limiting examples of a safety switch and its activator can include Caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV) ; and cytosine deaminase (CD) and 5-fluorocytosine (5-FC) .
  • Caspase 9 or caspase 3 or 7
  • AP1903 thymidine kinase
  • GCV ganciclovir
  • CD cytosine deaminase
  • 5-FC 5-fluorocytosine
  • modified epidermal growth factor receptor (EGFR) containing epitope recognized by an antibody e.g., anti-EGFR Ab, such as cetuximab
  • an antibody e.g., anti-EGFR Ab, such as cetuximab
  • the engineered immune cells e.g., the engineered NK cells
  • the engineered immune cells can comprise a safety switch protein selected from the group consisting of caspase 9 (caspase 3 or 7) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.3
  • the engineered immune cell can exhibit enhanced cytotoxicity against a target cell as compared to a control cell.
  • the engineered immune cell as disclosed herein can exhibit cytotoxicity (e.g., in vitro, ex vivo, or in vivo) against a target cell or a target population of cells that is greater than that of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or
  • the engineered immune cell can induce reduced immune response from separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host) as compared to a control cell.
  • separate immune cells e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host
  • the engineered immune cell as disclosed herein can reduce the immune response from the separate immune cells by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold,
  • the engineered immune cell can exhibit enhanced half-life upon exposure to separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host) as compared to a control cell.
  • separate immune cells e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune cell to the host
  • the half-life of the engineered immune cells can be greater than that of the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about
  • the engineered immune cell can effect enhanced function or pathological condition of a bodily tissue of a subject as compared to a control cell.
  • treatment with the engineered immune cell can effect enhanced function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold,
  • the engineered immune cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject as compared to a control cell.
  • treatment with the engineered immune cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up
  • the bodily tissue can comprise one or more members selected from the group consisting of blood, plasma, serum, urine, perilymph fluid, feces, saliva, semen, amniotic fluid, cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bone, heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine, liver, pancreas, spleen, kidney, gall bladder, thyroid gland, adrenal gland, mammary gland, ovary, prostate gland, testicle, skin, adipose, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.
  • the bodily tissue can comprise one or more members selected from the group consisting of blood, plasma, serum, urine, perilymph fluid, feces, saliva, semen, amniotic fluid, cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bone, heart,
  • the engineered immune cell can induce immune response towards a target cell.
  • the target can be, for example, a diseased cell, a cancer cell, a tumor cell, etc.
  • regulation of a target gene can be regulated by a gene editing moiety as disclosed herein.
  • the regulation can be enhancing (or activating) or reducing (or inhibiting) the expression level and/or the activity level of the target gene.
  • the regulation can comprise cleaving at least a portion of the target gene via action of the gene editing moiety (e.g., CRISPR-Cas system with a guide nucleic acid molecule, such as a guide RNA molecule) , to edit the at least the portion of the target gene.
  • the regulation may not require cleaving any portion of the target gene via action of the gene editing moiety.
  • the gene editing moiety e.g., dCas-activator or dCas-inhibitor fusion protein, along with a guide nucleic acid molecule
  • the gene editing moiety can form a complex with the target gene, to either (A) activate expression of the target gene or (B) hinder or inhibit expression of the target gene.
  • a heterologous gene can be operatively coupled to (e.g., for knock-in) a constitutive, inducible, temporal, tissue-specific, and/or cell type-specific promoter.
  • a promoter of interest can include CMV, EF1a, PGK, CAG, and UBC.
  • Non-limiting examples of an insertion site can include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • TME tumor microenvironment gene
  • having reduced expression or activity of a TME can enhance the engineered immune cell’s immune activity against a target cell.
  • a TME gene may be an immune checkpoint inhibitor.
  • Non-limiting examples of the TME can include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGF beta receptor, 2B4, and SHIP2.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in
  • enhanced function CD137, CD80, CD86, DAP10 (e.g., with or without point mutation) .
  • any one of the engineered immune cell e.g., the engineered NK cell
  • endogenous genes for, e.g., hypo-immunity: B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) .
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • heterologous genes e.g., knocked-in for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in: CD3, CD4, CD80, 41BBL, and CD131.
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can comprise a chimeric polypeptide receptor as disclosed herein (e.g., at least 1, 2, 3, 4, 5, or more different types of chimeric polypeptide receptors) .
  • the engineered immune cell can be engineered to express a chimeric polypeptide receptor transiently or permanently.
  • a recombinant chimeric polypeptide receptor can be delivered to the engineered immune cell via, e.g., a liposome, and be incorporated into the engineered immune cell via membrane fusion.
  • a heterologous polynucleotide construct encoding the chimeric polypeptide receptor can be delivered to the engineered immune cell.
  • the heterologous polynucleotide construct i.e., a gene
  • encoding the heterologous polynucleotide construct can be incorporated into the chromosome of the engineered immune cell (i.e., chromosomal gene) or, alternatively, may not or need not be integrated into the chromosome of the engineered immune cell as disclosed herein.
  • a chimeric polypeptide receptor can comprises a T cell receptor fusion protein (TFP) .
  • T cell receptor fusion protein or “TFP” generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of TCR extracellular domain, (iii) at least a portion of TCR transmembrane domain, and (iv) at least a portion of TCR intracellular domain.
  • an endogenous T cell receptor (TCR) of the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be inactivated.
  • a function of the endogenous TCR of the engineered immune cell can be inhibited by an inhibitor.
  • a gene encoding a subunit of the endogenous TCR can be inactivated (e.g., edited via action of the gene editing moiety as disclosed herein) such that the endogenous TCR is inactivated.
  • the gene encoding the subunit of endogenous TCR can be one or more of: TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • a chimeric polypeptide receptor can comprises a chimeric antigen receptor (CAR) .
  • CAR chimeric antigen receptor
  • the term “chimeric antigen receptor” or “CAR” generally refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular or intrinsic signaling domain” ) comprising a functional signaling domain derived from a stimulatory molecule.
  • the stimulatory molecule may be the zeta chain associated with the T cell receptor complex.
  • the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule.
  • the costimulatory molecule may comprise 4-1BB (i.e., CD137) , CD27, and/or CD28.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • a CAR may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof.
  • First-generation CARs include an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody) , a transmembrane domain derived from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor) , and a signaling domain derived from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ⁇ receptor or Fc ⁇ RI ⁇ ) .
  • an adaptive immune receptor e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ⁇ receptor or Fc ⁇ RI ⁇
  • Second-generation CARs modify the first-generation CAR by addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137/4-1BB, and CD134/OX40) , which abrogates the need for administration of a co-factor (e.g., IL-2) alongside a first-generation CAR.
  • Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3 ⁇ -CD28-OX40, or CD3 ⁇ -CD28-41BB) .
  • Fourth-generation CARs modify second-or third-generation CARs by the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3 ⁇ ITAM domain) or under the control of a CAR-induced promoter (e.g., the NFAT/IL-2 minimal promoter) .
  • a CAR may be a new generation CAR system that is different than the first-, second-, third-, or fourth-generation CAR system as disclosed herein.
  • a hinge domain (e.g., the linker between the extracellular antigen binding domain and the transmembrane domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • a transmembrane domain of a CAR in the engineered immune cell can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • the hinge domain and the transmembrane domain of a CAR as disclosed herein can be derived from the same protein (e.g., CD8) .
  • the hinge domain and the transmembrane domain of the CAR as disclosed herein can be derived from different proteins.
  • a signaling domain of a CAR can comprise at least or up to about 1 signaling domain, at least or up to about 2 signaling domains, at least or up to about 3 signaling domains, at least or up to about 4 signaling domains, at least or up to about 5 signaling domains, at least or up to about 6 signaling domains, at least or up to about 7 signaling domains, at least or up to about 8 signaling domains, at least or up to about 9 signaling domains, or at least or up to about 10 signaling domains.
  • a signaling domain (e.g., a signaling peptide of the intracellular signaling domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • the signaling domain CAR in the engineered immune cell can comprise a full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell comprises the chimeric polypeptide receptor (e.g., CAR) that comprises at least CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain.
  • the engineered cell e.g., the engineered NK cell
  • the chimeric polypeptide receptor e.g., TFP or CAR
  • the 2B4 signaling domain can be flanked by the CD8 transmembrane domain and the DAP10 signaling domain.
  • the DAP10 signaling domain can be flanked by the CD8 transmembrane domain and the 2B4 signaling domain.
  • the chimeric polypeptide receptor as disclosed herein can further comprise yet an additional signaling domain derived from CD3 ⁇ .
  • An antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor can be a cell surface marker, a secreted marker, or an intracellular marker.
  • Non-limiting examples of an antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) as disclosed herein can include ADGRE2, carbonic anhydrase IX (CA1X) , CCRI, CCR4, carcinoembryonic antigen (CEA) , CD3 ⁇ , CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD133, CD138, CD269 (BCMA) , CD S, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM)
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 1-40- ⁇ -amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1) , B7-H3, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphoma cell, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX) , cardiac myosin, CCL11 (eotaxin-1) , CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin) , CD15, CD152, CD154 (CD40L)
  • coli shiga toxin type-1 E. coli shiga toxin type-2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor ⁇ -chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54) , IFN- ⁇
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2) , abl-bcr alb-b4 (b3a2) , adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, b-Catenin, bcr-abl, bcr-abl p190 (e1a2) , bcr-abl p210 (b2a2) , bcr-abl p210 (b3a2) , BING-4, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an antibody, a fragment thereof, or a variant thereof.
  • antibody can be a natural antibody (e.g., naturally secreted by a subject’s immune cell, such as B cells) , a synthetic antibody, or a modified antibody.
  • the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an Fc domain of an antibody from the group comprising 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtu
  • the heterologous adaptor can exhibit specific binding to CD20.
  • the heterologous adaptor can comprise a light chain or a derivative thereof, such as, for example, a full length light chain, a fragment of the light chain, the CDRs of the light chain, or modifications thereof.
  • the heterologous adaptor can comprise a heavy chain or a derivative thereof, such as, for example, a full length light chain, a fragment of the light chain, the CDRs of the light chain, or modifications thereof.
  • the light chain of the heterologous adaptor can comprise an amino acid sequence that is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or
  • the heavy chain of the heterologous adaptor can comprise an amino acid sequence that is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain
  • the antigen binding domain can be capable of binding specifically and preferentially to an antigen comprising one or more members selected from the group comprising BCMA, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • the NKG2D ligand comprises one or more members selected from the group comprising of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain capable of specifically binding an antigen of a target cell, and the engineered immune cell can exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor.
  • a population of the engineered immune cells can avoid targeting and killing each other, e.g., upon administration to a subject in need thereof.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell ’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • the subject engineered immune cells comprising the chimeric polypeptide receptor against CD38 can be capable of targeting and effecting death (or degradation) of plasma cells.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell is an engineered NK cell that is derived from an isolated ESC or an induced stem cell (e.g., iPSC) .
  • the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • any one of the engineered immune cell e.g., the engineered NK cell disclosed herein can be derived from an isolated stem cell (e.g., an ESC) or an induced stem cell (iPSC) .
  • the isolated stem cell or the induced stem cell can be modified (e.g., genetically modified) to generate the engineered immune cell.
  • pluripotency of stem cells can be determined, in part, by assessing pluripotency characteristics of the cells.
  • Pluripotency characteristics can include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only) , SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm) ; (v) teratoma formation consisting of the three somatic lineages; and (
  • stem cells e.g., ESCs or iPSCs
  • the stem cells can be genetically modified to express any one of the heterologous polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation.
  • the stem cells can be genetically modified to reduce expression or activity of any one of the endogenous genes or polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation.
  • such genetically modified CD34+ hematopoietic stem cell is or is a source of any one of the engineered immune cell of the present disclosure.
  • stem cells as disclosed herein can be cultured in APEL media with ROCKi (Y-27632) (e.g., at about 10 micromolar ( ⁇ M) ) , SCF (e.g., at about 40 nanograms per milliner (ng/mL) of media) , VEGF (e.g., at about 20 ng/mL of media) , and BMP-4 (e.g., at about 20 ng/mL of media) to differentiate into CD34+ hematopoietic stem cells.
  • ROCKi Y-27632
  • SCF e.g., at about 40 nanograms per milliner (ng/mL) of media
  • VEGF e.g., at about 20 ng/mL of media
  • BMP-4 e.g., at about 20 ng/mL of media
  • the CD34+ hematopoietic stem cells (e.g., genetically modified with one or more features of any one of the engineered immune cell of the present disclosure) can be induced to differentiate in to a committed immune cell, such as T cells or NK cells.
  • a committed immune cell such as T cells or NK cells.
  • the induced differentiation process generates any one of the engineered NK cell of the present disclosure.
  • genetically modified CD34+ hematopoietic stem cells are cultured in the presence of IL-3 (e.g., about 5 ng/mL) , IL-7 (e.g., about 20 ng/mL) , IL-15 (e.g., about 10 ng/mL) , SCF (e.g., about 20 ng/mL) , and Flt3L (e.g., about 10 ng/mL) to differentiate into CD45+ NK cells.
  • IL-3 e.g., about 5 ng/mL
  • IL-7 e.g., about 20 ng/mL
  • IL-15 e.g., about 10 ng/mL
  • SCF e.g., about 20 ng/mL
  • Flt3L e.g., about 10 ng/mL
  • the CD45+ NK cells can be expanded in culture, e.g., in a media comprising IL-2, mbIL-21 aAPC using Gas Permeable Rapid Expansion (G-Rex) platform.
  • G-Rex Gas Permeable Rapid Expansion
  • iPSC-derived NK cells as disclosed herein can be cultured with one or more heterologous cytokines comprising Il-2, IL-15, or IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with (e.g., for cell expansion) one or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21.
  • iPSC-derived NK cells as disclosed herein can be cultured with two or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-2 and IL-21, or IL-15 and IL-21) , either simultaneously or sequentially in any order.
  • iPSC-derived NK cells as disclosed herein can be cultured with all of Il-2, IL-15, and IL-21, either simultaneous or sequentially in any order.
  • the gene editing moiety as disclosed herein can comprise a CRISPR-associated polypeptide (Cas) , zinc finger nuclease (ZFN) , zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN) , transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP) , an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.
  • Cas CRISPR-associated polypeptide
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding proteins
  • Argonaute protein any derivative thereof, any variant thereof, or any fragment thereof.
  • the actuator moiety comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein.
  • the actuator moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein.
  • the gRNA comprises a targeting segment which exhibits at least 80%sequence identity to a target polynucleotide.
  • the Cas protein substantially lacks DNA cleavage activity.
  • a suitable gene editing moiety comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN) ; transcription activator-like effector nucleases (TALEN) ; meganucleases; RNA-binding proteins (RBP) ; CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo) , archaeal Argonaute (aAgo) , and
  • Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD) , Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12) , Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA) , Cse2 (CasB) , Cse3 (CasE) , Cse4 (CasC) , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, C
  • the gene editing moiety as disclosed herein can be fused with an additional functional moiety (e.g., to form a fusion moiety) , and non-limiting examples of a function of the additional functional moiety can include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristo
  • gene editing e.g., knock in
  • delivery of heterologous genetic material can be achieved other viral and non-viral based gene transfer methods can be used to introduce nucleic acids in host cells (e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein) .
  • host cells e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein
  • Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism) .
  • Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell.
  • Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., a transcript of a vector described herein) , naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • RNA e.g., a transcript of a vector described herein
  • naked nucleic acid e.g., naked nucleic acid
  • nucleic acid complexed with a delivery vehicle such as a liposome.
  • RNA or DNA viral based systems can be used to target specific cells and traffick the viral payload to the nucleus of the cell.
  • Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo) . Alternatively, viral vectors can be administered directly (in vivo) to the subject.
  • Viral based systems can include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, which can result in long term expression of the inserted transgene.
  • Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.
  • antisense oligonucleotides can be utilized to suppress or silence a target gene expression.
  • Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA) , microRNA (miRNA) , and small interfering RNA (siRNA) .
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be combined with a co-therapeutic agent to treat a subject in need thereof.
  • the engineered immune cell can be administered to the subject prior to, concurrent with, or subsequent to administration of the co-therapeutic agent to the subject.
  • the present disclosure provides a composition
  • a composition comprising (a) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (b) a co-therapeutic agent (i.e., a separate therapeutic agent) (e.g., an antibody, such as anti-CD20 antibody or anti-PD1 antibody) .
  • a co-therapeutic agent i.e., a separate therapeutic agent
  • an antibody such as anti-CD20 antibody or anti-PD1 antibody
  • the engineered immune cell can comprise one or more of: (i) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein, (ii) a CD16 variant for enhanced CD16 signaling as disclosed herein, and (iii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclose herein.
  • the co-therapeutic agent comprises an anti-CD20 antibody.
  • the engineered immune cell can comprise the heterologous cytokine (e.g., IL-15) as disclosed herein and one or both of: (ii) the CD16 variant for enhanced CD16 signaling and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.
  • heterologous cytokine e.g., IL-15
  • the engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.
  • the heterologous cytokine e.g., IL-15
  • the chimeric polypeptide receptor comprising the antigen binding moiety.
  • the engineered immune cell can comprise the chimeric polypeptide receptor comprising the antigen binding moiety and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (ii) the CD16 variant for enhanced CD16 signaling.
  • the heterologous cytokine e.g., IL-15
  • the CD16 variant for enhanced CD16 signaling e.g., CD16 signaling.
  • Non-limiting examples of a co-therapeutic agent can include cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., Pembrolizumab) platelet derived growth factor inhibitors (e.g., GLEEVEC TM (imatinib mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR- ⁇ , BlyS, APRIL, BCMA receptor (s) , TRAIL/Apo2, other bioactive and organic chemical agents, and the like.
  • anti-CD20 antibodies e.g., Pembrolizumab
  • platelet derived growth factor inhibitors e.g
  • cytotoxic agent generally refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • a cytotoxic agent can include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu) , chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide) , doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.
  • radioactive isotopes e.g., At211, I131, I125,
  • Non-limiting examples of a chemotherapeutic agent can include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; delta-9-tetrahydrocannabinol (dronabinol, ); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan CPT-11 (irinotecan, ) , acetylcampto
  • ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill. ) , and docetaxel ( Rorer, Antony, France) ; chloranbucil; gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine oxaliplatin; leucovovin; vinorelbine novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoids such as retinoic acid; capecitabine pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as
  • chemotherapeutic agent can also include “anti-hormonal agents” or “endocrine therapeutics” that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves.
  • Examples include anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (including tamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs) ; agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example,
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, or ) , etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; as well as troxacitabine (a 1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR) ; vaccines such as vaccine and gene therapy vaccines, for example, vaccine, vaccine, and vaccine; topoisomerase 1 inhibitor; rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016)
  • Examples of a chemotherapeutic agent can also include antibodies such as alemtuzumab (Campath) , bevacizumab ( Genentech) ; cetuximab ( Imclone) ; panitumumab ( Amgen) , rituximab ( Genentech/Biogen Idec) , pertuzumab ( 2C4, Genentech) , trastuzumab ( Genentech) , tositumomab (Bexxar, Corixia) , and the antibody drug conjugate, gemtuzumab ozogamicin ( Wyeth) .
  • antibodies such as alemtuzumab (Campath) , bevacizumab ( Genentech) ; cetuximab ( Imclone) ; panitumumab ( Amgen) , rituximab ( Genentech/Biogen Idec) , pertuzumab ( 2C4, Genentech) ,
  • Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolov
  • Examples of a chemotherapeutic agent can also include “tyrosine kinase inhibitors” such as an EGFR-targeting agent (e.g., small molecule, antibody, etc. ) ; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724, 714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI) ; dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline) , an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis) ; pan-HER inhibitors such as canertinib (CI-1033; Pharmacia) ; Raf-1 inhibitors such as antis
  • Examples of a chemotherapeutic agent can also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, opr
  • Examples of a chemotherapeutic agent can also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene
  • growth inhibitory agent generally refers to a compound or composition which inhibits growth and/or proliferation of a cell (e.g., a cell whose growth is dependent on PD-L1 expression) either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) , such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ( (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy- ⁇ -L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthacenedione) , epirubicin, daunorubicin, etoposide, and bleomycin.
  • doxorubicin (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy- ⁇ -L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5
  • paclitaxel and docetaxel are anticancer drugs both derived from the yew tree.
  • Docetaxel Rhone-Poulenc Rorer
  • paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be generated from an isolated stem cell (e.g., isolated ESCs, iPSCs, etc. ) .
  • the heterologous adaptor, the heterologous receptor (for forming a complex with the heterologous adaptor) , and/or one or more members of the group consisting of (i) , (ii) , (iii) , (iv) , (v) , and (vi) ( “ (i) - (vi) ” ) , as disclosed herein, can be introduced during any stage (or cellular state) between (and including) (a) the isolated stem cell and (b) the differentiated immune cell state thereof (e.g., a terminally differentiated immune cell state, such as a terminally differentiated NK cell state) .
  • the engineered NK cell can be derived from iPSCs, and the heterologous adaptor, the heterologous receptor, and/or one or more members of (i) - (vi) can be introduced to the cell at (A) the iPSC state, (B) the hematopoietic stem cell state, and/or (C) the NK cell state.
  • the heterologous adaptor, the heterologous receptor, and/or one or more members of (i) - (vi) can be introduced to the cell once during one of (A) , (B) , and (C) .
  • the heterologous adaptor, the heterologous receptor, and/or one or more members of (i) - (vi) can be introduced to the cell multiple times during two or all of (A) , (B) , and (C) .
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be used (e.g., administered) to treat a subject in need thereof.
  • the subject can have or can be suspected of having a condition, such as a disease (e.g., cancer, tumor, tissue degeneration, fibrosis, etc. ) .
  • a cell e.g., a stem cell or a committed adult cell
  • the engineered immune cell can be administered to the subject for adaptive immunotherapy.
  • the subject can be treated (e.g., administered with) a population of engineered immune cells (e.g., engineered NK cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses.
  • engineered immune cells e.g., engineered NK cells
  • the present disclosure provides a method comprising (a) obtaining a cell from a subject; and (b) generating, from the cell, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein.
  • the cell obtained from the subject is ESC.
  • the cell e.g., a fibroblast, such as an adult skin fibroblast
  • the cell is modified and transformed into an iPSC.
  • the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein.
  • the method can further comprise administering to the subject a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
  • a co-therapeutic agent e.g., a chemotherapeutic agent, anti-CD20 antibody, etc.
  • the present disclosure provides a method comprising administering to a subject in need thereof any one of the composition disclosed herein.
  • the composition can comprise (i) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (ii) a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
  • Any one of the methods disclosed herein can be utilized to treat a target cell, a target tissue, a target condition, or a target disease of a subject.
  • a target disease can be a viral, bacterial, and/or parasitic infection; inflammatory and/or autoimmune disease; or neoplasm such as a cancer and/or tumor.
  • a target cell can be a diseased cell.
  • a diseased cell can have altered metabolic, gene expression, and/or morphologic features.
  • a diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell.
  • a diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
  • a variety of target cells can be killed using any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein.
  • a target cell can include a wide variety of cell types.
  • a target cell can be in vitro.
  • a target cell can be in vivo.
  • a target cell can be ex vivo.
  • a target cell can be an isolated cell.
  • a target cell can be a cell inside of an organism.
  • a target cell can be an organism.
  • a target cell can be a cell in a cell culture.
  • a target cell can be one of a collection of cells.
  • a target cell can be a mammalian cell or derived from a mammalian cell.
  • a target cell can be a rodent cell or derived from a rodent cell.
  • a target cell can be a human cell or derived from a human cell.
  • a target cell can be a prokaryotic cell or derived from a prokaryotic cell.
  • a target cell can be a bacterial cell or can be derived from a bacterial cell.
  • a target cell can be an archaeal cell or derived from an archaeal cell.
  • a target cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a target cell can be a pluripotent stem cell.
  • a target cell can be a plant cell or derived from a plant cell.
  • a target cell can be an animal cell or derived from an animal cell.
  • a target cell can be an invertebrate cell or derived from an invertebrate cell.
  • a target cell can be a vertebrate cell or derived from a vertebrate cell.
  • a target cell can be a microbe cell or derived from a microbe cell.
  • a target cell can be a fungi cell or derived from a fungi cell.
  • a target cell can be from a specific organ or tissue.
  • a target cell can be a stem cell or progenitor cell.
  • Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc. ) .
  • Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.
  • Clonal cells can comprise the progeny of a cell.
  • a target cell can comprise a target nucleic acid.
  • a target cell can be in a living organism.
  • a target cell can be a genetically modified cell.
  • a target cell can be a host cell.
  • a target cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell.
  • a target cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell.
  • a target cell can be a pluripotent cell.
  • a target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell.
  • a target cell may be able to develop into a whole organism.
  • a target cell may or may not be able to develop into a whole organism.
  • a target cell may be a whole organism.
  • a target cell can be a primary cell.
  • cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more.
  • Cells can be unicellular organisms. Cells can be grown in culture.
  • a target cell can be a diseased cell.
  • a diseased cell can have altered metabolic, gene expression, and/or morphologic features.
  • a diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell.
  • a diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
  • the target cells may be harvested from an individual by any method.
  • leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc.
  • Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy.
  • An appropriate solution may be used for dispersion or suspension of the harvested cells.
  • Such solution can generally be a balanced salt solution, (e.g., normal saline, phosphate-buffered saline (PBS) , Hank's balanced salt solution, etc.
  • PBS phosphate-buffered saline
  • Buffers can include HEPES, phosphate buffers, lactate buffers, etc.
  • Cells may be used immediately, or they may be stored (e.g., by freezing) . Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10%DMSO, 50%serum, 40%buffered medium) , and/or some other such common solution used to preserve cells at freezing temperatures.
  • Non-limiting examples of cells which can be target cells include, but are not limited to, lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell) , Natural killer cell, cytokine induced killer (CIK) cells (see e.g., US20080241194) ; myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil) , Monocyte/Macrophage, Red blood cell (Reticulocyte) , Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell) , parathyroid (Parathyroid chief cell, Oxyphil cell) , adrenal (Chromaffin cell) , pineal (Pinealocyte) cells; cells of the nervous system, including glial cells
  • Apocrine sweat gland cell odoriferous secretion, sex-hormone sensitive
  • Gland of Moll cell in eyelid specialized sweat gland
  • Sebaceous gland cell lipid-rich sebum secretion
  • Bowman's gland cell in nose washes olfactory epithelium
  • Brunner's gland cell in duodenum enzymes and alkaline mucus
  • Seminal vesicle cell secretes seminal fluid components, including fructose for swimming sperm
  • Prostate gland cell secretes seminal fluid components
  • Bulbourethral gland cell massbourethral gland cell
  • Bartholin's gland cell vaginal lubricant secretion
  • Gland of Littre cell Gland of Littre cell
  • Uterus endometrium cell (carbohydrate secretion)
  • Isolated goblet cell of respiratory and digestive tracts micus secretion
  • Duct cell (of seminal vesicle, prostate gland, etc. ) , Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell) , Megakaryocyte (platelet precursor) , Monocyte, Connective tissue macrophage (various types) , Epidermal Langerhans cell, Osteoclast (in bone) , Dendritic cell (in lymphoid tissues) , Microglial cell (in central nervous system) , Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types) ,
  • the target cell is a cancer cell.
  • cancer cells include cells of cancers including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma
  • the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell.
  • the cancer is of a hematopoietic lineage, such as a lymphoma.
  • the antigen can be a tumor associated antigen.
  • the target cell e.g., B cells
  • the target cell as disclosed herein is associated or is suspected of being associated with an autoimmune disease.
  • the subject being treated with any one of the engineered immune cell (e.g., engineered NK cell) of the present disclosure can have or can be suspected of having an autoimmune disease.
  • Non-limiting examples of an autoimmune disease can include acute disseminated encephalomyelitis (ADEM) , acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplantation rejection, anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome (APS) , autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED) , autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP) , autoimmune thyroid disease, autoimmune urticaria, axonal &neuronal neuropathies, Balo
  • the autoimmune disease comprises one or more members selected from the group comprising rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis, multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves' Disease, Wegener's Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplant
  • the target disease is acute myeloid leukemia (AML) .
  • AML acute myeloid leukemia
  • a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to an antigen (e.g., CD33) as disclosed herein
  • a heterologous cytokine e.g., IL-15
  • CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat AML.
  • the target disease is non-Hodgkin’s lymphoma (NHL) .
  • the target disease is chronic lymphocytic leukemia (CLL) .
  • CLL chronic lymphocytic leukemia
  • the target disease is B-cell leukemia (BCL) .
  • BCL B-cell leukemia
  • any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD19 as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat BCL.
  • the target disease is non-small-cell lung carcinoma (NSCLC) .
  • NSCLC non-small-cell lung carcinoma
  • the target cells form a tumor (i.e., a solid tumor) .
  • a tumor treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20%in size, and/or do not metastasize) .
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks.
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
  • the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more.
  • the tumor is completely eliminated, or reduced below a level of detection.
  • a subject remains tumor free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment.
  • a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment.
  • a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.
  • Example 1 Engineered immune cell for secreting a heterologous adaptor
  • FIG. 1A schematically illustrates use of the engineered immune cell as disclosed herein for targeting the target cell, to kill the target cell.
  • the engineered immune cell can secrete 110 the heterologous adaptor comprising (i) the coupling domain and (ii) the antigen binding moiety.
  • the heterologous adaptor can be an antibody (e.g., a monoclonal antibody) .
  • the engineered immune cell can specifically bind the target cell (or can be recruited towards the target cell) , e.g., to induce killing of the target cell.
  • FIG. 2 schematically illustrates an example use of the described features of FIG. 1.
  • the engineered immune cell is an engineered NK cell derived from a stem cell (e.g., iPSC) .
  • the surface receptor of the engineered NK cell is a heterologous receptor, such as hnCD16.
  • the heterologous adaptor is anti-CD20-IgG1 (or anti-CD20 scFv-Fc) .
  • the engineered NK cell can secrete 210 the heterologous adaptor comprising (i) the coupling domain (e.g., IgG1) and (ii) the antigen binding moiety (e.g., ani-CD20 antibody or scFv) .
  • the engineered NK cell can specifically bind the target cell (or can be recruited towards the target cell) , e.g., to induce killing of the target cell (e.g., tumor cell) .
  • utilizing isolated stem cells can yield mass-production of homogeneous engineered NK cells.
  • a continuous secretion of full-length anti- CD20-IgG1 antibody that targets the CD20 antigen displayed on the surface of malignant and/or normal cancer cells e.g., B lymphocytes
  • the surface receptor can be a heterologous hnCD16 receptor.
  • the heterologous hnCD17 receptor may be a high-affinity 158V, non-cleavable CD16 Fc receptor that is engineered to augment ADCC by preventing CD16 down-regulation and enhancing CD16 binding to the heterologous adaptor (e.g., anti-CD20 antibodies) .
  • the heterologous adaptor e.g., anti-CD20 antibodies
  • FIG. 3 schematically illustrates an example gene construct (e.g., expression cassette) 300 for expression of the heterologous adaptor (e.g., secretory heterologous adaptor) as disclosed herein.
  • the heterologous adaptor encoded by the gene construct 300 can be the anti-CD20 antibody, as described in FIG. 2.
  • the gene construct 300 can comprise a signal peptide (SP) .
  • SP signal peptide
  • the signal peptide for example, can have the following amino acid sequence (SEQ ID NO. 1) :
  • the gene construct 300 can comprise an antigen binding moiety comprising a light chain (e.g., Rituximab light chain) and a heavy chain (e.g., Rituximab heavy chain) that are separated by a self-cleavable peptide sequence (e.g., T2A) .
  • the gene construct 300 can comprise the following amino acid sequence (SEQ ID NO. 2) , which is referred to as “Rituximab heavy chain” domain and encodes the VH, C H 1, hinge, C H 2, and C H 3 of Rituximab:
  • the gene construct 300 can comprise the following amino acid sequence (SEQ ID NO. 3) , which is referred to as “Rituximab light chain” domain and encodes the VH and C L of Rituximab:
  • the self-cleavable peptide sequence (e.g., T2A) can comprise the following amino acid sequence (SEQ ID NO. 4) :
  • the gene construct 300 can comprise of a Fc receptor (e.g., hnCD16) with the following amino acid sequence (SEQ ID NO. 5) :
  • the gene construct 300 can comprise IgG signal peptide of different variations (e.g., IFN-gamma signal peptide, GM-CSF signal peptide, IgK VIII signal peptide) , e.g., with one or more of the following amino acid sequences:
  • IFN-gamma signal peptide SEQ ID NO. 6
  • GM-CSF signal peptide (SEQ ID NO. 7) :
  • IgK VIII signal peptide SEQ ID NO. 8
  • FIG. 15 schematically illustrates an example scFv-Fc construct (e.g., expression cassette) 400 for expression of the heterologous adaptor (e.g., secretory heterologous adaptor) as disclosed herein.
  • the heterologous adaptor encoded by the gene construct 400 can be the anti-CD20 antibody, as described in FIG. 2.
  • the scFv-Fc construct 400 can comprise a signal peptide (SP) .
  • the signal peptide for example, can comprise the following amino acid sequence (SEQ ID NO. 1) :
  • the gene construct 400 can comprise a Fc receptor (e.g., hnCD16) , an antigen binding moiety comprising a light chain (e.g., Rituximab light chain) and a heavy chain (e.g., Rituximab heavy chain) that are separated by a scFv peptide linker (e.g., (G4S) 3 linker) , followed by hinge domain and Fc fusion protein.
  • a Fc receptor e.g., hnCD16
  • an antigen binding moiety comprising a light chain (e.g., Rituximab light chain) and a heavy chain (e.g., Rituximab heavy chain) that are separated by a scFv peptide linker (e.g., (G4S) 3 linker) , followed by hinge domain and Fc fusion protein.
  • a scFv peptide linker e.g., (G4S) 3 linker
  • the Fc receptor (e.g., hnCD16) with the following amino acid sequence (SEQ ID NO. 5) :
  • the Rituximab light chain variable region can comprise the following amino acid sequence (SEQ ID NO. 9) :
  • the Rituximab heavy chain variable region can comprise the following amino acid sequence (SEQ ID NO. 10) :
  • the scFv peptide linker (e.g., (G4S) 3 linker) can comprise the following amino acid sequence (SEQ ID NO. 11) :
  • the hinge domain can comprise the following amino acid sequence (SEQ ID No. 12) :
  • the Fc fusion protein can comprise the following amino acid sequence (SEQ ID No. 13) :
  • Example 2 Antibody dependent cell-mediated cytotoxicity (ADCC) of the engineered immune cell
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell that is capable of secreting the heterologous adaptor, as disclosed herein, can be utilized to induce killing of a target cell via ADCC.
  • various cells e.g., iPSC, iPSC-derived immune cells, such as iPSC-derived NK cells, etc.
  • the heterologous adaptor e.g., anti-CD20-IgG1 antibody
  • a DNA sequence encoding the antigen binding moiety (e.g., anti-CD20 scFv) can be codon optimized for mammalian expression, synthesized with 5’ NdeI and 3’ ClaI restriction sites, and cloned in place of a placeholder (e.g., ZsGreen) in the human-Fc IgG1 or stabilized IgG4 lentiviral vector.
  • a placeholder e.g., ZsGreen
  • the antigen binding moiety e.g., anti-CD20 scFv
  • Lentiviruses can be produced by transient transfection of five plasmids into 293T cells using Polyethyleneimine (PEI) . After virus production by the 293T cells, the virus supernatant can be collected and concentrated using Lenti-X Concentrator, following the manufacturer instructions, and kept frozen at -80 °C.
  • PEI Polyethyleneimine
  • iPSCs or iPSC-derived immune cells can be transduced with the Lentiviruses at a multiplicity of infection of 20 and 10 ⁇ g/mL of Diethylaminoethyl.
  • the ability of iPSCs or derivatives to be transduced is determined to be variable, the number of cells added to the experiment can be corrected by the %of transduction to avoid this bias.
  • Lenti-transduced iPSCs or iPSC-derived immune cells can be maintained (e.g., for two days) . Afterwards, at least a portion of the medium (e.g., 100 microliter) can be removed to dose total IgG or the heterologous adaptor (e.g., comprising anti-CD20 IgG) . Total level of IgG secreted into the medium of transduced cells can be detected using Human IgG ELISA Quantitation Set.
  • recombinant antigen protein e.g., CD20 human-Fc protein
  • streptavidin-HRP e.g., for 1 h
  • the absorbance can be read in accordance with the staining kit (
  • Target cells expressing the antigen can be plated and cultured.
  • the engineered cells can be added to the target cells in various Effector cells: Target cells (E: T) ratio (e.g., 25: 1, 50: 1, 100: 1, etc. ) and incubated (e.g., overnight) .
  • the engineered cells can be removed, and the viability of the target cells can be assayed by MTT assay.
  • the target cells can be incubated (e.g., for 1 h, 37°C) with a solution comprising supernatant of the engineered cells (e.g., 50 ⁇ L) that is adjusted (e.g., adjusted to 0.5 ⁇ g/mL of the respective antibodies or heterologous adaptors) .
  • the cells can be incubated with various NK cells: Target cells (E: T) ratio (e.g., 12.5: 1, 25: 1, 50: 1, etc. ) (e.g., for 4 h, 37 °C) .
  • Lactate dehydrogenase (LDH) can be measured in the supernatant by CytoTox Non-Radioactive Cytotoxicity Assay.
  • Example 3 Antibody dependent cell-mediated cytotoxicity (ADCC) of mammalian cell-secreted heterologous adaptor (e.g., secreted heterologous antibody)
  • ADCC antibody dependent cell-mediated cytotoxicity
  • One or more cells can be engineered to collectively (i) secrete the heterologous adaptor as disclosed herein and (ii) a surface receptor (e.g., a heterologous receptor such as a chimeric antigen receptor or an engineered T cell receptor or NK cell receptor) capable of coupling to a coupling domain of the heterologous adaptor.
  • a surface receptor e.g., a heterologous receptor such as a chimeric antigen receptor or an engineered T cell receptor or NK cell receptor
  • at least one cell of the one or more cells expressing the surface receptor can bind a target cell via action of a complex comprising the secreted heterologous adaptor and the surface receptor.
  • a first cell can be engineered to secrete the heterologous adaptor (e.g., and not express the surface receptor)
  • a second cell can be engineered to express the surface receptor (e.g., and not express and/or secrete the heterologous adaptor) .
  • both the first cell and the second cell can be administered to a subject in need thereof, e.g., to treat cancer.
  • a single cell e.g., T cell, NK cell, etc.
  • such single cell upon such engineering, can be administered to a subject in need thereof, e.g., to treat cancer.
  • a heterologous adaptor can be an antibody or a modification thereof, such as anti-CD20 antibody.
  • 293FT human embryonal kidney cells were transfected (e.g., transiently transfected) with either (i) a negative control vector that does not encode anti-CD20 Ab (e.g., GGV8-hnCD16 or “hnCD16” ) , or (ii) a vector encoding anti-CD20 Ab (e.g., GGV8-hnCD16-anti- CD20 or “hnCD16-anti-CD20” ) , and the cultured media were subsequently collected (e.g., at 2 days after transfection) .
  • a negative control vector that does not encode anti-CD20 Ab e.g., GGV8-hnCD16 or “hnCD16”
  • a vector encoding anti-CD20 Ab e.g., GGV8-hnCD16-anti- CD20 or “hnCD16-anti-CD20”
  • each supernatant was then incubated with Raji cells that express CD20 (e.g., at 4°C for 1 hour) , followed by incubation with FITC-conjugated goat anti-human IgG H&L antibody (e.g., for 30 minutes) .
  • Control supernatants included supernatant from control 293FT cells that were not transfected ( “Ctrl” ) , a solution comprising commercially available anti-CD20 antibody ( “Rituximab (300 nM) ” ) , and a solution comprising a secondary, non-CD20 targeting antibody ( “Secondary AB” ) .
  • the number of detected Raji cells correlated with the amount of the anti-CD20 Ab secreted by the 293FT cells.
  • secreted anti-CD20 antibody was detected in the supernatant of transiently transfected 293FT cells, as measured by FACS binding assay with Raji cells.
  • ADCC mediated by the heterologous adaptor secreted by a mammalian cell
  • the supernatant of the transfected 293FT cells was collected and incubated with (i) effector cells, such as cord blood NK cells (CBNK) and (ii) target cells, such as Raji cells or Daudi cells (e.g., for 6 hours) at various E/T ratios (e.g., 2: 1 or 1: 2) .
  • effector cells such as cord blood NK cells (CBNK)
  • target cells such as Raji cells or Daudi cells
  • E/T ratios e.g., 2: 1 or 1: 2
  • the supernatant was diluted (e.g., 3 times or 15 times) consequentially.
  • a commercially available anti-CD23 antibody e.g., Rituximab at 5 microgram per milliliter
  • IgG1 isotype was used as a negative control.
  • CD16-anti-CD20 supernatant mediated a dose-dependent ADCC function of CBNK against both Raji cells and Daudi cells, at various E/T ratios.
  • the supernatant of the transfected 293FT cells was collected and incubated with (i) effector cells, such as expanded NK cells (eNK) and (ii) target cells, such as Raji cells at 1: 1 E/T ratio.
  • effector cells such as expanded NK cells (eNK)
  • target cells such as Raji cells at 1: 1 E/T ratio.
  • eNK cells mediated ADCC function against Raji cells, as measured by luminescence assay that detects cytotoxicity.
  • NK92 cells e.g., via transfection
  • Cultured media from transfected NK92 cells were collected and concentrated (e.g., by Ultra-4 Centrifugal Filter units with a cutoff at 50 kilodalton) .
  • the concentrated supernatants were then incubated with CD20-expressing Raji cells (e.g., at 4°C for 1 hour) , followed by incubation with FITC-conjugated goat anti-human IgG Fc antibody (e.g., for 30 minutes) .
  • scFv-Fv format of anti-CD20 antibody was detected in the supernatant of NK92 cells, but not the full-length format of anti-CD20 antibody, as measured by FACS binding assay with Raji cells.
  • anti-CD20 mRNA level in the scFv-Fc format was 3.5 times higher than that of the full-length format, which may have contributed to more antibody secretion.
  • iPSCs e.g., wt ANB iPSC clone or OI42 ANB iPSC clone expressing CAR19
  • a vector encoding anti-CD20 Ab e.g., GGV8-hnCD16-anti-CD20 or “hnCD16-anti-CD20”
  • some of the transfected iPSCs e.g., transfected ANB iPSC clones
  • the transfected iPSCs exhibited expression of the heterologous CD16, suggesting that the transfected iPSCs also expressed and secreted the heterologous anti-CD20 antibody.
  • the cultured media of the transfected iPSCs were collected (e.g., without changing medium for 2 days) , and concentrated (e.g., by Ultra-4 Centrifugal Filter units with a cutoff at 50 kilodalton) .
  • the concentrated supernatants were then incubated with CD20-expressing Raji cells (e.g., at 4°C for 1 hour) , followed by incubation with FITC-conjugated goat anti-human IgG Fc antibody (e.g., for 30 minutes) .
  • secreted anti- CD20 antibody was detected in the supernatant of transfected iPSCs, as measured by FACS binding assay with Raji cells.
  • ADCC mediated by the heterologous adaptor secreted by iPSCs
  • the transfected iPSCs cells as aforementioned can be differentiated into NK cells, thus generating engineered NK cells that are each capable of (i) expressing the anti-CD20 antibody and (ii) hnCD16.
  • engineered NK cells can be utilized as effector cells, and be incubated with target cells, such as Raji cells or Daudi cells (e.g., for 6 hours) at various E/T ratios (e.g., 2: 1 or 1: 2) .
  • a commercially available anti-CD23 antibody e.g., Rituximab at 5 microgram per milliliter
  • the engineered NK cells can exhibit enhanced targeting and killing of the target cells as compared to NK cells lacking the secretory anti-CD20 antibody.
  • Example 4 Antibody dependent cell-mediated cytotoxicity (ADCC) of heterologous adaptors secreted by engineered cells
  • Engineered immune cells as disclosed herein can be derived from stem cells, such as iPSCs. Accordingly, such stem cells engineered to secrete the heterologous adaptor can be tested, prior to being subject to immune cell differentiation, for (i) the ability to generate and secrete the heterologous adaptor and/or (ii) the ability of such secreted heterologous adaptor to effect ADCC.
  • iPSC-derived EB induced pluripotent stem cells derived embryoid bodies
  • iPSCs were transfected with a vector encoding anti-CD20 Ab (e.g., GGV8-hnCD16-anti-CD20 or “hnCD16-anti-CD20” , where GGV8 is a cloning vector in which hnCD16-anti-CD20 is cloned into) .
  • a vector encoding anti-CD20 Ab e.g., GGV8-hnCD16-anti-CD20 or “hnCD16-anti-CD20” , where GGV8 is a cloning vector in which hnCD16-anti-CD20 is cloned into
  • Transfected iPSCs expressing either low or high expression of the heterologous CD16 variant (e.g., hnCD16) , were differentiated into embryoid bodies (EB) .
  • Cultured media from iPSC-derived EB were collected and concentrated (e.g., by Ultra-4 Centrifugal Filter units with a
  • the concentrated supernatants were then incubated with CD20-expressing Raji cells (e.g., at 4°C for 1 hour) , followed by incubation with FITC-conjugated goat anti-human IgG Fc antibody (e.g., for 30 minutes) .
  • secreted anti-CD20 antibody was detected in the supernatant of EB differentiated from iPCS with high (H) and low (L) expression of heterologous CD16 variant, as measured by FACS binding assay with Raji cells.
  • ADCC mediated by the heterologous adaptor secreted by iPSC-derived EBs
  • the supernatant of the transfected iPSC-derived EB were collected and incubated with (i) effector cells, such as peripheral blood NK cells (PBNK) and (ii) target cells, such as Raji-GFP cells.
  • effector cells such as peripheral blood NK cells (PBNK)
  • target cells such as Raji-GFP cells.
  • PBNK peripheral blood NK cells
  • RTX Rituximab
  • PBNK with supernatant from hnCD16-antiCD20 EB performed ADCC against Raji-GFP cells across 24 hours, and green fluorescence area was calculated as a measure of tumor size.
  • the ability of the secreted anti-CD20 heterologous adaptor to induce cytotoxicity against the CD20-expressing target cells was comparable to the control anti-CD20 antibody (e.g., a recombinant monoclonal antibody against CD20) .
  • the secreted anti-CD20 heterologous adaptor nor the control anti-CD20 antibody exerted noticeable cytotoxicity against the target cells, demonstrating that the abovementioned cytotoxicity is due to ADCC in combination with the PBNK cells.
  • iPSCs expressing both high and low levels of heterologous CD16 variant e.g., hnCD16
  • iNK cells e.g., at least 95%of iNK cells
  • WT wildtype
  • the supernatant of iNK cells was collected at 2 differentiation stages (iNK at day 12 (D12) and at day 24 (D24) ) , and concentrated (e.g., by Ultra-4 Centrifugal Filter units with a cutoff at 50 kilodalton) .
  • secreted anti-CD20 antibody was detected in the supernatant of iNK D12 and D24, as measured by FACS binding assay with Raji cells..
  • the amount of the heterologous adaptor (e.g., anti-CD20 antibody) secreted by the engineered NK cell may be sufficient to effect ADCC against target cells (e.g., CD20 expressing cancer cells) .
  • ADCC mediated by the heterologous adaptor secreted by expanded iPSC-derived NK cells
  • Antibody e.g., anti-CD20 antibody
  • iNK cells were expanded with K562-41BBL-mlL21 aAPC, and then incubated with either Raji or K562 cells (e.g., for 5 hours) to test cytotoxicity against these target cells.
  • both expanded NK (eNK) cells differentiated from iPSCs with high or low heterologous CD16 variant (e.g., hnCD16) demonstrated higher cytotoxicity than that of WT (e.g., iPSC derived NK cells that do not express hnCD16-aCD20 IgG1) .
  • the anti-CD20 antibody-secreting iNK cells exhibited between about 10%and about 30% (e.g., about 20%for CD16- ⁇ CD20 high, and about 15%for CD16- ⁇ CD20 low) higher cytotoxicity against the CD20-expressing Raji cells as compared to control NK cells that do not express the anti-CD20 antibody, suggesting ADCC mediated by the engineered immune cell as disclosed herein.
  • the anti-CD20 antibody-secreting iNK cells and the control NK cells that do not express the anti-CD20 antibody exhibited comparable cytotoxicity against K562 cells that do not express CD20, further demonstrating the ability of the secreted anti-CD20 antibody by the iNK cell to promote ADCC.
  • compositions of matter including the engineered immune cells of the present disclosure may be utilized in the method section including methods of use and production disclosed herein, or vice versa.

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Abstract

La présente invention concerne des systèmes et des procédés d'immunothérapies. Les cellules immunitaires peuvent être modifiées pour présenter une demi-vie accrue par comparaison avec une cellule témoin (par exemple, une cellule immunitaire non modifiée). Les cellules immunitaires peuvent être modifiées pour présenter une prolifération améliorée par comparaison avec une cellule témoin. Les cellules immunitaires peuvent être modifiées pour cibler efficacement et particulièrement les cellules malades (p. ex., les cellules cancéreuses) qu'une cellule témoin ne peut pas ou ne sait pas cibler. Les cellules immunitaires peuvent être modifiées ex vivo, in vitro, et dans certains cas, in vivo. Les cellules immunitaires modifiées préparées ex vivo ou in vitro peuvent être administrées à un sujet pour traiter une maladie, par exemple un myélome ou des tumeurs solides. Les cellules immunitaires modifiées peuvent être autologues au sujet. En variante, les cellules immunitaires modifiées peuvent être allogéniques au sujet.
PCT/CN2022/133745 2021-11-24 2022-11-23 Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules WO2023093763A1 (fr)

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