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WO2022179562A1 - Récepteurs chimériques à l'antigène dans des cellules immunitaires - Google Patents

Récepteurs chimériques à l'antigène dans des cellules immunitaires Download PDF

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WO2022179562A1
WO2022179562A1 PCT/CN2022/077675 CN2022077675W WO2022179562A1 WO 2022179562 A1 WO2022179562 A1 WO 2022179562A1 CN 2022077675 W CN2022077675 W CN 2022077675W WO 2022179562 A1 WO2022179562 A1 WO 2022179562A1
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cell
fold
engineered
cells
antigen
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Luhan Yang
Yangbin Gao
Jiabiao HU
Xiaomeng BIAN
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Hangzhou Qihan Biotech Co Ltd
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Hangzhou Qihan Biotech 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/49—Breast
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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

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 comprising: a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen binding moiety comprises an ankyrin repeat domain.
  • the antigen comprises an immune cell antigen of an immune cell that is not a NK cell.
  • the immune cell is a T cell.
  • the antigen is selected from the group consisting of CD3, CD4, CD8, CCR7, CD45RO, CR45RA, and a fragment thereof.
  • the antigen is CD3.
  • the antigen is CD4.
  • the antigen is CD8.
  • the antigen is CCR7.
  • the antigen is CD45RO.
  • the antigen is CR45RA.
  • the antigen comprises NY-ESO-1 or a fragment thereof.
  • the antigen comprises CD33 or a fragment thereof.
  • the antigen comprises CD123 or a fragment thereof.
  • the antigen comprises CD4 or a fragment thereof.
  • the antigen binding moiety comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the antigen comprises HER2 or a fragment thereof.
  • the antigen binding moiety comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 5.
  • the antigen comprises EGFR or a fragment thereof.
  • the antigen binding moiety comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of a member selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • the chimeric polypeptide receptor comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 10.
  • the chimeric polypeptide receptor comprises a lead sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 11.
  • the chimeric polypeptide receptor comprises a linker between the antigen binding moiety and a transmembrane domain, wherein the linker is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 12.
  • the chimeric polypeptide receptor comprises a transmembrane domain that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 13.
  • the chimeric polypeptide receptor comprises at least one signaling domain that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
  • the chimeric polypeptide receptor comprises a first signaling domain that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 14, and a second signaling domain that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to the polypeptide sequence of SEQ ID NO: 15.
  • the antigen comprises a cytokine or a fragment thereof. In some embodiments of any one of the engineered NK cell disclosed herein, the antigen comprises a receptor of a cytokine or a fragment thereof. In some embodiments of any one of the engineered NK cell disclosed herein, the cytokine is an interleukin. In some embodiments, the interleukin is selected from the group consisting of IL2, IL3, IL7, IL12, IL15, IL18, and IL21. In some embodiments, the interleukin is IL2. In some embodiments, the interleukin is IL3. In some embodiments, the interleukin is IL7. In some embodiments, the interleukin is IL12. In some embodiments, the interleukin is IL15. In some embodiments, the interleukin is IL18. In some embodiments, the interleukin is IL21.
  • the antigen comprises an intracellular antigen peptide.
  • the intracellular antigen peptide is presented by a major histocompatibility complex.
  • the antigen binding moiety is part of an extracellular domain of the chimeric polypeptide receptor.
  • the chimeric polypeptide receptor comprises a plurality of different antigen binding moieties capable of binding to a plurality of different antigens.
  • the engineered NK cell further comprises an additional chimeric polypeptide receptor that comprises an additional antigen binding moiety capable of binding to an additional antigen, wherein the additional antigen and the antigen are different.
  • the chimeric polypeptide receptor or the one or more chimeric polypeptide receptors comprise a chimeric antigen receptor.
  • the engineered NK cell is derived from a hematopoietic cell obtained from a peripheral blood sample of a subject.
  • the engineered NK cell is derived from an NK cell obtained from a peripheral blood sample of a subject.
  • the engineered NK cell is derived from an isolated stem cell.
  • the isolated stem cell is an embryonic stem cell.
  • the isolated stem cell is an induced pluripotent stem cell.
  • the present disclosure provides a composition comprising any one of the engineered NK cell disclosed herein.
  • the composition further comprises a separate therapeutic agent.
  • the separate therapeutic agent comprises a different antigen binding moiety.
  • the separate therapeutic agent comprises a chemotherapeutic agent.
  • the present disclosure provides a method comprising administering to a subject in need thereof any one of the composition disclosed herein.
  • FIG. 1 schematically illustrates an example chimeric polypeptide receptor comprising an antigen binding moiety that comprises one or more ankyrin repeat domains (e.g., DARPin) , wherein the one or more ankyrin repeat domains are designed to specifically target (e.g., bind) one or more antigens.
  • ankyrin repeat domains e.g., DARPin
  • FIG. 2 schematically illustrates killing of an antigen expressing target cell by an NK cell expressing a CAR comprising a designed ankyrin repeat protein (DARPin) domain against the antigen.
  • DARPin ankyrin repeat protein
  • FIG. 3 schematically illustrates an example polynucleotide construct encoding a CAR comprising a DARPin domain against epidermal growth factor receptor (EGFR) .
  • EGFR epidermal growth factor receptor
  • FIG. 4 schematically illustrates example cell killing assay between various NK cells and various target cells.
  • FIG. 5 shows in vitro killing efficacy of engineered NK cells against target cancer cells.
  • 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.
  • algal cell e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like
  • seaweeds e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like
  • seaweeds e.g.
  • 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. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g.
  • rhodamine or flurescein linked to the sugar thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • genomic DNA 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. ) .
  • 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) .
  • 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, a functional variant thereof (e.g., a designed ankyrin repeat protein (DARPin) ) , 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.
  • designed ankyrin repeat protein generally refers to a synthetic polypeptide comprising one or more ankyrin repeat domains, wherein the one or more ankyrin repeat domains are capable of binding to one or more antigens.
  • the ankyrin repeat domains described herein generally comprise at least one ankyrin repeat motif.
  • the ankyrin repeat motif comprises of two anti-parallel ⁇ -helices followed by a beta-bulge and beta-hairpin containing loop connecting it to the next repeat, each of which has about 33 residues.
  • Recombinant proteins, or binding domains thereof, comprising designed ankyrin repeat motifs may be referred to as DARPin proteins or DARPin polypeptides.
  • the ankyrin repeat domains described herein may comprise (i) a core scaffold that provides structure and (ii) target binding residues that bind to a target (e.g., a target antigen) .
  • the structural core may comprise conserved amino acid residues, and the target binding surface may comprise amino acid residues that differ depending on the target.
  • an ankyrin repeat motif can comprise the following sequence: DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx (SEQ ID NO: 1) , wherein “x” denotes any amino acid.
  • an ankyrin repeat motif can comprise the following sequence: DxxGxTPLHLAxxxGxxxIVxVLLxxGADVNAx (SEQ ID NO: 2) , wherein “x” denotes any amino acid.
  • multiple ankyrin repeat domains can be linked (either through a covalent bond or non-covalent association) to form bispecific or multi-specific molecules (e.g., bispecific or multi-specific chimeric polypeptide receptors) .
  • 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
  • immune regulator polypeptide generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secretory polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc. ) capable of regulating or controlling one or more attributes of an immune cell, such as a NK cell.
  • One or more attributes of an immune cell can comprise differentiation of the immune cell, immune cell morphology, expression of a polynucleotide or polypeptide construct within the immune cell, or activity of the immune cell (e.g., cytotoxic activity of an engineered NK cell against a diseased cell, such as a cancer cell) .
  • An immune regulator polypeptide can be endogenous to a host cell.
  • an immune regulator polypeptide can be heterologous to a hots cell.
  • controlling the one or more attributes of the immune cell can be mediated by downregulating expression of the immune regulator polypeptide (e.g., suppression, knock-down or knock-out) .
  • controlling the one or more attributes of the immune cell can be mediated by upregulating expression of the immune regulator polypeptide (e.g., upregulation of an endogenous gene or knock-in of a heterologous gene encoding the immune regulator polypeptide) .
  • controlling the one or more attributes of the immune cell can be mediated by maintaining expression of the immune regulator polypeptide for time period that is longer than a natural or normal expression profile of the immune regulator polypeptide in a host cell.
  • an immune regulator polypeptide can comprise a hypo-immunity regulator.
  • an immune regulator polypeptide can comprise an immune checkpoint inhibitor.
  • 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
  • NK cells sourced and engineered to exhibit, for example, enhanced proliferation, half-life, and cytotoxic activity against target cells.
  • 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) .
  • the engineered immune cell can comprise a chimeric polypeptide receptor (e.g., at least 1, 2, 3, 4, 5, or more chimeric polypeptide receptors) or comprise a gene (e.g., a heterologous gene) encoding the chimeric polypeptide receptor.
  • the chimeric polypeptide receptor can comprise at least one antigen binding moiety capable of binding to an antigen.
  • the antigen binding moiety can comprise an ankyrin repeat domain.
  • the antigen binding moiety can be a designed ankyrin repeat protein (DARPin) that is configured to specifically bind to one or more antigens.
  • DARPin ankyrin repeat protein
  • the chimeric polypeptide receptor can comprise one antigen binding moiety capable of binding to an antigen.
  • the chimeric polypeptide receptor can comprise a plurality of antigen binding moieties (e.g., at least 2, 3, 4, 5, or more antigen binding moieties that are capable of binding to different antigens) .
  • the DARPin as disclosed herein may exhibit specific binding affinity against (or towards) a target antigen, and the binding affinity between the DARPin and the target antigen (e.g., equilibrium dissociation constants (K D ) , as ascertained by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) ) can be 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 1.1-fold, at least or up to about 1.2-fold, at least or up to about 1.3-fold, at least or up to about 1.4-fold, at least or up to
  • the equilibrium dissociation constant (K D ) between the DARPin as disclosed herein and the target antigen may be at least or up to about 1 x10 -12 molar (M) , at least or up to about 2 x10 -12 M, at least or up to about 3 x10 -12 M, at least or up to about 4 x10 -12 M, at least or up to about 5 x10 -12 M, at least or up to about 6 x10 -12 M, at least or up to about 7 x10 -12 M, at least or up to about 8 x10 -12 M, at least or up to about 9 x10 -12 M, at least or up to about 1 x10 -11 M, at least or up to about 2 x10 -11 M, at least or up to about 3 x10 -11 M, at least or up to about 4 x10 -11 M, at least or up to about 5 x10 -11 M, at least or
  • the ankyrin repeat domain (e.g., DARPin) can be configured to specifically bind an immune cell antigen.
  • the immune cell antigen can be presented by or derived from an immune cell. In some cases, the immune cell may not be an immune cell.
  • the engineered immune cell may be an engineered NK cell, and the ankyrin repeat domain of a chimeric polypeptide receptor of the engineered NK cell can be designed to specifically bind to an immune cell antigen of a T cell, such that the engineered NK cell can recruit the T cell.
  • Non-limiting examples of an immune cell antigen of a T cell can include CD3, CD4, CD8, CCR7, CD45RO, CR45RA, CD45RA, T-cell receptor (TCR) , modifications thereof, and fragments thereof.
  • the engineered immune cell e.g., NK cell
  • the one or more chimeric antigen receptors can comprise two different DARPins for specifically binding (i) an immune cell antigen of a different immune cell (e.g., T cell) and (ii) a different antigen (e.g., a disease-specific antigen, such as, for example, NY-ESO01, CD33, and/or CD123) .
  • the engineered immune cell can comprise one a CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • the engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • a cytokine e.g., a secretory cytokine
  • the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • IL heterologous interleukin
  • the engineered immune cell as disclosed herein can comprise a heterologous receptor or a heterologous gene encoding the heterologous receptor.
  • the heterologous receptor can be a respective receptor of the heterologous cytokine as disclosed herein (e.g., heterologous IL-15 receptor (IL-15R, such as IL-15 ⁇ or IL-15 ⁇ ) for heterologous IL-15) .
  • the engineered immune cell may not and need not comprise any heterologous receptor that is a respective receptor of the heterologous cytokine.
  • the engineered immune cell comprising a heterologous IL e.g., IL-15
  • a heterologous gene or a heterologous polynucleotide, as disclosed herein, can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) .
  • the heterologous gene or a 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 heterologous cytokine as disclosed herein can be a heterologous IL.
  • a heterologous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous ILs.
  • a heterologous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of heterologous ILs.
  • the heterologous IL can be a single type of heterologous IL.
  • Non-limiting examples of the heterologous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, and IL-38.
  • the heterologous IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof.
  • the engineered immune cell e.g., an engineered NK cell
  • the engineered immune cell can comprise at least a portion of heterologous human IL-15 (or a gene encoding thereof) .
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine.
  • 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 (e.g., a fusion comprising at least a portion of IL15 and at least a portion of IL15Ra) .
  • 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.
  • a respective receptor of the heterologous cytokine e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15
  • a different receptor such as a common gamma chain ( ⁇ C ) receptor or a modification thereof.
  • 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
  • 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.
  • 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 as disclosed herein can exhibit reduced expression or activity of an endogenous immune regulator polypeptide.
  • the expression or activity of the endogenous immune regulator polypeptide can be reduced in the engineered immune cell (e.g., the engineered NK cell) , for example, via action of a gene editing moiety as disclosed herein.
  • the reduced expression or activity of the endogenous immune regulator polypeptide in the engineered immune cell can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation or dephosphorylation of a downstream signaling protein (e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling) or (ii) expression of the endogenous immune regulator polypeptide (e.g., PD1) via Western blotting or PCR techniques.
  • a downstream signaling protein e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling
  • a downstream signaling protein e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling
  • expression of the endogenous immune regulator polypeptide e.g., PD1
  • reduced expression of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by a decrease in the expression of the endogenous immune regulator polypeptide (e.g., PD1) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about
  • reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by a decrease in phosphorylation of a downstream signaling protein (e.g., SHP2 for PD1/PDL1 signaling) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or
  • a downstream signaling protein e.g., SHP2 for PD
  • reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by an increase in phosphorylation of a downstream target signaling protein (e.g., Ig ⁇ / ⁇ or Syk for PD1/PDL1 signaling) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-
  • a downstream target signaling protein e.g., Ig ⁇ / ⁇
  • the engineered immune cell as disclosed herein can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) .
  • endogenous cytokine signaling e.g., endogenous IL signaling, such as endogenous IL-17 signaling
  • 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-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 as disclosed herein can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein.
  • 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 as disclosed herein can exhibit one or more of: (i) a heterologous transcription factor (e.g., a heterologous STAT) , and (ii) reduced expression or activity of endogenous enzyme (e.g., a ligase, such as CBL-B) .
  • a heterologous transcription factor e.g., a heterologous STAT
  • endogenous enzyme e.g., a ligase, such as CBL-B
  • the heterologous transcription factor can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factor.
  • the heterologous transcription factor can comprise at most 5, 4, 3, or 2 different types of transcription factor.
  • the heterologous transcription factor can have a single type of transcription factor.
  • the transcription factor can be involved in the engineered immune cell’s immune activity, proliferation, apoptosis, and/or differentiation.
  • the heterologous transcription factor for the engineered immune cell e.g., the engineered NK cell
  • STAT can include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof.
  • STAT can comprise STAT3.
  • STAT can comprise STAT5B.
  • 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 without (i) the heterologous transcription factor (e.g., the heterologous STAT) or (ii) the reduced activity of endogenous cytokine signaling (e.g., endogenous IL-17 signaling) .
  • the heterologous transcription factor e.g., the heterologous STAT
  • endogenous cytokine signaling e.g., endogenous IL-17 signaling
  • 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 as disclosed herein can exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell as disclosed herein (e.g., a NK cell marker, such as KIR) as compared to a control cell.
  • a specific endogenous cell marker is KIR.
  • the reduced expression or activity of the specific endogenous cell marker for the committed immune cell e.g., KIR for NK cells
  • the expression of the specific endogenous cell marker for a committed immune cell (e.g., KIR or NK cells) in the engineered immune cell of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about
  • the engineered immune cell as disclosed herein can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) .
  • the engineered immune cell can exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD94 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD96 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous TGF beta receptor and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous SHIP (e.g., SHIP2) and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iii) endogenous TGF beta receptor.
  • endogenous SHIP e.g., SHIP2
  • endogenous CD94 e.g., 1, 2, or all of
  • the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about
  • the reduced expression or activity of the endogenous CD94 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the reduced expression or activity of the endogenous CD96 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the reduced expression or activity of the endogenous TGF beta receptor in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-
  • the reduced expression or activity of the endogenous SHIP (e.g., SHIP2) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up
  • the engineered immune cell as disclosed herein can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise one or more hypo-immunity regulators.
  • the engineered immune cell exhibits reduced expression or activity of one or more hypo-immunity regulators from: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL.
  • the reduced expression or activity of the endogenous hypo-immunity regulator (e.g., CD80, CD86, ICOSL, CD40L, MICA, MICB, NKG2DL, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-
  • the reduced expression or activity of the endogenous CD80 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the reduced expression or activity of the endogenous CD86 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the reduced expression or activity of the endogenous ICOSL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2- fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold
  • the reduced expression or activity of the endogenous hypo-immunity regulator CD40L in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to
  • the reduced expression or activity of the endogenous MICA or MICB in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about
  • the reduced expression or activity of the endogenous NKG2DL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) .
  • the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • the immune regulator polypeptide comprises a hypo-immunity regulator.
  • the hypo-immunity regulator can be PDL2.
  • the hypo-immunity regulator can be TGF-beta.
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • the immune regulator polypeptide comprises a hypo-immunity regulator.
  • the hypo-immunity regulator can comprise one or more members from: (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CCL21 and one or more of (e.g., 1, 2, 3, or all of) : (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous IL-10 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD46 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD55 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD59 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (iv) a heterologous CD55.
  • the engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell comprises a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) , as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced signaling of the endogenous signaling pathway induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R) as disclosed herein.
  • the heterologous cytokine and/or the heterologous receptor e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R
  • 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 can comprise a heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be secreted by the engineered immune cell.
  • the engineered immune cell can comprise a heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be bound to a cell surface membrane of the engineered immune cell.
  • the engineered immune cell can comprise at least one chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as provided in the present disclosure.
  • 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.
  • the engineered immune cell can comprise heterologous immune receptor polypeptide.
  • the immune regulator polypeptide can comprise one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the engineered immune cell can exhibits reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator (or both) .
  • the immune checkpoint inhibitor as disclosed herein can comprise one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • the immune checkpoint inhibitor can comprise SHIP2.
  • the hypo-immunity regulator as disclosed herein can comprise one or more members selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the engineered immune cell can comprise a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered immune cell.
  • 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.
  • 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, DARPin, etc. ) during cellular processing and localization of the CAR to the cellular membrane.
  • the antigen recognition domain e.g., a scFv, DARPin, etc.
  • 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 a DARPin, 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
  • 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 transmembrane of the CAR as disclosed herein can comprise at least a portion of the transmembrane region of (or a modified variant thereof) CD3d, CD3e, CD3g, CD3c, CD4, CD8a, CD8b, CD27, CD28, CD40, CD80, CD86, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, 2B4, CD16, NKp30, NKp44, NKp46, NKG2C, and/or NKG2D.
  • CD3d CD3d, CD3e, CD3g, CD3c, CD4, CD8a, CD8b, CD27, CD28, CD40, CD80, CD86, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, 2B4, CD16, NKp30, NKp44, NKp46, NKG2C, and/or NKG2D.
  • 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 of the CAR as disclosed herein can comprise one or more (immunoreceptor tyrosine-based activation motif (ITAM) sequences of (or modified variant (s) thereof) CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, 4-1BB, NKp30, NKp44, NKp46, NKG2C, and/or NKG2D.
  • ITAM immunosorbent tyrosine-based activation motif
  • 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.
  • he antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an Fc domain of an antibody from the group comprising 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemt
  • 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 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 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 (a1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR) ; vaccines such as vaccine and gene therapy vaccines, for example, vaccine, vaccine, and vaccine; topoisomerase 1 inhibitor; rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016)
  • 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 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.
  • myeloid cells such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil) , Monocyte/Macrophage, Red blood cell (Reticulocyte) , Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell) , parathyroid (Parathyroid chief cell, Oxyphil cell) , adrenal (Chromaffin cell) , pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia) , Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph) ; cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, granulocyte,
  • 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 cells configured to target immune cell antigens
  • the engineered immune cells of the present disclosure can be configured to specifically target and bind immune cells (e.g., diseased immune cells) of a subject, e.g., to treat leukemia or lymphoma.
  • the engineered immune cells can comprise one or more chimeric polypeptide receptors (e.g., chimeric antigen receptors) exhibiting specific binding to at least one immune cell antigen, e.g., CD3, CD4, CD8, CCR7, CD45RO, CR45RA, CD45RA, T-cell receptor (TCR) , modifications thereof, and fragments thereof.
  • the one or more chimeric polypeptide receptors can comprise an antigen binding domain against the at least one immune cell antigen, and the antigen binding domain can comprise an ankyrin repeat domain.
  • the antigen binding domain can be based on a DARPIn polypeptide or a modification thereof.
  • an engineered NK Cell can comprise a CAR comprising an anti-CD4 DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3.
  • an engineered NK Cell can comprise a CAR comprising an anti-CD4 DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 4.
  • Example 2 Engineered immune cells configured to target cancer cell antigens
  • the engineered immune cells of the present disclosure can be configured to specifically target and bind immune cells (e.g., diseased immune cells) of a subject, e.g., to treat leukemia or lymphoma.
  • the engineered immune cells can comprise one or more chimeric polypeptide receptors (e.g., chimeric antigen receptors) exhibiting specific binding to at least one cancer cell antigen, e.g., CD19, HER2, EGFR, etc.
  • the one or more chimeric polypeptide receptors can comprise an antigen binding domain against the at least one immune cell antigen, and the antigen binding domain can comprise an ankyrin repeat domain.
  • the antigen binding domain can be based on a DARPIn polypeptide or a modification thereof.
  • an engineered NK Cell can comprise a CAR comprising an anti-HER2 DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 5.
  • an engineered NK Cell can comprise a CAR comprising an anti-EGFR DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%) sequence identity to the polynucleotide sequence of a member selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • Example 3 Engineered NK cells
  • Human iPSC cells can be engineered by knocking in gene edits such as a chimeric antigen receptor (CAR) comprising a DARPin domain (e.g., anti-HER2 DARPin) .
  • CAR chimeric antigen receptor
  • DARPin domain e.g., anti-HER2 DARPin
  • Such engineered iPSC cells can be differentiated into NK cells.
  • peripheral blood (PB) -NK cells e.g., human PB-NK cells
  • AAV system encoding at least the CAR comprising the DARPin domain.
  • the cells as disclosed herein can be engineered to further comprise (i) a heterologous CD16 variant (e.g., hnCD16) and/or (ii) a heterologous cytokine and/or a receptor thereof (e.g., IL15-IL15Ra fusion) .
  • a heterologous CD16 variant e.g., hnCD16
  • a heterologous cytokine and/or a receptor thereof e.g., IL15-IL15Ra fusion
  • a variety of breast cancer cell lines including, HER2-negative lines (LCL lymphoma, MDA-MB-468, U87 glioma) , low-HER2 expressing lines (MDA-MB-361, 231BR) and high-HER2 expressing lines (SKBR3, BT474, BBM1) can be used to characterize in vitro anti-tumor activity of the engineered NK cells expressing a CAR comprising anti-HER2 DARPin.
  • Flow cytometry can be used to assess tumor cell killing following co-culture (e.g., a 72h co-culture) of the engineered NK cell comprising anti-HER2 DARPin CAR, or control NK cells without the CAR or with a CAR comprising anti-HER2 scFv with tumor targets.
  • Tumor cell killing with Effector: HER2 positive cells (i.e., tumor cell) (E: T) ratio ranging from 0.25: 1 to 2: 1 can be measured for the CAR NK cells as disclosed herein.
  • the engineered NK cells comprising the anti-HER2 DARPin CAR can be more effective in tumor cell killing in vitro than the control NK cells without the CAR.
  • the engineered NK cells comprising the anti-HER2 DARPin CAR can be as effective, if not more effective, in tumor cell killing in vitro than the control NK cells with the CAR comprising anti-HER2 scFv.
  • the activity of the engineered NK cells comprising anti-HER2 DARPin CAR can be assessed in a patient-derived breast-to-brain metastasis model.
  • Mice can be treated by administration of engineered NK cells comprising anti-HER2 DARPin CAR, or control NK cells without the CAR or with a CAR comprising anti-HER2 scFv.
  • Optical imaging of the tumors and Kaplan-Meier survival curves can be used to analyze mice that are treated (e.g., treated locally or intravenously, with either at day 3, 8 or 14 post administration of the engineered NK cells) .
  • BBM1 cells e.g., 0.2M
  • BT474 e.g., 0.15M
  • the engineered NK cells herein can be administered (e.g., administered intratumorally)
  • BBM1 and/or BT474 tumors can be monitored by luciferase-based optical imaging. Kaplan Meier curves can be used to represent the optical imaging data.
  • Example 4 Engineered NK cells
  • NK cells e.g., NK92 cell line
  • NK92 cell line can be engineered by knocking in gene edits (e.g., via AAV delivery) , such as a chimeric antigen receptor (CAR) comprising a DARPin domain (e.g., anti-EGFR DARPin, such as the polynucleotide sequence of SEQ ID NO: 6) .
  • CAR chimeric antigen receptor
  • DARPin domain e.g., anti-EGFR DARPin, such as the polynucleotide sequence of SEQ ID NO: 6
  • the cells as disclosed herein can be engineered to further comprise (i) a heterologous CD16 variant (e.g., hnCD16) and/or (ii) a heterologous cytokine and/or a receptor thereof (e.g., IL15-IL15Ra fusion) .
  • a heterologous CD16 variant e.g., hnCD
  • the ability of the engineered NK cells to induce killing (e.g., in vitro killing) of target cells can be assayed.
  • the target cells for the killing assays can include K562 cells expressing the target antigen (e.g., EGFR+ K562 cells) .
  • the E: T (Effector: Target) ratio for the killing assays can arrange from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, to 10. After the target cells are incubated with the engineered NK cells (e.g., for 4 hours) , the respective percentage of specific killing (target cell lysis) can be calculated.
  • NK92 cells were engineered to express a CAR comprising an extracellular domain that comprises anti-EGFR DARPin comprising the polynucleotide sequence of SEQ ID NO: 6 (i.e., NK92-E01) , in order to specifically target and kill EGFR-expressing cells such as EGFR+ K562 cells. See illustration in FIG. 2.
  • the NK92 cells were engineered by knocking in the gene construct as shown as “DNA structure” in FIG. 3.
  • the gene construct encoded a CAR under the control of a constitutive promoter for mammalian systems (e.g., SV40, CMV, UBC, EF1a, PGK and CAGG) , such as human EF1a promoter.
  • a constitutive promoter for mammalian systems e.g., SV40, CMV, UBC, EF1a, PGK and CAGG
  • human EF1a promoter e.g., SV40, CMV, UBC, EF1a, PGK and CAGG
  • the gene construct further encoded a CAR comprising a lead sequence (e.g., CD8 lead sequence) , the anti-EGFR DARpin (i.e., E01-DARPin) , a linker (e.g., GGGS e linker such as 2xGGGS) , a transmembrane domain (e.g., CD8 transmembrane domain) , and two intracellular signaling domains (e.g., 2B4 and CD3zeta) .
  • the gene construct further encoded a reporter gene (e.g., EGFP reporter) under the control of an internal ribosome entry site (IRES) , such that the reporter would be cleaved from the CAR upon transcription and translation.
  • a reporter gene e.g., EGFP reporter
  • the polynucleotide sequence of the CAR comprising the anti-EGFR DARPin is shown in SEQ ID NO: 10.
  • the polynucleotide sequence of SEQ ID NO: 10 comprises CD8 lead sequence (SEQ ID NO: 11) , anti-EGFR DARPin (SEQ ID NO: 6) , a linker (SEQ ID NO: 12) , CD8 transmembrane domain (SEQ ID NO: 13) , 2B4 cytoplastic signaling domain (SEQ ID NO: 14) , and CD3zeta signaling domain (SEQ ID NO: 15) .
  • NK92-E01 In vitro cell killing efficacy of the engineered NK92-E01 cells was assessed, as illustrated in FIG. 4.
  • the engineered NK92-E01 cells were cultured with EGFR+ K562 cancer cells (i.e., NK92-E01: K562-EGFR) .
  • the engineered NK92-E01 cells were cultured with K562 cells that are substantially free of EGFR expression (i.e., NK92-E01: K562) .
  • NK92 cells that do not express the anti-EGFR DARPin CAR were cultured with either the EGFR+ K562 cells (i.e., NK92: K562-EGFR) or with K562 cells that are substantially free of EGFR expression (i.e., NK92: K562) .
  • the E: T (Effector: Target) ratios tested for the killing assays were 4: 1, 2: 1, 1: 1, 0.5: 1, and 0.25: 1. As shown in FIG.
  • the highest degree of target cell killing (i.e., lysis cells %) was observed between the engineered NK92 cells expressing the anti-EGFR DARpin CAR (i.e., NK92-E01) and the EGFR+ K562 cancer cells (i.e., K562-EGFR) across multiple E: T ratios, such as 4: 1, 2: 1, 1: 1, and 0.5: 1, indicating (i) that NK cells expressing a CAR comprising EGFR-specific DARPin can efficiently kill EGFR-expressing cancer/tumor cells, and (ii) that NK cells expressing a CAR comprising a DARPin against a specific antigen can efficiently bind and kill cancer/tumor cells that express the specific antigen.

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Abstract

L'invention concerne des cellules immunitaires et des méthodes d'immunothérapie. Les cellules immunitaires peuvent être modifiées pour présenter une demi-vie améliorée 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.
PCT/CN2022/077675 2021-02-24 2022-02-24 Récepteurs chimériques à l'antigène dans des cellules immunitaires Ceased WO2022179562A1 (fr)

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Publication number Priority date Publication date Assignee Title
US12331090B2 (en) 2020-05-14 2025-06-17 Molecular Partners Ag Multispecific proteins
WO2024238832A3 (fr) * 2023-05-16 2025-01-02 Fred Hutchinson Cancer Center Récepteurs recombinants steap1 améliorés par cytokine pro-inflammatoire

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